HK1168125A - Fermentation process - Google Patents

Description

Fermentation process

[ technical field ] A method for producing a semiconductor device

The present invention relates to fermentation processes that allow for improved cell growth and improved expression of polypeptides in prokaryotic host cells. In particular, the invention relates to a fermentation process for culturing a prokaryotic host cell comprising an expression vector encoding a polypeptide under the control of a mannose-inducible promoter.

[ background of the invention ]

Fermentation processes for culturing cells are of great importance in the production of active substances for biological and pharmaceutical applications. In particular, the fermentation process should be suitable for producing the desired substance in an amount sufficient for practical use, such as clinical or commercial use.

Various strategies have been developed for achieving efficient expression of a targeting polypeptide by culturing prokaryotic host cells containing an expressible nucleic acid sequence encoding the targeting polypeptide. The efficiency of expression is strongly dependent on the promoter controlling the expression of the nucleic acid sequence encoding the targeting polypeptide.

In particular, promoters with high transcription rates that allow for the production of high copy numbers of the targeting polypeptide are desired.

Furthermore, in fermentation processes, it is desirable to control the expression of the targeted polypeptide. Control of expression can be achieved, for example, by operably linking a nucleic acid sequence encoding a targeting polypeptide to an inducible promoter that initiates expression only in the presence of a suitable inducer.

A promoter is a nucleic acid sequence that causes transcription of a nucleic acid sequence (structural gene) encoding a targeting polypeptide.

In particular, the present invention relates to fermentation processes utilizing novel vectors for heterologous expression in a host comprising a promoter region of the mannose operon operably linked to a transcription unit comprising a nucleic acid sequence encoding a polypeptide, whereas the expression of said nucleic acid sequence is controlled by the promoter region of said mannose operon.

By suitable induction, the promoter is activated and allows transcription of the structural gene. The induction can be under negative or positive control.

In negative control, the repressor binds to the promoter and prevents transcription of the structural gene. If a suitable inducer is present, the repressor is inactivated and transcription is allowed.

In positive induction, the promoter is activated upon binding of an activator, wherein binding of the activator to the promoter is mediated by a suitable inducer.

Typical inducers may be substrates required for the metabolism of the prokaryotic host, e.g. different types of sugars.

The present invention relates to a positive inducible system in which an activator binds to a promoter that initiates transcription of a gene operably linked to the promoter in the presence of a suitable substrate, i.e., an inducer.

To date, most heterologous gene expression systems in prokaryotic host systems have relied entirely on a limited set of bacterial promoters. Thus, the number of substrates available as inducers is also limited.

Moreover, the yield of heterologous expression systems depends on the number of transformed prokaryotic hosts available. Thus, there is a need for prokaryotic host systems that can grow to high cell densities, i.e., allow rapid propagation without release of the vector during cell division.

[ SUMMARY OF THE INVENTION ]

According to the present invention, these and other objects, which will become apparent from the following description, have been achieved by a fermentation process for culturing a prokaryotic host cell transformed with a novel vector comprising a transcription unit operably linked to a nucleic acid sequence heterologous to said host, whereas the expression of said nucleic acid sequence is controlled by the promoter region of the mannose operon.

According to a particular aspect, the invention provides a method of culturing a bacterial host cell, which method allows the bacterial host cell to grow to a high cell density.

Also provided is the use of the novel vector for the regulated expression of nucleic acid sequences in a prokaryotic host; an isolated and purified nucleic acid sequence expressible in a host comprising the promoter region of the mannose operon; a prokaryotic host transformed with said vector or said isolated and purified nucleic acid sequence; methods of producing a polypeptide in a host using said vector or said isolated and purified nucleic acid sequence; and the use of a prokaryotic host transformed with said vector or said isolated and purified nucleic acid sequence in fermentation, especially in high cell density fermentation.

Other objects and advantages will be apparent to those skilled in the art from the following detailed description, taken together with the accompanying drawings and the appended claims.

[ description of the drawings ]

Which is shown in

The nucleic acid sequence of B.subtilis used in the mapping of the transcription start site from the manP promoter highlights the transcription start site at adenine nucleotides, the deduced-35 and-10 boxes are shown in italics, the end of manR and the start of the lys gene are marked with arrows, and the restriction sites BglII, XbaI, AflII and NdeI are underlined;

FIG. 2A nucleic acid sequence comprising the promoter region of the manR promoter, will be highlighted at the transcription start site of guanine nucleotides, the deduced boxes-10 and-35 are indicated in italics, the start of the manR gene is indicated by an arrow, and the HindIII restriction site and the deduced cre sequence are underlined;

fig. 3 nucleic acid sequences obtained from bacillus subtilis (b.subtilis) comprising the promoter regions of the manR promoters as contained in pSUN291, pSUN384.1 and pSUN385.2, respectively, with arrows indicating the start of lacZ, and the restriction sites underlined;

