HK1125118A - Novel gene products from bacillus licheniformis forming or decomposing polyamino acids and improved biotechnological production methods based thereon - Google Patents

Description

Novel gene products of Bacillus licheniformis forming or decomposing polyamino acids and improved biotechnological production methods therefor

The present invention relates to 5 or 4 novel genes from Bacillus licheniformis (Bacillus licheniformis), gene products thereof, and sufficiently similar genes and proteins which are involved in the formation, modification and/or degradation of polyamino acids in vivo and which can be used for this purpose, and to improved biotechnological production methods using microorganisms which are characterized by the inactivation or activation of these genes.

The present invention is in the field of biotechnology, specifically the preparation of suitable products of interest by fermentation of microorganisms capable of forming such suitable products. This includes, for example, the preparation of low molecular weight compounds such as dietary supplements or pharmaceutically relevant compounds, or the preparation of proteins which, due to their diversity, have a wide range of industrial applications. In the first case, the metabolic properties of the relevant microorganism are exploited or modified to produce the appropriate product; in the second case, cells capable of expressing the gene of the desired protein are used. In both cases, therefore, Genetically Modified Organisms (GMOs) are mostly involved.

Microbial fermentation exists a wide range of prior art, particularly on an industrial scale; it ranges from the optimization of the rate of formation and nutrient utilization of the relevant strains by the technical design of the fermenter until the isolation of the valuable product from the relevant cells themselves and/or the fermentation medium. Both genetic and microbial, as well as process engineering and biochemical methods are applicable here. The object of the present invention is to improve the general properties of the microorganisms used in the process, which properties impair the actual fermentation step, in particular at the level of the genetic properties of the strains used.

For industrial biotechnological production, the relevant microorganisms are cultivated in fermenters whose design is adapted to their metabolic properties. During the cultivation they metabolize the supplied substrate, which then usually forms a large amount of other substances in addition to the actual product, which substances are generally not required and/or, as will be explained later, may lead to difficulties in the fermentation or in the subsequent processes.

In general, fermentation is a very complex process in which a large number of different parameters have to be adjusted or monitored. Thus, for example, aerobic processes are often involved, meaning that the microorganisms used must be supplied with sufficient oxygen throughout the fermentation (control of the aeration rate). Examples of other such parameters are the geometry of the reactor, the composition of the nutrient medium, the pH or the CO₂A continuous change in speed is generated. A parameter which is particularly important for both economics and process management per se is the necessary energy input, for example by means of a stirring system to ensure that the contents of the reactor are as thoroughly mixed as possible. Furthermore, in addition to the distribution of the substrate, it is also necessary to ensure an adequate supply of oxygen to the organisms.

After the fermentation is completed, in addition to the removal of the producer organisms, it is generally necessary to purify and/or concentrate the desired valuable product from the so-called fermenter slurry. Subsequent processes may include, for example, various chromatographic and/or filtration steps. Thus, besides the content of valuable products, also decisive for the success of the overall subsequent process is the biophysical properties of the fermenter slurry, in particular its viscosity immediately after the fermentation is complete.

Its properties are also influenced by the metabolic activity of the chosen microorganism, and undesirable effects may also occur. This includes, for example, the fact that the viscosity of the nutrient medium often increases during the fermentation. This weakens the mixing and thus the transport of material and the oxygen supply inside the reactor. Other difficulties mostly occur in the subsequent process, since the increased viscosity has a considerable impairment of, for example, the efficiency of the filtration process.

It is known in particular that slime-producing Bacillus species are essentially composed of poly-gamma-glutamic acid (PGA) and/or-aspartic acid, meaning that the polyamino acids are linked by related gamma peptide bonds. In scientific research on Bacillus subtilis, there are mainly 3 genes, ywsC, ywtA and ywtB, and gene products derived therefrom, which are involved in the production of polyglutamic acid; the product of the ywtD gene is involved in degradation. The general gene marker symbol "ywt" has the same meaning as the abbreviations "cap" and "pgs" commonly used for the same function. This will be explained below.

M. Ashiuchi et al, "Physiological and biochemical characterization of poly gamma-glutamate synthase complex of Bacillus subtilis" (2001), Eur.J. biochem., vol268, p5321-5328, describe the PgsBCA (poly-gamma-glutamate synthase complex) enzyme complex from Bacillus subtilis, which consists of 3 subunits PgsB, PgsC and PgsA. According to this paper, the complex is an atypical amide ligase which converts both the D and L enantiomers of glutamate into the corresponding polymers. According to this paper, the gene disruption experiments described therein will serve as evidence, indicating that in B.subtilis this complex is the only enzyme complex catalyzing the reaction.

U.Urushibata et al, in the article "Characterization of the Bacillus subtilis C gene, immersed in gamma-polyglutamic acid production" (2002), J.Bacteriol., vol.184, 337-343p, confirm that the three gene products responsible for PGA production in Bacillus subtilis are encoded by these three genes by deletion mutations of the three genes ywsC, ywtA and ywtB. They form a contiguous operon in this order in this microorganism together with the subsequent gene ywtC.

Suzuki and Y.Tahara articles "mutation of the Bacillus subtilis recombined gene" (2003), while product is involved in gamma-polyglutamic acid differentiation ", J.Bacteriol., vol185, p2379-2382, reveal the fact that another gene involved in PGA metabolism is located downstream of the ywtC gene in the ywtC self-operon of the Bacillus subtilis genome. This gene encodes a DL-endopeptidase capable of hydrolyzing PGA and can therefore be referred to as γ -DL-glutamyl hydrolase.

Furthermore, the article "Biochemistry and molecular diagnostics" of poly-gamma-glutamate synthesis ", appl. Microbiol. Biotechnol., 2002, vol59, p9-14, by M.Ashiuchi and H.Misono, also provides recent investigations regarding these enzymes. The genes homologous to pgsB, pgsC and pgsA and encoding the PGA synthase complex in B.anthracis are referred to herein as capB, capC and capA. According to this article, the gene located downstream is called dep (referred to as "D-PGA lyase") in Bacillus anthracis and pgdS (referred to as "PGA lyase") in Bacillus subtilis.

In the current state of the art, the activity of these enzymes has been actively used, mainly for the preparation of poly-gamma-glutamic acid as a starting material for e.g. cosmetics, although their exact DNA sequence and amino acid sequence have not been known so far-especially for bacillus licheniformis. Thus, for example, patent application JP 08308590A discloses the production of PGA by fermentation of PGA-producing bacteria per se, i.e., Bacillus species such as Bacillus subtilis and Bacillus licheniformis; the isolation of the feedstock from the culture medium is also described. According to patent application WO 02/055671A 1, the Bacillus subtilis variant Chunkokjang (B.subtilis var. chunkokjang) represents a microorganism which is particularly suitable for this purpose.

Thus, GLA is of interest in certain fermentations as a valuable product produced by the fermentation.

However, in all other fermentations, there is interest in producing other valuable products; in this connection, the formation of polyamino acids, for the reasons mentioned above, means side reactions. A typical way to control the increase in viscosity of the fermentation medium resulting from this formation is to increase the stirring speed. However, this has an effect on the energy input. Furthermore, the fermented microorganisms are thus exposed to increased shear forces, which represent a considerable stress effect for them. Finally, even so, the very high viscosity cannot be overcome, so that the fermentation must be terminated prematurely, if at all, and production can continue.

As a side reaction of the extensive fermentation process, the formation of slime may thus negatively affect the overall outcome of the fermentation for a number of reasons. Conventional methods may only be shown to be inadequate in terms of being able to continue the fermentation process despite an increase in the viscosity of the nutrient medium, in particular because they do not represent a causal control.

Therefore, a more urgent problem is to suppress as much as possible the formation of undesirable slime, particularly slime produced by poly-gamma-amino acids such as poly-gamma-glutamic acid, during the fermentation of microorganisms. It is particularly desirable to be able to find solutions for the control representing causal relationships. Another aspect of the problem is to provide related genes for the positive utilization of the gene product of GLA synthesis and for its degradation and/or modification.

The following proteins, each of which is involved in the formation or degradation of polyamino acids, represent in each case a partial solution which is of equal value in principle for the problem:

-YwsC (CapB, PgsB) encoded by the nucleotide sequence YwsC, which is at least 80% identical to the nucleotide sequence shown in SEQ ID No. 1;

-YwsC '(truncated variant of YwsC) encoded by a nucleotide sequence YwsC' having at least 83% identity with the nucleotide sequence shown in SEQ ID No. 3;

YwtA (CapC, PgsC), encoded by a nucleotide sequence ywtA, which is at least 82% identical to the nucleotide sequence shown in SEQ ID No. 5;

-YwtB (CapA, PgdA) encoded by a nucleotide sequence YwtB, which is at least 72% identical to the nucleotide sequence shown in SEQ ID No. 7; and

YwtD (Dep, PgdS), encoded by the nucleotide sequence ywtD, which is at least 67% identical to the nucleotide sequence shown in SEQ ID No. 9.

