WO2016030373A1

WO2016030373A1 - Modified microorganism for improved production of fine chemicals on sucrose

Info

Publication number: WO2016030373A1
Application number: PCT/EP2015/069445
Authority: WO
Inventors: Joanna Martyna KRAWCZYK; Stefan Haefner; Hartwig Schröder; Esther DANTAS COSTA; Oskar Zelder; Gregory Von Abendroth
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2014-08-28
Filing date: 2015-08-25
Publication date: 2016-03-03
Anticipated expiration: 2017-02-28

Abstract

The present invention relates to a modified microorganism having, compared to its wildtype, a reduced activity of the enzyme that is encoded by the pfsG-gene, and wherein the modified microorganism expresses an enzyme having the activity of sucrose hydrolase and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pasteurellaceae. The present invention also relates to a method for producing succinic acid and to the use of modified microorganisms.

Description

Modified microorganism for improved production of fine chemicals on sucrose

The present invention relates to a modified microorganism, to a method for producing organic compounds and to the use of modified microorganisms.

Organic compounds such as small dicarboxylic acids having 6 or fewer carbons are commercially significant chemicals with many uses. For example, the small diacids include 1 ,4-diacids, such as succinic acid, malic acid and tartaric acid, and the 5-carbon molecule itaconic acid. Other diacids include the two carbon oxalic acid, three carbon malonic acid, five carbon glutaric acid and the 6 carbon adipic acid and there are many derivatives of such diacids as well.

As a group the small diacids have some chemical similarity and their uses in polymer production can provide specialized properties to the resin. Such versatility enables them to fit into the downstream chemical infrastructure markets easily. For example, the 1 ,4-diacid molecules fulfill many of the uses of the large scale chemical maleic anhydride in that they are converted to a variety of industrial chemicals (tetrahydrofuran, butyrolactone, 1 ,4-butanediol, 2-pyrrolidone) and the succinate derivatives succindiamide, succinonitrile, diaminobutane and esters of succinate. Tartaric acid has a number of uses in the food, leather, metal and printing industries. Itaconic acid forms the starting material for production of 3-methylpyrrolidone, methyl-BDO, me- thyl-THF and others.

In particular, succinic acid or succinate - these terms are used interchangeably herein - has drawn considerable interest because it has been used as a precursor of many industrially im- portant chemicals in the food, chemical and pharmaceutical industries. In fact, a report from the U.S. Department of Energy reports that succinic acid is one of 12 top chemical building blocks manufactured from biomass. Thus, the ability to make diacids in bacteria would be of significant commercial importance. WO-A-2009/024294 discloses a succinic acid producing bacterial strain, being a member of the family of Pasteurellaceae, originally isolated from rumen, and capable of utilizing glycerol as a carbon source and variant and mutant strains derived there from retaining said capability, in particular, a bacterial strain designated DD1 as deposited with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) having the deposit number DSM 18541 (ID 06-614) and having the ability to produce succinic acid. The DD1 -strain belongs to the species Basfia succiniciproducens and the family of Pasteurellaceae as classified by Kuhnert et a/., 2010. Mutations of these strains, in which the Idh- gene and/or the pfID- or the pflA-gene have been disrupted, are disclosed in WO-A- 2010/092155, these mutant strains being characterized by a significantly increased production of succinic acid from carbon sources such as glycerol or mixtures of glycerol and carbohydrates such as maltose, under anaerobic conditions compared to the DD1 -wildtype disclosed in WO-A- 2009/024294. However, bio-based succinate still faces the challenge of becoming cost competitive against petrochemical-based alternatives. In order to develop the bio-based industrial production of succinic acid, it will be important to grow the cells in a low cost medium, and the working strain optimally should be able to metabolize a wide range of low-cost sugar feedstock to produce succinic acid in good yields so that the cheapest available raw materials can be used.

Sucrose (commonly known as sugar) is a disaccharide consisting of glucose and fructose, and it is a carbon source that is very abundant in nature and is produced from all plants having photosynthesis ability. Particularly, sugarcane and sugar beet contain large amounts of sucrose, and more than 60% of the world's sucrose is currently being produced from sugarcane. Particularly, sucrose is produced at a very low cost, because it can be industrially produced through a simple process of evaporating/concentrating extracts obtained by mechanical pressing of sug- arcanes. Sucrose as a raw material for producing chemical compounds through microbial fermentation is thus inexpensive and it also functions to protect the cell membrane from an exter- nal environment containing large amounts of desired metabolites, thus producing high- concentrations of desired metabolites as shown by Kilimann et al. (Biochimica et Biophysica Acta, 1764, 2006).

Even though sucrose is an excellent raw material having the above-described advantages, in- eluding low price and a function to protect microorganisms from an external environment, the disadvantage of this carbon source can be seen in the fact a large number of microorganisms do not have a complete mechanism of transporting sucrose into cell, degrading the transported sucrose and linking the degraded products to glycolysis, and thus cannot use sucrose as a carbon source. Even in the case of microorganisms having a mechanism capable of using sucrose, they cannot efficiently produce desired metabolites, because the rate of ingestion and degradation of sucrose as a carbon source is very low.

It was therefore an object of the present invention to overcome the disadvantages of the prior art.

In particular, it was an object of the present invention to provide microorganisms which can be used for the fermentative production of organic compounds such as succinic acid and that can efficiently utilize sucrose as a predominant carbon source without sacrificing growth rates or yields. Preferably said microorganisms would be able to use a number of low cost carbon sources and produce excellent yields of organic compounds such as succinic acid. Compared to the recombinant microorganisms of the prior art that are used for the fermentative production of succinic acid, the microorganisms of the present invention should be characterized by an increased succinic acid yield and an increased carbon yield during growth of the cells on sucrose as the predominant carbon source.

A contribution to achieving the above mentioned aims is provided by a modified microorganism having, compared to its wildtype, a reduced activity of the enzyme that is encoded by the ptsG- gene, and wherein the modified microorganism expresses an enzyme having the activity of sucrose hydrolase and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pasteurellaceae. A contribution to achieving the above mentioned aims is in particular provided by a modified microorganism in which the pfsG-gene or parts thereof have been deleted, wherein the modified microorganism expresses an enzyme encoded by a preferably heterologous cscA-gene or expresses an enzyme encoded by at least a fragment of a preferably heterologous cscA-gene or expression a fusion enzyme encoded by a fusion gene comprising at least a fragment of a preferably heterologous cscA-gene, and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pasteurellaceae.

As used herein the term "heterologous" refers to a structural gene not found in wild type microorganism and to polypeptide sequences not produced by such microorganism.

Surprisingly, it has been discovered that a reduction of the activity of the enzyme that is encoded by the pfsG-gene (this enzyme encodes a PEP-dependent sucrose-permease (EIIBC- component; EC:2.7.1 .69) of the bacterial phosphotransferase system which mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation), for example by a deletion of the pfsG-gene, in a microorganism that belongs to the family of Pasteurellaceae, wherein it is ensured that the microorganism expresses an enzyme having the activity of sucrose hydrolase (EC:3.2.1 .26), results in a recombinant Pasteurellaceae-stram that, compared to the corresponding microorganism in which the activity of the enzyme encoded by the pfsG-gene has not been decreased and in which an enzyme having the activity of sucrose hydrolase is not expressed, is characterized by an increased yield of organic compounds such as succinic acid, and also by an increased carbon yield, preferably if these modified microorganisms are grown on sucrose as the assimilable carbon source. This is indeed surprising as according to Lee et al. ^'Mannheimia succiniciproducens Phosphotransferase System for Sucrose Utilization", Applied and Environmental Microbiology (2010), Vol. 76 (5), pages 1.699- 1 .703) a deletion of the sucrose pfs-gene in Manheimia succiniciproducens leads to a strong reduction of the growth of the cells on sucrose, compared to the wildtype.

In context with the expression "a modified microorganism having, compared to its wildtype, a reduced activity of the enzyme that is encoded by the x-gene", wherein the x-gene is the pfsG- gene and optionally, as described later, the IdhA-gene, the pflA-gene, the pflD-gene, the wcaJ- gene, the pykA-gene and/or the fruA-gene, the term "wildtype" refers to a microorganism in which the activity of the enzyme that is encoded by the x-gene has not been decreased, i. e. to a microorganism whose genome is present in a state as before the introduction of a genetic modification of the x-gene (or the IdhA-gene, the pflA-gene, the pflD-gene, the wcaJ-gene, the pykA-gene or the fruA-gene, respectively). Preferably, the expression "wildtype" refers to a mi- croorganism whose genome, in particular whose x-gene, is present in a state as generated naturally as the result of evolution. The term may be used both for the entire microorganism but preferably for individual genes, e.g. the pfsG-gene, the IdhA-gene, the pflA-gene, the pflD-gene, the wcaJ-ge e, the py/oA-gene or the fruA-ge e). The term "modified microorganism" thus includes a microorganism which has been genetically altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype and/or phe- notype (e. g., when the genetic modification affects coding nucleic acid sequences of the micro- organism) as compared to the naturally-occurring wildtype microorganism from which it was derived. According to a particular preferred embodiment of the modified microorganism according to the present invention the modified microorganism is a recombinant microorganism, which means that the microorganism has been obtained using recombinant DNA. The expression "recombinant DNA" as used herein refers to DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. An example of such a recombinant DNA is a plasmid into which a heterologous DNA-sequence has been inserted. For example a heterologous DNA may be the combination of a gene with a non-natural promoter. The wildtype from which the microorganisms according to the present invention are derived (i. e. from which the the microorganisms according to the present invention have been obtained by genetic alteration, modification or engineering) belongs to the family of Pasteurellaceae. Pas- teurellaceae comprise a large family of Gram-negative Proteobacteria with members ranging from bacteria such as Haemophilus influenzae to commensals of the animal and human muco- sa. Most members live as commensals on mucosal surfaces of birds and mammals, especially in the upper respiratory tract. Pasteurellaceae are typically rod-shaped, and are a notable group of facultative anaerobes. They can be distinguished from the related Enterobacteriaceae by the presence of oxidase, and from most other similar bacteria by the absence of flagella. Bacteria in the family Pasteurellaceae have been classified into a number of genera based on metabolic properties and there sequences of the 16S RNA and 23S RNA. Many of the Pasteurellaceae contain pyruvate-formate-lyase genes and are capable of anaerobically fermenting carbon sources to organic acids.

According to a particular preferred embodiment of the modified microorganism according to the present invention the wildtype from which the modified microorganism has been derived belongs to the genus Basfia and it is particularly preferred that the wildtype from which the modified microorganism has been derived belongs to the species Basfia succiniciproducens.

Most preferably, the wildtype from which the modified microorganism according to the present invention as been derived is Basfia succiniciproducens-straln DD1 deposited under the Budapest Treaty with DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenst^e 7B, D-38124 Braunschweig, Germany), having the deposit number DSM 18541 and being deposited on August 1 1 , 2006. This strain has been originally isolated from the rumen of a cow of German origin. Pasteurella bacteria can be isolated from the gastro-intestinal tract of animals and, preferably, mammals. The bacterial strain DD1 , in particular, can be isolated from bovine rumen and is capable of utilizing glycerol (including crude glycerol) as a carbon source. Further strains of the genus Basfia that can be used for preparing the modified microor- ganism according to the present invention are the Sasf/a-strain that has been deposited under the deposit number DSM 22022 with DSZM or the Sasf/a-strains that have been deposited with the Culture Collection of the University of Goteborg (CCUG), Sweden, having the deposit numbers CCUG 57335, CCUG 57762, CCUG 57763, CCUG 57764, CCUG 57765 or CCUG 57766. Said strains have been originally isolated from the rumen of cows of German or Swiss origin.

In this context it is particularly preferred that the wildtype from which the modified microorganism according to the present invention has been derived has a 16S rDNA of SEQ ID NO: 1 or a sequence, which shows a sequence homology of at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99,5%, at least 99,6%, at least 99.7%, at least 99.8% or at least 99.9 % with SEQ ID NO: 1. It is also preferred that the wildtype from which the modified microorganism according to the present invention has been derived has a 23S rDNA of SEQ ID NO: 2 or a sequence, which shows a sequence homology of at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99,5%, at least 99,6%, at least 99.7%, at least 99.8% or at least 99.9 % with SEQ ID NO: 2.

The identity in percentage values referred to in connection with the various polypeptides or polynucleotides to be used for the modified microorganism according to the present invention is, preferably, calculated as identity of the residues over the complete length of the aligned se- quences, such as, for example, the identity calculated (for rather similar sequences) with the aid of the program needle from the bioinformatics software package EMBOSS (Version 5.0.0, http://emboss.source-forge.net/what ) with the default parameters which are, i.e. gap open (penalty to open a gap): 10.0, gap extend (penalty to extend a gap): 0.5, and data file (scoring matrix file included in package): EDNAFUL.

It should be noted that the modified microorganism according to the present invention can not only be derived from the above mentioned wildtype-microorganisms being present in a state as generated naturally as the result of evolution, especially from Basfia succiniciproducens-straln DD1 , but also from variants of these strains. In this context the expression "a variant of a strain" comprises every strain having the same or essentially the same characteristics as the wildtype- strain. In this context it is particularly preferred that the 16 S rDNA of the variant has an identity of at least 90 %, preferably at least 95 %, more preferably at least 99 %, more preferably at least 99.5 %, more preferably at least 99.6 %, more preferably at least 99.7 %, more preferably at least 99.8 % and most preferably at least 99.9 % with the wildtype from which the variant has been derived. It is also particularly preferred that the 23 S rDNA of the variant has an identity of at least 90 %, preferably at least 95 %, more preferably at least 99 %, more preferably at least 99.5 %, more preferably at least 99.6 %, more preferably at least 99.7 %, more preferably at least 99.8 % and most preferably at least 99.9 % with the wildtype from which the variant has been derived. A variant of a strain in the sense of this definition can, for example, be obtained by treating the wildtype-strain with a mutagenizing chemical agent, X-rays, or UV light. The modified microorganism according to the present invention is characterized in that, compared to its wildtype, the activity of the enzyme that is encoded by the pfsG-gene is reduced.

The reduction of the enzyme activity (A_activit_y) is preferably defined as follows: activity of the modified microorganism

Aactivity = 100% 100%

activity of the wildtype wherein, when determining A_activit_y, the activity in the wildtype and the activity in the modified microorganism are determined under exactly the same conditions. Methods for the detection and determination of the activity of the enzyme that is encoded by the pfsG-gene can be found, for example, in Lee et al., "Mannheimia succiniciproducens Phosphotransferase System for Sucrose Utilization", Applied and Environmental Microbiology (2010), Vol. 76 (5), pages 1.699- 1 .703. The reduced activity of the enzymes disclosed herein, in particular the reduced activity of the enzyme encoded by the pfsG-gene, the lactate dehydrogenase, the pyruvate formate lyase, the enzyme encoded by the wcaJ-gene and/or the enzyme encoded by the fruA-gene, can be a reduction of the enzymatic activity by at least 50%, compared to the activity of said enzyme in the wildtype of the microorganism, or a reduction of the enzymatic activity by at least 90%, or more preferably a reduction of the enzymatic activity by at least 95%, or more preferably a reduction of the enzymatic activity by at least 98%, or even more preferably a reduction of the enzymatic activity by at least 99% or even more preferably a reduction of the enzymatic activity by at least 99.9%. In connection with the pykA-gene, the reduced activity can be a reduction a reduction of the activity of the enzyme encoded by the pykA-gene in the range of 15 to 99 %, more preferably in the range of 50 to 95 % and even more preferably in the range of 90 to 99 %. The term "reduced activity of the enzyme that is encoded by the ptsG-gene" or - as described below - "a reduced lactate dehydrogenase activity, "a reduced pyruvate formate lyase activity, "a reduced activity of the enzyme encoded by the wcaJ-gene", "a reduced activity of the enzyme encoded by the pykA-gene" and/or "a reduced activity of the enzyme encoded by the fruA-gene"a\so encompasses a modified microorganism which has no detectable activity of these enzymes.

The term "reduced activity of an enzyme" includes, for example, the expression of the enzyme by said genetically modified (e.g., genetically engineered) microorganism at a lower level than that expressed by the wildtype of said microorganism. Genetic manipulations for reducing the expression of an enzyme can include, but are not limited to, altering or modifying regulatory sequences or sites associated with expression of the gene encoding the enzyme (e.g., by removing strong promoters or repressible promoters), modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of the gene encoding the enzyme and/or the translation of the gene product, or any other conventional means of decreasing expression of a particular gene routine in the art (including, but not limited to, the use of antisense nucleic acid molecules or other methods to knock-out or block expression of the target protein). Further on, one may introduce destabilizing elements into the mRNA or introduce genetic modifications leading to deterioration of ribosomal binding sites (RBS) of the RNA. It is also possible to change the codon usage of the gene in a way, that the translation efficiency and speed is decreased.

A reduced activity of an enzyme can also be obtained by introducing one or more gene mutations which lead to a reduced activity of the enzyme. Furthermore, a reduction of the activity of an enzyme may also include an inactivation (or the reduced expression) of activating enzymes which are necessary in order to convert an enzyme in its active form. By the latter approach the enzyme the activity of which is to be reduced is preferably kept in an inactivated state.

Microorganisms having a reduced activity of the enzyme encoded by the ptsG-gene may occur naturally, i.e. due to spontaneous mutations. A microorganism can be modified to lack or to have significantly reduced activity of the enzyme that is encoded by the pfsG-gene by various techniques, such as chemical treatment or radiation. To this end, microorganisms will be treated by, e.g., a mutagenizing chemical agent, X-rays, or UV light. In a subsequent step, those microorganisms which have a reduced activity of the enzyme that is encoded by the pfsG-gene will be selected. Modified microorganisms are also obtainable by homologous recombination tech- niques which aim to mutate, disrupt or excise the pfsG-gene in the genome of the microorganism or to substitute the gene with a corresponding gene that encodes for an enzyme which, compared to the enzyme encoded by the wildtype-gene, has a reduced activity.

According to a preferred embodiment of the modified microorganism according to the present invention a reduction of the activity of the enzyme encoded by the pfsG-gene is achieved by a modification of the pfsG-gene, wherein this gene modification is preferably realized by a deletion of the pfsG-gene or at least a part thereof, a deletion of a regulatory element of the pfsG- gene or at least a part thereof, such as a promotor sequence, by replacing the regulatory element of the pfsG-gene with a different regulatory element leading to a reduced transcription or translation of the pfsG gene, by antisense technologies, by RNAi-technologies, by an introduction of at least one mutation into the pfsG-gene or by substitution of the pfsG-gene by an inactive pfs-gene. In the following, a suitable technique for recombination, in particular for introducing a mutation or for deleting sequences, is described. Particularly preferred is the at least partial substitution of the pfsG-gene by a preferably heterologous cscA-gene, but it is also possible to delete the ptsG-gene or at least a part thereof in a different manner and to introduce the preferably heterologous cscA-gene in a different location of the genome of the microorganism.

This technique is also sometimes referred to as the "Campbell recombination" herein (Leen- houts et al., Appl Env Microbiol. (1989), Vol. 55, pages 394-400). "Campbell in", as used herein, refers to a transformant of an original host cell in which an entire circular double stranded DNA molecule (for example a plasmid) has integrated into a chromosome by a single homologous recombination event (a cross in event), and that effectively results in the insertion of a linearized version of said circular DNA molecule into a first DNA sequence of the chromosome that is homologous to a first DNA sequence of the said circular DNA molecule. "Campbelled in" refers to the linearized DNA sequence that has been integrated into the chromosome of a "Campbell in" transformant. A "Campbell in" contains a duplication of the first homologous DNA sequence, each copy of which includes and surrounds a copy of the homologous recombination crossover point.