FIG. 4A plasmid map of the expression vector pSUN279.2 according to the present invention;

fig. 5. beta-galactosidase activity of bacillus subtilis 3NA containing plasmids pSUN279.2, pSUN284.1 and pSUN291, respectively, according to the present invention;

fig. 6 a nucleic acid sequence obtained from bacillus subtilis (b.subtilis) comprising the promoter region of the manP promoter from bacillus subtilis (b.subtilis) including the C-terminus of manR, the intergenic region between manR and manP where it is replaced by the reporter gene lacZ, the transcription start site, the-35 and-10 cassettes are shown in bold, the end of manR and the start of lacZ are indicated by arrows, and the restriction sites are underlined;

figure 7. beta-galactosidase activity of bacillus subtilis 3NA containing plasmid pSUN279.2 and other plasmids containing fragments of the nucleic acid sequences of different lengths shown in figure 6;

fig. 8. beta. -galactosidase activity of bacillus subtilis 3NA containing vectors pSUN291, pSUN384.1 and pSUN345.2 having the nucleic acid sequences shown in fig. 3;

FIG. 9A plasmid map of the expression vector pMW168.1 according to the present invention;

figure 10 is a graph of the results of plasmid stability testing of pMW168.1 in bacillus subtilis (b.subtilis)3NA, plotting the propental part of the cells containing the plasmid against the passage number;

FIGS. 11-14 logarithmically show plots of dry biomass concentration plotted against the duration of fermentation runs 1-4, and plots of fluorescence signal (RFU) plotted against the duration of fermentation runs 1-4; and

figures 15 and 16 are graphs of fluorescence signals plotted against the duration of the fermentations of fermentation runs 5 and 6.

[ detailed description of the invention ]

As used herein, the following definitions are provided to facilitate an understanding of the present invention.

A "vector expressible in a host" or "expression vector" is a polynucleic acid construct of a series of specific polynucleic acid elements produced recombinantly or synthetically, allowing transcription of a specific nucleic acid sequence in a host cell. Generally, such vectors include a transcription unit comprising a particular nucleic acid sequence to be transcribed operably linked to a promoter. The vector expressible in the host may be, for example, an autonomous or self-replicating plasmid, a cosmid, a phage, a virus or a retrovirus.

The terms "host", "host cell" and "recombinant host cell" are used interchangeably herein to refer to a prokaryotic cell into which one or more vectors or isolated and purified nucleic acid sequences of the present invention have been introduced. It is understood that the term refers not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term "comprising" is generally used in an inclusive sense, which is to say that one or more other features or components are permitted to be present.

As used herein, "promoter" refers to a nucleic acid sequence that controls the expression of a transcriptional unit. A "promoter region" is a regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Within the promoter region, protein binding domains (consensus sequences) responsible for the binding of RNA polymerases such as the putative-35 box and the Pribnow box (-10 box) will be found. Furthermore, the promoter region may comprise a transcription initiation site and a binding site for regulatory proteins.

"mannose operon" refers to the mannose operon of Bacillus subtilis.

3 genes have been identified in the mannose operon (Kunst F.N.et al, "the complete genome sequence of gram-positive bacteria Bacillus subtilis", Nature 390, p249-256 (1997)).

The 1 st gene, manP, encodes a mannose-specific enzyme component (transporter) belonging to the fructose-permease family. The 2 nd gene, manA, encodes mannose-6-phosphate isomerase, whereas the 3 rd gene, yjdF, is of unknown function. The regulatory gene, manR, located upstream of these 3 genes and in the same orientation, encodes the manR, an activator of the mannose operon.

The mannose operon, consisting of 3 genes manP-manA-yjdF (joined in series, referred to as "manP"), under the control of a manP promoter which itself is positively regulated.

The other promoter, the manR promoter, is responsible for the mannose-dependent induction of the essential manR expression of the manP-promoter.

The manR promoter region also contains the catabolite regulator protein binding site (catabolite response element (cre)) of the manR gene.

"Cre sequence" refers to a nucleic acid sequence located upstream (5' to) of a catabolic gene. The cre sequence binds to a Catabolite Control Protein (CCP) that prevents Carbon Catabolite Repression (CCR) of expression of catabolic genes.

By "promoter region of the mannose operon" is meant a promoter region that regulates the expression of manP and manR with or without cre sequences.

The "manP promoter" as referred to herein comprises at least the-35 region, the Pribnow box, and the ManR binding site.