It is evident from the above-mentioned papers such as Urushibata et al that 4 or 3 genes involved in GLA formation occur sequentially on the same operon in Bacillus subtilis. ywtD is located directly downstream thereof. These components act together in vivo in the form of a complex, with downstream components acting on the polyamino acid formed thereby, and it is expected that this organization will also be found in many other microorganisms, in particular in Bacillus. Thus, in addition to common biochemical functions, there are also features at the genetic level that give rise to the uniformity of the invention.

Further partial solutions are represented by the relevant nucleic acids ywsC, ywsC', ywtA, ywtB and ywtD, and on this basis the use of the relevant nucleic acids for reducing the slime formation caused by polyamino acids in microbial fermentation processes and for corresponding methods of microbial fermentation. According to the invention, at least one gene from ywsC, ywtA or ywtB is functionally inactivated and/or the activity of ywtD is increased in the genetic reduction of mucus formation. In addition, these genes or derived gene products can be used positively for the preparation, modification or degradation of poly-gamma-glutamic acid.

The invention can in principle be used for all fermentable microorganisms, in particular of the genus Bacillus, so that these microorganisms are used for the fermentative production of valuable products other than polyamino acids, in particular of pharmaceutically relevant low molecular weight compounds or proteins, the formation of polyamino acids, in particular GLA, being prevented at the genetic level or being immediately degraded again. On the other hand, this has a favorable effect on the viscosity of the culture medium, in addition to the mixing capacity, oxygen input and energy consumption, and on the other hand the subsequent processing of the desired product is considerably facilitated. Furthermore, most of the used raw materials, such as the N source, are not converted into undesired products, so a higher overall fermentation yield can be expected.

According to another aspect of the invention, said genes can now be used for the positive use of the GLA synthesis gene products or for their degradation and/or modification, in particular using the derived proteins YwsC, YwsC', YwtA, YwtB and/or YwtD, which are produced biotechnologically and introduced into the cells producing them or independently as catalysts in a suitable reaction mixture.

The first partial solution shows a protein YwsC (CapB, PgsB) which is involved in the formation of polyamino acids and is encoded by the nucleotide sequence ywsC, which has at least 80% identity with the nucleotide sequence shown in SEQ ID No. 1.

The specific enzyme was obtained by analysis of the genome of B.licheniformis DSM13 (see example 1). This protein can be replicated by the nucleotide and amino acid sequences in SEQ ID NO.1 and 2 of the present application (see example 1).

In line with the literature information mentioned in the introduction, this takes the form of a subunit of the poly-gamma-glutamate synthetase complex. The protein currently known in the art and most similar to it, found to be homologous YwsC from bacillus subtilis, has been registered in the GenBank database (National Center for Biotechnology Information NCBI, National institutes of Health, Bethesda, MD, USA) under accession number AB046355.1 with homology identity of 75.4% at the nucleic acid level and identity of 86.1% at the amino acid level (see example 2). These significant agreement states not only the same biochemical function, but also the presence of a large number of related proteins with the same function within the scope of the claims, which are likewise included in the protection scope of the present application.

The following embodiments will be assigned to this first partial solution:

any corresponding protein YwsC, the nucleotide sequence coding for which, with increasing preference, has at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence shown in SEQ ID No. 1. This is because the conclusion drawn from the increase in sequence identity is that the identity of function and mutual replaceability increases at the genetic level.

Any protein YwsC (CapB, PgsB) which is involved in the formation of polyamino acids and whose amino acid sequence has at least 91% identity, with increasing preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the amino acid sequence indicated in SEQ ID No. 2.

For purposes of this application, the expression "at least X%" means "X% to 100%, inclusive of the extreme values X and 100, and all integer and non-integer percentages therebetween".

The specific protein obtained from bacillus licheniformis DSM13 is most preferred in each case because it is specifically described in the present application and is 100% reproducible.

The second partial solution shows the protein YwsC '(as a truncated variant of YwsC) which is involved in the formation of polyamino acids and which codes for a nucleotide sequence ywsC' which has at least 83% identity with the nucleotide sequence shown in SEQ ID NO. 3.

This specific enzyme was obtained by analysis of the genome of B.licheniformis DSM13 (see example 1). This protein can be reproduced by the nucleotide and amino acid sequences shown in SEQ ID NO.3 and 4 in the present application (see example 1).

As explained additionally in example 1, FIG. 6 shows that a comparison between the YwsC sequences of Bacillus licheniformis and Bacillus subtilis shows that the first 16 amino acids of YwsC of Bacillus licheniformis are not important for its function as subunit C of the poly-gamma-glutamate synthetase complex. The invention can therefore also be carried out with this truncated variant.

Reference to "5 or 4 genes" in this application means that ywsC and ywsC' are considered two genes and the derived protein is considered two proteins according to the invention. On the other hand, it may be assumed that the two "genes" are not present in each case in the relevant organism, but only one in each case, and that therefore only one corresponding gene product YwsC or YwsC' may also be present. Thus, the first and second partial solutions represent, to some extent, two aspects of the same subject matter. However, because of the difference in amino acid levels, a split into two partial solutions is justified.

The protein currently known in the art, which is most similar to the protein, is once again found to be the bacillus subtilis homolog YwsC, which has been deposited in the GenBank database under accession No. AB046355.1, with 78.5% homology at the nucleic acid level; the identity at the amino acid level was 89.6% (see example 2).

In accordance with the above statement, the following embodiment is designated as a second partial solution method:

any corresponding protein YwsC', which encodes a nucleotide sequence which, with increasing preference, is at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identical to the nucleotide sequence shown in SEQ ID No. 3.

Any protein YwsC' (truncated variant of YwsC) involved in the formation of polyamino acids, the amino acid sequence of which has at least 94% identity, with increasing preference at least 95%, 96%, 97%, 98%, 99% and preferably 100% identity with the amino acid sequence shown in SEQ ID No. 4.

The specific protein obtained from bacillus licheniformis DSM13 is most preferred in each case because it is specifically described in the present application and can replicate 100%.

The third partial solution shows a protein YwtA (CapC, PgsC) which is involved in the formation of polyamino acids and which codes for a nucleotide sequence ywtA which has at least 82% identity with the nucleotide sequence indicated in SEQ ID No. 5.

This specific enzyme was obtained by analysis of the genome of bacillus licheniformis DSM13 (see example 1). This protein can be replicated by the nucleotide and amino acid sequences shown in SEQ ID NO.5 and 6 in the present application (see example 1).

In accordance with the literature information mentioned in the introduction, this takes the form of another subunit of the poly-gamma-glutamate synthetase complex. The protein currently known in the art, which is most similar to the protein found in the present technical field, is found to be the homologue YwsA of Bacillus subtilis, which has been registered in the GenBank database under accession number AB046355.1, with a homology of 77.8% at the nucleic acid level and 89.9% at the amino acid level (see example 2). These significant agreements suggest not only the same biochemical function, but also the presence within the scope of the claims of a large number of related proteins having the same function, which are likewise included in the protection conferred by the present application.

The following embodiments are specified as this third partial solution:

any corresponding protein YwtA, the nucleotide sequence encoded for which has, with increasing preference, at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the nucleotide sequence shown in SEQ ID No. 5.

Any protein YwtA (CapC, PgsC) involved in the formation of polyamino acids, the amino acid sequence of which has at least 94% identity, with increasing preference at least 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the amino acid sequence indicated in SEQ ID No. 6.

The specific protein obtained from bacillus licheniformis DSM13 is most preferred in each case because it is specifically described in the present application and can replicate 100%.

The fourth partial solution shows a protein YwtB (CapA, PgsA) which is involved in the formation of polyamino acids and which codes for a nucleotide sequence ywtB which has at least 72% identity with the nucleotide sequence indicated in SEQ ID NO. 7.

This specific enzyme was obtained by analysis of the genome of B.licheniformis DSM13 (see example 1). This protein can be replicated by the nucleotide and amino acid sequences shown in SEQ ID NO.7 and 8 in the present application (see example 1).

In line with the literature information mentioned in the introduction, this takes the form of the third subunit of the poly-gamma-glutamate synthetase complex. The protein currently known in the art, which is most similar to the protein found in the present technical field, is found as the homologue of Bacillus subtilis YwsA, which has been registered in the GenBank database under accession number AB046355.1, with a homology of 67.1% at the nucleic acid level and 65.8% at the amino acid level (see example 2). These significant agreement states not only the same biochemical function, but also the presence of a large number of related proteins with the same function within the scope of the claims, which are likewise included in the protection scope of the present application.

The following embodiment is designated as this fourth partial solution:

any corresponding protein YwtB, the nucleotide sequence which encodes it, with increasing preference, has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated under SEQ ID No. 7.

Any protein YwtB (CapA, PgsA) involved in the formation of polyamino acids, the amino acid sequence of which has at least 70% identity, and with increasing preference at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity, with the amino acid sequence indicated in SEQ ID No. 8.

The specific protein obtained from bacillus licheniformis DSM13 is most preferred in each case because it is specifically described in the present application and is 100% reproducible.

The fifth partial solution shows the protein YwtD (Dep, PgdS), which is involved in the degradation of polyamino acids and which codes for a nucleotide sequence ywtD which has at least 67% identity with the nucleotide sequence indicated in SEQ ID No. 9.