"Campbell out", as used herein, refers to a cell descending from a "Campbell in" transformant, in which a second homologous recombination event (a cross out event) has occurred between a second DNA sequence that is contained on the linearized inserted DNA of the "Campbelled in" DNA, and a second DNA sequence of chromosomal origin, which is homologous to the second DNA sequence of said linearized insert, the second recombination event resulting in the deletion (jettisoning) of a portion of the integrated DNA sequence, but, importantly, also resulting in a portion (this can be as little as a single base) of the integrated Campbelled in DNA remaining in the chromosome, such that compared to the original host cell, the "Campbell out" cell contains one or more intentional changes in the chromosome (for example, a single base substitution, multiple base substitutions, insertion of a heterologous gene or DNA sequence, insertion of an additional copy or copies of a homologous gene or a modified homologous gene, or insertion of a DNA sequence comprising more than one of these aforementioned examples listed above). A "Campbell out" cell is, preferably, obtained by a counter-selection against a gene that is contained in a portion (the portion that is desired to be jettisoned) of the "Campbelled in" DNA sequence, for example the Bacillus subtilis sacB gene, which is lethal when expressed in a cell that is grown in the presence of about 5% to 10% sucrose. Either with or without a counter- selection, a desired "Campbell out" cell can be obtained or identified by screening for the de- sired cell, using any screenable phenotype, such as, but not limited to, colony morphology, colony color, presence or absence of antibiotic resistance, presence or absence of a given DNA sequence by polymerase chain reaction, presence or absence of an auxotrophy, presence or absence of an enzyme, colony nucleic acid hybridization, antibody screening, etc. The term "Campbell in" and "Campbell out" can also be used as verbs in various tenses to refer to the method or process described above.

It is understood that the homologous recombination events that leads to a "Campbell in" or "Campbell out" can occur over a range of DNA bases within the homologous DNA sequence, and since the homologous sequences will be identical to each other for at least part of this range, it is not usually possible to specify exactly where the crossover event occurred. In other words, it is not possible to specify precisely which sequence was originally from the inserted DNA, and which was originally from the chromosomal DNA. Moreover, the first homologous DNA sequence and the second homologous DNA sequence are usually separated by a region of partial non-homology, and it is this region of non-homology that remains deposited in a chro- mosome of the "Campbell out" cell. Preferably, first and second homologous DNA sequence are at least about 200 base pairs in length, and can be up to several thousand base pairs in length. However, the procedure can be made to work with shorter or longer sequences. For example, a length for the first and second homologous sequences can range from about 500 to 2000 bases, and the obtaining of a "Campbell out" from a "Campbell in" is facilitated by arranging the first and second homologous sequences to be approximately the same length, preferably with a difference of less than 200 base pairs and most preferably with the shorter of the two being at least 70% of the length of the longer in base pairs. The pfsG-gene the activity of which is reduced in the modified microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: nucleic acids having the nucleotide sequence of SEQ ID NO: 3; b1 ) nucleic acids encoding the amino acid sequence of SEQ ID NO: 4; c1 ) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identi- cal to the nucleic acid of a1 ) or b1 ), the identity being the identity over the total length of the nucleic acids of a1 ) or b1 ); d1 ) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least

99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of a1 ) or b1 ), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a1 ) or b1 ); e1 ) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a1 ) or b1 ); and f1 ) nucleic acids encoding the same protein as any of the nucleic acids of a1 ) or b1 ), but differing from the nucleic acids of a1 ) or b1 ) above due to the degeneracy of the genetic code.

The term "hybridization" as used herein includes "any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing" (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules.

As used herein, the term "Tm" is used in reference to the "melting temperature". The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm = 81.5 + 0.41 (% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCI (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent conditions, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. In particular, the term "stringency conditions" refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 2 x SSC, 0.1 % SDS at 50°C or 65°C, preferably at 65°C, with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably, the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 1 x SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C, more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1 * SSC, 0.1 % SDS at 50°C or 65°C, preferably 65°C. Preferably, the complementary nucleotides hybridize with a fragment or the whole pykA nucleic acids. Alternatively, preferred hybridization conditions encompass hybridisation at 65°C in 1 ^χ SSC or at 42°C in 1 ^χ SSC and 50% formamide, followed by washing at 65°C in 0.3 * SSC or hybridisation at 50°C in 4 x SSC or at 40°C in 6 * SSC and 50% formamide, followed by washing at 50°C in 2 x SSC. Further preferred hybridization conditions are 0.1 % SDS, 0.1 SSD and 65°C.

The pfsG-gene or parts of which that may be deleted by the above mentioned "Campbell recombination" or in which at least one mutation is introduced by the above mentioned "Campbell recombination" preferably comprises a nucleic acid as defined above.

Nucleic acid having the nucleotide sequence of SEQ ID NO: 3 corresponds to the pfsG-gene of Basfia succiniciproducens-stra^'m DDL

The modified microorganism according to the present invention is also characterized in that it expresses an enzyme having the activity of sucrose hydrolase, preferably having the activity of sucrose hydrolase encoded by the cscA-gene. Generally, the modified microorganism according to the present invention can be characterized in that the wildtype of the microorganism al- ready expresses an enzyme having the activity of sucrose hydrolase (in this case the reduced activity of the enzyme encoded by the pfsG-gene can be the sole genetic modification), or in that the wildtype of the microorganism does not express an enzyme having the activity of sucrose hydrolase (in this case the reduced activity of the enzyme encoded by the pfsG-gene is not the sole genetic modification and the microorganism is furthermore characterized by an expression of a heterologous enzyme having the activity of sucrose hydrolase).

According to a preferred embodiment of the modified microorganism according to the present invention the wildtype of the microorganism does not express an enzyme having the activity of sucrose hydrolase, in particular an enzyme having the activity of sucrose hydrolase encoded by the cscA-gene, and an expression of an enzyme having the activity of a sucrose hydrolase (EC:3.2.1 .26) is ensured by the introduction of a heterologous cscA-gene or at least a fragment thereof. In this particular embodiment the modified microorganism therefore has, compared to its wildtype, an increased activity of sucrose hydrolase and it is particularly preferred that the increased activity of sucrose hydrolase is accomplished by the expression of an enzyme encoded by a cscA-gene, by the expression of an enzyme encoded by a fragment of a cscA-gene or by the expression of a fusion enzyme encoded by a fusion gene comprising at least a fragment of a cscA-gene, provided that the enzyme encoded by the fragment of the cscA-gene and the fusion enzyme encoded by the fusion gene comprising at least a fragment of a cscA-gene have the activity of sucrose hydrolase. In this context it is furthermore preferred that the ptsG- gene or at least a part of this gene is replaced by the heterologous cscA-gene or by at the fragment of the cscA-gene, which leads to a reduced activity of the enzyme encoded by the ptsG- gene (preferably to a complete inactivation of this enzyme) and, at the same time, to the expression of a heterologous enzyme encoded by the cscA-gene or to the expression of at least a part of a heterologous enzyme encoded by the cscA-gene.

The increase of the enzyme activity (A_activit_y) is - in case of a wildtype which already has a certain activity of sucrose hydrolase - preferably defined as follows: activity of the modified microorganism

^activity x 100 /o -100%

activity of the wildtype wherein, when determining A_activit_y, the activity in the wildtype and the activity in the modified microorganism are determined under exactly the same conditions. The increased activity of activity of sucrose hydrolase can be an increase of the enzymatic activity by 1 to 10000%, compared to the activity of said enzyme in the wildtype of the microorganism, or an increase of the enzymatic activity by at least 50 %, or at least 100 %, or at least 200 %, or at least 300 %, or at least 400 %, or at least 500 %, or at least 600 % or at least 700 %, or at least 800 %, or at least 900 %, or at least 1000 %, or at least 5000 %. Preferably, the increase of the activity of an enzyme is in the range of 10 to 1000 %, more preferably in the range of 100 to 500 %. The cscA-gene encoding for the sucrose hydrolase that is expressed by the recombinant microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of:

a2) nucleic acids having the nucleotide sequence of SEQ ID NO: 5; b2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 6; c2) nucleic acids which are at least 70% identical to the nucleic acid of a2) or b2), the identity being the identity over the total length of the nucleic acids of a2) or b2); d2) nucleic acids encoding an amino acid sequence which is at least 70% identical to the amino acid sequence encoded by the nucleic acid of a2) or b2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a2) or b2); e2) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a2) or b2); and f2) nucleic acids encoding the same protein as any of the nucleic acids of a2) or b2), but differing from the nucleic acids of a2) or b2) above due to the degeneracy of the genetic code.

SEQ ID NO: 5 corresponds to the cscA-gene of E. co//^' W (ATCC 9637).

In the case in which only a fragment of the heterologous cscA-gene is introduced into the microorganisms, it is preferred that this part comprises at least 500 nucleotides (e. g. nucleotide 935 to 1434 of SEQ ID NO: 5) of the cscA-gene, preferably at least 600 nucleotides (e. g. nucleotide 835 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably at least 700 nucleo- tides (e. g. nucleotide 735 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably at least 800 nucleotides (e. g. nucleotide 635 to 1434 of SEQ ID NO: 5) of the cscA-gene and most preferably at least 900 nucleotides (e. g. nucleotide 535 to 1434 of SEQ ID NO: 5) of the cscA- gene. Furthermore, in the case in which only a fragment of the heterologous cscA-gene is introduced into the microorganisms, it is also possible that this fragment of the cscA-gene is fused with a fragment of the argD-gene (encoding for N-acetylornithin-aminotransferase; EC:2.6.1 .1 1 and/or EC 2.6.1 .17) such that the modified microorganism expresses a fusion protein consisting of two parts, a first part being a part of the N-acetylornithin-aminotransferase encoded by the argD- gene and a second part being a part of the sucrose hydrolase encoded by the cscA-gene. The argD-gene encoding for the N-acetylornithin-aminotransferase a part of which may be the first part of a fusion protein preferably comprises a nucleic acid selected from the group consisting of: a3) nucleic acids having the nucleotide sequence of SEQ ID NO: 7;

b3) nucleic acids encoding the amino acid sequence of SEQ ID NO: 8; c3) nucleic acids which are at least 70% identical to the nucleic acid of a3) or b3), the identity being the identity over the total length of the nucleic acids of a3) or b3); d3) nucleic acids encoding an amino acid sequence which is at least 70% identical to the amino acid sequence encoded by the nucleic acid of a3) or b3), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a3) or b3); e3) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a3) or b3); and f3) nucleic acids encoding the same protein as any of the nucleic acids of a3) or b3), but differing from the nucleic acids of a3) or b3) above due to the degeneracy of the genetic code.

SEQ ID NO: 7 corresponds to the argD-gene of Basfia succiniciproducens-straln DDL The part being the first part of the fusion protein is preferably encoded by at least 500 nucleotides (e. g. nucleotide 1 to 500 of SEQ ID NO: 7) of the argD-gene, preferably by at least 600 nucleotides (e. g. nucleotide 1 to 600 of SEQ ID NO: 7) of the argD-gene, more preferably by at least 700 nucleotides (e. g. nucleotide 1 to 700 of SEQ ID NO: 7) of the argD-gene, more preferably by at least 800 nucleotides (e. g. nucleotide 1 to 800 of SEQ ID NO: 7) of the argD-gene and most preferably by at least 900 nucleotides (e. g. nucleotide 1 to 900 of SEQ ID NO: 7) of the argD-gene, whereas the part being the second part of the fusion protein is preferably encoded by at least 500 nucleotides (e. g. nucleotide 935 to 1434 of SEQ ID NO: 5) of the cscA- gene, preferably by at least 600 nucleotides (e. g. nucleotide 835 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably by at least 700 nucleotides (e. g. nucleotide 735 to 1434 of SEQ ID NO: 5) of the cscA-gene, more preferably by at least 800 nucleotides (e. g. nucleotide 635 to 1434 of SEQ ID NO: 5) of the cscA-gene and most preferably by at least 900 nucleotides (e. g. nucleotide 535 to 1434 of SEQ ID NO: 5) of the cscA-gene.

In case where the wildtype of the microorganism does not express an enzyme having the activi- ty of sucrose hydrolase, the expression of such an enzyme in particular by introducing a heterologous cscA-gene or a part thereof can be ensured by recombinant methods known to the person skilled in the art, for example by transformation, transduction, conjugation, or a combination of these methods with a vector containing the desired gene or the desired part of the gene, an allele of this gene or parts thereof and ensuring an expression of the gene in the microorganism. Heterologous expression can be achieved in particular by integration of the gene or of a part thereof into the chromosome of the cell or an extrachromosomally replicating vector. In view of the fact that the reduced activity of the enzyme that is encoded by the pfsG-gene is preferably accomplished by the above described "Campbell recombination", the heterologous cscA-gene can be integrated into the vector that is used for this recombination or can be introduced before or after the "Campbell recombination" in a separate vector. In this context it is also preferred that the modified microorganism according to the present invention is not characterized by an increased activity of sucrose permease (EC 2.7.1.69) encoded by the cscS-gene and/or an increased activity of fructokinase (EC 2.7.1 .4) encoded by the cscK-gene. In this context it is particularly preferred that neither sucrose permease encoded by the cscS-gene nor fructokinase encoded by the cscK-gene are expressed in an increased amount compared to the wildtype.

According to a preferred embodiment of the modified microorganism according to the present invention, this microorganism is not only characterized by a reduced activity of the enzyme encoded by the pfsG-gene and by the expression of an enzyme having the activity of sucrose hy- drolase, but also, compared to its wildtype, i) by a reduced pyruvate formate lyase activity, and/or ii) by a reduced lactate dehydrogenase activity, and/or iii) by a reduced activity of an enzyme encoded by the wcaJ-gene, and/or iv) by a reduced activity of an enzyme encoded by the pykA-gene, and/or v) by a reduced activity of an enzyme encoded by the fruA-gene.

Modified microorganisms being deficient in lactate dehydrogenase and/or being deficient in pyruvate formate lyase activity are disclosed in WO-A-2010/092155, US 2010/0159543 and WO- A-2005/052135, the disclosure of which with respect to the different approaches of reducing the activity of lactate dehydrogenase and/or pyruvate formate lyase in a microorganism, preferably in a bacterial cell of the genus Pasteurella, particular preferred in Basfia succiniciproducens strain DD1 , is incorporated herein by reference. Methods for determining the pyruvate formate lyase activity are, for example, disclosed by Asanum N. and Hino T. in "Effects of pH and Energy Supply on Activity and Amount of Pyruvate-Formate-Lyase in Streptococcus bovis", Appl. Environ. Microbiol. (2000), Vol. 66, pages 3773-3777 and methods for determining the lactate dehydrogenase activity are, for example, disclosed by Bergmeyer, H.U., Bergmeyer J. and Grassl, M. (1983-1986) in "Methods of Enzymatic Analysis", 3^rd Edition, Volume III, pages 126- 133, Verlag Chemie, Weinheim. In this context it is preferred that the reduction of the activity of lactate dehydrogenase is achieved by an inactivation of the IdhA-gene (which encodes the lactate dehydrogenase LdhA; EC 1 .1 .1.27 or EC 1.1 .1 .28) and the reduction of the pyruvate formate lyase is achieved by an inactivation of the pflA-gene (which encodes for an activator of pyruvate formate lyase PfIA; EC 1.97.1.4) or the pflD-gene (which encodes the pyruvate formate lyase PfID; EC 2.3.1 .54), wherein the inactivation of these genes (i. e. IdhA, pfIA and pfID) is preferably achieved by a deletion of theses genes or parts thereof, by a deletion of a regulatory element of these genes or at least a part thereof or by an introduction of at least one mutation into these genes, particu- lar preferred by means of the "Campbell recombination" as described above.

The IdhA-gene the activity of which may be reduced in the modified microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: a1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 9; a2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 10; a3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least

99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of a1 ) or a2), the identity being the identity over the total length of the nucleic acids of a1 ) or a2); a4) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of a1 ) or a2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a1 ) or a2); a5) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a1 ) or a2); and a6) nucleic acids encoding the same protein as any of the nucleic acids of a1 ) or a2), but differing from the nucleic acids of a1 ) or a2) above due to the degeneracy of the genetic code.

Nucleic acid having the nucleotide sequence of SEQ ID NO: 9 corresponds to the IdhA-gene of Basfia succiniciproducens-straln DD1 . The pf/A-gene the activity of which may be reduced in the modified microorganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: β1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 11 ; β2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 12; β3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of β1 ) or β2), the identity being the identity over the total length of the nucleic acids of β1 ) or β2); β4) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of β1 ) or β2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of β1 ) or β2); β5) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to β1 ) or β2); and β6) nucleic acids encoding the same protein as any of the nucleic acids of β1 ) or β2), but differing from the nucleic acids of β1 ) or β2) above due to the degeneracy of the genetic code.

Nucleic acid having the nucleotide sequence of SEQ ID NO: 11 corresponds to the pf/A-gene of Basfia succiniciproducens-stra^'m DDL

The pf/D-gene the activity of which may be reduced in the modified microroganism according to the present invention preferably comprises a nucleic acid selected from the group consisting of: γ1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 13; γ2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 14; γ3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of γ1 ) or y2), the identity being the identity over the total length of the nucleic acids of γ1 ) or y2); nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of γ1 ) or γ2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of γ1 ) or y2); nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to γ1 ) or γ2); and nucleic acids encoding the same protein as any of the nucleic acids of γ1 ) or y2), but differing from the nucleic acids of γ1 ) or γ2) above due to the degeneracy of the genetic code.

Nucleic acid having the nucleotide sequence of SEQ ID NO: 13 corresponds to the pflD-gene of Basfia succiniciproducens-stra^'m DDL

The wcaJ-gene the activity of which may be reduced in the modified microorganism according to the present invention presumably encodes for an enzyme being a glucose transferase, whereas the pykA-gene encodes for a pyruvate kinase catalyzing the conversion of phosphoe- nolpyruvate (PEP) to pyruvate (EC 2.7.1.40). The fruA-gene the activity of which may be reduced in the modified microorganism according to the present invention presumably encodes for a fructose-specific phosphotransferase.

Microorganisms having a reduced activity of the enzyme encoded by the wcaJ-gene or the enzyme encoded by the pykA-gene may occur naturally, i.e. due to spontaneous mutations. A microorganism can be modified to lack or to have significantly reduced activity of the enzyme that is encoded by the wcaJ-gene or the pykA-gene by various techniques, such as chemical treat- ment or radiation. To this end, microorganisms will be treated by, e.g., a mutagenizing chemical agent, X-rays, or UV light. In a subsequent step, those microorganisms which have a reduced activity of the enzyme that is encoded by the wcaJ-gene or the pykA-gene will be selected. Modified microorganisms are also obtainable by homologous recombination techniques which aim to mutate, disrupt or excise the wcaJ-gene or the pykA-gene in the genome of the microor- ganism or to substitute the gene with a corresponding gene that encodes for an enzyme which, compared to the enzyme encoded by the wildtype-gene, has a reduced activity.

In the modified microorganism according to the present invention a reduction of the activity of the enzyme encoded by the wcaJ-gene or a reduction of the activity of the enzyme encoded by the fruA-gene is preferably achieved by a modification of the wcaJ-gene and the fruA-gene, respectively, wherein this gene modification is preferably realized by a deletion of the wcaJ-gene and/or the fruA-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ- gene and/or the fruA-gene or at least a part thereof, such as a promotor sequence, or by an introduction of at least one mutation into the wcaJ-gene and/or into the fruA-gene. Particularly preferred in this context is a deletion of the wcaJ-gene and/or the fruA-gene by "Campbell recombination" as described above. Also particularly preferred is an introduction of at least one mutation into the wcaJ-gene, wherein this mutation preferably leads to the expression of a truncated enzyme encoded by the wcaJ-gene. In this context it is furthermore preferred that in the truncated enzyme at least 100 amino acids, preferably at least 125 amino acids, more preferred at least 150 amino acids and most preferred at least 160 amino acids of the wildtyp enzyme encoded by the wcaJ-gene are deleted from the C-terminal end. Such a truncated enzyme en- coded the wcaJ-gene can, for example, be obtained by inserting or deleting nucleotides at appropriate positions within the wcaJ-gene gene which leads to a frame shift mutation, wherein by means of this frame shift mutation a stop codon introduced. For example, insertion of a nucleotide in the codon that encodes of lysine between thymine at position 81 and adenine at position 82 leads to a frame shift mutation by means of which a stop codon is introduced as shown in SEQ ID NO: 17. Such mutations of the wcaJ-gene can be introduced, for example, by site- directed or random mutagenesis, followed by an introduction of the modified gene into the genome of the microorganism by recombination. Variants of the wcaJ -gene can be are generated by mutating the wcaJ-gene sequence SEQ ID NO: 15 by means of PCR. The "Quickchange Site-directed Mutagenesis Kit' (Stratagene) can be used to carry out a directed mutagenesis. A random mutagenesis over the entire coding sequence, or else only part thereof, of

SEQ ID NO: 15 can be performed with the aid of the "GeneMorph II Random Mutagenesis Kit' (Stratagene).