The "manR promoter" as referred to herein comprises at least the putative-35 region, the Pribnow box, the ManR binding site and, optionally, the cre sequence.

D-mannose, also referred to as "mannose," is a 2-epimer of glucose and is present in mannan and heteromannan polysaccharides, glycoproteins, and a variety of other complex carbohydrates.

"CcpA" refers to "catabolite control protein A," which is a global regulator protein and can activate or repress the activation of some catabolic operons. In the case of the mannose operon, CcpA represses by binding to the cre-sequence.

An "enhancer" is a nucleic acid sequence that functions to enhance transcription of a transcriptional unit independently of the identity of the transcriptional unit, with respect to the sequence position, or sequence orientation, of the transcriptional unit. The vectors of the present invention optionally may include enhancers.

As used herein, "transcriptional unit" refers to a nucleic acid sequence that is normally transcribed as a single RNA molecule. The transcriptional unit may contain one gene (monocistronic) or 2 (dicistronic) or more genes (polycistronic) that encode functionally related polypeptide molecules.

A nucleic acid sequence is "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a transcription initiation region, such as a ribosome binding site, is operably linked to a nucleic acid sequence encoding, for example, a polypeptide, if it is positioned to facilitate translation of the polypeptide. Ligation may be achieved by ligation at convenient restriction sites. If this site is not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

As referred to herein, a "nucleic acid" or "nucleic acid sequence" or "isolated and purified nucleic acid or nucleic acid sequence" may be DNA, RNA or a DNA/RNA hybrid. In the case where the nucleic acid or nucleic acid sequence is located in a vector, it is typically DNA. The DNA herein can be any polydeoxynucleotide sequence, including, for example, double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands consist of 2 or more fragments, double-stranded DNA wherein one or both strands have an unbroken phosphodiester backbone, DNA comprising one or more single-stranded portions and one or more double-stranded portions, double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically-synthesized DNA, semi-synthesized DNA, biosynthetic DNA, naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled DNA, such as radiolabeled DNA and fluorochrome-labeled DNA, DNA comprising one or more non-naturally occurring nucleic acid species. The DNA sequence may be synthesized by standard chemical techniques, for example, the phosphotriester method or via automated synthesis methods and PCR methods. Purified and isolated DNA sequences can also be generated by enzymatic techniques.

The RNA herein can be, for example, single-stranded RNA, cRNA, double-stranded RNA with one or both strands consisting of 2 or more fragments, double-stranded RNA with one or both strands having an unbroken phosphodiester backbone, RNA containing one or more single-stranded portions and one or more double-stranded portions, double-stranded RNA with the RNA strands fully complementary, double-stranded RNA with the RNA strands only partially complementary, covalently cross-linked RNA, enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi-synthesized RNA, biosynthetic RNA, naturally-isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non-naturally occurring nucleic acid species.

"variant" or "sequence variant" means a nucleic acid sequence that is altered from a reference sequence by conservative nucleic acid substitutions, wherein one or more nucleic acids are substituted with another having the same characteristics. Variants also include degenerate sequences, sequences with deletions and insertions, as long as the modified sequence exhibits the same function as the reference sequence (functionally equivalent).

As used herein, the terms "polypeptide", "peptide", "protein", "polypeptide" and "peptide" are used interchangeably to designate a series of amino acid residues linked to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues.

The term "isolated and purified nucleic acid sequence" refers to the state in which a nucleic acid sequence will be in accordance with the present invention. Nucleic acid sequences will be free or substantially free of materials with which they are naturally associated, such as other nucleic acids found in their natural environment, or the environment in which they are prepared (e.g., cell culture) when such preparation is practiced in vitro or in vivo by recombinant techniques.

The terms "transformation", "transformed" or "directing a nucleic acid into a host cell" refer to any process by which an extracellular nucleic acid, such as a vector, with or without accompanying material, enters a host cell. The term "transformed cell" or "transformed cell" refers to a cell or progeny thereof into which an extracellular nucleic acid has been introduced, thereby carrying the extracellular nucleic acid. The nucleic acid can be introduced into a cell such that the nucleic acid can replicate as a chromosomal integrant or as an extra-chromosomal element. Transformation of suitable host cells with, for example, expression vectors can be accomplished by well-known methods such as microinjection, electroporation, particle bombardment, or by chemical methods such as calcium phosphate-mediated transformation, as described, for example, in Maniatis et al 1982, Molecular Cloning, A laboratory Manual, Cold Spring harbor laboratory or in Austubel et al 1994, Current protocols in Molecular biology, John Wiley and Sons.