This specific enzyme was obtained by analysis of the genome of B.licheniformis DSM13 (see example 1). This protein can be reproduced by the nucleotide and amino acid sequences shown in SEQ ID Nos. 9 and 10 of the present application (see example 1).

In accordance with the literature information mentioned in the introduction, this takes the form of a gamma-DL-glutamyl hydrolase, a D-PGA lyase or a PGA lyase. The protein currently known in the art, which is most similar to the protein found in the present technical field, is found to be the homologue YwtD of Bacillus subtilis, which has been deposited in the GenBank database under accession number AB080748, with a homology of 62.3% at the nucleic acid level and 57.3% at the amino acid level (see example 2). These significant agreement states not only the same biochemical function, but also the presence of a large number of related proteins with the same function within the scope of the claims, which are likewise included in the protection scope of the present application.

The following embodiment is designated as this fifth partial solution:

any corresponding protein YwtD, the nucleotide sequence encoded for which has, with increasing preference, at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated under SEQ ID No. 9.

Any protein YwtD (Dep, PgdS) involved in the degradation of polyamino acids, the amino acid sequence of which has at least 62% identity, with increasing preference at least 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the amino acid sequence indicated in SEQ ID No. 10.

The specific protein obtained from bacillus licheniformis DSM13 is most preferred in each case because it is specifically described in the present application and is 100% reproducible.

In each case preference is given to the aforementioned proteins of the invention which, in each case, are involved in the formation or degradation of polyamino acids and are naturally produced by a microorganism, preferably a bacterium, particularly preferably a gram-positive bacterium, preferably one of the genera Bacillus, particularly preferably one of the species Bacillus licheniformis, very particularly preferably Bacillus licheniformis DSM 13.

This is because, in accordance with the problems faced, it is advantageous to improve the fermentation of microorganisms, since the bacteria of these particular gram-positive bacteria are frequently used, in particular those capable of secreting bacteria which produce valuable products and proteins, such as bacillus. In addition, there is a rich clinical experience in this regard. In addition, as previously described, it is possible to detect the proteins indicated in the Bacillus licheniformis sequence Listing, in particular Bacillus licheniformis DSM 13. It is expected that the degree of relatedness of related organisms will increase, involving an increased degree of identity of nucleotide and amino acid sequences, and hence interchangeability.

In accordance with the statements so far, the relevant nucleic acids in each of the following cases will be designated as further representations of the partial solutions stated in the present invention:

-a nucleic acid ywsC (capB, pgsB) which codes for a gene product which is involved in the formation of polyamino acids and whose nucleotide sequence has at least 80% identity with the nucleotide sequence shown in SEQ ID No. 1;

-a corresponding nucleic acid ywsC, the nucleotide sequence of which, with increasing preference, is at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identical to the nucleotide sequence shown in SEQ ID No. 1;

-a nucleic acid ywsC' (truncated ywsC variant) encoding a gene product which is involved in the formation of a polyamino acid and which has a nucleotide sequence which has at least 83% identity with the nucleotide sequence shown in SEQ ID No. 3;

-a corresponding nucleic acid ywsC' whose nucleotide sequence, with increasing preference, is at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identical to the nucleotide sequence shown in SEQ ID No. 3;

a nucleic acid ywtA (capC, pgsC) which codes for a gene product which is involved in the formation of polyamino acids, whose nucleotide sequence exhibits at least 82% identity with the nucleotide sequence indicated under SEQ ID No. 5;

the corresponding nucleic acid ywtA, the nucleotide sequence of which increases with preference, corresponds to SEQ ID

NO.5 shows a nucleotide sequence which is at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identical;

a nucleic acid ywtB (capA, pgsA) which codes for a gene product which is involved in the formation of polyamino acids and whose nucleotide sequence has at least 72% identity with the nucleotide sequence indicated under SEQ ID No. 7;

a corresponding nucleic acid ywtB whose nucleotide sequence, with increasing preference, is at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identical to the nucleotide sequence indicated under SEQ ID No. 7;

-a nucleic acid ywtD (dep, pgdS) encoding a gene product involved in the degradation of polyamino acids, the nucleotide sequence of which has at least 67% identity with the nucleotide sequence shown in SEQ ID No. 9; and

the corresponding nucleic acid ywtD, whose nucleotide sequence increases with preference, has at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated under SEQ ID No. 9.

The nucleic acids provided herein can be used by existing molecular biology methods to inactivate or enhance the activity of the associated protein. Thus, inactivation becomes possible, for example, by appropriate deletion of the vector (see below); the enhancement of the activity can be carried out by overexpression, which can be achieved with the aid of an expression vector (see below). The problem posed can therefore be solved using these nucleic acids by inactivation of ywsC, ywsC', ywtA and/or ywtB and/or enhancement of the activity of the ywtD gene.

The corresponding genes within the homology range shown in each case can be obtained from the desired organism, for example with the aid of probes prepared on the basis of sequences 1, 3, 5, 7 or 9. These complete genes can also be used as a model for generating PCR primers by which the relevant genes can be readily prepared from the corresponding total DNA preparations; these genes then provide the aforementioned proteins. The success rate here generally increases with the proximity of the relevant strain, in this case B.licheniformis, to the strain used to construct the probes or PCR primers.

Preference is given in each case to the nucleic acids according to the invention which, in each case, occur naturally in microorganisms which are preferably bacteria, particularly preferably gram-positive bacteria, preferably of the genus Bacillus, particularly preferably of the species Bacillus licheniformis, very particularly preferably Bacillus licheniformis DSM 13.

This is because, as mentioned above, it is particularly advantageous to use these genes for the fermentation of these microorganisms. In another aspect, the invention also relates to the possibility of regulating the metabolism of polyamino acids, in particular gamma-glutamate, at least in part by means of the genes and/or proteins described herein. The success rate generally increases, particularly in appropriately transgenic host cells, with the degree of identity between the gene of interest and the gene of the native cell.

Furthermore, it is possible in principle to easily isolate alternative embodiments of genes and proteins from all natural organisms.

Another embodiment of the present invention shows all nucleic acids encoding the above-described proteins of the invention.

Thus, there are differences in the use of synonymous codons to encode the corresponding amino acids, particularly between distantly related species, whereby the protein biosynthetic elements are also adapted thereto by e.g. the available number of appropriately loaded tRNAs. One of the genes is transferred into a less relevant species and can be used particularly successfully, for example, for deletion mutations or for the synthesis of related proteins if the gene is appropriately optimized with respect to the codon. Thus, it is possible to introduce an increased percentage difference at the DNA level, without an effect at the amino acid level. Thus, such nucleic acids also represent an implementation of the present invention.

The invention also relates to vectors containing the nucleic acid regions of the invention indicated above.

This is because they are suitably ligated into vectors for the manipulation of nucleic acids relevant to the present invention, such as, in particular, for the preparation of protein products of the present invention. Such vectors and associated methods of operation are described in detail in the prior art. There are a large number and a wide variety of vectors commercially available for cloning and expression. They include, for example, vectors derived from bacterial plasmids, bacteriophages or viruses, or mainly synthetic vectors. They may also be distinguished by the nature of the cell type in which they are capable of self-construction into a vector, for example a gram-negative bacterial vector, a gram-positive bacterial vector, a yeast vector or a higher eukaryotic vector. They form suitable starting points for, for example, molecular biological and biochemical studies and expression of related genes or related proteins. As is evident from the prior art in this connection, they are in fact indispensable, in particular in the preparation of constructs for deletion or enhancement of expression.

Among the preferred vectors are those containing two or more of the above-described nucleic acids of the present invention.

This is because, on the other hand, the relevant genes can be stored simultaneously or can be expressed under the control of the same promoter. According to another application, vectors containing simultaneously complete copies of two or more genes of the invention can be used to maintain the survival (rescue) of deletion mutants simultaneously missing a plurality of these genes. Subsequent directed removal of the vector results in the simultaneous shutdown of these multiple genes.

In another embodiment, the vector of the invention is a cloning vector.

This is because cloning vectors are suitable for their molecular biological characteristics in addition to the storage, biological amplification or selection of the desired gene. At the same time, they represent transportable and storable forms of the claimed nucleic acids and are also starting points for cell-independent molecular biological techniques, such as PCR or in vitro mutagenesis methods.

The vector of the present invention is preferably an expression vector.

This is because such expression vectors are the basis for supplying the corresponding nucleic acids and producing the relevant proteins in biological production systems. A preferred embodiment of this subject matter of the invention is an expression vector consisting of the genetic elements necessary for expression, for example the native promoter originally located in front of the gene, or a promoter from a different organism. These elements may be arranged, for example, in the form of so-called expression cassettes. Another alternative possibility is also provided for one or all of the regulatory elements by the corresponding host cell. Expression vectors which are associated with other properties, such as an optimum copy number which is compatible with the chosen expression system, in particular the host cell, are particularly preferred (see below).

The possibility of forming a complete gene product on the basis of a vector in the form of a replicon is particularly important for the rescue described above and for the shutting down of specific genes. In contrast, the use of an expression vector is most likely to increase the production of the protein of the invention, and thus the activity involved.