Furthermore, in the modified microorganism according to the present invention a reduction of the activity of the enzyme encoded by the pykA-gene is preferably also achieved by a modification of the pykA-gene, wherein this gene modification is preferably realized by a deletion of the pykA-gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof, such as a promotor sequence, or by an introduction of at least one mutation into the pykA-gene. It is, however, particularly preferred that the reduction of the activity of the enzyme encoded by the pykA-gene is achieved by introducing at least one mutation into the wildtype-py/oA-gene. A mutation into the pykA-gene can be introduced, for example, by site- directed or random mutagenesis, followed by an introduction of the modified gene into the genome of the microorganism by recombination. Variants of the pykA-gene can be are generated by mutating the pykA-gene sequence by means of PCR. The "Quickchange Site-directed Muta- genesis Kit' (Stratagene) can be used to carry out a directed mutagenesis. A random mutagenesis over the entire coding sequence, or else only part thereof, of the pykA-gene can be performed with the aid of the "GeneMorph II Random Mutagenesis Kit' (Stratagene). The mutagenesis rate is set to the desired amount of mutations via the amount of the template DNA used. Multiple mutations are generated by the targeted combination of individual mutations or by the sequential performance of several mutagenesis cycles. Introduction of the modified gene into the genome of the microorganism can again be accomplished by "Campbell recombination" as described above. Preferably, the wcaJ-ge e comprises a nucleic acid selected from the group consisting of: δ1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 15; δ2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 16; δ3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of δ1 ) or δ2), the identity being the identity over the total length of the nucleic acids of δ1 ) or δ2); δ4) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of δ1 ) or δ2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of δ) or δ2); δ5) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to δ1 ) or δ2); and δ6) nucleic acids encoding the same protein as any of the nucleic acids of δ1 ) or δ2), but differing from the nucleic acids of δ1 ) or δ2) above due to the degeneracy of the genetic code.

Nucleic acid having the nucleotide sequence of SEQ ID NO: 15 corresponds to the wcaJ-gene of Basfia succiniciproducens-stra^'m DDL

Preferably, the pykA-gene comprises a nucleic acid selected from the group consisting of: ε1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 18; s2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 19; s3) nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of ε1 ) or ε2), the identity being the identity over the total length of the nucleic acids of ε1 ) or s2); nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of ε1 ) or ε2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of ε) or s2); nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to ε1 ) or s2); and nucleic acids encoding the same protein as any of the nucleic acids of ε1 ) or ε2), but differing from the nucleic acids of ε1 ) or ε2) above due to the degeneracy of the genetic code. Nucleic acid having the nucleotide sequence of SEQ ID NO: 18 corresponds to the pykA-gene of Basfia succiniciproducens-stra^'m DDL

Examples of modified microorganisms having a reduced activity of the enzyme encoded by the pykA-gene are microorganisms of the genus Basfia and in particular of the species Basfia suc- ciniciproducens, in which at least one mutation has been introduced in the pykA-gene, preferably at least one mutation the results in the substitution of at least one amino acid in the enzyme encoded by the pykA-gene, most preferred a mutation that results at least in a substitution of glycine by cysteine a position 167, or a substitution of cysteine by tyrosine at position 417 or a substitution of alanine by glycine at position 171 , or a substitution glycine by cysteine a position 167 and a substitution of cysteine by tyrosine at position 417, or a substitution of glycine by cysteine a position 167 and a substitution of alanine by glycine at position 171 , or a substitution of cysteine by tyrosine at position 417 and a substitution of alanine by glycine at position 171 , or a substitution glycine by cysteine a position 167, a substitution of cysteine by tyrosine at position 417 and a substitution of alanine by glycine at position 171 in the enzyme encoded by the pykA- gene.

Preferably, the fruA-gene comprises a nucleic acid selected from the group consisting of: φ1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 20; φ2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 21 ; nucleic acids which are at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the nucleic acid of φ1 ) or φ2), the identity being the identity over the total length of the nucleic acids of φ1 ) or φ2); φ4) nucleic acids encoding an amino acid sequence which is at least 70 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, at least 99.5 %, at least 99.6 %, at least 99.7 %, at least 99.8 % or at least 99.9 %, most preferably 100 % identical to the amino acid sequence encoded by the nucleic acid of φ1 ) or φ2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of φ) or φ2); φ5) nucleic acids capable of hybridizing under stringent conditions with a complementary se- quence of any of the nucleic acids according to φ1 ) or φ2); and φ6) nucleic acids encoding the same protein as any of the nucleic acids of φ1 ) or φ2), but differing from the nucleic acids of φ1 ) or φ2) above due to the degeneracy of the genetic code.

Nucleic acid having the nucleotide sequence of SEQ ID NO: 20 corresponds to the fruA-gene of Basfia succiniciproducens-stra^'m DDL

In this context it is preferred that the modified microorganism according to the present invention further comprises:

A) a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mutation into the IdhA-gene;

B) a deletion of the pflD-gene or at least a part thereof, a deletion of a regulatory element of the pflD-gene or at least a part thereof or an introduction of at least one mutation into the pflD-gene; C) a deletion of the pflA-gene or at least a part thereof, a deletion of a regulatory element of the pflA-gene or at least a part thereof or an introduction of at least one mutation into the pflA-gene;

D) a deletion of the wcaJ-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ-gene or at least a part thereof or an introduction of at least one mutation into the wcaJ-gene;

E) a deletion of the pykA-gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof or an introduction of at least one mutation into the pykA-gene; a deletion of the fruA-gene or at least a part thereof, a deletion of a regulatory element of the fruA-gene or at least a part thereof or an introduction of at least one mutation into the pflA-gene; a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mutation into the IdhA-gene and a deletion of the pflD-gene or at least a part thereof, a deletion of a regulatory element of the pflD-gene or at least a part thereof or an introduction of at least one mutation into the pflD-gene and a deletion of the wcaJ-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ-gene or at least a part thereof or an introduction of at least one mutation into the wcaJ-gene and a deletion of the pykA-gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof or an introduction of at least one mutation into the pykA-gene; or a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mutation into the IdhA-gene and a deletion of the pflA-gene or at least a part thereof, a deletion of a regulatory element of the pflA-gene or at least a part thereof or an introduction of at least one mutation into the pflA-gene, and a deletion of the wcaJ-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ-gene or at least a part thereof or an introduction of at least one mutation into the wcaJ-gene and a deletion of the pykA-gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof or an introduction of at least one mutation into the pykA-gene.

Particular preferred embodiments of the modified microorganisms according to the present invention are: modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted and in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced, and in which, compared to the wildtype, the activity of the lactate dehydrogenase is reduced, preferably by a modification of the IdhA- gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the pyruvate formate lyase is reduced, preferably by a modification of the pflA-gene or the pflD-gene, in particular by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12 or by a modification of the pflD- gene encoding for PfID having the amino acid sequence according to SEQ ID NO: 14; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase and the pyruvate formate lyase is reduced, preferably by a modification of the IdhA-gene and the pflA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10 or by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12, or a modification of the IdhA-gene and the pflD- gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10 or by a modification of the pflD-gene encoding for PfID having the amino acid sequence according to SEQ ID NO: 14; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase, the pyruvate formate lyase and the enzyme encoded by the wcaJ-gene is reduced, preferably by a modification of the IdhA-gene, the pflA-gene and the wcaJ-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12 and by a modification of the wcaJ-gene encoding for an enzyme having the amino sequence according to SEQ ID NO: 16, or a modification of the IdhA-gene, the pflD-gene and the wcaJ-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflD-gene encoding for PfID having the amino acid sequence according to SEQ ID NO: 14 and by a modification of the wcaJ-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 16; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E. coli according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase, the pyruvate formate lyase and the enzyme encoded by the pykA-gene is reduced, preferably by a modification of the IdhA-gene, the pflA-gene and the pykA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12 and by a modification of the pykA-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 19, or a modification of the IdhA-gene, the pflD-gene and the pykA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflD-gene encoding for PfID having the amino acid sequence according to SEQ ID NO: 14 and by a modification of the pykA-gene encoding for an enzyme having the aminoacid sequence according to SEQ ID NO: 19; modified bacterial cells of the genus Basfia and particular preferred of the species Basfia succiniciproducens, in which the pfsG-gene or at least a part thereof has been deleted, in which the cscA-gene of E. coll according to sequence according to SEQ ID NO: 5 or at least a part thereof has been introduced and in which, compared to the wildtype, the activity of the lactate dehydrogenase, the pyruvate formate lyase, the enzyme encoded by the wcaJ-gene and the enzyme encoded by the pykA-gene is reduced, preferably by a mod if i- cation of the IdhA-gene, the pflA-gene, the wcaJ-gene and the pykA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflA-gene encoding for PfIA having the amino acid sequence according to SEQ ID NO: 12, by a modification of the wcaJ-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 16 and by a modification of the pykA-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 19, or a modification of the IdhA-gene, the pflD-gene, the wcaJ-gene and the pykA-gene, in particular by a modification of the IdhA-gene encoding for LdhA having the amino acid sequence according to SEQ ID NO: 10, by a modification of the pflD-gene encoding for PAD having the amino acid sequence according to SEQ ID NO: 14, by a modification of the wcaJ-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 16 and by a modification of the pykA-gene encoding for an enzyme having the amino acid sequence according to SEQ ID NO: 19.

A contribution to solving the problems mentioned at the outset is furthermore provided by a method of producing an organic compound comprising:

I) cultivating the modified microorganism according to the present invention in a culture medium comprising at least one assimilable carbon source to allow the modified microorganism to produce the organic compound, thereby obtaining a fermentation broth comprising the organic compound;

II) recovering the organic compound from the fermentation broth obtained in process step I).

In process step I) the modified microorganism according to the present invention is cultured in a culture medium comprising at least one assimilable carbon source to allow the modified microorganism to produce the organic compound, thereby obtaining a fermentation broth comprising the organic compound. Preferred organic compounds that can be produced by the process according to the present invention comprise carboxylic acids such as formic acid, lactic acid, propionic acid, 2-hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, acrylic acid, pyruvic acid or salts of these carboxylic acids, dicarboxylic acids such as malonic acid, succinic acid, malic acid, tartaric acid, glutaric acid, itaconic acid, adipic acid or salts thereof, tricarboxylic acids such as citric acid or salts thereof, alcohols such as methanol or ethanol, amino acids such as L-asparagine, L-aspartic acid, L-arginine, L-isoleucine, L-glycine, L- glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, L-tryptophan, L-threonine, L-valine, L-histidine, L-proline, L-methionine, L-lysine, L-leucine, etc.. According to a preferred embodiment of the process according to the present invention the organic compound is succinic acid. The term "succinic acid', as used in the context of the present invention, has to be understood in its broadest sense and also encompasses salts thereof (i. e. succinate), as for example alkali metal salts, like Na⁺ and K⁺-salts, or earth alkali salts, like Mg²⁺ and Ca²⁺-salts, or ammonium salts or anhydrides of succinic acid.

The modified microorganism according to the present invention is preferably incubated in the culture medium at a temperature in the range of about 10 to 60°C or 20 to 50°C or 30 to 45°C at a pH of 5.0 to 9.0 or 5.5 to 8.0 or 6.0 to 7.0.

Preferably, the organic compound, especially succinic acid, is produced under anaerobic conditions. Anaerobic conditions may be established by means of conventional techniques, as for example by degassing the constituents of the reaction medium and maintaining anaerobic conditions by introducing carbon dioxide or nitrogen or mixtures thereof and optionally hydrogen at a flow rate of, for example, 0.1 to 1 or 0.2 to 0.5 vvm. Aerobic conditions may be established by means of conventional techniques, as for example by introducing air or oxygen at a flow rate of, for example, 0.1 to 1 or 0.2 to 0.5 vvm. If appropriate, a slight over pressure of 0.1 to 1.5 bar may be applied in the process. The assimilable carbon source is preferably selected from sucrose, maltose, maltotriose, malto- tetraose, maltopentaose, maltohexaose, maltoheptaose, D-fructose, D-glucose, D-xylose, L- arabinose, D-galactose, D-mannose, glycerol and mixtures thereof or compositions containing at least one of said compounds, or is selected from decomposition products of starch, cellulose, hemicellulose and/or lignocellulose. A preferred assimiable carbon source is sucrose. Further preferred mixtures are a mixture of sucrose and at least one further assimilable carbon source, such as a mixture of sucrose and maltose, sucrose and D-fructose, sucrose and D-glucose, sucrose and D-xylose, sucrose and L-arabinose, sucrose and D-galactose, sucrose and D- mannose. According to a preferred embodiment of the process according to the present invention at least 50 wt.-%, preferably at least 75 wt.-%, more preferably at least 90 wt.-%, even more preferably at least 95 wt.-% and most preferably at least 99 wt.-% of the assimilable carbon source, based on the total weight of the assimilable carbon source with the exception of carbon dioxide, is sucrose.

The initial concentration of the assimilable carbon source is preferably adjusted to a value in a range of 5 to 100 g/l, preferably 5 to 75 g/l and more preferably 5 to 50 g/l and may be maintained in said range during cultivation. The pH of the reaction medium may be controlled by addition of suitable bases as for example, gaseous ammonia, NH4HCO3, (NhU^COs, NaOH, Na₂C0₃, NaHCOs, KOH, K2CO3, KHCOs, Mg(OH)₂, MgCOs, Mg(HC0₃)₂, Ca(OH)₂, CaCOs, Ca(HC03)₂, CaO, CH6N₂0₂, C₂H₇N and/or mixtures thereof. These alkaline neutralization agents are especially required if the organic compounds that are formed in the course of the fermentation process are carboxylic acids or dicarboxylic acids. In the case of succinic acid as the organic compound, Mg(OH)2 and MgC03 are particular preferred bases.

The fermentation step I) according to the present invention can, for example, be performed in stirred fermenters, bubble columns and loop reactors. A comprehensive overview of the possible method types including stirrer types and geometric designs can be found in Chmiel: "Bio- prozesstechnik: Einfuhrung in die Bioverfahrenstechnik' , Volume 1. In the process according to the present invention, typical variants available are the following variants known to those skilled in the art or explained, for example, in Chmiel, Hammes and Bailey: "Biochemical Engineering": such as batch, fed-batch, repeated fed-batch or else continuous fermentation with and without recycling of the biomass. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or appropriate gas mixtures may be effected in order to achieve good yield (YP/S). Particularly preferred conditions for producing the organic acid, especially succinic acid, in process step I) are:

Assimilable carbon source sucrose

Temperature: 30 to 45°C

pH: 5.5 to 7.0

Supplied gas: C0₂

It is furthermore preferred in process step I) that the assimilable carbon source is converted to the organic compound, preferably to succinic acid, with a carbon yield YP/S of at least 0.5 g/g up to about 1 .28 g/g; as for example a carbon yield YP/S of at least 0,6 g/g, of at least 0.7 g/g, of at least 0.75 g/g, of at least 0.8 g/g, of at least 0.85 g/g, of at least 0.9 g/g, of at least 0.95 g/g, of at least 1.0 g/g, of at least 1 .05 g/g, of at least 1.1 g/g, of at least 1 .15 g/g, of at least 1 .20 g/g, of at least 1 .22 g/g, or of at least 1 .24 g/g (organic compound/carbon, preferably succinic acid/carbon).

It is furthermore preferred in process step I) that the assimilable carbon source is converted to the organic compound, preferably to succinic acid, with a specific productivity yield of at least 0.6 g g DCW-¹ IT¹ organic compound, preferably succinic acid, or of at least of at least

0.65 g g DCW-¹h-¹, of at least 0.7 g g DCW-¹lr¹, of at least 0.75 g g DCW-¹h^"1 or of at least 0.77 g g DCW-¹lr¹ organic compound, preferably succinic acid.

It is furthermore preferred in process step I) that the assimilable carbon source is converted to the organic compound, preferably to succinic acid, with a space time yield for the organic compound, preferably for succinic acid, of at least 2.2 g/(l_xh) or of at least 2.5 g/(Lxh) , at least 2.75 g/(Lxh), at least 3 g/(Lxh), at least 3.25 g/(Lxh), at least 3.5 g/(Lxh), at least 3.7 g/(Lxh), at least 4.0 g/(Lxh) at least 4.5 g/(Lxh) or at least 5.0 g/(Lxh) of the organic compound, preferably succinic acid. According to another preferred embodiment of the process according to the pre- sent invention in process step I) the modified microorganism is converting at least 20 g/L, more preferably at least 25 g/l and even more preferably at least 30 g/l of the assimilable carbon source to at least 20 g/l, more preferably to at least 25 g/l and even more preferably at least 30 g/l of the organic compound, preferably succinic acid.

The different yield parameters as described herein (^'carbon yield' or "YP/S"; "specific productivity yield'; or "space-time-yield (STY)") are well known in the art and are determined as described for example by Song and Lee, 2006. "Carbon yield' and "YP/S" (each expressed in mass of organic compound produced/mass of assimilable carbon source consumed) are herein used as synonyms. The specific productivity yield describes the amount of a product, like succinic acid, that is produced per h and L fermentation broth per g of dry biomass. The amount of dry cell weight stated as "DCW describes the quantity of biologically active microorganism in a biochemical reaction. The value is given as g product per g DCW per h (i.e. g g DCW-¹h-¹). The space-time-yield (STY) is defined as the ratio of the total amount of organic compound formed in the fermentation process to the volume of the culture, regarded over the entire time of cultivation. The space-time yield is also known as the "volumetric productivity.

In process step II) the organic compound, preferably succinic acid, is recovered from the fermentation broth obtained in process step I).

Usually, the recovery process comprises the step of separating the recombinant microorganism from the fermentation broth as the so called "biomass". Processes for removing the biomass are known to those skilled in the art, and comprise filtration, sedimentation, flotation or combinations thereof. Consequently, the biomass can be removed, for example, with centrifuges, separators, decanters, filters or in a flotation apparatus. For maximum recovery of the product of value, washing of the biomass is often advisable, for example in the form of a diafiltration. The selection of the method is dependent upon the biomass content in the fermentation broth and the properties of the biomass, and also the interaction of the biomass with the organic compound (e. the product of value). In one embodiment, the fermentation broth can be sterilized or pas- teurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirement, this concentration can be done batch wise or continuously. The pressure and temperature range should be selected such that firstly no product damage occurs, and secondly minimal use of apparatus and energy is necessary. The skillful selection of pressure and temperature levels for a multistage evaporation in particular enables saving of energy.

The recovery process may further comprise additional purification steps in which the organic compound, preferably succinic acid, is further purified. If, however, the organic compound is converted into a secondary organic product by chemical reactions as described below, a further purification of the organic compound is, depending on the kind of reaction and the reaction con- ditions, not necessarily required. For the purification of the organic compound obtained in process step II), preferably for the purification of succinic acid, methods known to the person skilled in the art can be used, as for example crystallization, filtration, electrodialysis and chromatog- raphy. In the case of succinic acid as the organic compound, for example, succinic acid may be isolated by precipitating it as a calcium succinate product by using calcium hydroxide, -oxide, - carbonate or hydrogen carbonate for neutralization and filtration of the precipitate. The succinic acid is recovered from the precipitated calcium succinate by acidification with sulfuric acid fol- lowed by filtration to remove the calcium sulfate (gypsum) which precipitates. The resulting solution may be further purified by means of ion exchange chromatography in order to remove un- desired residual ions. Alternatively, if magnesium hydroxide, magnesium carbonate or mixtures thereof have been used to neutralize the fermentation broth, the fermentation broth obtained in process step l)may be acidified to transform the magnesium succinate contained in the medium into the acid form (i. e. succinic acid), which subsequently can be crystallized by cooling down the acidified medium. Examples of further suitable purification processes are disclosed in EP-A- 1 005 562, WO-A-2008/010373, WO-A-201 1/082378, WO-A-201 1/043443, WO-A- 2005/030973, WO-A-201 1/123268 and WO-A-201 1/064151 and EP-A-2 360 137. According to a preferred embodiment of the process according to the present invention the process further comprises the process step:

III) conversion of the organic compound contained in the fermentation broth obtained in process step I) or conversion of the recovered organic compound obtained in process step II) into a secondary organic product being different from the organic compound by at least one chemical reaction.

In case of succinic acid as the organic compound preferred secondary organic products are selected from the group consisting of succinic acid esters and polymers thereof, tetrahydrofuran (THF), 1 ,4-butanediol (BDO), gamma-butyrolactone (GBL), pyrrolidones, polyols and polyure- thanes.

According to a preferred embodiment for the production of THF, BDO and/or GBL this process comprises: b1 ) either the direct catalytic hydrogenation of the succinic acid obtained in process steps I) or II) to THF and/or BDO and/or GBL or b2) the chemical esterification of succinic acid and/or succinic acid salts obtained in process steps I) or II) into its corresponding di-lower alkyl ester and subsequent catalytic hydrogenation of said ester to THF and/or BDO and/or GBL.