"heterologous nucleic acid sequence" or "nucleic acid sequence heterologous to the host" means a nucleic acid sequence encoding, for example, an expression product such as a polypeptide which is foreign to the host ("heterologous expression" or "heterologous product"), i.e. a nucleic acid sequence originating from a donor which is different from the host, or a chemically synthesized nucleic acid sequence encoding, for example, an expression product such as a polypeptide which is foreign to the host, or a nucleic acid sequence originating from the host and encoding a polypeptide which is naturally expressed by said host, wherein the nucleic acid sequence is inserted into a vector and is under the control of the promoter region of the mannose operon of the invention.

In case the host is a particular prokaryotic species, the heterologous nucleic acid sequences may originate from different genera or families of organisms, from different orders or classes, from different phyla (parts) or from different domains (kingdoms).

The heterologous nucleic acid sequence may be modified by mutation, insertion, deletion or substitution of a single nucleic acid or a portion of the heterologous nucleic acid sequence prior to its introduction into the host cell, provided that the modified sequence exhibits the same function (functional equivalent) as the reference sequence. Heterologous nucleic acid sequences as referred to herein also include sequences derived from organisms such as different domains (kingdoms) from eukaryotes (eukaryotic origin), such as for example human antibodies that have been used in phage display libraries and single nucleic acids or parts of nucleic acid sequences that have been modified according to the "codon usage" of a prokaryotic host.

A "transcription initiation region" is a signal region that facilitates transcription initiation and contains sequences for ribosome binding sites such as the Shine Dalgarno sequence.

Typically, the transcriptional initiation region is located downstream of the transcriptional initiation site and is operably linked to the gene to be expressed.

"transcription termination region" refers to a sequence that results in the termination of transcription by RNA polymerase. Transcription termination regions are generally portions of transcriptional units that can prevent unwanted transcription of other nearby genes or transcription from other potential promoters, and can increase the stability of the mRNA.

"antibody" refers to a class of plasma proteins produced by the B cells of the immune system following stimulation by an antigen. Mammalian (i.e., human) antibodies are immunoglobulins of the Ig G, M, a, E or D classes. The term "antibody" as used for the purposes of the present invention includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, anti-idiotypic antibodies and auto-antibodies as well as chimeric antibodies present in autoimmune diseases such as diabetes, multiple sclerosis and rheumatoid arthritis.

In one aspect, the invention utilizes a vector expressible in a host comprising a promoter region of the mannose operon operably linked to a transcription unit comprising a nucleic acid sequence heterologous to the host, whereas expression of said nucleic acid sequence is controlled by the promoter region of said mannose operon.

The vectors of the invention are preferably autonomous or self-replicating plasmids, cosmids, phages, viruses or retroviruses. A wide variety of host/vector combinations may be employed in expressing the nucleic acid sequences of the present invention.

Useful expression vectors, for example, can be comprised of chromosomal, non-chromosomal, and/or synthetic nucleic acid segments.

Suitable vectors include vectors with a specific host range, such as vectors specific for, e.g., bacillus subtilis and escherichia coli (e.coli), respectively, as well as vectors with a broad-host-range, such as vectors useful for gram-positive and gram-negative bacteria.

"Low-copy", "medium-copy" and "high-copy" plasmids may be used.

For example, in Bacillus subtilis, the low copy plasmid is pAMbeta1, the medium copy plasmid is a pBS72 derivative, and the high copy plasmid is pUB 110.

According to the present invention, the promoter regions of the mannose operon comprise a manR promoter region and a manP promoter region, respectively.

The nucleic acid sequence from bacillus subtilis (b.subtilis) includes the C-terminus of manR, the intergenic region between manR and manP, followed by the lysostaphin gene lys as a reporter gene, shown in fig. 1.

The nucleic acid sequence of the invention comprises the promoter region of manP, preferably the nucleic acid sequence comprising the start codon from bp-80 to lys in FIG. 1 (SEQ ID NO: 1) and more preferably the nucleic acid sequence from bp-80 and comprising bp-1, i.e.upstream of the transcription start site A in bp +1 (SEQ ID NO: 2) in FIG. 1.

Nucleic acid sequences from bacillus subtilis (b. subtilis) include the promoter region of manR, the transcription start site G at bp +1, the putative cre sequence, the transcription start region between bp +1 and manR, and portions of manR, shown in fig. 2 and 3, where manR is replaced by lacZ.

The nucleic acid sequence of the invention comprises the promoter region of manR, preferably the nucleic acid sequence comprising the start codon of FIG. 3 from bp-122 to lacZ (SEQ ID NO: 3), more preferably the nucleic acid sequence of FIG. 3 from bp-122 and bp +7, i.e.comprising the putative cre-sequence, (SEQ ID NO: 4), and, in particular, the nucleic acid sequence of FIG. 3 from bp-122 and bp-1, i.e.upstream of the transcription start site G of pb +1 (SEQ ID NO: 5).