The cells, after being genetically modified, contain one of the nucleic acids of the invention specified above, thus forming an isolated subject matter of the invention.

This is because these cells contain the genetic information for synthesizing the proteins of the present invention. These cells refer to specific cells that have been provided with the nucleic acid of the present invention by existing methods, or cells derived from such cells. Host cells appropriately selected for this purpose are cells which can be cultured relatively simply and/or provide high product yields.

In countries where human embryonic stem cells may not be patented, it is in principle necessary to exclude these human embryonic stem cells of the invention from the protection granted.

The cells of the invention make it possible, for example, to amplify the corresponding genes and also to mutate or transcribe and translate these genes and finally to produce the relevant proteins biotechnologically. This genetic information can either be present extrachromosomally as an isolated genetic element, i.e.in the case of bacteria, meaning located in a plasmid, or integrated into the chromosome. The choice of a suitable system depends on a number of issues, such as the nature and storage time of the gene or organism, or the nature of the mutation or selection.

In addition to cells which are overexpressed in a particular YwtD, this includes, in particular, those which contain one of the genes ywsC, ywsC', ywtA and ywtB via a trans-vector and can therefore be used for the corresponding deletion (see below).

This explains the preferred embodiment, wherein the nucleic acid is part of a vector, in particular the previously described vector, in such a cell.

Among these, bacteria are preferred host cells.

This is because bacteria have the characteristics of short propagation time and low requirements for culture conditions. It is thus possible to establish a cost-effective method. In addition, there is a great deal of experience in the fermentation technology of bacteria. Gram-negative or gram-positive bacteria may be suitable for a particular production due to a variety of factors, which should be individually determined experimentally, such as nutrient source, rate of product formation, time required, etc.

Preferred embodiments comprise gram-negative bacteria, in particular bacteria of the genus Escherichia coli (Escherichia coli), Klebsiella pneumoniae (Klebsiella), Pseudomonas (Pseudomonas) or Xanthomonas (Xanthomonas), in particular strains of Escherichia coli K12(E.coli K12), Escherichia coli B (E.coli B) or Klebsiella planticola, most particularly derivatives of Escherichia coli BL21(DE3), Escherichia coli RV308, Escherichia coli DH5 alpha, Escherichia coli JM109, Escherichia coli XL-1 or Klebsiella planticola (Rf).

This is because for gram-negative bacteria such as E.coli, a large amount of protein is secreted into the periplasmic space. This may be advantageous for certain applications. The patent application WO01/81597A1 also discloses methods for excreting expressed proteins using gram-negative bacteria. The preferred gram-negative bacteria mentioned above are generally readily available, i.e.are commercially available or available through public culture collections, and can be optimized for the particular conditions of preparation in combination with other components, such as vectors which are likewise available in large quantities.

Alternative but not less preferred embodiments comprise gram-positive bacteria, in particular bacteria of the Bacillus, staphylococcal or corynebacterium genus, more in particular strains of Bacillus lentus (Bacillus lentus), Bacillus licheniformis (b. licheniformis), Bacillus amyloliquefaciens (b. amyloliquefaciens), Bacillus subtilis (b. subtilis), Bacillus sphaericus (b. globigii) or Bacillus alcalophilus (b. alcalophilus), Staphylococcus carnosus (Staphylococcus) or corynebacterium glutamicum (corynebacterium glutamicum), of which the strain DSM13 derivative is very particularly preferred.

This is because gram-positive bacteria are fundamentally different from gram-negative bacteria in that they are capable of releasing secreted proteins immediately into the nutrient medium surrounding the cells, from which the expressed proteins of the invention can be purified directly, if desired. In addition, they are related to or consistent with most industrially important enzyme source organisms and they themselves mostly produce similar enzymes, so they have similar codon usage and their protein synthesis elements are naturally properly constructed. Derivatives of Bacillus licheniformis DSM13 are highly preferred, since on the one hand they are also widely used in the art as biotechnological production strains and on the other hand the genes and proteins of the invention in this application are derived from Bacillus licheniformis DSM13, and therefore the practice of the invention in such strains should most likely be successful.

Another embodiment of the invention consists of a process for preparing one or more of the gene products YwsC, YwsC', YwtA, YwtB and YwtD described above.

This includes any method for preparing the above-described proteins of the invention, such as chemical synthesis. In this connection, however, all molecular-biological, microbiological and biotechnological production processes which have already been discussed above in various aspects and which are established in the art are preferred. The aim is mainly to obtain the proteins of the invention so that they can be subjected to suitable applications, for example for the synthesis, modification or degradation of poly-gamma-glutamic acid.

Preferred methods in this connection are methods using the nucleic acids specified above, preferably using the vectors specified above, particularly preferably using the cells specified above.

This is because the nucleic acids, in particular the nucleic acids identified in the sequence listing SEQ ID No.1, 3, 5, 7 and 9, make the corresponding preferred genetic information available for the means available to the microorganism, i.e.the genetic production process. It is even more preferred to provide vectors which can be used particularly successfully by the host cell, or the cells themselves. The relevant production methods are familiar to the skilled worker.

Embodiments of the invention may be based on related nucleic acid sequences and are cell-free expression systems in which the biosynthesis of proteins is replicated in vitro. All of the mentioned elements can also be combined with the novel process to prepare the proteins of the invention. Moreover, a large number of possible combinations of method steps can be envisaged for each protein of the invention, so that the optimum method needs to be determined experimentally for each particular individual case.

This type of inventive method is more preferred when the nucleic acid sequence is adapted to one or more codons for the host strain in which the codons are used.

This is because, according to the statements above, the transfer of one of the genes into a less relevant species can be used particularly successfully if the relevant codon usage is appropriately optimized.

A further expression of the invention is the use of a nucleic acid ywsC according to the invention described above, a nucleic acid ywsC 'according to the invention described above, a nucleic acid ywtA according to the invention described above, a nucleic acid ywtB according to the invention described above or corresponding nucleic acids which encode one of the proteins according to the invention described above or parts thereof in each case serve for the functional inactivation of the respective relevant gene ywsC, ywsC', ywtA or ywtB in a microorganism. .

In the present application, functional inactivation means any form of modification or mutation by which the function of the relevant protein as an enzyme involved in the formation of polyamino acids or as a subunit of this enzyme is inhibited. The embodiments included form a virtually intact but inactive protein, whereas the inactive part of the protein is present in the cell, depending on the possibility that the relevant gene is no longer translated or even completely deleted. The specific "use" of these factors or genes in this embodiment is therefore that they no longer function precisely in the manner in which they are native in the relevant cells. This is achieved at the genetic level by switching off the relevant genes according to the subject matter of the invention.

An alternative embodiment for inactivating the gene ywsC, ywsC', ywtA or ywtB is the use of the aforementioned nucleic acid ywtD according to the invention or of a corresponding nucleic acid which encodes one of the aforementioned proteins according to the invention for increasing the activity of the associated gene ywtD in a microorganism.

This is because, as described by way of introduction, the enzyme functions in vivo to degrade GLA. Increasing this activity thus leads to a reduction in the concentration of polyamino acids in the culture medium, which, according to the invention, has a positive effect on the industrial fermentation of the relevant microorganisms. This increase in activity advantageously occurs at the genetic level. Such a method is known per se. For example, this can be carried out by transferring the gene into an expression vector: such vectors can be introduced into the cells for fermentation by transformation and are suitably activated under certain conditions so that the derived protein can function in addition to the endogenously formed ywtD.

In a preferred embodiment, both usages are a functional deactivation or an increase in activity during the fermentation of the microorganism, preferably a reduction of the slime caused by polyamino acids to 50%, particularly preferably less than 20%, very particularly preferably less than 5%, all intermediate integers or percentages again being understood as suitable preferred gradations.

To determine these values, the cells of the untreated and treated strains were fermented under otherwise identical conditions, the viscosity of the respective medium being appropriately determined during the fermentation. Since the other conditions of the strains are the same, the difference in viscosity is caused by the different contents of polyamino acids. According to the present invention, a reduction in viscosity is desired. By taking samples from both fermentations and determining the amount of polyamino acid-containing mucilage by known methods, a comparison value, expressed as a percentage, can be obtained. With increasing preference, the values determined in the samples in the transition from the stationary growth phase according to the invention are less than 50%, 40%, 30%, 20%, 10%, 5%, very particularly preferably less than 1%, of the corresponding values in the control fermentations.

This is because the problem of the present invention is to improve the fermentation of microorganisms for biotechnological production. This therefore makes it desirable to carry out the relevant molecular biological construction, either, in particular when a plurality of genes is affected, generally on a laboratory scale, and, where appropriate, on host cells which represent only an intermediate stage, for example the construction of deletion vectors in E.coli. However, according to the invention, it is necessary to inactivate the genes ywsC, ywsC', ywtA or ywtB in order to show the desired effect, in particular during the fermentation. The increase in the activity of the ywtD gene can be controlled, for example, by inducible promoters, such as the relevant transgene. Thus, the activity of the gene can be deliberately switched on by adding the inducer at a suitable time during the fermentation. Alternatively, the gene can also be coupled to a promoter which responds to a stress signal, for example, with too low an oxygen content, which can also occur in fermenters which are blocked by slime when mixing is insufficient.