According to a preferred embodiment for the production of pyrrolidones this process comprises: b) the chemical conversion of succinic acid ammonium salts obtained in process steps I) or II) to pyrrolidones in a manner known per se. For details of preparing these compounds reference is made to US-A-2010/0159543 and WO- A-2010/092155.

A contribution to solving the problems mentioned at the outset is furthermore provided by the use of the modified microorganism according to the present invention for the fermentative production of organic compounds. Preferred organic compounds are those compounds that have already been mentioned in connection with the process according to the present invention, succinic acid being the most preferred organic compound. Furthermore, preferred conditions for the fermentative production of organic compounds, preferably of succinic acid, are those conditions that have already been described in connection with process step I) of the process according to the present invention.

The invention is now explained in more detail with the aid of figures and non-limiting examples. Figure 1 shows a schematic map of plasmid pSacB (SEQ ID NO: 22).

Figure 2 shows a schematic map of plasmid pSacB AldhA (SEQ ID NO: 23). Figure 3 shows a schematic map of plasmid pSacB ApfIA (SEQ ID NO: 24).

Figure 4 shows a schematic map of plasmid pSacB wcaJ^* (SEQ ID NO: 25). Figure 5 shows a schematic map of plasmid pSacB AptsG cscA (SEQ ID NO: 26). Figure 6 shows a schematic map of plasmid pSacB AptsG argD::cscA-fusion (SEQ ID NO: 27). Figure 7 shows a schematic map of plasmid pSacB pykA1 (SEQ ID NO: 29). EXAMPLES

Example 1 : General method for the transformation of Basfia succiniciproducens

Strain

DD1 AldhA AldhA ApfIA wcaJ^*

DD1 AldhA ApfIA AptsG argD.vcscA-fusion

DD1 AldhA ApfIA AptsG pykA1 argD::cscA-fusion

DD1 AldhA ApfIA AptsG pykA1 cscA

DD1 AptsG cscA

Table 1 : Nomenclature of the DD1 -mutants referred to in the examples The expression "wcaJ^* " as used herein refers to a truncated wcaJ-gene Basfia succiniciproducens DD1 (wildtype) was transformed with DNA by electroporation using the following protocol:

For preparing a pre-culture DD1 was inoculated from frozen stock into 40 ml BHI (brain heart infusion; Becton, Dickinson and Company) in 100 ml shake flask. Incubation was performed over night at 37°C; 200 rpm. For preparing the main-culture 100 ml BHI were placed in a 250 ml shake flask and inoculated to a final OD (600 nm) of 0.2 with the pre-culture. Incubation was performed at 37°C, 200 rpm. The cells were harvested at an OD of approximately 0.5, 0.6 and 0.7, pellet was washed once with 10% cold glycerol at 4°C and re-suspended in 2 ml 10% glyc- erol (4°C).

100 μΙ of competent cells were the mixed with 2-8 μg Plasmid-DNA and kept on ice for 2 min in an electroporation cuvette with a width of 0.2 cm. Electroporation under the following conditions: 400 Ω; 25 \ F; 2.5 kV (Gene Pulser, Bio-Rad). 1 ml of chilled BHI was added immediately after electroporation and incubation was performed for approximately 2 h at 37°C.

Cells were plated on BHI with 5 mg/L chloramphenicol and incubated for 2-5 d at 37°C until the colonies of the transformants were visible. Clones were isolated and restreaked onto BHI with 5 mg/l chloramphenicol until purity of clones was obtained.

Example 2: Generation of deletion constructs

Mutation/deletion plasmids were constructed based on the vector pSacB (SEQ ID NO: 22). Figure 1 shows a schematic map of plasmid pSacB. 5'- and 3'- flanking regions (approx. 1500 bp each) of the chromosomal fragment, which should be deleted were amplified by PCR from chromosomal DNA of Basfia succiniciproducens and introduced into said vector using standard techniques. Normally, at least 80 % of the ORF were targeted for a deletion. In such a way, the mutation/deletion plasmids for the lactate dehydrogenase IdhA, pSacB_delta_/c/M

(SEQ ID NO: 23), the pyruvate formate lyase activating enzyme pfIA, pSacB_delta_ pflA1

(SEQ ID NO: 24), pSacB_ wcaJ^* (SEQ ID NO: 25), pSacB_delta_pfsG_cscA (SEQ ID NO: 26), pSacB_delta_pfsG_ argD.vcscA-fusion (SEQ ID NO: 27) and pSacB pykA1 (SEQ ID NO: 29) were constructed. Figures 1 , 2, 3, 4, 5 and 6 show schematic maps of plasmid pSacB, pSacB_delta_/c/M, pSacB_delta_p/7A pSacB_wcaJ\ and pSacB_delta_pfsG_cscA

pSacB_delta_pfsG_ argD::cscA-fusion and pSacB pykA1 respectively.

A transformation of Basfia succiniciproducens with pSacB_ivcaJ^* leads to the expression of a truncated enzyme encoded by the wcaJ-gene, whereas a transformation with pSacB pykA1 leads to the expression of a pyruvate kinase in which at amino acid position 167 glycine is substituted by cysteine.

A transformation of Basfia succiniciproducens with pSacB_delta_pfsG_cscA leads to a deletion of the pfsG-gene and an introduction of the cscA-gene from E. coli \N (ATCC 9637) into the cell, whereas a transformation with pSacB_delta_pfsG_

leads to a deletion of the pfsG-gene and an introduction of a fusion gene comprising nuelceotides 1 to 869 of the argD- gene and nucleotides 524 to 1434 of the cscA-gene from E. co//^' W (ATCC 9637) into the cell. In the plasmid sequence of pSacB (SEQ ID NO: 22) the sacS-gene is contained from bases 2380-3801 . The sacS-promotor is contained from bases 3802-4264. The chloramphenicol gene is contained from base 526-984. The origin of replication for E. coli (ori EC) is contained from base 1477-2337 (see fig. 1 ). In the plasmid sequence of pSacB_delta_/c/M (SEQ ID NO: 23) the 5' flanking region of the IdhA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1519-2850, while the 3' flanking region of the IdhA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 62-1518. The sacS-gene is contained from bases 5169-6590. The sacS-promoter is contained from bases 6591 -7053. The chloramphenicol gene is contained from base 3315-3773. The origin of replication for E. coli (ori EC) is contained from base 4266-5126 (see fig. 2).

In the plasmid sequence of pSacB_delta_pf/A (SEQ ID NO: 24) the 5' flanking region of the pflA- gene, which is homologous to the genome of Basfia succiniciproducens, is contained from ba- ses 1506-3005, while the 3' flanking region of the pflA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1505. The sacS-gene is contained from bases 5278-6699. The sacS-promoter is contained from bases 6700-7162. The chloramphenicol gene is contained from base 3424-3882. The origin of replication for E. coli (ori EC) is contained from base 4375-5235 (see fig. 3).

In the plasmid sequence of pSacB_ivcaJ^* (SEQ ID NO: 25) the 5' flanking region of the wcaJ- gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1838-3379, while the 3' flanking region of the wcaJ-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1236. The sacS-gene is con- tained from bases 5652-7073. The sacS-promoter is contained from bases 7074-7536. The chloramphenicol gene is contained from base 3798-4256. The origin of replication for E. coli (ori EC) is contained from base 4749-5609. The wcaJ-gene is contained from bases 1237-1837 with an insertion of a nucleotide in the codon that encodes of lysine between thymine at position 81 and adenine at position 82 of at position of the wcaJ-gene (which corresponds to position 1756 of plasmid pSacB-ivcaJ^*. This insertion leads to a frame shift mutation, wherein by means of this frame shift mutation a stop codon is introduced, leading to the expression of a truncated enzyme.

In the plasmid sequence of pSacB_delta_pfsG cscA (SEQ ID NO: 26) the 5' flanking region of the pfsG-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 4138-5698, while the 3' flanking region of the pfsG-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 7133-8692. The sacS-gene is contained from bases 2272-3693. The sacS-promoter is contained from bases 3694-4156. The chloramphenicol gene is contained from base 418-876. The origin of replication for E. coli (ori EC) is contained from base 1369-2229 and the cscA-gene from E. coli \N is contained from bases 5699-7132 (see fig. 5).

In the plasmid sequence of pSacB_delta_pfsG argD cscA- us\on (SEQ ID NO: 27) the 5' flanking region of the pfsG-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 3384-4838, while the 3' flanking region of the pfsG-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 5738-7300. The sacS-gene is contained from bases 1473-2894. The sacS-promoter is contained from bases

2895-3357. The chloramphenicol gene is contained from base 7719-77. The origin of replication for E. coli (ori EC) is contained from base 570-1430 and the fusion gene (SEQ ID NO: 28) argDv.cscA is contained from bases 3959-5737, consisting of a truncated version of the argD- gene (3959-4826) and a truncated version of the cscA-gene (4827-5737) (see fig. 6).

In the plasmid sequence of pSacB_py/o4i (SEQ ID NO: 29) the part of the py/oA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1 185. The sacS-gene is contained from bases 3458-4879. The sacS-promoter is contained from bases 4880-5342. The chloramphenicol gene is contained from bases 1604-2062. The origin of repli- cation for E. coli (ori EC) is contained from bases 2555-3415 (see fig. 7).

Example 3: Generation of improved succinate producing strains a) Basfia succiniciproducens DD1 was transformed as described above with the

pSacB_delta_/c/M and "Campbelled in" to yield a "Campbell in" strain. Transformation and integration into the genome of Basfia succiniciproducens was confirmed by PCR yielding bands for the integrational event of the plasmid into the genome of Basfia succiniciproducens. The "Campbell in" strain was then "Campbelled out" using agar plates containing sucrose as a counter selection medium, selecting for the loss (of function) of the sacB gene.

Therefore, the "Campbell in" strains were incubated in 25-35 ml of non selective medium (BHI containing no antibiotic) at 37°C, 220 rpm over night. The overnight culture was then streaked onto freshly prepared BHI containing sucrose plates (10%, no antibiotics) and in- cubated overnight at 37°C ("first sucrose transfer"). Single colony obtained from first transfer were again streaked onto freshly prepared BHI containing sucrose plates (10%) and incubated overnight at 37°C ("second sucrose transfer"). This procedure was repeated until a minimal completion of five transfers ("third, forth, fifth sucrose transfer") in sucrose. The term "first to fifth sucrose transfer" refers to the transfer of a strain after chromosomal integration of a vector containing a sacB levan-sucrase gene onto sucrose and growth medium containing agar plates for the purpose of selecting for strains with the loss of the sacB gene and the surrounding plasmid sequences. Single colony from the fifth transfer plates were inoculated onto 25-35 ml of non selective medium (BHI containing no antibiotic) and incubated at 37°C, 220 rpm over night. The overnight culture was serially diluted and plated onto BHI plates to obtain isolated single colonies. The "Campbelled out" strains containing the mutation/deletion of the IdhA-ge e were confirmed by chloramphenicol sensitivity. The mutation/deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the /c/M-deletion mutant Basfia succiniciproducens DD1 AldhA. b) Basfia succiniciproducens AldhA was transformed with pSacB_delta_pf/A as described above and "Campbelled in" to yield a "Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the IdhA pfIA double deletion mutant Basfia succiniciproducens DD1 AldhA ApfIA. c) Basfia succiniciproducens AldhA ApfIA was transformed with pSacB_ivcaJ^* as described above and "Campbelled in" to yield a "Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The deletion mutants among these strains were identified and confirmed by

PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* in which IdhA and pfIA are deleted and which expresses a truncated enzyme encoded by the wcaJ-gene. d) Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* was transformed with pSacB_pykA1 as described above and "Campbelled in" to yield a "Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* pykA1 in which IdhA and pfIA are deleted, which expresses a truncated enzyme encoded by the wcaJ-gene and which expresses a pyruvate kinase in which at amino acid position 167 glycine is substituted by cysteine. e) Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* was transformed with

pSacB_delta_pfsG argD.vcscA-fusion as described above and "Campbelled in" to yield a

"Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* AptsGargD.vcscA-fusion in which IdhA, pfIA and ptsG are deleted, which expresses a truncated enzyme encoded by the wcaJ-gene and which expresses the heterologous fusion protein ArgD::CscA. Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* pykA1 was transformed with pSacB_delta_pfsG argD::cscA- us\on as described above and "Campbelled in" to yield a "Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* pykA1 AptsGargD;;cscA-fusion in which IdhA, pfIA and ptsG are deleted, which expresses a truncated enzyme encoded by the wcaJ-gene, which expresses a pyruvate kinase in which at amino acid position 167 glycine is substituted by cysteine and which expresses the heterologous fusion protein ArgD::CscA.

Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* pykA1 was transformed with pSacB_delta_pfsG cscA as described above and "Campbelled in" to yield a "Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA in which IdhA, pfIA and ptsG are deleted, which expresses a truncated enzyme encoded by the wcaJ-gene, which expresses a pyruvate kinase in which at amino acid position 167 glycine is substituted by cysteine and which expresses a heterologous sucrase encoded by the cscA-gene from E. CO// W.

Basfia succiniciproducens DD1 was transformed with pSacB_delta_pfsG cscA to yield a "Campbell in" strain. Transformation and integration was confirmed by PCR. The "Campbell in" strain was then "Campbelled out" as described previously. The mutants among these strains were identified and confirmed by PCR analysis. This led to the mutant Basfia succiniciproducens DD1 AptsG cscA.

Example 4: Cultivation of various DD1 -strains on sucrose

The productivity of the DD1 AldhA ApfIA wcaJ^* was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* AptsG argD::cscA-fusion in the presence of sucrose as a carbon source. Furthermore, the productivity of the DD1 AldhA ApfIA wcaJ^* pykA1 was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG argD::cscA-fusion in the presence of sucrose as a carbon source.

Also, the productivity of the DD1 AldhA ApfIA wcaJ^* pykA1 was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA in the presence of sucrose as a carbon source. Furthermore, the productivity of the DD1 strain was compared with the productivity of the mutant strain DD1 Δ ptsG cscA in the presence of sucrose as a carbon source.

Productivity was analyzed utilizing media and incubation conditions described below.

Table 3: Composition of trace element solution Vitamin solution

Compound Final concentration

Thiamin HCI (B1 ) 1 .0 g/L

Nicotinic acid (B3) 1 .0 g/L

Riboflavin (B2) 20 mg/L

Biotin (B7) 50 mg/L

Pantothenic acid (B5) 1 .0 g/L

Pyridoxine (B6) 1 .0 g/L

Cyanocobalamin (B12) 50 mg/L

Lipoic acid 5 mg/L

Table 4: Composition of vitamin solution

Table 5: Composition of LSM_3 medium for cultivation on sucrose

Compound Volume/ Mass Stock concentration Final concentration

Medium 1

MgCOs 2.5 g 100% 50.00 g/L

Water 36.28 mL - -

Medium 2

Succinic acid 2.5 mL 50 g/L 2.50 g/L

Sucrose 4.00 mL 650 g/L 52.00 g/L

(NH₄)₂S0₄ 0.30 mL 500 g/L 3.00 g/L

Beta in 0.50 mL 23 g/L 0.23 g/L

KH₂P0₄ 0.75 mL 100 g/L 1 .50 g/L

(NH₄)₂HP0₄ 0.63 ml 200 g/L 2.50 g/L

Na₂C0₃ 0.50 mL 200 g/L 2.00 g/L

Yeast extract 1 .50 ml 100 g/L 3.00 g/L

vitamin solution 0.50 mL 4 g/L 0.04 g/L

trace element solution 0.05 mL 21 g/L 0.02 g/L

Table 6: Composition of LSM_3_YE medium for cultivation on sucrose

Cultivations and analytics

For growing the main culture bacteria from a freshly grown BHI-agar plate (incubated overnight at 37°C under anaerobic conditions) was used to inoculate to OD600 = 0.75 a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the CGM liquid medium described in table 2 with a C0₂-atmosphere. The bottles were incubated at 37°C and 160 rpm (shaking diameter: 2.5 cm). For growing the main culture 2.5 ml of the bacterial culture in the CGM medium (after 1 1 hours of incubation; when comparing the productivity of the DD1 strain with the productivity of the mutant strain DD1 Δ ptsG cscA after 13 hours of incubation for DDI and after 35 hours of incubation for DD1 AptsG cscA) was used to inoculate a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the CGM liquid medium (or the LSM_3 medium, or the LSM_3_YE medium) described in table 2 (LSM_3 medium: Table 5, LSM_3_YE medium: Table 6) with a C0₂- atmosphere. Consumption of the C-sources and production of carboxylic acids was quantified via HPLC (HPLC methods are described in tables 13 and 14) after 24h (for glucose) and 48 h (for fructose and sucrose). Cell growth was measured by measuring the absorb- ance at 600 nm (OD600) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden).

Results

The results of the cultivation experiments with for different DD1 -strains are shown in tables 7, 8, 9, 10, 1 1 and 12. a) The productivity of the DD1 AldhA ApfIA wcaJ^* strain was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* AptsG argD::cscA-fusion in the presence of sucrose as a carbon source (medium LSM_3_YE). The results of the cultivation experiments with DD1 AldhA ApfIA wcaJ^* and strain DD1 AldhA ApfIA wcaJ^* AptsG argD::cscA-fusion are shown in Table 7. The deletion of the pfsG-gene encoding a putative sucrose-specific transporter, and heterologous expression of sucrase from E. coli \N (encoded by the cscA-gene) in the DD1 AldhA ApfIA wcaJ^* strain results in increase of succinic acid yield on sucrose. b) The productivity of the DD1 AldhA ApfIA wcaJ^* pykA1 strain was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* pykA^* AptsG argDv.cscA- fusion in the presence of sucrose as a carbon source (medium LSM_3_YE). The results of the cultivation experiments with DD1 AldhA ΔρίΙΑ wcaJ^* pykA1 and strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG argD::cscA-fusion are shown in Table 8. The deletion of the pfsG-gene encoding a putative sucrose-specific transporter, and heterologous expression of a part of the sucrase from E. coll W (encoded by the cscA- gene) in the form of a fusion protein in the DD1 AldhA ApfIA wcaJ^* pykA1 strain results in increase of succinic acid yield on sucrose. c) The productivity of the DD1 AldhA ApfIA wcaJ^* pykA1 strain was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG argDv.cscA- fusion in the presence of sucrose as a carbon source (medium CGM). The results of the cultivation experiments with DD1 AldhA ΔρίΙΑ wcaJ^* pykA1 and strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG argD::cscA-fusion are shown in Table 9. The deletion of the ptsG gene encoding a putative sucrose-specific transporter, and heterologous expression of a part of the sucrase from E. coli W (encoded by the cscA gene) in the form of a fusion protein in the DD1 AldhA ApfIA wcaJ^* pykA\ strain results in increase of succinic acid yield on sucrose. d) The productivity of the DD1 AldhA ApfIA wcaJ^* pykA1 strain was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA in the presence of sucrose as a carbon source (medium LSM_3). The results of the cultivation experiments with DD1 AldhA ApfIA wcaJ^* pykA1 and strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA are shown in Table 10. The deletion of the pfsG-gene en- coding a putative sucrose-specific transporter, and heterologous expression of sucrase from E. coli \N (encoded by the cscA-gene) in the DD1 AldhA ApfIA wcaJ^* pykA1 strain results in increase of succinic acid yield on sucrose. e) The productivity of the DD1 AldhA ApfIA wcaJ^* pykA1 strain was compared with the productivity of the mutant strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA in the presence of sucrose as a carbon source (medium CGM). The results of the cultivation experiments with DD1 AldhA ApfIA wcaJ^* pykA1 and strain DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA are shown in Table 1 1 . The deletion of the ptsG-gene encoding a putative sucrose-specific transporter, and heterologous expression of su- crase from E. coli \N (encoded by the cscA-gene) in the DD1 AldhA ApfIA wcaJ^* pykA1 strain results in increase of succinic acid yield on sucrose. f) The productivity of the DD1 strain was compared with the productivity of the mutant strain DD1 AptsG cscA in the presence of sucrose as a carbon source (medium LSM_3). The results of the cultivation experiments with DD1 AptsG cscA and strain DD1 are shown in Table 12. The deletion of the pfsG-gene encoding a putative sucrose-specific transporter, and heterologous expression of sucrase from E. co// W (encoded by the cscA-gene) in DD1 results in increase of succinic acid yield on sucrose.