The two promoter regions manP and manR comprise the binding site for manR.

The invention also includes polypeptides similar to SEQ ID NO: 1 to 5, and variants thereof.

The mannose operon used in the present invention, such as the manP promoter region, the manR promoter region (with or without cre sequence) and the mannose operon of SEQ ID NO: 1-5, the sequence complementarity or variants thereof usually being derived from the mannose operon of bacillus subtilis (b.subtilis) or from functionally equivalent promoter regions of other prokaryotes, especially of Bacillaceae (bacillus). Other prokaryotes include functionally equivalent promoter regions that can be induced by mannose, i.e., promoter regions that have higher expression activity in the presence of mannose than in the absence of mannose.

In many prokaryotes, such as Firmicutes such as bacillus subtilis, the mannose operon is involved in D-mannose metabolism.

Bacillus subtilis (b.subtilis) can use many different sugars as carbon sources. Hexoses such as glucose and D-mannose are predominantly via phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) uptake. In PTS, the corresponding hexose is simultaneously phosphorylated and transported to the cell during uptake. Uptake and utilization of specific sugar substrates is subject to Carbon Catabolite Repression (CCR). Preferred sugar substrates of bacillus subtilis (b.subtilis) repress transcription of genes such as the mannose operon for uptake and utilization of other substrates in the presence of glucose.

The mechanism of glucose-dependent CCR in Bacillus subtilis (B.subtilis) has been extensively studied and known in the art (St. lke J.et al, "Regulation of carbon metabolism in Bacillus species," in Annu. Rev. Microbiol.54, 2000, pages 849-880).

A transcriptional unit according to the invention typically also comprises a translation initiation region upstream of the start of translation (start codon) of the transcriptional unit, whereas the translation initiation region is operably linked to the nucleic acid sequence. The translation initiation region is generally located immediately upstream of the start of translation of the transcriptional unit (which may be ATG, GTG or TTG).

The translation initiation region may be the translation initiation region of the transcriptional unit of the manP gene or manR gene in the mannose operon.

The translation initiation region of the manP or manR gene of the mannose operon may be partially or completely replaced by other translation initiation regions.

For example, the translation initiation regions of tufA (elongation factor Tu) and gsiB (stress protein; Jurgen et al, 1998, mol.Gen.Genet.258, 538-545), both from Bacillus subtilis (B.subtilis), can be used.

The corresponding nucleic acid sequences of tufA and gsiB with the transcription initiation region and the initiation of the corresponding gene are shown below, the initiation codon is shown in bold, the restriction sites are underlined, and the Shine-Dalgarno-sequence is highlighted.

tufA：

5′-cttaaqGAGGATTTTAGAGCTAAAGAAAAATTCggatcc-3′

AflII SD start codon BamHI

gsiB：

5′-cttaagAATTAAAGGAGGAATTCAAAGCAGACAATAACAAAggatcc-3′

AflII SD start codon BamHI

By suitable selection of the transcription initiation region, the stability of the mRNA can be enhanced, which is an important feature in gene expression. The stability of mRNA is characterized by its specific half-life.

In addition to the transcription initiation region, a translation initiation site can also be substituted, and, optionally, a number of codons of the gene following the initiation site, e.g., about 5-6 codons, such as shown in the nucleic acid sequences of tufA and gsiB, respectively, above.

The translation initiation region may further comprise a sequence encoding a signal sequence operably linked to the nucleic acid sequence to be expressed. The signal sequence is typically located immediately downstream of the translation initiation point.

In the case of using a bicistronic or polycistronic transcription unit, different or identical signal sequences operably linked to each cistron may be used. Different signal sequences are preferred for this case. The signal sequence used may be a prokaryotic or eukaryotic signal sequence. Prokaryotic signal sequences are generally used.

The DNA sequence encoding the signal sequence to be employed in the expression vector of the present invention may be obtained commercially or chemically synthesized. For example, the signal sequence may be synthesized according to the solid phase phosphoramidite triester method, as described, for example, in Beaucage & Caruthers, 1981, Tetrahedron LeHs.22, 1859-S1862 as described in Van Devanter et al, Nucleic Acids Res.12: 6159-6168(1984). The oligonucleotides can be purified by native acrylamide gel electrophoresis or by electrophoresis as described in Pearson & Reanier, j.chrom.255: 137-149(1983) by anion-exchange HPLC.

Usually the transcriptional unit also comprises a transcriptional termination region.