In another preferred embodiment, the use according to the invention is such that, with increasing preference, 2, 3 or 4 of the ywsC, ywsC', ywtA and ywtB genes are inactivated, preferably in combination with an increase in activity mediated by the ywtD gene.

It can be recalled at this point that in the relevant organisms it is possible that the genes ywsC and ywsC' do not occur simultaneously, but only one in each case. In these cases it is possible that a maximum of 3 of the genes are inactivated, and in this case this represents the most preferred embodiment.

In the case where the inactivated molecular biological form is selected for inactivation of one of these genes, this embodiment is incomplete as a safeguard and the cell still has a corresponding residual activity. This applies in particular to host cells other than B.subtilis, which, according to the Ashiuchi et al article (see above), have been shown to be present in each case in only one copy. It would be particularly valuable to combine deletion methods with methods that enhance ywtD-mediated activity, thereby combining two systems that in principle function differently.

In one embodiment for functionally inactivating one or more of the genes ywsC, ywsC', ywtA and ywtB, a nucleic acid encoding an inactivated protein and having a point mutation is used.

Nucleic acids of this type can be produced by known point mutation methods. Such methods are described, for example, in the relevant handbooks, such as Fritsch, Sambrook and Maniatis. "Molecular cloning: a laboratory Manual", Cold Spring harbor laboratory Press, New York, 1989. In addition, a large number of commercially available construction kits are available, for example from Stratagene (La Jolla, USA)A kit. The principle is to synthesize oligonucleotides (mismatch primers) with a single crossover, then hybridize with the gene in single-stranded form; subsequent DNA polymerization produces the corresponding point mutations. To achieve this, it is possible to use the corresponding species-specific sequences of these genes. Due to the high degree of homology, it is possible and particularly advantageous in the present invention to carry out this reaction on the basis of the sequences provided by SEQ ID NO.1, 3, 5 and 7. These sequences can also be used to design suitable mismatch primers for related species, particularly in the alignment of FIGS. 6 to 10 and FIGS. 1 to 5, based on the conserved regions identified.

In one embodiment of this use, functional inactivation is carried out in each case using nucleic acids having deletion mutations or insertion mutations, preferably including the border sequences of the protein-coding region, in each case comprising at least 70 to 150 nucleic acid positions.

These methods are also known per se to the skilled worker. It is thus possible to prevent the formation of one or more of the factors YwsC, YwsC', YwtA and YwtB in the host cell by cutting off a part of the relevant gene in a suitable transformation vector using restriction endonucleases, and then transferring the vector into the desired host, where the active gene is replaced by an inactive copy by homologous recombination which is still possible at that time. In the case of insertional mutagenesis, it may only be necessary to introduce the entire gene, or another gene, for example a selectable marker, instead of a part of the gene, by disruption. It is thus possible to carry out a phenotypic check of the mutational events in a known manner.

In order to ensure that such recombination events, which are necessary in each case, are between the defective gene introduced into the cell and the complete gene copy endogenously present, for example, on the chromosome, it is necessary, according to the state of the art, that at least 70 to 150 successive nucleic acid positions in the sequences on both sides of the non-identical sequence section are identical in each case, the section lying in between being unimportant. Thus, a preferred embodiment comprises only two flanking regions, at least one of which has such a size.

In an alternative embodiment of this use, nucleic acids are used which contain in total two nucleic acid fragments which in each case comprise at least 70 to 150 nucleic acid positions and are at least partially, preferably entirely, flanking the coding region of the protein. In this connection, it can be determined by known methods that the flanking regions start from a known sequence, for example with the aid of PCR primers in the outward direction, and use the genomic DNA preparation as template (anchored PCR). This is because the fragments do not have to be protein-encoded in order to enable the exchange of the two gene copies by homologous recombination. According to the invention, the primers required for this purpose can be designed on the basis of SEQ ID NO.1, 3, 5 and 7, but also for other species of gram-positive bacteria, in particular of the genus Bacillus. As an alternative to this experimental approach, it is possible to obtain genes at least partially not encoding a plurality of such genes from related species, such as B.subtilis database entries, e.g.subtilista database of the institutes Pasteur, Paris, France (a)http://genolist.pasteur.fr/SubtiList/genome cgi)。

Another preferred embodiment comprises one of the described uses of the invention, wherein an expression vector is used for said increase in the activity mediated by the ywtD gene, preferably a vector comprising the gene and a nucleic acid fragment regulating the gene.

As already stated above, the increase in the activity of the gene and of the derived protein can thus be regulated manually from the outside or automatically adapted by the conditions prevailing in the fermentation medium in order to achieve the requirement for a reduction in the concentration of polyamino acids. The use here is particularly advantageous for the nucleic acids described in the present invention which code for ywtD, very particularly advantageously using the nucleic acid of SEQ ID NO. 9.

The invention can also be carried out in the form of genetically modified microorganisms, to which the above statements apply accordingly.

They are very generally microorganisms in which at least one of the genes ywsC, ywsC', ywtA or ywtB is functionally inactivated or ywtD has an increased activity.

They are preferably microorganisms in which, with increasing preference, 2, 3 or 4 of the genes ywsC, ywsC', ywtA or ywtB are inactivated and preferably accompanied by an increase in the activity mediated by the ywtD gene.

Preferred microorganisms are in the form of bacteria.

Wherein preferred microorganisms according to the description herein are gram-negative bacteria, in particular bacteria of the genus Escherichia coli, Klebsiella, Pseudomonas or Xanthomonas, in particular strains of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, most particularly derivatives of the strains Escherichia coli BL21(DE3), Escherichia coli RV308, Escherichia coli DH5 alpha, Escherichia coli JM109, Escherichia coli XL-1 or Klebsiella planticola (Rf).

According to the statements so far, microorganisms of considerable preference are gram-positive bacteria, in particular bacteria of the genus Bacillus, Staphylococcus or Corynebacterium, more particularly strains of Bacillus lentus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus sphaericus or Bacillus alkalophilus, Staphylococcus carnosus or Corynebacterium glutamicum, very particularly preferably Bacillus licheniformis DSM 13.

In light of the problems on which this application is based, the aim is primarily to improve industrial fermentation processes. The invention is therefore carried out in particular in a corresponding inventive fermentation process.

These methods are very general methods for fermenting the above-mentioned microorganisms of the present invention.

In accordance with the statements made hereinbefore, the processes which are characterized in this respect are correspondingly preferred. These include in particular embodiments in which one or more of the genes ywsC, ywsC', ywtA or ywtB is functionally inactivated or in which the activity of ywtD is increased, in particular a combination of these two methods. Particularly preferred sources for this are the nucleic acids according to the invention described above, in particular the nucleic acids shown in SEQ ID NO.1, 3, 5, 7 or 9. This applies correspondingly to the selected species suitable for the respective fermentation. According to the previous statement, among these the species with increased preference are those with increased relevance to B.licheniformis DSM13, since this increases the success prospects for the use of the nucleic acids.

In the fermentation process of the invention, preference is given to processes for preparing valuable products, in particular for preparing low molecular weight compounds or proteins.

This is because it is the most important field of application for industrial fermentation.

In a preferred method the low molecular weight compound is a natural product, a dietary supplement or a pharmaceutical related compound.

In this way, for example amino acids or vitamins are produced which are used in particular as dietary supplements. The drug related compound may be a precursor or intermediate of a drug, or even the subsequent drug itself. In all these cases, the term biotransformation is also used, whereby the metabolic properties of the microorganism are used to replace the complex chemical synthesis of the whole or at least individual steps.

Also preferably, the protein produced in this way is an enzyme, in particular one of an alpha-amylase, a protease, a cellulase, a lipase, an oxidoreductase, a peroxidase, a laccase, an oxidase and a hemicellulase.

Industrial enzymes prepared by such methods are used, for example, in the food industry. Thus, alpha-amylases are used, for example, to prevent staling of bread or to clarify juice. Proteases are used for the breakdown of proteins. All these enzymes have been described for use in detergent or detergent compositions, which are the prominent sites specifically occupied by subtilisin enzymes naturally produced by gram-positive bacteria. They are used in particular in the textile and leather industries for the processing of natural raw materials. Another possibility for all these enzymes is the use as catalysts for chemical reactions in biotransformations.

Most of these enzymes are derived from bacillus strains and can therefore be produced particularly successfully in gram-positive bacteria, in particular bacteria of the genus bacillus, in many cases also bacillus licheniformis DSM 13. In particular production processes based on these microbial systems can be improved with the aid of the present invention, since the sequences indicated in particular by SEQ ID NO.1, 3, 5, 7 and 9 are derived specifically from this species.

Finally, the factors that can be used in the present application can also be used positively, i.e. in the sense of their natural function, meaning in connection with the directed preparation, modification or degradation of poly-gamma-glutamic acid.

An embodiment of the formation is therefore the preparation, modification or degradation of poly-gamma-glutamic acid by microbial methods, wherein one of the above-described nucleic acids ywsC, ywsC', ywtA, ywtB and/or ywtD according to the invention or one of the corresponding nucleic acids encoding the above-described proteins according to the invention is used for the transgene, preferably for the formation of the above-described corresponding proteins according to the invention.