Table 7: Cultivation of the DD1 AldhA ApfIA wcaJ^*-strain and the DD1 AldhA ApfIA wcaJ^*

AptsG argD::cscA-fusion-strain on sucrose (medium LSM_3_YE) ^a cultivation time

b consumption of substrate (sucrose)

c formation of succinic acid, lactic acid, formic acid, acetic acid and pyruvic acid ^d SA yield (ratio of SA per consumed substrate)

e detection limits for acetic acid, lactic acid and formic acid were found to be lower than 0.01 g/l in the given HPLC method

Table 8: Cultivation of the DD1 AldhA ApfIA wcaJ^* py/oAi-strain and the DD1 AldhA ApfIA wcaJ^* pykA1 AptsG argD: :cscA-fusion-strain on sucrose (medium LSM_3_YE) cultivation time

consumption of substrate (sucrose)

formation of succinic acid, lactic acid, formic acid, acetic acid and pyruvic acid SA yield (ratio of SA per consumed substrate)

detection limits for acetic acid, lactic acid and formic acid were found to be lower than 0.01 g/l in the given HPLC method

Table 9: Cultivation of the DD1 AldhA ApfIA wcaJ^* py/oAi-strain and the DD1 AldhA ApfIA wcaJ^* pykA1 AptsG argD: :cscA-fusion-strain on sucrose (medium CGM) cultivation time

consumption of substrate (sucrose)

Table 10: Cultivation of the DD1 AldhA ApfIA wcaJ^* py/oAi-strain and the DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA-strain on sucrose (medium LSM_3) cultivation time

consumption of substrate (sucrose)

Table 1 1 : Cultivation of the DD1 AldhA ApfIA wcaJ^* py/oAi-strain and the DD1 AldhA ApfIA wcaJ^* pykA1 AptsG cscA-strain on sucrose (medium CGM) cultivation time

consumption of substrate (sucrose)

Table 12: Cultivation of the DD1 -strain and the DDIApfsG cscA-strain on sucrose (medium LSM_3) cultivation time

consumption of substrate (sucrose)

HPLC column Aminex HPX-87 H, 300 ^χ 7.8 mm (BioRad)

Precolumn Cation H

Temperature 50°C

Eluent flow rate 0.50 ml/min

Injection volume 5.0 μΙ

Diode array detector Rl-Detector

Runtime 28 min

max. pressure 140 bar

Eluent A 5 mM H2SO4

Eluent B 5 mM H2SO4

Time [min] A[%] B[%] Flow [ml/min]

Gradient 0.0 50 50 0.50

28.0 50 50 0.50

Table 13: HPLC method (ZX-THF50) for analysis of succinic acid, formic acid, lactic acid, acetic acid and pyruvic acid

HPLC column Fast Carbohydrate, 100 * 7.8 mm (Biorad)

Precolumn Deashing Refill Cartridges (30°C)

Temperature 75°C

Eluent flow rate 1 .00 ml/min

Injection volume 1 .0 μΙ

Diode array detector Rl-Detector

Runtime 8 min

max. pressure 150 bar

Eluent A water

Eluent B water

Gradient Time [min] A[%] B[%] Flow [ml/min]

0.0 50 50 1 .00

8.0 50 50 1 .00

Table 14: HPLC method (Fast-CH) for analysis of sucrose SEQUENCES

SEQ ID NO: 1 (nucleotide sequence of 16 S rDNA of strain DD1 )

tttgatcctggctcagattgaacgctggcggcaggcttaacacatgcaagtcgaacggtagcgggaggaaagcttgctttctttgccga cgagtggcggacgggtgagtaatgcttggggatctggcttatggagggggataacgacgggaaactgtcgctaataccgcgtaatat cttcggattaaagggtgggactttcgggccacccgccataagatgagcccaagtgggattaggtagttggtggggtaaaggcctacc aagccgacgatctctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagca gtggggaatattgcacaatggggggaaccctgatgcagccatgccgcgtgaatgaagaaggccttcgggttgtaaagttctttcggtg acgaggaaggtgtttgttttaataggacaagcaattgacgttaatcacagaagaagcaccggctaactccgtgccagcagccgcggt aatacggagggtgcgagcgttaatcggaataactgggcgtaaagggcatgcaggcggacttttaagtgagatgtgaaagccccgg gcttaacctgggaattgcatttcagactgggagtctagagtactttagggaggggtagaattccacgtgtagcggtgaaatgcgtagag atgtggaggaataccgaaggcgaaggcagccccttgggaagatactgacgctcatatgcgaaagcgtggggagcaaacaggatt agataccctggtagtccacgcggtaaacgctgtcgatttggggattgggctttaggcctggtgctcgtagctaacgtgataaatcgacc gcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatg caacgcgaagaaccttacctactcttgacatccagagaatcctgtagagatacgggagtgccttcgggagctctgagacaggtgctg catggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcatgtaaagatgg gaactcaaaggagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctaca cacgtgctacaatggtgcatacagagggcggcgataccgcgaggtagagcgaatctcagaaagtgcatcgtagtccggattggagt ctgcaactcgactccatgaagtcggaatcgctagtaatcgcaaatcagaatgttgcggtgaatacgttcccgggccttgtacacaccg cccgtcacaccatgggagtgggttgtaccagaagtagatagcttaaccttcggggggggcgtttaccacggtatgattcatgactggg gtgaagtcgtaacaaggtaaccgtaggggaacctgcgg

SEQ ID NO: 2 (nucleotide sequence of 23 S rDNA of strain DD1 )

agtaataacgaacgacacaggtataagaatacttgaggttgtatggttaagtgactaagcgtacaaggtggatgccttggcaatcaga ggcgaagaaggacgtgctaatctgcgaaaagcttgggtgagttgataagaagcgtctaacccaagatatccgaatggggcaaccc agtagatgaagaatctactatcaataaccgaatccataggttattgaggcaaaccgggagaactgaaacatctaagtaccccgagg aaaagaaatcaaccgagattacgtcagtagcggcgagcgaaagcgtaagagccggcaagtgatagcatgaggattagaggaat cggctgggaagccgggcggcacagggtgatagccccgtacttgaaaatcattgtgtggtactgagcttgcgagaagtagggcggga cacgagaaatcctgtttgaagaaggggggaccatcctccaaggctaaatactcctgattgaccgatagtgaaccagtactgtgaagg aaaggcgaaaagaaccccggtgaggggagtgaaatagaacctgaaaccttgtacgtacaagcagtgggagcccgcgagggtga ctgcgtaccttttgtataatgggtcagcgacttatattatgtagcgaggttaaccgaataggggagccgaagggaaaccgagtcttaact gggcgtcgagttgcatgatatagacccgaaacccggtgatctagccatgggcaggttgaaggttgggtaacactaactggaggacc gaaccgactaatgttgaaaaattagcggatgacctgtggctgggggtgaaaggccaatcaaaccgggagatagctggttctccccg aaatctatttaggtagagccttatgtgaataccttcgggggtagagcactgtttcggctagggggccatcccggcttaccaacccgatgc aaactgcgaataccgaagagtaatgcataggagacacacggcgggtgctaacgttcgtcgtggagagggaaacaacccagacc gccagctaaggtcccaaagtttatattaagtgggaaacgaagtgggaaggcttagacagctaggatgttggcttagaagcagccatc atttaaagaaagcgtaatagctcactagtcgagtcggcctgcgcggaagatgtaacggggctcaaatatagcaccgaagctgcggc atcaggcgtaagcctgttgggtaggggagcgtcgtgtaagcggaagaaggtggttcgagagggctgctggacgtatcacgagtgcg aatgctgacataagtaacgataaaacgggtgaaaaacccgttcgccggaagaccaagggttcctgtccaacgttaatcggggcag ggtgagtcggcccctaaggcgaggctgaagagcgtagtcgatgggaaacgggttaatattcccgtacttgttataattgcgatgtggg gacggagtaggttaggttatcgacctgttggaaaaggtcgtttaagttggtaggtggagcgtttaggcaaatccggacgcttatcaaca ccgagagatgatgacgaggcgctaaggtgccgaagtaaccgataccacacttccaggaaaagccactaagcgtcagattataata aaccgtactataaaccgacacaggtggtcaggtagagaatactcaggcgcttgagagaactcgggtgaaggaactaggcaaaata gcaccgtaacttcgggagaaggtgcgccggcgtagattgtagaggtatacccttgaaggttgaaccggtcgaagtgacccgctggct gcaactgtttattaaaaacacagcactctgcaaacacgaaagtggacgtatagggtgtgatgcctgcccggtgctggaaggttaattg atggcgttatcgcaagagaagcgcctgatcgaagccccagtaaacggcggccgtaactataacggtcctaaggtagcgaaattcctt gtcgggtaagttccgacctgcacgaatggcataatgatggccaggctgtctccacccgagactcagtgaaattgaaatcgccgtgaa gatgcggtgtacccgcggctagacggaaagaccccgtgaacctttactatagcttgacactgaaccttgaattttgatgtgtaggatag gtgggaggctttgaagcggtaacgccagttatcgtggagccatccttgaaataccaccctttaacgtttgatgttctaacgaagtgcccg gaacgggtactcggacagtgtctggtgggtagtttgactggggcggtctcctcccaaagagtaacggaggagcacgaaggtttgcta atgacggtcggacatcgtcaggttagtgcaatggtataagcaagcttaactgcgagacggacaagtcgagcaggtgcgaaagcag gtcatagtgatccggtggttctgaatggaagggccatcgctcaacggataaaaggtactccggggataacaggctgataccgccca agagttcatatcgacggcggtgtttggcacctcgatgtcggctcatcacatcctggggctgaagtaggtcccaagggtatggctgttcgc catttaaagtggtacgcgagctgggtttaaaacgtcgtgagacagtttggtccctatctgccgtgggcgttggagaattgagaggggct gctcctagtacgagaggaccggagtggacgcatcactggtgttccggttgtgtcgccagacgcattgccgggtagctacatgcggaa gagataagtgctgaaagcatctaagcacgaaacttgcctcgagatgagttctcccagtatttaatactgtaagggttgttggagacgac gacgtagataggccgggtgtgtaagcgttgcgagacgttgagctaaccggtactaattgcccgagaggcttagccatacaacgctca agtgtttttggtagtgaaagttattacggaataagtaagtagtcagggaatcggct

SEQ ID NO: 3 (nucleotide sequence of pfsG-gene from strain DD1 )

ttgctcgttttagctagaattggcgaaaatttttgcttaatttataaacgaggagtcgctatgaactaccctaaaattgcccaacaggttattg aaaaacttggcgggaaagaaaatatcgccaatcttgcgcattgtgcaacgcgtttgcgcttgacaatgaatgacgaaagtaaaatcg acaaacaggccattgaagatattgagggcgtaaaagggcagttttcaacctccggtcaataccaaattattttcggttcaggtacggtg aataaagtttacgccgaaatgaataccattatgaacggttcaccgtcggcggattccaccggggaaagtcaacaggcgaaagggc cgcagcaaggtttgattcaacgattaattaaaggtctggctgatattttcgttcccattattccggctattgtcgccggcggtttgttaatggg gattaataatgtctttaccgcaaaagatttattcgaagaagggaagacattactcgacctttatccgcaatacaaagatttagcggattta attaatacctttgctaacgcgccttttgtgtttctgcccgtattgttaggtttttcggcaaccagaaaattcggtggcaatccgttcttaggagc gacattaggtatgttgctcgttcaccccgctttaaccaatgcttacggttatgcggaagcgttagccggcggcaatcttcaattatggaata ttttcgggctagagattgaaaaagtcggctatcaaggcacggttattcccgttttaattgccgcctgggtattggcgactttagaaaaattct tagtgaaagtagtgccttccgtattaaataatttagtcacgccgttattttcattatttatcaccggttttttggctttcaccgtaatcggacctttc ggtcgtgaagcgggggaatttttaagtcagggtttaacctggttatatgatactttaggttttatcggcggcggcgtgttcggcgcattatac gcacctatcgtgattaccggtatgcaccaaacctttatcgccattgaaacgcaattgctggcaagcactgcggcaacttttatcttcccga ttgccgccatgtcgaatattgcgcagggtgccgcttgtttagccgttgccgtgttaaataaagatgccaaaacccgaggtctggcgttgc cttccggcatttccgcattattagggattaccgaacctgccatgttcggggtgaatttgcgcttccgttatccgttctatgcggctatgttaggt gccggttccgccgcggcgtttattgcgttcttcaatgttaaagccactgcgcttggcgcggcgggcttaatcggtatcgcatcaattcgtgc cggcgactggggaatgtattccgtggggatggtaatttcgttttgtgtggctttcgctgcggcattagtgctgggcgcaagagctaacgca aaagaatag

SEQ ID NO: 4 (amino acid sequence of PtsG from strain DD1 )

MLVLARIGENFCLIYKRGVAMNYPKIAQQVIEKLGGKENIANLAHCATRLRLTMNDESKIDKQAIE DIEGVKGQFSTSGQYQIIFGSGTVNKVYAEMNTIMNGSPSADSTGESQQAKGPQQGLIQRLIKG LADIFVPIIPAIVAGGLLMGINNVFTAKDLFEEGKTLLDLYPQYKDLADLINTFANAPFVFLPVLLGF SATRKFGGNPFLGATLGMLLVHPALTNAYGYAEALAGGNLQLWNIFGLEIEKVGYQGTVIPVLIA AVWLATLEKFLVKVVPSVLNNLVTPLFSLFITGFLAFTVIGPFGREAGEFLSQGLTWLYDTLGFIG GGVFGALYAPIVITGMHQTFIAIETQLLASTAATFIFPIAAMSNIAQGAACLAVAVLNKDAKTRGLA LPSGISALLGITEPAMFGVNLRFRYPFYAAMLGAGSAAAFIAFFNVKATALGAAGLIGIASIRAGD WG M YSVG MVIS FCVAFAAALVLGARAN AKE SEQ ID NO: 5 (nucleotide sequence of cscA-gene from E. coli \N)

atgacgcaatctcgattgcatgcggcgcaaaacgccctagcaaaacttcatgagcaccggggtaacactttctatccccattttcacct cgcgcctcctgccgggtggatgaacgatccaaacggcctgatctggtttaacgatcgttatcacgcgttttatcaacatcatccgatgag cgaacactgggggccaatgcactggggacatgccaccagcgacgatatgatccactggcagcatgagcctattgcgctagcgcca ggagacgataatgacaaagacgggtgtttttcaggtagtgctgtcgatgacaatggtgtcctctcacttatctacaccggacacgtctgg ctcgatggtgcaggtaatgacgatgcaattcgcgaagtacaatgtctggctaccagtcgggatggtattcatttcgagaaacagggtgt gatcctcactccaccagaaggaatcatgcacttccgcgatcctaaagtgtggcgtgaagccgacacatggtggatggtagtcggggc gaaagatccaggcaacacggggcagatcctgctttatcgcggcagttcgttgcgtgaatggaccttcgatcgcgtactggcccacgct gatgcgggtgaaagctatatgtgggaatgtccggactttttcagccttggcgatcagcattatctgatgttttccccgcagggaatgaatg ccgagggatacagttaccgaaatcgctttcaaagtggcgtaatacccggaatgtggtcgccaggacgactttttgcacaatccgggca ttttactgaacttgataacgggcatgacttttatgcaccacaaagctttttagcgaaggatggtcggcgtattgttatcggctggatggatat gtgggaatcgccaatgccctcaaaacgtgaaggatgggcaggctgcatgacgctggcgcgcgagctatcagagagcaatggcaa acttctacaacgcccggtacacgaagctgagtcgttacgccagcagcatcaatctgtctctccccgcacaatcagcaataaatatgtttt gcaggaaaacgcgcaagcagttgagattcagttgcagtgggcgctgaagaacagtgatgccgaacattacggattacagctcggc actggaatgcggctgtatattgataaccaatctgagcgacttgttttgtggcggtattacccacacgagaatttagacggctaccgtagta ttcccctcccgcagcgtgacacgctcgccctaaggatatttatcgatacatcatccgtggaagtatttattaacgacggggaagcggtg atgagtagtcgaatctatccgcagccagaagaacgggaactgtcgctttatgcctcccacggagtggctgtgctgcaacatggagca ctctggctactgggttaa

SEQ ID NO: 6 (amino acid sequence of CscA from E. coli W)

MTQSRLHAAQNALAKLHEHRGNTFYPHFHLAPPAGWMNDPNGLIWFNDRYHAFYQHHPMSE HWGPMHWGHATSDDMIHWQHEPIALAPGDDNDKDGCFSGSAVDDNGVLSLIYTGHVWLDGA GNDDAIREVQCLATSRDGIHFEKQGVILTPPEGIMHFRDPKVWREADTWWMVVGAKDPGNTG QILLYRGSSLREWTFDRVLAHADAGESYMWECPDFFSLGDQHYLMFSPQGMNAEGYSYRNR FQSGVIPGMWSPGRLFAQSGHFTELDNGHDFYAPQSFLAKDGRRIVIGWMDMWESPMPSKR EGWAGCMTLARELSESNGKLLQRPVHEAESLRQQHQSVSPRTISNKYVLQENAQAVEIQLQW ALKNSDAEHYGLQLGTGMRLYIDNQSERLVLWRYYPHENLDGYRSIPLPQRDTLALRIFIDTSS VEVFINDGEAVMSSRIYPQPEERELSLYASHGVAVLQHGALWLLG

SEQ ID NO: 7 (nucleotide sequence of argD-gene from strain DD1 )

atgagccaatatactcgcaaaacattcgatgaagtgatgatccaaaactacgtacctgcggattttattcctgtgaaaggcaaaggttg caaagtttgggatcagcaagggcgagattatattgattttaccagcggaattgccgtaaatgcattgggacattgtcctgacgaaatcgt cgatgtgctgaaaaaacaaggcgaaactctatggcattcaagcaactggttcaccagtgaaccgactctggaacttgcctccaaattg gttgaacatacctttgccgaacgtgtgatgtttgccaattccggcggtgaagccaatgaggcggcgcttaaattggcgcgtcgttatgcg gtggataattacggctatcaaaaagataccattatttcattcaaaaaaagtttccacggacgtaccttatttaccgtcagcgtcggcggtc aggcgaaatattcggacggattcggcccgaaacctgccggaatcgtgcatttgccgtttaatgatcttgatgcggttaaagcgatgattg atgatcacagctgtgcggtaatcgttgagccgattcagggagaaagcgggattattccggctaccaaagaatttttacagggtttgcgtc gactttgcgacgaaaataacgctctattgatttttgacgaagtacaaacgggggtagggcgtaccggttatttatacgcatatgaaagtt atgatgtagtgccggatattctgacctcgtcaaaagcccttgctaacggttttccgattagcgcaatgttaaccaccacaaaaattgccg ccagtttcaaaccgggcgtgcacggcaccacattcggcggtaatccgttagcctgtgcggtcggggcgaaagtgattgagacgattg caaatccggcgtttcttgaaaatgtacaaaaaacatccgcactttttatcagcgaattaaacaaacttaatgagaaatatcacttatttaa cgaagttcgcggtcagggattattaatcggggcggaattaattgaaaaatatcagggcaaagcctcagagtttgtcaaagcctgtgcg gataatcaacttatgattttagtcgctgggccgaatgtgctacgttttgcgccggcattaaatattagtcaacaggaagtggcggaagga tttaaacgtttagatcaggctctgcaaaaatttgcttaa

SEQ ID NO: 8 (amino acid sequence of ArgD from strain DD1 )

MSQYTRKTFDEVMIQNYVPADFIPVKGKGCKVWDQQGRDYIDFTSGIAVNALGHCPDEIVDVL KKQGETLWHSSNWFTSEPTLELASKLVEHTFAERVMFANSGGEANEAALKLARRYAVDNYGY QKDTIISFKKSFHGRTLFTVSVGGQAKYSDGFGPKPAGIVHLPFNDLDAVKAMIDDHSCAVIVEP IQGESGIIPATKEFLQGLRRLCDENNALLIFDEVQTGVGRTGYLYAYESYDWPDILTSSKALAN GFPISAMLTTTKIAASFKPGVHGTTFGGNPLACAVGAKVIETIANPAFLENVQKTSALFISELNKL NEKYHLFNEVRGQGLLIGAELIEKYQGKASEFVKACADNQLMILVAGPNVLRFAPALNISQQEV AEGFKRLDQALQKFA