Preferably, a strong transcription termination region is used to avoid "read-through" of the transcriptional unit by the promoter into the flanking plasmid sequences, as well as into the transcriptional unit from other plasmid promoters. Further stabilization of the mRNA was observed in the presence of this transcription termination region.

A suitable example for a strong transcription termination region has the nucleic acid sequence 5'-CGAGACCCCTGTGGGTCTCG-3' from the 3 ' -region of tufA of commercially available bacillus subtilis 168.

According to the present invention, heterologous nucleic acid sequences encoding expression products foreign to the host may be used. In case the host is a prokaryotic species such as bacillus subtilis or escherichia coli (e.coli), the nucleic acid sequence of interest may be from another class, such as a gamma-proteobacteria, such as from e.g. Burkholderia sp, especially from a different phylum, such as archaea, or, most particularly, from a eukaryotic organism, such as a mammal, especially from a human. However, the heterologous nucleic acid sequence may be modified according to the "codon usage" of the host. The heterologous sequence of the invention is typically a gene of interest. The gene of interest preferably comprises a heterologous polypeptide such as a structural, regulatory or therapeutic protein, or an N-or C-terminal fusion or other fusion protein of a structural, regulatory or therapeutic protein with another protein ("Tag"), such as green fluorescent protein. The heterologous nucleic acid sequence may also encode a transcript that may be used in the form of RNA, such as, for example, antisense-RNA.

The protein may be produced as insoluble aggregates or as soluble protein present in the cytoplasmic or periplasmic space of the host cell and/or in the extracellular medium. Preferably, the protein is produced as a soluble protein present in the periplasmic space of the host cell and/or in the extracellular medium.

The heterologous protein of interest may be of human, mammalian or prokaryotic origin. Other proteins are antigens, such as glycoproteins and carbohydrates from microbial pathogens, both viral and antibacterial agents, and from tumors. Other proteins are enzymes such as rennin, proteases, polymerases, dehydrogenases, nucleases, glucanases, oxidases, alpha-amylases, oxidoreductases, lipases, amidases, nitrile hydratases, esterases or nitrilases.

In the present invention, the order and distance in which the signal sequence and the heterologous nucleic acid sequence are arranged within the expression vector may vary. In a preferred embodiment, the signal sequence is 5' (upstream) to a nucleic acid sequence encoding, for example, a polypeptide of interest. The signal sequence and the nucleic acid sequence encoding, for example, a polypeptide of interest may be separated by 0 to about 1000 amino acids. In a preferred embodiment, the signal sequence and the nucleic acid sequence encoding, for example, a polypeptide of interest are directly adjacent to each other, i.e.0 nucleic acids apart.

Preferably, the vector of the invention comprises a sequence according to SEQ ID NO: 1 to 5, the sequence complementarity and variants thereof.

The invention also encompasses the use of the vector of the invention in a fermentation process of the invention for the regulated heterologous expression of a nucleic acid sequence in a prokaryotic host.

In yet another aspect, the present invention provides isolated and purified nucleic acid sequences comprising a promoter region of the mannose operon. Preferably, the isolated and purified nucleic acid sequence comprises the manP promoter and/or the manR promoter of the mannose operon. More preferably, the isolated and purified nucleic acid sequence comprises SEQ ID NO: 1 to 5.

The isolated and purified nucleic acid sequence comprising the promoter region of the mannose operon may be operably linked to a transcription unit comprising a nucleic acid sequence encoding a polypeptide, wherein expression of the nucleic acid sequence encoding the polypeptide is under the control of the promoter region of the mannose operon.

The isolated and purified nucleic acid sequences of the invention can be isolated according to standard PCR protocols and methods well known in the art. The purified and isolated DNA sequence may further comprise one or more regulatory sequences, as known in the art, such as enhancers, typically used for expression of the product encoded by the nucleic acid sequence.

To select host cells that are successfully and stably transformed with the vectors of the invention or the isolated and purified nucleic acid sequences, a gene encoding a selectable marker (e.g., antibiotic resistance) can be introduced into the host cell with the nucleic acid sequence of interest. The gene encoding the selectable marker may be located on a vector or on an isolated and purified nucleic acid sequence or may optionally be co-introduced in an isolated form, such as an isolated vector. Various selectable markers that can be used include those that confer antibiotic resistance, such as spectinomycin, hygromycin, ampicillin, and tetracycline. The amount of antibiotic can be adapted as desired in order to create selective conditions. A selectable marker is typically used.

In the case where the vector is a shuttle vector, markers common to the appropriate host may be used. For example, in the case where the vector is a shuttle vector replicable in escherichia coli (e.coli) and bacillus subtilis (b.subtilis), a resistance marker gene encoding a spectinomycin-adenylyltransferase enzyme of Enterococcus faecalis (Enterococcus faecalis) that confers resistance to spectinomycin may be used.