Among them, the preferred method uses microorganisms from the genus Bacillus, particularly Bacillus subtilis or Bacillus licheniformis.

It is thus possible to produce GLA by microorganisms, in particular in Bacillus subtilis and Bacillus licheniformis, as described, for example, in patent application JP 08308590A or WO 02/055671A 1. The DNA sequences provided herein can be used, for example, to increase the activity of the corresponding gene in a suitable cell, thus increasing yield.

As an alternative to this, cell-free processes for preparing, modifying or degrading poly-gamma-glutamic acid are now also possible, which comprise the above-described gene products YwsC, YwsC', YwtA, YwtB and/or YwtD of the invention which are involved in the formation of polyamino acids, preferably using the corresponding nucleic acids of the invention described above.

Thus, these factors can be reacted in, for example, a bioreactor. The design of such enzyme reactors is known in the prior art.

Particular preference is given to using 2, preferably 3, particularly preferably 4, different such gene products or nucleic acids in a corresponding method of this type.

This is because, as described in the introduction, the factors YwsC, YwtA and YwtB in particular often form tightly bound complexes and therefore a common activity must be mentioned. Simultaneous or subsequent YwtD activity can be used, for example, to influence the biophysical properties of the formed polyamino acids, and, for example, to adapt for use in cosmetic products.

The following examples further illustrate the invention.

Examples

All Molecular biological work steps are carried out according to standard methods as indicated, for example, in Fritsch, Sambrook and Maniatis, "Molecular cloning: a Laboratory Manual" (Cold spring harbor Laboratory Press, New York, 1989) or similar related works. The enzymes, construction kits and instruments were used according to the instructions of the respective manufacturers.

Example 1

Identification of the genes ywsC, ywsC', ywtA, ywtB and ywtD from Bacillus licheniformis DSM13

Preparation of genomic DNA from Bacillus licheniformis DSM13 Strain by standard methods anyone can obtain it from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig (R) ((R))http://www.dsmz.de) The strain was obtained, and genomic DNA was mechanically separated and separated by electrophoresis in 0.8% agarose gel. For shotgun cloning of the smaller fragments, fragments of 2 to 2.5kb in size were eluted from the agarose gel, dephosphorylated and ligated as blunt-ended fragments into the SmaI restriction site of the vector pTZ 19R-Cm. This vector is a derivative of plasmid pTZ19R obtained from Fermentas (St.Leon-Rot) and is endowed with chloramphenicol resistance. Thus, a gene library of smaller fragments was obtained. In a second shotgun cloning, the genomic fragment obtained by partial digestion with the enzyme SauIIIaI was ligated into the SuperCos 1 Vector system ("Cosmid Vector Kit") from Stratagene, La Jolla, USA, resulting in a gene library covering predominantly larger fragments.

Relevant recombinant plasmids were isolated from E.coli DH 5. alpha. obtained by transformation with relevant gene libraries and sequenced (D.Hannahan (1983): Study on transformation on Escherichia coli;. J.mol.Microbiol., vol.166P 557-580). The dye termination method (dye terminator chemistry) was used in this case by means of the automated sequencer MegaBACE1000/4000(Amersham Bioscience, Piscataway, USA) and ABI Prism 377(Applied Biosystems, Foster City, USA).

By this method, the sequences SEQ ID NO.1, 3, 5, 7 and 9 indicated in the sequence listing of the present application were obtained, on the basis of which the genes ywsC, ywsC' (truncated variant of ywsC), ywtA, ywtB and ywtD were obtained. The amino acid sequences deduced therefrom are shown in the corresponding sequences in SEQ ID NO.2, 4, 6, 8 and 10, respectively. The truncated variant ywsC '(or YwsC') corresponds to the gene or protein ywsC (or YwsC), since a comparison of the amino acid sequences of homologous proteins in Bacillus subtilis shows that the polypeptide is shorter by 16 amino acids at the N-terminus, while the other parts have a considerably higher homology and therefore a similar activity, as shown in FIG. 6.

Reproducibility of reproduction

These genes and gene products can now be synthesized artificially by existing methods without the need to replicate the described sequences in a targeted manner on the basis of these sequences. As a further alternative thereto, it is possible to isolate the relevant genes from Bacillus strains, in particular Bacillus licheniformis strain DSM13 obtainable from DSMZ, by PCR, by which it is possible to synthesize primers using the corresponding border sequences shown in the sequence listing. If other strains were used, to obtain genes homologous thereto in each case, the success rate of PCR would increase as the selected strain was closely related to B.licheniformis DSM13, as this could be correlated with an increase in sequence identity in the primer binding region.

Example 2

Sequence homology

After the DNA and amino acid sequences in example 1 were determined, the DNA and amino acid sequences were determined by searching the database GenBank (National Center for Biotechnology Information NCBI, National institutes of Health, Bethesda MD, USA;http://www.ncbi.nlm.nih.gov) And a Subtilist of the Institute Pasteu, Paris, France (A), (B), (C)http://genolist.pasteur.fr/Subtilist/genome.cgi) In which a search is carried out, it is possible to determine the most similar homologous sequences in each case disclosed so far.

The determined DNA and amino acid sequences are compared with each other by means of the alignment shown in FIGS. 1 to 10; the computer program used is vectorrSuite, seventh edition, available from InformatxInc. (Bethesda, USA). In this case, the standard parameters of the program are used, meaning that for the comparison of the DNA sequences: the K-tuple size is 2, the optimal number of diagonals is 4, the window size is 4, the gap penalty is 5, the gap opening penalty is 15, and the gap extension penalty is 6.66. For comparison of amino acid sequences, the following standard parameters were used: the K-tuple size is 1, the optimal number of diagonals is 5, the window size is 5, the gap penalty is 3, the gap opening penalty is 10, and the gap extension penalty is 0.1. The results of these sequence comparisons are compiled in table 1 below, and the accession numbers indicate the accession numbers in the NCBI database.

TABLE 1: genes and proteins most similar to those found in example 1

Gene or protein found in bacillus licheniformis/SEQ ID NO.	Most closely related genes or proteins	Database accession numbers for the most closely related genes or proteins	Homology identity%
				ywsC/1	ywsC of Bacillus subtilis	AB046355.1	75.4
ywsC′/3	ywsC of Bacillus subtilis	AB046355.1	78.5
				ywtA/5	ywsA of Bacillus subtilis	AB046355.1	77.8
ywtB/7	ywsB of Bacillus subtilis	AB046355.1	67.1
				ywtD/9	ywtD of Bacillus subtilis	AB080748	62.3
YwsC/2	YwsC of Bacillus subtilis	AB046355.1	86.1
				YwsC′/4	YwsC of Bacillus subtilis	AB046355.1	89.6
YwtA/6	YwsA of Bacillus subtilis	AB046355.1	89.9
				YwtB/8	YwsB of Bacillus subtilis	AB046355.1	65.8
YwtD/10	YwsD of Bacillus subtilis	AB046355.1	57.3

It is clear that the genes discovered and the gene products obtained therefrom are novel genes and proteins, which are significantly different from the prior art genes and proteins disclosed so far.

Example 3

Functional inactivation of one or more of the ywsC, ywsC', ywtA and ywtB genes in Bacillus licheniformis

Principle of preparation of deletion vectors

Each of these genes can be functionally inactivated by, for example, a so-called deletion vector. The method is described, for example, in J.Et al (1991) in "geneticmanipulating of Bacillus amyloqueforces"; j.biotechnol, vol19，p221-240。

A suitable vector for this is pE194, whose characteristics are described in the article "Replication and compatibility properties of plasmid pE194 in Bacillus subtilis", J.bacteriol., vol.152P 722-735. The advantage of this deletion vector is that it has a temperature-dependent origin of replication. pE194 is able to replicate at 33 ℃ in the transformed cells, so successful transformations were initially selected at this temperature. The cells containing the vector were then cultured at 42 ℃. The deletion vector no longer replicates at this temperature and selection pressure is applied to integrate the plasmid into the chromosome through the previously selected homologous region. A second homologous recombination, which then occurs through a second homologous region, results in excision of the vector from the chromosome together with the complete copy of the gene, thereby deleting the gene located on the chromosome in vivo. Another possibility for the second recombination is the reverse reaction of integration, meaning that the vector recombines out of the chromosome and thus the genes on the chromosome will remain intact. Thus, gene deletions must be detected by conventional methods, for example southern hybridization after restriction digestion of chromosomal DNA with appropriate enzymes, or on the basis of the size of the region to be amplified with the aid of PCR techniques.

It is therefore necessary to select two homologous regions of the deleted gene, each region comprising in each case 70 base pairs, for example in the 5 'region and in the 3' region of the selected gene. They are cloned into the vector in such a way that they flank the coding part of the inactivated protein, or are directly linked without intermediate regions. Thus, a deletion vector was obtained.