SEQ ID NO: 9 (nucleotide sequence of IdhA-gene from strain DD1 )

ttgacaaaatcagtatgtttaaataaggagctaactatgaaagttgccgtttacagtactaaaaattatgatcgcaaacatctggatttgg cgaataaaaaatttaattttgagcttcatttctttgattttttacttgatgaacaaaccgcgaaaatggcggagggcgccgatgccgtctgta ttttcgtcaatgatgatgcgagccgcccggtgttaacaaagttggcgcaaatcggagtgaaaattatcgctttacgttgtgccggttttaat aatgtggatttggaggcggcaaaagagctgggattaaaagtcgtacgggtgcctgcgtattcgccggaagccgttgccgagcatgcg atcggattaatgctgactttaaaccgccgtatccataaggcttatcagcgtacccgcgatgcgaatttttctctggaaggattggtcggtttt aatatgttcggcaaaaccgccggagtgattggtacgggaaaaatcggcttggcggctattcgcattttaaaaggcttcggtatggacgtt ctggcgtttgatccttttaaaaatccggcggcggaagcgttgggcgcaaaatatgtcggtttagacgagctttatgcaaaatcccatgtta tcactttgcattgcccggctacggcggataattatcatttattaaatgaagcggcttttaataaaatgcgcgacggtgtaatgattattaata ccagccgcggcgttttaattgacagccgggcggcaatcgaagcgttaaaacggcagaaaatcggcgctctcggtatggatgtttatg aaaatgaacgggatttgtttttcgaggataaatctaacgatgttattacggatgatgtattccgtcgcctttcttcctgtcataatgtgctttttac cggtcatcaggcgtttttaacggaagaagcgctgaataatatcgccgatgtgactttatcgaatattcaggcggtttccaaaaatgcaac gtgcgaaaatagcgttgaaggctaa

SEQ ID NO: 10 (amino acid sequence of LdhA from strain DD1 )

MTKSVCLNKELTMKVAVYSTKNYDRKHLDLANKKFNFELHFFDFLLDEQTAKMAEGADAVCIFV NDDASRPVLTKLAQIGVKIIALRCAGFNNVDLEAAKELGLKWRVPAYSPEAVAEHAIGLMLTLN RRIHKAYQRTRDANFSLEGLVGFNMFGKTAGVIGTGKIGLAAIRILKGFGMDVLAFDPFKNPAAE ALGAKYVGLDELYAKSHVITLHCPATADNYHLLNEAAFNKMRDGVMIINTSRGVLIDSRAAI EAL KRQKIGALGMDVYENERDLFFEDKSNDVITDDVFRRLSSCHNVLFTGHQAFLTEEALNNIADVT LSNIQAVSKNATCENSVEG SEQ ID NO: 11 (nucleotide sequence of pflA-gene from strain DD1 )

atgtcggttttaggacgaattcattcatttgaaacctgcgggacagttgacgggccgggaatccgctttattttatttttacaaggctgcttaa tgcgttgtaaatactgccataatagagacacctgggatttgcacggcggtaaagaaatttccgttgaagaattaatgaaagaagtggtg acctatcgccattttatgaacgcctcgggcggcggagttaccgcttccggcggtgaagctattttacaggcggaatttgtacgggactgg ttcagagcctgccataaagaaggaattaatacttgcttggataccaacggtttcgtccgtcatcatgatcatattattgatgaattgattgat gacacggatcttgtgttgcttgacctgaaagaaatgaatgaacgggttcacgaaagcctgattggcgtgccgaataaaagagtgctcg aattcgcaaaatatttagcggatcgaaatcagcgtacctggatccgccatgttgtagtgccgggttatacagatagtgacgaagatttgc acatgctggggaatttcattaaagatatgaagaatatcgaaaaagtggaattattaccttatcaccgtctaggcgcccataaatgggaa gtactcggcgataaatacgagcttgaagatgtaaaaccgccgacaaaagaattaatggagcatgttaaggggttgcttgcaggctac gggcttaatgtgacatattag

SEQ ID NO: 12 (amino acid sequence of PflA from strain DD1 )

MSVLGRIHSFETCGTVDGPGIRFILFLQGCLMRCKYCHNRDTWDLHGGKEISVEELMKEVVTY RHFMNASGGGVTASGGEAI LQAEFVRDWFRACHKEGINTCLDTNGFVRHHDHIIDELIDDTDLV LLDLKEMNERVHESLIGVPNKRVLEFAKYLADRNQRTWIRHVWPGYTDSDEDLHMLGNFIKD MKNIEKVELLPYHRLGAHKWEVLGDKYELEDVKPPTKELMEHVKGLLAGYGLNVTY

SEQ ID NO: 13 (nucleotide sequence of pflD-gene from strain DD1 )

atggctgaattaacagaagctcaaaaaaaagcatgggaaggattcgttcccggtgaatggcaaaacggcgtaaatttacgtgacttt atccaaaaaaactatactccgtatgaaggtgacgaatcattcttagctgatgcgactcctgcaaccagcgagttgtggaacagcgtga tggaaggcatcaaaatcgaaaacaaaactcacgcacctttagatttcgacgaacatactccgtcaactatcacttctcacaagcctgg ttatatcaataaagatttagaaaaaatcgttggtcttcaaacagacgctccgttaaaacgtgcaattatgccgtacggcggtatcaaaat gatcaaaggttcttgcgaagtttacggtcgtaaattagatccgcaagtagaatttattttcaccgaatatcgtaaaacccataaccaagg cgtattcgacgtttatacgccggatattttacgctgccgtaaatcaggcgtgttaaccggtttaccggatgcttacggtcgtggtcgtattatc ggtgactaccgtcgtttagcggtatacggtattgattacctgatgaaagataaaaaagcccaattcgattcattacaaccgcgtttggaa gcgggcgaagacattcaggcaactatccaattacgtgaagaaattgccgaacaacaccgcgctttaggcaaaatcaaagaaatgg cggcatcttacggttacgacatttccggccctgcgacaaacgcacaggaagcaatccaatggacatattttgcttatctggcagcggtt aaatcacaaaacggtgcggcaatgtcattcggtcgtacgtctacattcttagatatctatatcgaacgtgacttaaaacgcggtttaatca ctgaacaacaggcgcaggaattaatggaccacttagtaatgaaattacgtatggttcgtttcttacgtacgccggaatacgatcaattatt ctcaggcgacccgatgtgggcaaccgaaactatcgccggtatgggcttagacggtcgtccgttggtaactaaaaacagcttccgcgt attacatactttatacactatgggtacttctccggaaccaaacttaactattctttggtccgaacaattacctgaagcgttcaaacgtttctgt gcgaaagtatctattgatacttcctccgtacaatacgaaaatgatgacttaatgcgtcctgacttcaacaacgatgactatgcaatcgcat gctgcgtatcaccgatggtcgtaggtaaacaaatgcaattcttcggtgcgcgcgcaaacttagctaaaactatgttatacgcaattaac ggcggtatcgatgagaaaaatggtatgcaagtcggtcctaaaactgcgccgattacagacgaagtattgaatttcgataccgtaatcg aacgtatggacagtttcatggactggttggcgactcaatatgtaaccgcattgaacatcatccacttcatgcacgataaatatgcatatg aagcggcattgatggcgttccacgatcgcgacgtattccgtacaatggcttgcggtatcgcgggtctttccgtggctgcggactcattatc cgcaatcaaatatgcgaaagttaaaccgattcgcggcgacatcaaagataaagacggtaatgtcgtggcctcgaatgttgctatcga cttcgaaattgaaggcgaatatccgcaattcggtaacaatgatccgcgtgttgatgatttagcggtagacttagttgaacgtttcatgaaa aaagttcaaaaacacaaaacttaccgcaacgcaactccgacacaatctatcctgactatcacttctaacgtggtatacggtaagaaa accggtaatactccggacggtcgtcgagcaggcgcgccattcggaccgggtgcaaacccaatgcacggtcgtgaccaaaaaggt gcggttgcttcacttacttctgtggctaaacttccgttcgcttacgcgaaagacggtatttcatataccttctctatcgtaccgaacgcattag gtaaagatgacgaagcgcaaaaacgcaaccttgccggtttaatggacggttatttccatcatgaagcgacagtggaaggcggtcaa cacttgaatgttaacgttcttaaccgtgaaatgttgttagacgcgatggaaaatccggaaaaatacccgcaattaaccattcgtgtttcag gttacgcggttcgtttcaactcattaactaaagagcaacaacaagacgtcatcactcgtacgtttacacaatcaatgtaa

SEQ ID NO: 14 (amino acid of PfID from strain DD1 )

MAELTEAQKKAWEGFVPGEWQNGVNLRDFIQKNYTPYEGDESFLADATPATSELWNSVMEGI KIENKTHAPLDFDEHTPSTITSHKPGYINKDLEKIVGLQTDAPLKRAIMPYGGIKMIKGSCEVYGR KLDPQVEFIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGRGRIIGDYRRLAVYGIDYL MKDKKAQFDSLQPRLEAGEDIQATIQLREEIAEQHRALGKIKEMAASYGYDISGPATNAQEAIQ WTYFAYLAAVKSQNGAAMSFGRTSTFLDIYIERDLKRGLITEQQAQELMDHLVMKLRMVRFLRT PEYDQLFSGDPMWATETIAGMGLDGRPLVTKNSFRVLHTLYTMGTSPEPNLTILWSEQLPEAF KRFCAKVSIDTSSVQYENDDLMRPDFNNDDYAIACCVSPMVVGKQMQFFGARANLAKTMLYAI NGGIDEKNGMQVGPKTAPITDEVLNFDTVIERMDSFMDWLATQYVTALNIIHFMHDKYAYEAAL MAFHDRDVFRTMACGIAGLSVAADSLSAI KYAKVKPIRGDIKDKDGNWASNVAIDFEIEGEYPQ FGNNDPRVDDLAVDLVERFMKKVQKHKTYRNATPTQSILTITSNVVYGKKTGNTPDGRRAGAP FGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDDEAQKRNLAGLMDG YFHHEATVEGGQHLNVNVLNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRT FTQSM

SEQ ID NO: 15 (nucleotide sequence of wcaJ-gene from strain DD1 )

atgataaaacgccttttcgatattgttgtcgcattgatagcattgattttgttttcgcccttatatttgtttgtggcttataaggtaaaacaaaattt gggatcaccggtgttatttaaacaaacccgccccggattgcatggtaaaccctttgagatgattaagttcagaacaatgaaagacggc gcagatgaaaacggtaatattttgccggatgcggagcgcttaacacctttcggcaaaatgttgcgcgctaccagtctggacgagttgcc ggaactttggaatgtattaaaaggtgatatgagtctggtggggccgcgtcctctactgatggaatatttgccgctgtataacgaaagaca ggctaagcgccatgaagtgaaacccggaattaccggttatgcacaggtaaacggtcgcaatgccatcagttgggagcagaaatttg aattggatgcctggtatgttgaacatcaatccttgtggctggatttgaaaattatcgcaaagaccatccaaaaagtgatcgcaaaagac gatattaatgcggcagatgatgccaccatgcctaaatttgaagggaataaaaaatcatga

SEQ ID NO: 16 (amino acid sequence of the enzyme encoded by the above wcaJ-gene) MIKRLFDIVVALIALILFSPLYLFVAYKVKQNLGSPVLFKQTRPGLHGKPFEMIKFRTMKDGADEN GNILPDAERLTPFGKMLRATSLDELPELWNVLKGDMSLVGPRPLLMEYLPLYNERQAKRHEVK PGITGYAQVNGRNAISWEQKFELDAWYVEHQSLWLDLKIIAKTIQKVIAKDDINAADDATMPKFE GNKKS

SEQ ID NO: 17 (nucleotide sequence of wcaJ-gene from strain DD1 with insertion of cytosine between nucleotides 81 and 82) atgataaaacgccttttcgatattgttgtcgcattgatagcattgattttgttttcgcccttatatttgtttgtggcttatcaaggtaaaacaaaatt tgggatcaccggtgttatttaaacaaacccgccccggattgcatggtaaaccctttgagatgattaagttcagaacaatgaaagacgg cgcagatgaaaacggtaatattttgccggatgcggagcgcttaacacctttcggcaaaatgttgcgcgctaccagtctggacgagttgc cggaactttggaatgtattaaaaggtgatatgagtctggtggggccgcgtcctctactgatggaatatttgccgctgtataacgaaagac aggctaagcgccatgaagtgaaacccggaattaccggttatgcacaggtaaacggtcgcaatgccatcagttgggagcagaaattt gaattggatgcctggtatgttgaacatcaatccttgtggctggatttgaaaattatcgcaaagaccatccaaaaagtgatcgcaaaaga cgatattaatgcggcagatgatgccaccatgcctaaatttgaagggaataaaaaatcatga SEQ ID NO: 18 (nucleotide sequence of pykA-gene from strain DD1 )

atgtccagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattgc tgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgctc ataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaat atcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcggcggtttatctgccgatgcattaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacattaaacttatgc gcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaaga cgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattgtt gaataa

SEQ ID NO: 19 (amino acid sequence of PykA from strain DD1 )

MSRRLRRTKIVCTMGPATDKGNNLEKIIAAGANVVRMNFSHGTPEDHIGRAEKVREIAHKLGKH VAILGDLQGPKIRVSTFKEGKIFLNIGDKFILDAEMPKGEGNQEAVGLDYKTLPQDWPGDILLLD DGRVQLKVLATEGAKVFTEVTVGGPLSNNKGINKLGGGLSADALTEKDKADIITAARIGVDYLAV SFPRSSADLNYARQLAKDAGLDAKIVAKVERAETVETDEAMDDIINAADVIMVARGDLGVEIGDP ELVGVQKKLIRRSRQLNRVVITATQMMESMISNPMPTRAEVMDVANAVLDGTDAVMLSAETAA GQYPAETVAAMAKVALGAEKMPSI NVSKH RMNVQFESI EESVAMSAMYAANH MRGVAAI ITLT SSGRTARLMSRISSGLPIFALSRNESTLNLCALYRGVTPVHFDKDSRTSEGATAAVQLLKDEGF LVSGDLVLLTQGDASSSSGTNLCRTLIVE

SEQ ID NO: 20 (nucleotide sequence of fruA-gene from strain DD1 )

ttgaaggataagccgatgaatatttttcttacgcaatcaccaaatttaggtcgtgcaaaagcgtttttattgcaccaggttttggctgccgca gtaaaacaacaaaatcatcaactggtagaaaatgccgaacaagcggatttagcgattgttttcggtaaaactttgccgaatttgaccgc acttttaggtaaaaaagtgtatttggcggatgaagaacaagcgttgaatgcgcctgaaaataccgtcgcgcaggcattaaccgaggct gtggattatgttcaaccggcgcaacaggacgtgcaacccgcaactgcttccggtatgaaaaatatcgtggcggttaccgcttgtccga ccggggtggcgcacacctttatgtctgccgaggcgattacaacctactgccaacagcaaggttggaatgtaaaagtggaaaccaga ggtcaagtcggtgcgaacaatattatttctgcggaagatgtggcggcggccgatttagtctttatcgctacggatattaatgtggatttaag caaattcaaaggaaaaccgatgtatcgtacttcaacgggcttagcattgaagaaaaccgcacaggaatttgataaagcctttaaaga agcgacgatttatcagggtgaagaaactacaaccaccacagaaacacaaacttcaggcgagaaaaaaggtgtatataaacatctt atgaccggggtttcccatatgttaccgcttgtcgttgccggcggtttattgattgctatttcgtttatgttcggtattgaggcgtttaaagacgaa aacatcgcaggcggcttgccgaaagcattaatggatatcggcggcggtgcggcgttccacttaatgattgccgtatttgcaggttatgtt gcattctctattgcagaccgtccggggttagccgtaggtcttatcggcggtatgcttgccacatccgccggtgccggtattttgggcggtat tatcgcgggttttcttgccggttatgtagtgaaattcctgaatgatgccattcaactgccagccagtttaacttcgttaaaaccgattttaattc tgcctttattaggttcggcgatcgtcggcttggccatgatttatttattaaatccaccggttgctgcggcaatgaatgcgctaaccgaatggt taaaaggtttgggctcggcaaacgcgctggtgttgggtgcgattcttggcggtatgatgtgtatcgatatgggcggtccggtaaacaaa gccgcttatgtattcggtacgggcatgattggttcacaggtttatacgccgatggctgcggtaatggctgcgggtatggtaccgcctttag gaatggcgattgccacctggattgcgcgcgctaaatttaacgcaagccaacgtgatgcgggtaaagcttcattcgtactaggtttatgct ttatttccgaaggtgcgttaccgtttgttgccgccgaccctgtacgcgtgattgtttcaagtgtaattggcggagccattgccggcgcaattt ctatgagccttgccattacgctgcaagcgcctcacggcggtttattcgtgattccgtttgtgtcgcaaccgttaatgtatttgggtgcgattgc cgtaggcgccttaacaaccggcgttctttacgcaattatcaaaccgaaacaagctgcggaataa

SEQ ID NO: 21 (amino acid sequence of the enzyme encoded by the above fruA-gene) MKDKPMNIFLTQSPNLGRAKAFLLHQVLAAAVKQQNHQLVENAEQADLAIVFGKTLPNLTALLG KKVYLADEEQALNAPENTVAQALTEAVDYVQPAQQDVQPATASGMKNIVAVTACPTGVAHTF MSAEAITTYCQQQGWNVKVETRGQVGANNIISAEDVAAADLVFIATDINVDLSKFKGKPMYRTS TGLALKKTAQEFDKAFKEATIYQGEETTTTTETQTSGEKKGVYKHLMTGVSHMLPLVVAGGLLI AISFMFGIEAFKDENIAGGLPKALMDIGGGAAFHLMIAVFAGYVAFSIADRPGLAVGLIGGMLATS AGAGILGGIIAGFLAGYVVKFLNDAIQLPASLTSLKPILILPLLGSAIVGLAMIYLLNPPVAAAMNAL TEWLKGLGSANALVLGAILGGMMCIDMGGPVNKAAYVFGTGMIGSQVYTPMAAVMAAGMVPP LGMAIATWIARAKFNASQRDAGKASFVLGLCFISEGALPFVAADPVRVIVSSVIGGAIAGAISMSL AITLQAPHGGLFVI PFVSQPLMYLGAIAVGALTTGVLYAI I KPKQAAE

SEQ ID NO: 22 (complete nucleotide sequence of plasmid pSacB)

tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttcggacctagggatatcgtcgacatcgatgctcttctgcgtt aattaacaattgggatcctctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttatt ttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgca aacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcgg atgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggagg acggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccg gattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcg atattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttcc ggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaa cattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgatacca gattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatca ccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttc atcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctg tatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcg cttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtg agaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcg ctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgc gttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagg actataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcac gaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca ctagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacc accgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaagg ccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttct tgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacatt gtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgtt cagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgat ccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttga agatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc

SEQ ID NO: 23 (complete nucleotide sequence of plasmid pSacB_delta_/c/M)

tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttcggacctagggatgggtcagcctgaacgaaccgcactt gtatgtaggtagttttgaccgcccgaatattcgttataccttggtggaaaaattcaaaccgatggagcaattatacaattttgtggcggcgc aaaaaggtaaaagcggtatcgtctattgcaacagccgtagcaaagtggagcgcattgcggaagccctgaagaaaagaggcatttc cgcagccgcttatcatgcgggcatggagccgtcgcagcgggaagcggtgcaacaggcgtttcaacgggataatattcaagtggtgg tggcgaccattgcttttggtatggggatcaacaaatctaatgtgcgttttgtggcgcattttgatttatctcgcagcattgaggcgtattatcag gaaaccgggcgcgcggggcgggacgacctgccggcggaagcggtactgttttacgagccggcggattatgcctggttgcataaaat tttattggaagagccggaaagcccgcaacgggatattaaacggcataagctggaagccatcggcgaatttgccgaaagccagacc tgccgtcgtttagtgctgttaaattatttcggcgaaaaccgccaaacgccatgtaataactgtgatatctgcctcgatccgccgaaaaaat atgacggattattagacgcgcagaaaatcctttcgaccatttatcgcaccgggcaacgtttcggcacgcaatacgtaatcggcgtaatg cgcggtttgcagaatcagaaaataaaagaaaatcaacatgatgagttgaaagtctacggaattggcaaagataaaagcaaagaat actggcaatcggtaattcgtcagctgattcatttgggctttgtgcaacaaatcatcagcgatttcggcatggggaccagattacagctcac cgaaagcgcgcgtcccgtgctgcgcggcgaagtgtctttggaactggccatgccgagattatcttccattaccatggtacaggctccgc aacgcaatgcggtaaccaactacgacaaagatttatttgcccgcctgcgtttcctgcgcaaacagattgccgacaaagaaaacattc cgccttatattgtgttcagtgacgcgaccttgcaggaaatgtcgttgtatcagccgaccagcaaagtggaaatgctgcaaatcaacggt gtcggcgccatcaaatggcagcgcttcggacagccttttatggcgattattaaagaacatcaggctttgcgtaaagcgggtaagaatc cgttggaattgcaatcttaaaatttttaactttttgaccgcacttttaaggttagcaaattccaataaaaagtgcggtgggttttcgggaattttt aacgcgctgatttcctcgtcttttcaatttyttcgyctccatttgttcggyggttgccggatcctttcttgactgagatccataagagagtagaa tagcgccgcttatatttttaatagcgtacctaatcgggtacgctttttttatgcggaaaatccatatttttctaccgcactttttctttaaagatttat acttaagtctgtttgattcaatttatttggaggttttatgcaacacattcaactggctcccgatttaacattcagtcgcttaattcaaggattctg gcggttaaaaagctggcggaaatcgccgcaggaattgcttacattcgttaagcaaggattagaattaggcgttgatacgctggatcat gccgcttgttacggggcttttacttccgaggcggaattcggacgggcgctggcgctggataaatccttgcgcgcacagcttactttggtg accaaatgcgggattttgtatcctaatgaagaattacccgatataaaatcccatcactatgacaacagctaccgccatattatgtggtcg gcgcaacgttccattgaaaaactgcaatgcgactatttagatgtattgctgattcaccgwctttctccctgtgcggatcccgaacaaatcg cgcgggcttttgatgaactttatcaaaccggraaagtacgttatttcggggtatctaactatacgccggctaagttcgccatgttgcaatctt atgtgaatcagccgttaatcactaatcaaattgagatttcgcctcttcatcgtcaggcttttgatgacggtaccctggattttttactggaaaa acgtattcaaccgatggcatggtcgccacttgccggcggtcgtttattcaatcaggatgagaacagtcgggcggtgcaaaaaacatta ctcgaaatcggtgaaacgaaaggagaaacccgtttagatacattggcttatgcctggttattggcgcatccggcaaaaattatgccggt tatggggtccggtaaaattgaacgggtaaaaagcgcggcggatgcgttacgaatttccttcactgaggaagaatggattaaggtttatg ttgccgcacagggacgggatattccgtaacatcatccgtctaatcctgcgtatctggggaaagatgcgtcatcgtaagaggtctataat attcgtcgttttgataagggtgccatatccggcacccgttaaaatcacattgcgttcgcaacaaaattattccttacgaatagcattcacct cttttaacagatgttgaatatccgtatcggcaaaaatatcctctatatttgcggttaaacggcgccgccagttagcatattgagtgctggttc ccggaatattgacgggttcggtcataccgagccagtcttcaggttggaatccccatcgtcgacatcgatgctcttctgcgttaattaacaa ttgggatcctctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcaca aatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtca gatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggc cggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggaggacggaatg ttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccggattgacct gaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgatattaccg ctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccggagttcc ggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaacattctctg cactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgataccagattgtttc cgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccggaaa tgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatcatgca gtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtatttaagc caccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttggactc ctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatcca agcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgct tcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacaga atcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcg tttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaag ataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg gaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagcc actggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagg acagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggt agcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacg ctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggccggccgc ggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgat gttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcg cttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttcagcggctt gtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcggga gtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatc acttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagttttt gactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttg cttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatg aactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctga tacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaat gtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttc cagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagac gatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagat atttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaa atgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatact gttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatgg caagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtctt gcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgttt gatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgt tgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtg atgggttaaaaaggatcggcggccgctcgatttaaatc SEQ ID NO: 24 (complete nucleotide sequence of plasmid pSacB_delta_pf/A)

tcgagtcaatgcggatttgacttatgatgtggcaaacaaccgatttccgattattactacacgtaaaagttattggaaagcggcgattgcg gagtttctgggttatatccgcggctacgataatgcggcggatttccgtaaattaggagcaaaaacctgggatgccaacgctaatgaaa atcaggtatggctgaataaccctcatcgcaaaggcaccgacgacatggggcgcgtttacggcgtacagggcagagcctggcgtaa gcctaacggcgaaaccgttgatcaattacgcaaaattgtcaacaatttaagtcgcggcattgatgatcgcggcgaaattctgaccttttt aaacccgggcgaattcgatctcggttgtctgcgcccttgtatgtacaatcacacgttttctttgctgggcgatacgctttatttaaccagttat caacgctcctgtgacgtacctttaggcttgaatttcaatcaaattcaagtatttacattcttagctttaatggcgcagattaccggtaaaaaa gccggtcaggcatatcacaaaatcgtcaatgcgcatatttacgaagaccagctggaactaatgcgcgacgtgcagttaaaacgcga accgttcccgtcgccaaaactggaaattaatccggacattaaaacccttgaagatttagaaacctgggtaaccatggatgatttcaacg tcgttggttaccaatgccacgaaccgataaaatatccgttctcggtataaaccgacaaaagtgcggtcaaaaatttaatattttcatctgtt atagaaaatatttttcaacataaaatctagggatgcctgtttggcgtccgtaaatacgcagaaaaatattaaatttttgaccgcacttttttc atctcaattaacagcctgataattcttatggatcaacaaattagctttgacgaaaaaatgatgaatcgagctcttttccttgccgacaagg cggaagctttaggggaaattcccgtaggtgccgtattggtggatgaacggggcaatatcattggtgaaggctggaacctctctattgtg aactcggatcccaccgcccatgccgaaattattgcgttgcgtaacgccgcgcagaaaatccaaaattaccgcctgctcaataccactt tatacgtgactttagaaccctgcaccatgtgcgccggcgcgattttacacagccgaatcaaacgcttggtattcggggcgtccgattac aaaaccggtgcggtgggttccagatttcatttttttgaggattataaaatgaatcatggggttgagatcacaagcggtgtcttacaggatc aatgcagtcagaagttaagccgctttttccaaaagcgcagggaacagaaaaaacaacaaaaagctaccgcacttttacaacaccc ccggcttaactcctctgaaaaatagtgacaaaaaaaccgtcataatgtttacgacggtttttttatttcttaatatgcccttaaataatcaac aaaatatagcaagaagattatagcaaagaatttcgtttttttcagagaatagtcaaatcttcgcaaaaaactaccgcacttttatccgcttt aatcaggggaattaaaacaaaaaaattccgcctattgaggcggaatttattaagcaataagacaaactctcaattacattgattgtgta aacgtacgagtgatgacgtcttgttgttgctctttagttaatgagttgaaacgaaccgcgtaacctgaaacacgaatggttaattgcgggt atttttccggattttccatcgcgtctaacaacatttcacggttaagaacgttaacattcaagtgttgaccgccttccactgtcgcttcatgatg gaaataaccgtccattaaaccggcaaggttgcgtttttgcgcttcgtcatctttacctaatgcgttcggtacgatagagaaggtatatgaa ataccgtctttcgcgtaagcgaacggaagtttagccacagaagtaagtgaagcaaccgcacctttttggtcacgaccgtgcattgggttt gcacccggtccgaatggcgcgcctgctcgacgaccgtccggagtattaccggttttcttaccgtataccacgttagaagtgatagtcag gatagattgtgtcggagttgcgttgcggtaagttttgtgtttttgaacttttttcatgaaacgttcaactaagtctaccgctaaatcatcaacac gcggatcattgttaccgaattgcggatattcgccttcaatttcgaagtcgatagcaacattcgaggccacgacattaccgtctttatctttga tgtcgccgcgaatcggtttaactttcgcatatttgattgcggataatgagtccgcagccacggaaagacccgcgataccgcaagccatt gtacggaatacgtcgcgatcgtggaacgccatcaatgccgcttcatatgcatatttatcgtgcatgaagtggatgatgttcaatgcggtta catattgagtcgccaaccagtccatgaaactgtccatacgttcgattacggtatcgaaattcaatacttcgtctgtaatcggcgcagtttta ggaccgacttgcataccatttttctcatcgataccgccgttaattgcgtataacatagttttagctaagtttgcgcgcgcaccgaagaattg catttgtttacctacgaccatcggtgatacgcagcatgcgattgcatagtcatcgttgttgaagtcaggacgcattaagtcatcattttcgta ttgtacggaggaagtatcaatagatactttcgcacagaaacgtttgaacgcttcaggtaattgttcggaccaaagaatagttaagtttggt tccggagaagtacccatagtgtataaagtatgtaatacgcggaagctgtttttagttaccaacggacgaccgtctaagcccataccggc gatagtttcggttgccctctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttc ggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaa cctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggat gattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggaggac ggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccggat tgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgata ttaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccgg agttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaaca ttctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgataccag attgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcac cggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcat catgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgta tttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgctt ggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgag aatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctc ttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatc cacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt gctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggac tataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctc ccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacg aaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggc agcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacac tagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggc cggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttctt gcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattg tttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttc agcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatc cgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttga agatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc

SEQ ID NO: 25 (complete nucleotide sequence of plasmid pSacB wcaJ^*)

tcgagtaagccgattcagctgatccgccacatggggaaaaagcctaatctgcggaatatgaaaccgataccagtccagtaaagttg acaaatcgacatcatattgctcaaccaagtattgaaaagcgttttcaccgcgatgatacaattcgaccagccggttaaataacgtttcac tccgttccggtgccaaacgagacgcaatatgcttataggcggaatacagaaaatcgatttccgcataaagcgtatcgtccaaatctaa aaccaacgctttatttttcataatgatgagccagtacttccgcgtcataccgcaacattaataaatccgcttcccagtcttcgaacgcagg aatcggctgattaaacaaatattcctgaatcaaccaacggggataattggctcccgccagataactcaacggataaccgccgccga acctagggttaatttcaataccaagaatttcagcggtggattccttataaaatacttggattgttaagcaaccgcgcgcccccggtaaac gggacaatttttccgataattgcgtcacgatggcattttttctggtcacacctttgttaatttcccccgctctgacaaaaattctctttctcggta ccgcacttttcagttcggaatttttatcaaaataacaatccacggtatattcgtcgtattccgccggcgaaatatattgcataaacattaattc gggattttccaattgctccggtgaaatatcttccggtttctccgccacaaaaattcctttacttaaactaccgttgtaaggcttcacaaaaac aggatattcaaattgacctttttcaaactgcttcggtaccgcaatattatgttcaataaacagttgattggttaatcgtttgtcgcgacattttct gacaaactctgtatcactaacggaaataaaaatacctttttctttaaaccgttgcagatgttcgcttaaaataagcaattccgtatcaatag tcggaataatcaatttcacgttattttcttcacagattttaagtaaggtcggaatatactccgcatcagtgacccggggtacaggaaaatgt ccgtcggccacataacaagccggcgccaactcgggatttaaatctacggttaacacttttccgtcacttactaactgcgataattccttttt aaacgcctgaacgagagaaacacgttgtccggccgatgtaacaagaatattcatgattttttattcccttcaaatttaggcatggtggcat catctgccgcattaatatcgtcttttgcgatcactttttggatggtctttgcgataattttcaaatccagccacaaggattgatgttcaacatac caggcatccaattcaaatttctgctcccaactgatggcattgcgaccgtttacctgtgcataaccggtaattccgggtttcacttcatggcg cttagcctgtctttcgttatacagcggcaaatattccatcagtagaggacgcggccccaccagactcatatcaccttttaatacattccaa agttccggcaactcgtccagactggtagcgcgcaacattttgccgaaaggtgttaagcgctccgcatccggcaaaatattaccgttttc atctgcgccgtctttcattgttctgaacttaatcatctcaaagggtttaccatgcaatccggggcgggtttgtttaaataacaccggtgatcc caaattttgttttaccttgataagccacaaacaaatataagggcgaaaacaaaatcaatgctatcaatgcgacaacaatatcgaaaag gcgttttatcatgaaaatctcctacgaccgaccaatttggggctgacaaaagtgccgtttttcaccagaaccgtataaactaaaaccag gaaaagcggataccagactaacggcagtcttaatatggaagacggcacccaaacgatccaacaaaaattaataaaaataaataa aataaatatacgttctttccacgccaacaattgggtattcatcacatagataattgaatttaataaaaaatatataaatgccaaaaatgca aaaatgccaaatgccaaataaagttctataaagaagctatgcggattagtgtaacccaaagggaaacttaactttatttgatcgaaata ctgaatgtagtcccgcggtccataacccaaccataaaattttaaaattatctaaaaatgtcgtataaatttccgtccggtaacctacggac ttatcatcgcccatagaaaatattaccaatgaaaaacgttcaatcggacgctccagccaatcaattttggcgagcagaataaacacttc ctgtaaccaagaaagattaaatataaataatgcgatcacgcaggcgaaaaacagatataccgccttaaaataggtagatgcgtttaa aaacaagatcagcatcaacataatcaaatagctcagtaataccgaacgggaggcactgatcacaatagctaaccccataataaaa ataagagcatagccgattaacttaattttccagttgttttctctgatgatgtaaaaaaatcccaccgccaccgcaagagaaatcataatta cggactggtcattggtattaaagaaaaaacctttaaacgccttatcagttacggttaattcttcattacccgaaaccaactggaacccca ataaagcctcaataaaaaagcccgccagcacaattaatgatattcccaacaaaaggtgcctaatccctgcttctccatcaccccggtt aaacgtcaaaaaagaataatgaaataaaaacatcacaattccgaagaaaaacaaatcaactaatttttctgtagaaaacgcatttaa taccgataaaaagccgaagaaaaacaacacgtacaccggaaactgtaatttaaaaaaatcagtttccatatcccttaaaaaagggg ttattaccgctaagaaaaagaacaaaaaacacaacgcactatctaacctcggcactcctatttgtgtcgatagtgcgggagaaagtat caccagtcccaacgcaaagagtaatagcaacttaaaaatgctgataacattaatattcatatcaaataatatttttgattaatttctcaattt ctttataagaacgctcgcgcagaaacttctcttttgccagcgataaattcacttgcgacattttgtctaaaaccgttctgtcttcggccaattt attcaacgtctcagccaactcccgataatctcccgccgtatattgaattccaccgcctttcgccagtagtttttccacttcaggatgtttctga cagcttacaatcggtaatgcgcaacagatataatcggatctagactccataggccgctttcctggctttgcttccagatgtatgctctcctc cggagagtaccgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtac cggcggaagacaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcaga actgatccgctatgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactga ataccaaacagcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaag gaataaccatgaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggat tcagcctgaccaccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgat ctcccgggctgttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttact gtctttcataaagaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacg gcagaatatcagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttg acgggatttaacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgta ttattacctgtttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacata ctgaaataaattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttca gactggttcaggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttg ccgccgggcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaagg agaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcca ggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcaga ggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctt accggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgc tccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtg gtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaaga agatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatct tcacctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgct gtctttgacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcat atagcttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttt taacacaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaa tgccgtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgtt actgtgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcag ccgtgcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcg ccttggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgt cgcctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccg ttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtaga ataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgc tgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcat ttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtccca aacgtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttg cagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgc cagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtct gcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagt atacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgttt ggattgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatct gtttcttttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcact aaataatagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc

SEQ ID NO: 26 (complete nucleotide sequence of plasmid pSacB_delta_pfsG_cscA) ctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcacaaatacaggg gtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtcagatggagatt gatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggccggaataaat aaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggaggacggaatgttacccattga gacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccggattgacctgaatacctgg aatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgatattaccgctttgcgtacc gcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccggagttccggatggcact gaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaacattctctgcactgtcctgc cgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgataccagattgtttccgcagggaa atttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccggaaatgatgattatttt gccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatcatgcagtctgtgatgg ctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtatttaagccaccgtatcc ggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttggactcctgttgatag atccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactag cggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctc actgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggg gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccat aggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccag gcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgt ggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcag cccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggta acaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtat ttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtg gaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggccggccgcggccgcc atcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgatgttcagca ggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcgcttgaggt acagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttcagcggcttgtatggg ccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcgggagtcagtg aacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcacttttttca gtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttcttg acggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaata ctaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgt acattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaac ttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaa cctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtca atagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggta gccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattg tggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtat gtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgtt ttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagt tacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgcttta tcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgtttgatagaaa atcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgttgcatgggc ataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtgatgggttaaa aaggatcggcggccgctcgatttaaatctcgagggataaaactatgagccaatatactcgcaaaacattcgatgaagtgatgatcca aaactacgtacctgcggattttattcctgtgaaaggcaaaggttgcaaagtttgggatcagcaagggcgagattatattgattttaccag cggaattgccgtaaatgcattgggacattgtcctgacgaaatcgtcgatgtgctgaaaaaacaaggcgaaactctatggcattcaagc aactggttcaccagtgaaccgactctggaacttgcctccaaattggttgaacatacctttgccgaacgtgtgatgtttgccaattccggcg gtgaagccaatgaggcggcgcttaaattggcgcgtcgttatgcggtggataattacggctatcaaaaagataccattatttcattcaaaa aaagtttccacggacgtaccttatttaccgtcagcgtcggcggtcaggcgaaatattcggacggattcggcccgaaacctgccggaat cgtgcatttgccgtttaatgatcttgatgcggttaaagcgatgattgatgatcacagctgtgcggtaatcgttgagccgattcagggagaa agcgggattattccggctaccaaagaatttttacagggtttgcgtcgactttgcgacgaaaataacgctctattgatttttgacgaagtaca aacgggggtagggcgtaccggttatttatacgcatatgaaagttatgatgtagtgccggatattctgacctcgtcaaaagcccttgctaa cggttttccgattagcgcaatgttaaccaccacaaaaattgccgccagtttcaaaccgggcgtgcacggcaccacattcggcggtaat ccgttagcctgtgcggtcggggcgaaagtgattgagacgattgcaaatccggcgtttcttgaaaatgtacaaaaaacatccgcacttttt atcagcgaattaaacaaacttaatgagaaatatcacttatttaacgaagttcgcggtcagggattattaatcggggcggaattaattgaa aaatatcagggcaaagcctcagagtttgtcaaagcctgtgcggataatcaacttatgattttagtcgctgggccgaatgtgctacgttttg cgccggcattaaatattagtcaacaggaagtggcggaaggatttaaacgtttagatcaggctctgcaaaaatttgcttaaaattgtgaa gaaaagcaaattatttaaaaattcaatattgacataaatagtaaataactttacacttcttgacgttttatctaagagtgactttgatagagct aaactttaaatattttattcataaaacccttttagccagagtatttttgctctggctcttttctttttaaacttccgtttcttttgaatttgatctaaate^ aggaaatttacctaatttttagatttttctagtgttgcttgctcgttttagctagaattggcgaaaatttttgcttaatttataaacgaggagtcgct atgacgcaatctcgattgcatgcggcgcaaaacgccctagcaaaacttcatgagcaccggggtaacactttctatccccattttcacct cgcgcctcctgccgggtggatgaacgatccaaacggcctgatctggtttaacgatcgttatcacgcgttttatcaacatcatccgatgag cgaacactgggggccaatgcactggggacatgccaccagcgacgatatgatccactggcagcatgagcctattgcgctagcgcca ggagacgataatgacaaagacgggtgtttttcaggtagtgctgtcgatgacaatggtgtcctctcacttatctacaccggacacgtctgg ctcgatggtgcaggtaatgacgatgcaattcgcgaagtacaatgtctggctaccagtcgggatggtattcatttcgagaaacagggtgt gatcctcactccaccagaaggaatcatgcacttccgcgatcctaaagtgtggcgtgaagccgacacatggtggatggtagtcggggc gaaagatccaggcaacacggggcagatcctgctttatcgcggcagttcgttgcgtgaatggaccttcgatcgcgtactggcccacgct gatgcgggtgaaagctatatgtgggaatgtccggactttttcagccttggcgatcagcattatctgatgttttccccgcagggaatgaatg ccgagggatacagttaccgaaatcgctttcaaagtggcgtaatacccggaatgtggtcgccaggacgactttttgcacaatccgggca ttttactgaacttgataacgggcatgacttttatgcaccacaaagctttttagcgaaggatggtcggcgtattgttatcggctggatggatat gtgggaatcgccaatgccctcaaaacgtgaaggatgggcaggctgcatgacgctggcgcgcgagctatcagagagcaatggcaa acttctacaacgcccggtacacgaagctgagtcgttacgccagcagcatcaatctgtctctccccgcacaatcagcaataaatatgtttt gcaggaaaacgcgcaagcagttgagattcagttgcagtgggcgctgaagaacagtgatgccgaacattacggattacagctcggc actggaatgcggctgtatattgataaccaatctgagcgacttgttttgtggcggtattacccacacgagaatttagacggctaccgtagta ttcccctcccgcagcgtgacacgctcgccctaaggatatttatcgatacatcatccgtggaagtatttattaacgacggggaagcggtg atgagtagtcgaatctatccgcagccagaagaacgggaactgtcgctttatgcctcccacggagtggctgtgctgcaacatggagca ctctggctactgggttaacataatatcaggtggaacaacggatcaacagcgggcaagggatccgtttaaaaaatcagtcaaaaaag accgcacttttaacgcaaagttcggtcttttttttgctattttttctccggtttttccgtatcggctatttttaaccggtaggaaaatattaatcccaa caggcttaaaacggataaggtctgcaaggcgatttgcagaccgtaatgatctgcaatccagcccacaattggtgaaaatatcccgcc catagttattcccagcccaagggtaatgccggctgcaaagccgacgcttttggcaagataagcctgaccaagcacgacaataggac tatattgagtaaaaacgcccagtcccaacggaattagcaaaataaatgagagccataagttttcgctttgagtaaaaattaaaatagtg ggtaaaaaaatcaaataagcgtagcggataattctgacataacccaggcgatcggaaagcaacccgccgataaaggtaatggcg acccccattgataaaaatattgtgagcgcaaagttagcgtcggtttcttgttgatgaagtatatgaatccaatagataggaataaaagcg ttaagtactgtgaaattggtggcgcggacaaaaataatcaccgacaatttggcaaaactgcgccaatcgttttgtaatgtggtagaagc ggtgttttttgctttattaaccacattttcgactgttaattttggcaactgtaagaaaataatgagcgcgatgatcgtgttaatcagggcaaaa atacttaaggtttgcgcaccgaataaataggcgagccccgcgaacatcggtcctatagcaaaaccggcattgccgccgacggcaa aaatccccatagctttgcctttttctccgccggacattcggttaaccagtctcgctccttccgggtgaaacagagcggaacctatgccgg caatcatggcaaaaaagaataaacccgggtaggaatggacaaagcccatagcggcaatacagcagcctgacagcatcattccca aaggaattaaccaaggcatagaacgtttatccgccaaatagccgaaaaacggttgagcaattgaggcgagaaccgtgttggcgaa aattaatccgccggcttcctgataggtcaatccgtaattttttataaaaagcggaagcagggcggggagcgctccttgcgccatatcggt gacgagatgcccgattgccgcaagataaacagcgaatttgttttgcataataagttcgttaaaagataaataagttctattataatcccg aagtttccggtgtattttaataaaacgaattattttgcattttattacttaagatcaaaagacgggtatcgggaaattaaaagtgcggtgaa aatttgcgtttttttggcgggtagaaaaaagccctttcatcatgaaagggcaaaatacatttggagttatattttttaaagtatgtttgtattata tatgcggaattttaacttttacaacatatttttaacatttacttttggtgaggattgcgcgtt