Other reporter genes, such as fluorescent proteins, can be introduced into the host cell along with the nucleic acid sequence of interest in order to determine transformation efficiency.

Suitable reporter genes are, for example, those which code for enhanced green fluorescent protein (eGFP) and lacZ which codes for β -galactosidase. Two reporter genes are commercially available and widely used.

Another aspect of the invention provides a prokaryotic host transformed with a vector of the invention, wherein the vector comprises a promoter region of the mannose operon. Preferably the vector comprises SEQ ID NO: 1 to 5, a sequence complementary thereto or a variant thereof.

A wide variety of prokaryotic host cells are useful for transformation with the mannose-inducible promoter region of the mannose operon of the present invention. These hosts may include gram-positive cell strains such as Bacillus and Streptomyces. Preferably, the host cell is Thellungicutes (Firmictites), more preferably the host cell is Bacillus (Bacillus).

Usable Bacillus (Bacillus) are, for example, Bacillus subtilis (b.subtilis), Bacillus amyloliquefaciens (b.amyloliquefaciens), Bacillus licheniformis (b.licheniformis), Bacillus natto (b.natto), Bacillus megaterium (b.megaterium) strain, and the like. Preferred host cells are bacillus subtilis, such as bacillus subtilis 3NA and bacillus subtilis 168.

Escherichia coli (E.coli) which can be used are, for example, the commercially available strains TG1, W3110, DH1, XL1-Blue and Origami.

Suitable host cells are commercially available, for example from a reservation center such as DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).

For example, Bacillus (Bacillus) is available from the Bacillus genetic Stock Center.

The host cell may or may not metabolize mannose. Host cells that normally take up and metabolize mannose, such as bacillus subtilis, can be modified to be deficient in one or more functions associated with the uptake and/or metabolism of mannose. A deficiency in one or more functions associated with the uptake and/or metabolism of mannose can be achieved, for example, by inhibiting or blocking the expression of a gene encoding a protein, such as the manA gene encoding mannose-6-phosphate-isomerase. This can be done by known techniques such as transposon-supported mutagenesis or knockout mutation.

Typically, the prokaryotic host corresponds to a selected signal sequence, for example in case a signal sequence of the genus Bacillus (Bacillus) is used, the host cell is typically a member of the same family of the family Bacillus (Bacillus), more preferably the host cell is a strain of the genus Bacillus (Bacillus).

Preferably, a compound having phosphoenolpyruvate: a host of the carbohydrate phosphotransferase system (PTS). In particular, the host having the PTS system is a microorganism of the order Bacillus (bacillals), in particular Bacillus (Bacillus), and more preferably the species Bacillus subtilis or Enterobacteriales (enterobacteriaceae), preferably the species Escherichia (Escherichia) and more preferably the species Escherichia coli (e.

The main element of CCR is the catabolite control protein a (ccpa), which binds to cre-sequences such as SEQ ID NO: 3 and 4. In the CcpA bound state, transcription of the corresponding gene, here manR, is repressed.

Transcription of the gene of the mannose operon is initiated by the absence of glucose and in the presence of an inducer, such as D-mannose, without repression of binding by CcpA, and by binding of a regulatory protein (ManR) to the corresponding binding site of the promoter region of the mannose operon.

Surprisingly, the inventors found that ManR is not only a regulatory protein of the manP promoter region, but also a self regulator of ManR itself.

The present invention provides a fermentation process for culturing a prokaryotic host cell comprising a vector comprising a mannose-inducible promoter of the mannose operon of Bacillus subtilis operably linked to a transcriptional unit comprising a nucleic acid sequence encoding a polypeptide, wherein in step 1 the prokaryotic host cell is cultured in the presence of a1 st carbon source which is different from an inducer of the inducible promoter, and in step 2 the prokaryotic host cell is cultured in the presence of said inducer.

The carbon source other than the inducer is generally the primary carbon source of the host cell, whereas the inducer is generally the secondary carbon source of the host cell.

The principal carbon source is preferably consumed by the host cell and, thus, is useful as a substrate in a fermentation process.

For many prokaryotic host cells, glucose is the predominant carbon source and may be suitably used as a substrate in the fermentation process of those prokaryotic cells.

The present invention also provides a method of producing a polypeptide in a host cell, comprising the steps of:

(a) constructing a carrier, and constructing a vector,

(b) transforming a prokaryotic host with said vector,

(c) allowing the polypeptide to be expressed in a cell culture system under suitable conditions,

(d) recovering the polypeptide from the cell culture system.