Deletion of the ywsC, ywsC', ywtA and ywtB genes referred to herein

The deletion vector of the present invention is constructed by PCR amplification of the 5 'and 3' regions of these 4 or 3 genes. The sequences SEQ ID No.1, 3, 5 and 7 shown in the sequence Listing can be used to design suitable primers which are derived from Bacillus licheniformis, but should be suitable for other strains, in particular of Bacillus, due to the expected homology.

The two amplified regions are directly ligated, via appropriate intermediate cloning, into a vector for this manipulation, for example, in the pUC18 vector suitable for the cloning procedure of E.coli.

The next step is subcloning into the vector pE194 selected for deletion and transformation into Bacillus subtilis DB104, which can be obtained, for example, by protoplast transformation, see Chang& Cohen(1979；“High Frenquency Transformation of Bacillussubtilis Protoplasts by Plasmid DNA”；Molec.Gen.Genet.(1979)，Vol168P 111-115). All working steps must be carried out at 33 ℃ to ensure replication of the vector.

The intermediate cloned vector is then transformed into the desired host strain, in this case B.licheniformis, by protoplast transformation. Transformants which were obtained by this method and identified as positive by conventional methods (selection by resistance markers of the plasmid; by plasmid preparation and PCR examination of the insert) were then cultivated at 42 ℃ and a selection pressure on the presence of the plasmid was created by the addition of erythromycin. The deletion plasmid cannot replicate at this temperature and only cells in which the vector has integrated into the chromosome survive, this integration being most likely in a homologous or consensus region. Excision of the deletion vector can then be induced by culturing at 33 ℃ under erythromycin-free selection pressure, and the gene encoded by the chromosome is completely deleted from the chromosome. The success of the deletion is checked by southern hybridization after restriction digestion of the chromosomal DNA with the appropriate enzyme, or with the aid of PCR techniques.

Transformants in which the relevant gene has been deleted can also be identified by a limited or even complete loss of the ability to form GLA.

Description of the drawings

FIG. 1 alignment of the ywsC gene (SEQ ID NO.1) (B.l.ywsC) of Bacillus licheniformis DSM13 with the homologous gene ywsC (B.s.ywsC) of Bacillus subtilis.

FIG. 2 alignment of the ywsC 'gene (SEQ ID NO.3) (B.l.ywsC') from Bacillus licheniformis DSM13 with the homologous gene ywsC (B.s.ywsC) from Bacillus subtilis.

FIG. 3 alignment of the ywtA gene of Bacillus licheniformis DSM13 (SEQ ID NO.5) (B.l.ywtA) with the homologous gene of Bacillus subtilis ywtA (B.s.ywtA).

FIG. 4 alignment of the ywtB gene of Bacillus licheniformis DSM13 (SEQ ID NO.7) (B.l.ywtB) with the homologous gene of Bacillus subtilis ywtB (B.s.ywtB).

FIG. 5 alignment of the ywtD gene of Bacillus licheniformis DSM13 (SEQ ID NO.9) (B.l.ywtD) with the homologous gene of Bacillus subtilis ywtD (B.s.ywtD).

FIG. 6 alignment of the YwsC protein of Bacillus licheniformis DSM13 (SEQ ID NO.2) (B.l.YwsC) with the homologous protein of Bacillus subtilis YwsC (B.s.YwsC).

FIG. 7 alignment of the YwsC 'protein of Bacillus licheniformis DSM13 (SEQ ID NO.4) (B.l.YwsC') with the homologous protein of Bacillus subtilis YwsC (B.s.YwsC).

FIG. 8 alignment of the YwtA protein of Bacillus licheniformis DSM13 (SEQ ID NO.6) (B.l.YwtA) with the homologous protein of Bacillus subtilis YwtA (B.s.YwtA).

FIG. 9 alignment of the YwtB protein of Bacillus licheniformis DSM13 (SEQ ID NO.8) (B.l.YwtB) with the homologous protein of Bacillus subtilis YwtB (B.s.YwtB).

FIG. 10 alignment of the YwtD protein of Bacillus licheniformis DSM13 (SEQ ID NO.10) (B.l.YwtD) with the homologous protein of Bacillus subtilis YwtD (B.s.YwtD).

Sequence listing

<110> Hangao two-ply company (Henkel Kommanditgesellschaft auf Aktien)

<120> Novel gene products of Bacillus licheniformis forming or degrading polyamino acids and improved biotechnological production methods therefor (Novel gene products from Bacillus licheniformis for forming or decomposing polyaminoacids and improved biotechnological production methods based thereon)

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<211>409

<212>PRT

<213>Bacillus licheniformis DSM 13

<400>2

<210>3

<211>1182

<212>DNA

<213>Bacillus licheniformis DSM 13

<220>

<221>gene

<222>(1)..(1182)

<223>ywsC’

<220>

<221>CDS

<222>(1)..(1182)

<223>

<400>3

<210>4

<211>393

<212>PRT

<213>Bacillus licheniformis DSM 13

<400>4

<210>5

<211>450

<212>DNA

<213>Bacillus licheniformis DSM 13

<220>

<221>gene

<222>(1)..(450)

<223>ywtA

<220>

<221>CDS

<222>(1)..(450)

<223>

<400>5

<210>6

<211>149

<212>PRT

<213>Bacillus licheniformis DSM 13

<400>6

<210>7

<211>1170

<212>DNA

<213>Bacillus licheniformis DSM 13

<220>

<221>gene

<222>(1)..(1170)

<223>ywtB

<220>

<221>CDS

<222>(1)..(1170)

<223>

<400>7

<210>8

<211>389

<212>PRT

<213>Bacillus licheniformis DSM 13

<400>8

<210>9

<211>1245

<212>DNA

<213>Bacillus licheniformis DSM 13

<220>

<221>gene

<222>(1)..(1245)

<223>ywtD

<220>

<221>CDS

<222>(1)..(1245)

<223>

<220>

<221>misc_feature

<222>(1)..(3)

<223>First codon translated as Met.

<400>9

<210>10

<211>414

<212>PRT

<213>Bacillus licheniformis DSM 13

<220>

<221>misc_feature

<222>(1)..(3)

<223>First codon translated as Met.

<400>10

Claims (60)

1. A protein YwsC (CapB, PgsB) which is involved in the formation of polyamino acids and which encodes a nucleotide sequence YwsC which shows at least 80% identity with the nucleotide sequence indicated in SEQ ID No. 1.

2. Protein YwsC according to claim 1, which encodes a nucleotide sequence which, with increasing preference, shows at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 1.

3. A protein YwsC (CapB, PgsB) which is involved in the formation of polyamino acids, whose amino acid sequence shows at least 91% identity with the amino acid sequence represented in SEQ ID No.2, with increasing preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity.

4. A protein YwsC '(truncated variant of YwsC) which is involved in the formation of polyamino acids and which encodes a nucleotide sequence YwsC' which exhibits at least 83% identity with the nucleotide sequence indicated in SEQ ID No. 3.

5. The protein YwsC' according to claim 4, which encodes a nucleotide sequence which, with increasing preference, exhibits at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 3.

6. A protein YwsC' (truncated variant of YwsC) which is involved in the formation of polyamino acids and whose amino acid sequence shows at least 94% identity with the amino acid sequence depicted in SEQ ID No.4, with increasing preference at least 95%, 96%, 97%, 98%, 99% and particularly preferably 100% identity.

7. A protein YwtA (CapC, PgsC) which is involved in the formation of polyamino acids and for which the nucleotide sequence YwtA which is encoded exhibits at least 82% identity with the nucleotide sequence represented in SEQ ID No. 5.

8. The protein YwtA according to claim 7, wherein the nucleotide sequence encoded thereby shows, with increasing preference, at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 5.

9. A protein YwtA (CapC, PgsC) which is involved in the formation of polyamino acids and whose amino acid sequence shows at least 94% identity with the amino acid sequence represented in SEQ ID No.6, with increasing preference at least 95%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity.

10. A protein YwtB (CapA, PgsA) which is involved in the formation of polyamino acids and which encodes a nucleotide sequence ywtB which shows at least 72% identity with the nucleotide sequence indicated in SEQ ID No. 7.

11. The protein YwtB according to claim 10, wherein the nucleotide sequence coding therefor shows, with increasing preference, at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 7.

12. A protein YwtB (CapA, PgsA) which is involved in the formation of polyamino acids and whose amino acid sequence shows at least 70% identity with the amino acid sequence represented in SEQ ID No.8, with increasing preference at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity.

13. A protein YwtD (Dep, PgdS) which is involved in the degradation of polyamino acids and which encodes a nucleotide sequence YwtD which shows at least 67% identity with the nucleotide sequence represented in SEQ ID No. 9.

14. The protein YwtD according to claim 13, wherein the nucleotide sequence encoded thereby shows with increasing preference at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence represented in SEQ ID No. 9.

15. A protein YwtD (Dep, PgdS) which is involved in degradation of polyamino acids and whose amino acid sequence shows at least 62% identity with the amino acid sequence represented in SEQ ID No.10, with increasing preference at least 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity.