SEQ ID NO: 27 (complete nucleotide sequence of plasmid pSacB_delta_pfsG_argD::cscA- fusion))

tgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccg gaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatca tgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtattt aagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttg gactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgaga atccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctctt ccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatcc acagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttg ctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcc cttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacga accccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggca gcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacact agaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggc cggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttctt gcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattg tttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttc agcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatc cgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttga agatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatctcgagtaaaatgaatgcccgcctgatggatgtagcgccgaccg gaaacgggcgccgcgaatcttatgcgaatttgccgatgccccgcatgaccaatacttatatgttgtcgggcgacagcaagtttgaggat ttgatcggttcaattgatagaggtatttttgcctcgcatttcggcggcgggcaagtggatattacttccggaaaattcactttctccaccaca gaagcctaccttattgaaaaggggaaaatcactcgtccggtaaaaggcgctactttaatcggcagcggtattgaagtgatgcagcag gtttcaatggtggcggataatatggaaatcgaccatggtatcggcgtgtgcggtaaagagggacaaagtgtgccggtcggcgtggga caacccgcattaaaaatcgaaaaaatcaccgttggcggtacaaattaaacaataacgaagaccgtacataaaagtgcggtctttttta tgtgaatttttattcattatatttgactaattattcaataagttctatgctaaatccaatttaacacaacataagaaagaaaaggataaaacta tgagccaatatactcgcaaaacattcgatgaagtgatgatccaaaactacgtacctgcggattttattcctgtgaaaggcaaaggttgc aaagtttgggatcagcaagggcgagattatattgattttaccagcggaattgccgtaaatgcattgggacattgtcctgacgaaatcgtc gatgtgctgaaaaaacaaggcgaaactctatggcattcaagcaactggttcaccagtgaaccgactctggaacttgcctccaaattgg ttgaacatacctttgccgaacgtgtgatgtttgccaattccggcggtgaagccaatgaggcggcgcttaaattggcgcgtcgttatgcgg tggataattacggctatcaaaaagataccattatttcattcaaaaaaagtttccacggacgtaccttatttaccgtcagcgtcggcggtca ggcgaaatattcggacggattcggcccgaaacctgccggaatcgtgcatttgccgtttaatgatcttgatgcggttaaagcgatgattga tgatcacagctgtgcggtaatcgttgagccgattcagggagaaagcgggattattccggctaccaaagaatttttacagggtttgcgtcg actttgcgacgaaaataacgctctattgatttttgacgaagtacaaacgggggtagggcgtaccggttatttatacgcatatgaaagttat gatgtagtgccggatattctgacctcgtcaaaagcccttgctaacggttttccgattagcgcaatgttaaccaccacaaaaattgccgcc agtttcaaaccgggcgtgcacggcaccacattcggcggtaatccgttagcctgtgcggtcggggcgaaagatccaggcaacacgg ggcagatcctgctttatcgcggcagttcgttgcgtgaatggaccttcgatcgcgtactggcccacgctgatgcgggtgaaagctatatgt gggaatgtccggactttttcagccttggcgatcagcattatctgatgttttccccgcagggaatgaatgccgagggatacagttaccgaa atcgctttcaaagtggcgtaatacccggaatgtggtcgccaggacgactttttgcacaatccgggcattttactgaacttgataacgggc atgacttttatgcaccacaaagctttttagcgaaggatggtcggcgtattgttatcggctggatggatatgtgggaatcgccaatgccctc aaaacgtgaaggatgggcaggctgcatgacgctggcgcgcgagctatcagagagcaatggcaaacttctacaacgcccggtaca cgaagctgagtcgttacgccagcagcatcaatctgtctctccccgcacaatcagcaataaatatgttttgcaggaaaacgcgcaagc agttgagattcagttgcagtgggcgctgaagaacagtgatgccgaacattacggattacagctcggcactggaatgcggctgtatattg ataaccaatctgagcgacttgttttgtggcggtattacccacacgagaatttagacggctaccgtagtattcccctcccgcagcgtgaca cgctcgccctaaggatatttatcgatacatcatccgtggaagtatttattaacgacggggaagcggtgatgagtagtcgaatctatccgc agccagaagaacgggaactgtcgctttatgcctcccacggagtggctgtgctgcaacatggagcactctggctactgggttaacataa tatcaggtggaacaacggatcaacagcgggcaagggatccgtttaaaaaatcagtcaaaaaagaccgcacttttaacgcaaagttc ggtcttttttttgctattttttctccggtttttccgtatcggctatttttaaccggtaggaaaatattaatcccaacaggcttaaaacggataaggt ctgcaaggcgatttgcagaccgtaatgatctgcaatccagcccacaattggtgaaaatatcccgcccatagttattcccagcccaagg gtaatgccggctgcaaagccgacgcttttggcaagataagcctgaccaagcacgacaataggactatattgagtaaaaacgcccag tcccaacggaattagcaaaataaatgagagccataagttttcgctttgagtaaaaattaaaatagtgggtaaaaaaatcaaataagcg tagcggataattctgacataacccaggcgatcggaaagcaacccgccgataaaggtaatggcgacccccattgataaaaatattgt gagcgcaaagttagcgtcggtttcttgttgatgaagtatatgaatccaatagataggaataaaagcgttaagtactgtgaaattggtggc gcggacaaaaataatcaccgacaatttggcaaaactgcgccaatcgttttgtaatgtggtagaagcggtgttttttgctttattaaccacat tttcgactgttaattttggcaactgtaagaaaataatgagcgcgatgatcgtgttaatcagggcaaaaatacttaaggtttgcgcaccga ataaataggcgagccccgcgaacatcggtcctatagcaaaaccggcattgccgccgacggcaaaaatccccatagctttgcctttttc tccgccggacattcggttaaccagtctcgctccttccgggtgaaacagagcggaacctatgccggcaatcatggcaaaaaagaata aacccgggtaggaatggacaaagcccatagcggcaatacagcagcctgacagcatcattcccaaaggaattaaccaaggcatag aacgtttatccgccaaatagccgaaaaacggttgagcaattgaggcgagaaccgtgttggcgaaaattaatccgccggcttcctgat aggtcaatccgtaattttttataaaaagcggaagcagggcggggagcgctccttgcgccatatcggtgacgagatgcccgattgccgc aagataaacagcgaatttgttttgcataataagttcgttaaaagataaataagttctattataatcccgaagtttccggtgtattttaataaa acgaattattttgcattttattacttaagatcaaaagacgggtatcgggaaattaaaagtgcggtgaaaatttgcgtttttttggcgggtaga aaaaagccctttcatcatgaaagggcaaaatacatttggagttatattttttaaagtatgtttgtattatatatgcggaattttaacttttacaac atatttttaacatttacttttggtgaggattgcgcgtatttctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccgg agagtaccgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccgg cggaagacaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactg atccgctatgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaatacc aaacagcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaata accatgaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagc ctgaccaccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccg ggctgttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttc ataaagaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcaga atatcagcatgataccagat

SEQ ID NO: 28 (nucleotide sequence of fusion-gene argD::cscA

atgagccaatatactcgcaaaacattcgatgaagtgatgatccaaaactacgtacctgcggattttattcctgtgaaaggcaaaggttg caaagtttgggatcagcaagggcgagattatattgattttaccagcggaattgccgtaaatgcattgggacattgtcctgacgaaatcgt cgatgtgctgaaaaaacaaggcgaaactctatggcattcaagcaactggttcaccagtgaaccgactctggaacttgcctccaaattg gttgaacatacctttgccgaacgtgtgatgtttgccaattccggcggtgaagccaatgaggcggcgcttaaattggcgcgtcgttatgcg gtggataattacggctatcaaaaagataccattatttcattcaaaaaaagtttccacggacgtaccttatttaccgtcagcgtcggcggtc aggcgaaatattcggacggattcggcccgaaacctgccggaatcgtgcatttgccgtttaatgatcttgatgcggttaaagcgatgattg atgatcacagctgtgcggtaatcgttgagccgattcagggagaaagcgggattattccggctaccaaagaatttttacagggtttgcgtc gactttgcgacgaaaataacgctctattgatttttgacgaagtacaaacgggggtagggcgtaccggttatttatacgcatatgaaagtt atgatgtagtgccggatattctgacctcgtcaaaagcccttgctaacggttttccgattagcgcaatgttaaccaccacaaaaattgccg ccagtttcaaaccgggcgtgcacggcaccacattcggcggtaatccgttagcctgtgcggteggggcgaaagatecaggcaacacg gggcagatcctgctttatcgcggcagttcgttgcgtgaatggaccttcgatcgcgtactggcccacg

tgggaatgtccggactttttcagccttggcgatcagcattatctga^

aatcgctttcaaagtggcgtaatacccggaatgtggtcgccaggacgactt^

catgadtttatgcaaacaaagdttttagcgaagga

caaaacgtgaaggatgggcaggdgcatgacgrtggcgcgcgagdatcagagagcaatggcaaacttctacaacgcccggtac acgaagctgagtcgttacgccagcagcatcaatctgtctetc(xx^cacaat∞

cagttgagattcagttgcagtgggcgdgaagaacagtgatgccgaacattacggattacagdcggra

gataaccaatdgagcgacttgttttgtggcggtattacccacacgagaattta^

acgctcgccctaaggatatttatcgatacatcatccgtggaagtatttatta^

cagccagaagaacgggaadgtcgctttatgcctcccacggagtggdgtgctgcaac^ underlined: nucleotides 1 to 869 of SEQ ID NO: 7

bold: nucelotides 525 to 1434 of SEQ ID NO: 5

SEQ ID NO: 29 (complete nucleotide sequence of plasmid pSacB_py/oAi)

tcgagcagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattg ctgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgct cataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaa tatcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggctgcggtttatctgccgatgcattaaccgaaaaaga taaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccgt caattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtac cgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaag acaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgct atgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaaca gcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccat gaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgac caccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggct gttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataa agaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatat cagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggattt aacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctg tttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaata aattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttc aggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgg gcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg atacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacac gacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat cctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtcttt gacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatag cttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaac acaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgcc gtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactg tgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgt gcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgcctt ggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgc ctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttga tgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataa acggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtct ttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcattttta ggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaac gtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcag catatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccag cagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgctt cttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatac actttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggat tgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttctt ttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaata atagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc bold and underlined: mutation in the pykA-gene that leads to a substitution of glycine by cysteine at amino acid position 167

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Indications are Made

FOR RECEIVING OFFICE USE ONLY

0-4 This form was received with the

international application: YES

(yes or no)

0-4-1 Authorized officer

Wilson, Patrick

FOR INTERNATIONAL BUREAU USE ONLY

0-5 This form was received by the

international Bureau on:

0-5-1 Authorized officer

Claims

A modified microorganism having, compared to its wildtype, a reduced activity of the enzyme that is encoded by the pfsG-gene and wherein the modified microorganism expresses an enzyme having the activity of sucrose hydrolase and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pas- teurellaceae.

Modified microorganism according to claim 2, wherein the wildtype from which the modified microorganism has been derived has a 16S rDNA of SEQ ID NO: 1 or a sequence, which shows a sequence homology of at least 96, 97, 98, 99 or 99.9% with SEQ ID NO: 1.

Modified microorganism according to claim 1 or 2, wherein the wildtype from which the modified microorganism has been derived belongs to the genus Basfia.

Modified microorganism according to anyone of claims 1 to 3, wherein the pfsG-gene comprises a nucleic acid selected from the group consisting of:

a1 ) nucleic acids having the nucleotide sequence of SEQ ID NO: 3;

b1 ) nucleic acids encoding the amino acid sequence of SEQ ID NO: 4;

c1 ) nucleic acids which are at least 70% identical to the nucleic acid of a1 ) or b1 ), the identity being the identity over the total length of the nucleic acids of a1 ) or b1 ); d1 ) nucleic acids encoding an amino acid sequence which is at least 70% identical to the amino acid sequence encoded by the nucleic acid of a2) or b2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a2) or b2);

e1 ) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a1 ) or b1 ); and

f1 ) nucleic acids encoding the same protein as any of the nucleic acids of a1 ) or b1 ), but differing from the nucleic acids of a1 ) or b1 ) above due to the degeneracy of the genetic code.

Modified microorganism according to anyone of claims 1 to 4, wherein the pfsG-gene is modified and wherein the modification of the pfsG-gene is achieved by a deletion of the pfsG-gene or at least a part thereof, a deletion of a regulatory element of the pfsG-gene or at least a part thereof or by an introduction of at least one mutation into the pfsG-gene.

Modified microorganism according to anyone of claims 1 to 5, wherein the modified microorganism has, compared to its wildtype, an increased activity of sucrose hydrolase.

Modified microorganism according to claim 6, wherein the increased activity of sucrose hydrolase is accomplished by the expression of an enzyme encoded by a cscA-gene, by the expression of an enzyme encoded by a fragment of a cscA-gene or by the expression of a fusion enzyme encoded by a fusion gene comprising at least a fragment of a cscA- gene, provided that the enzyme encoded by the fragment of the cscA-gene and the fusion enzyme encoded by the fusion gene comprising at least a fragment of a cscA-gene have the activity of sucrose-6-phosphate hydrolase.

Modified microorganism according to claim 6 or 7, wherein the cscA-gene comprises a nucleic acid selected from the group consisting of:

a2) nucleic acids having the nucleotide sequence of SEQ ID NO: 5;

b2) nucleic acids encoding the amino acid sequence of SEQ ID NO: 6;

c2) nucleic acids which are at least 70% identical to the nucleic acid of a2) or b2), the identity being the identity over the total length of the nucleic acids of a2) or b2); d2) nucleic acids encoding an amino acid sequence which is at least 70% identical to the amino acid sequence encoded by the nucleic acid of a2) or b2), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acids of a2) or b2);

e2) nucleic acids capable of hybridizing under stringent conditions with a complementary sequence of any of the nucleic acids according to a2) or b2); and

f2) nucleic acids encoding the same protein as any of the nucleic acids of a2) or b2), but differing from the nucleic acids of a2) or b2) above due to the degeneracy of the genetic code.

Modified microorganism according to anyone of claims 1 to 8, wherein the microorganism further has, compared to its wildtype,

i) a reduced pyruvate formate lyase activity, and/or

ii) a reduced lactate dehydrogenase activity, and/or

iii) a reduced activity of an enzyme encoded by the wcaJ-gene, and/or

iv) a reduced activity of an enzyme encoded by the pykA-gene, and/or

v) a reduced activity of an enzyme encoded by the fruA-gene.

Modified microorganism according to claim 9, wherein the microorganism comprises:

B) a deletion of the pflD-gene or at least a part thereof, a deletion of a regulatory element of the pflD-gene or at least a part thereof or an introduction of at least one mutation into the pflD-gene;

C) a deletion of the pflA-gene or at least a part thereof, a deletion of a regulatory element of the pflA-gene or at least a part thereof or an introduction of at least one mutation into the pflA-gene; a deletion of the wcaJ-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ-gene or at least a part thereof or an introduction of at least one mutation into the wcaJ-gene;

a deletion of the pykA -gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof or an introduction of at least one mutation into the pykA-gene;

a deletion of the fruA-gene or at least a part thereof, a deletion of a regulatory element of the fruA-gene or at least a part thereof or an introduction of at least one mutation into the fruA-gene;

a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mu^¬ tation into the IdhA-gene

and

a deletion of the pflD-gene or at least a part thereof, a deletion of a regulatory element of the pflD-gene or at least a part thereof or an introduction of at least one mutation into the pflD-gene

and

a deletion of the wcaJ-gene or at least a part thereof, a deletion of a regulatory element of the wcaJ-gene or at least a part thereof or an introduction of at least one mutation into the wcaJ-gene

and

or

a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mu tation into the IdhA-gene

and

a deletion of the pflA-gene or at least a part thereof, a deletion of a regulatory element of the pflA-gene or at least a part thereof or an introduction of at least one mutation into the pflA-gene,

and

a deletion of the pykA -gene or at least a part thereof, a deletion of a regulatory element of the pykA-gene or at least a part thereof or an introduction of at least one mutation into the pykA-gene.

1 1 . A method of producing an organic compound comprising:

I) cultivating the modified microorganism according to anyone of claims 1 to 10 in a culture medium comprising at least one assimilable carbon source to allow the modified microorganism to produce the organic compound, thereby obtaining a fermenta- tion broth comprising the organic compound;

12. Method according to claim 1 1 , wherein the organic compound is succinic acid.

13. Method according to claims 1 1 and 12, wherein at least 50 wt.-% of the assimilable carbon source, based on the total weight of the assimilable carbon source with the exception of carbon dioxide, is sucrose. 14. Method according to anyone of claims 1 1 to 13, wherein in process step I) the organic compound is produced under anaerobic conditions.

Method according to anyone of claims 1 1 to 14, wherein the process further comprises the process step:

Method according to claim 15, wherein the organic compound is succinic acid and wherein the secondary organic product is selected from the group consisting of succinic acid esters or polymers thereof, tetrahydrofuran (THF), 1 ,4-butanediol (BDO), gamma- butyrolactone (GBL), pyrrolidones, polyols and polyurethanes.

Use of a modified microorganism according to anyone of claims 1 to 10 for the fermentative production of an organic compound

18. Use according to claim 17, wherein the organic compound is succinic acid and wherein sucrose is used as an assimilable carbon source.