16. The protein involved in the formation or degradation of polyamino acids according to one or more of claims 1 to 3, 4 to 6, 7 to 9, 10 to 12, or 13 to 15, which is naturally produced by a microorganism, which is preferably a bacterium, particularly preferably a gram-positive bacterium, and wherein preferably a bacterium of the genus bacillus, wherein particularly preferred is bacillus licheniformis (b.licheniformis), and wherein very particularly preferred is bacillus licheniformis DSM 13.

17. A nucleic acid ywsC (capB, pgsB), which encodes a gene product which is involved in the formation of polyamino acids and whose nucleotide sequence exhibits at least 80% identity with the nucleotide sequence indicated in SEQ ID No. 1.

18. Nucleic acid ywsC according to claim 17, whose nucleotide sequence shows, with increasing preference, at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 1.

19. A nucleic acid ywsC' (truncated variant of ywsC) which encodes a gene product which is involved in the formation of polyamino acids and whose nucleotide sequence exhibits at least 83% identity with the nucleotide sequence indicated in SEQ ID No. 3.

20. The nucleic acid ywsC' according to claim 19, whose nucleotide sequence shows, with increasing preference, at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99% and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 3.

21. A nucleic acid ywtA (capC, pgsC), which encodes a gene product which is involved in the formation of polyamino acids and whose nucleotide sequence shows at least 82% identity with the nucleotide sequence indicated in SEQ ID No. 5.

22. The nucleic acid ywtA according to claim 21, whose nucleotide sequence shows, with increasing preference, at least 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 5.

23. A nucleic acid ywtB (capA, pgsA), which codes for a gene product which is involved in the formation of polyamino acids and whose nucleotide sequence exhibits at least 72% identity with the nucleotide sequence indicated in SEQ ID No. 7.

24. The nucleic acid ywtB according to claim 23, whose nucleotide sequence shows, with increasing preference, at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 7.

25. A nucleic acid ywtD (dep, pgdS), which codes for a gene product which is involved in the degradation of polyamino acids and whose nucleotide sequence exhibits at least 67% identity with the nucleotide sequence indicated in SEQ ID No. 9.

26. The nucleic acid ywtD according to claim 25, whose nucleotide sequence shows, with increasing preference, at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, 99%, and particularly preferably 100% identity with the nucleotide sequence indicated in SEQ ID No. 9.

27. The nucleic acid according to any of claims 17 and/or 18, 19 and/or 20, 21 and/or 22, 23 and/or 24, or 25 and/or 26, which is naturally present in a microorganism, preferably a bacterium, particularly preferably a gram-positive bacterium, and wherein preferably a bacterium of the genus bacillus, wherein bacillus licheniformis is particularly preferred, and wherein very particularly preferred is bacillus licheniformis DSM 13.

28. A nucleic acid encoding the protein of any one of claims 1 to 16.

29. A vector comprising one of the nucleic acids of claims 17 to 28.

30. The vector of claim 29, containing two or more of the nucleic acids of claims 17-28.

31. A cloning vector according to claim 29 or 30.

32. The expression vector of claim 29 or 30.

33. A cell which, after genetic modification, contains one of the nucleic acids of claims 17 to 28.

34. The cell according to claim 33, wherein the nucleic acid is part of a vector, in particular of any one of claims 29 to 32.

35. The cell of claim 33 or 34, which is a bacterium.

36. The cell according to claim 35, which is a gram-negative bacterium, in particular one of the bacteria of the genera escherichia coli, klebsiella, pseudomonas or xanthomonas, in particular escherichia coli K12, escherichia coli B or klebsiella phyta (klebsiella) strain, very particularly a derivative of escherichia coli BL21(DE3), escherichia coli RV308, escherichia coli DH5 α, escherichia coli JM109, escherichia coli XL-1 or klebsiella phytate (Rf) strain.

37. The cell according to claim 35, which is a gram-positive bacterium, in particular one of the genera Bacillus, Staphylococcus or corynebacterium, very particularly a strain of Bacillus lentus (b. lentus), Bacillus licheniformis (b. licheniformis), Bacillus amyloliquefaciens (b. myloliquefaciens), Bacillus subtilis (b. subtilis), Bacillus sphaericus (b. globigii) or Bacillus alcalophilus (b. alcalophilus), Staphylococcus carnosus (Staphylococcus carnosus) or corynebacterium glutamicum (corynebacterium glutamicum), wherein a very particularly preferred strain is again a derivative of Bacillus licheniformis DSM 13.

38. A process for the preparation of one or more of the gene products YwsC, YwsC', YwtA, YwtB and YwtD according to claims 1 to 16.

39. The method according to claim 38, wherein the nucleic acid of any one of claims 17 to 28 is used, preferably the vector of any one of claims 29 to 32 is used, particularly preferably the cell of any one of claims 33 to 37 is used.

40. The method of claim 38 or 39, wherein one, or preferably more, of the codons of the nucleotide sequence are changed to adapt to the codon usage of the host strain.

41. Use of the nucleic acid ywsC according to claim 17 and/or 18, of the nucleic acid ywsC 'according to claim 19 and/or 20, of the nucleic acid ywtA according to claim 21 and/or 22, of the nucleic acid ywtB according to claim 23 and/or 24, or of the corresponding nucleic acid according to claim 28, or of a partial nucleic acid thereof in each case, for the functional inactivation of the relevant genes in microorganisms, these genes in each case being ywsC, ywsC', ywtA and ywtB, respectively.

42. Use of the nucleic acid ywtD according to claim 25 and/or 26 or of the corresponding nucleic acid according to claim 28 for enhancing the activity of the relevant gene ywtD in a microorganism.

43. Use according to claim 41 or 42, wherein the functional inactivation or increase in activity occurs during fermentation of the microorganism, preferably reducing the slime caused by polyamino acids to 50%, particularly preferably to less than 20%, very particularly preferably to less than 5%.

44. Use according to any one of claims 41 to 43, wherein with increasing preference 2, 3 or 4 of the ywsC, ywsC', ywtA and ywtB genes are inactivated, preferably in combination with an enhancement of the activity mediated by the ywtD gene.

45. The use according to any one of claims 41 to 44, wherein in each case a nucleic acid encoding an inactive protein and having a point mutation is used for functional inactivation.

46. The use according to any one of claims 41 to 45, wherein the nucleic acids which are in each case used for functional inactivation have deletion mutations or insertion mutations, preferably comprising a border sequence which in each case comprises at least 70 to 150 nucleic acid positions of the protein-coding region.

47. Use according to any one of claims 41 to 46, wherein the expression vector is for enhancement of ywtD gene-mediated activity, preferably the expression vector comprises the gene and a nucleic acid fragment regulating the gene.

48. A microorganism in which at least one of the genes ywsC, ywsC', ywtA or ywtB is functionally inactivated or ywtD has an enhanced activity.

49. The microorganism of claim 48, wherein with increasing preference 2, 3 or 4 of the ywsC, ywsC', ywtA or ywtB genes are inactivated, preferably in combination with an enhancement of the activity mediated by the ywtD gene.

50. The microorganism of claim 48 or 49, which is a bacterium.

51. The microorganism according to claim 50, which is a gram-negative bacterium, in particular one of the genera Escherichia coli, Klebsiella, Pseudomonas or Xanthomonas, in particular a strain of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, very particularly a derivative of the strain Escherichia coli BL21(DE3), Escherichia coli RV308, Escherichia coli DH5 α, Escherichia coli JM109, Escherichia coli XL-1 or Klebsiella planticola (Rf).

52. The microorganism according to claim 50, which is a gram-positive bacterium, in particular one of the genera Bacillus, Staphylococcus or Corynebacterium, very particularly a strain of Bacillus lentus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus sphaericus or Bacillus alkalophilus, Staphylococcus carnosus or Corynebacterium glutamicum, wherein very particularly preferred is in turn Bacillus licheniformis DSM 13.

53. A method for fermenting the microorganism of any one of claims 48 to 52.

54. The process according to claim 53, for the preparation of a product of value, in particular a low molecular weight compound or protein.

55. The method of claim 54 wherein the low molecular weight compound is a natural product, a dietary supplement, or a pharmaceutical-related compound.

56. The method according to claim 54, wherein the protein is an enzyme, in particular one of an alpha-amylase, a protease, a cellulase, a lipase, an oxidoreductase, a peroxidase, a laccase, an oxidase and a hemicellulase.

57. A microbial method for the preparation, modification or degradation of poly-gamma-glutamic acid, wherein one of the nucleic acids according to any one of claims 17 and/or 18, 19 and/or 20, 21 and/or 22, 23 and/or 24, or 25 and/or 26, or the corresponding nucleic acid according to claim 28, is used for transgenesis, preferably to form a corresponding protein according to one or more of claims 1 to 16.

58. The method according to claim 57, wherein a microorganism of the genus Bacillus, in particular Bacillus subtilis or Bacillus licheniformis, is used.

59. A cell-free method for the preparation, modification or degradation of poly-gamma-glutamic acid comprising a gene product involved in the formation of a polyamino acid according to any one of claims 1 to 3, 4 to 6, 7 to 9, 10 to 12, or 13 to 15, preferably using a corresponding nucleic acid according to any one of claims 17 to 28.

60. The method according to any one of claims 57 to 59, wherein 2, preferably 3, particularly preferably 4, different gene products and nucleic acids are used.