The vector used, as well as its construction and transformation of a prokaryotic host, is as defined above, however the heterologous nucleic acid sequence comprised by the vector encodes a polypeptide.

As the cell culture system, continuous or discontinuous culture such as batch culture or fed-batch culture can be applied in a culture tube, a shake flask or a bacterial fermenter.

The preferred fermentation process of the present invention is fed-batch fermentation. The fed-batch process is characterized by a time-limited replenishment of the culture broth with the substrate without withdrawal of solution. Replenishment may be continuous or intermittent. Generally, a fed-batch process comprises a batch phase during which cells are initially grown to a desired concentration. During this time, cell growth is high and generally no target polypeptide is produced unless an inducer is added. After the desired cell concentration is reached, a replenishment period of substrate is initiated. Generally, the inducer is added during the feeding period.

For culturing the host cells, conventional media as known in the art may be used, such as complex media like "nutrient yeast broth", Kortz et al 1995, J.Biotechnol.39, 59-65 described glycerol-containing media, mineral salts media as described by Kulla et al 1983, Arch.Microbiol 135, 1, Wilms et al 2001, Biotechnol.Bioeng.73, 95-103 described batch media for fermentation of Bacillus subtilis (B.subtilis) or LB-media as described by Bertram et al 1051, J.Bacteriol.62, 293-.

The culture medium comprises a suitable carbon source, for example a sugar such as glucose, as a substrate for the host cell to be grown. The carbon source used as substrate is different from the inducer.

The medium may be modified as appropriate, for example by adding further ingredients such as buffers, salts, vitamins, amino acids, antibiotics or other micronutrients as is commonly known to the person skilled in the art.

Likewise, different media or combinations of media can be used during cell culture.

Preferably, the medium used as basal medium should not comprise an inducer in order to achieve a tight regulation of the mannose promoter region.

In the fermentation process of the invention, expression is initiated by addition of a suitable inducer. The inducer of the mannose operon is mannose. In addition, derivatives of mannose that induce the manR promoter region or manP promoter region of the mannose operon can be used. Expression can be regulated by the amount of inducer available to the prokaryotic host.

Addition of the inducer can be initiated after the culture reaches the assay parameters. Examples of such assay parameters are Optical Density (OD) indicating the concentration of cells in culture or the concentration of a substrate such as a carbon source different from the inducer.

For example, in the present method, the inducer may be added after the culture reaches the appropriate OD, depending on the particular culture system. Typical OD as a measured parameter for batch cultures in shake flasks₆₀₀Is about 0.3 or higher.

In the batch phase, according to one embodiment of the fed-batch culture of the present invention, when only the main carbon source is available in the culture medium, the cells are grown to 20-30 OD₆₀₀And, then, the culture is switched to a feeding phase with the addition of a mixture of a principal carbon source and an inducer. In the supply phase, the main carbon source: lureThe ratio of leads may vary, with a suitable ratio being from 3: 1 to 1: 3. By the main carbon source: the expression rate can be controlled by changing the ratio of the inducer.

The amount of inducer added may be selected depending on the particular conditions of the fermentation.

The mode of addition of the inducer (induction protocol) can be selected according to the particular culture system. By adding a mode, the growth rate and the expression speed of the host cell can be further regulated. For example, the inducer may be added discontinuously or continuously over a suitable period of time. In a discontinuous mode (shock induction), the addition may be only once at the point of induction, or twice or even several times at suitable intervals. Suitable modes depend on the culture system and can be readily determined by one skilled in the art.

For example, in continuous mode, the inducer can be added at a constant rate or at a decreasing/increasing rate.

The continuous addition may further be within a selected time interval of the culturing, for example a selected time interval during exponential growth of the culturing.

Also, a combination of discontinuous and continuous induction schemes is possible.

If the inducer is added in 2 or more portions, the addition of further inducer may be initiated after the culture reaches the 2 nd assay parameter. The 2 nd assay parameter may be, for example, optical density OD, concentration of expressed polypeptide, concentration of inducer in solution or signal intensity of expression of reporter gene.

Generally, the amount of inducer in the culture medium of the prokaryotic host is adjusted to about 10g/l, preferably about 5g/l, more preferably about 2 g/l.

The amount of inducer added during the feed can vary over the period of supplementation. As described above, the main carbon source: variation of the ratio of the inducer allows for modulation of the expression rate, i.e., cell density.

A suitable pH range is, for example, 6-8, preferably 7-7.5, and a suitable cultivation temperature is between 10 and 40 ℃, preferably between 30 and 37 ℃.

The cells are generally incubated until a maximum amount of expressed product and/or biomass has accumulated, preferably between 1 hour and 20 days, more preferably between 5 hours and 3 days.

The amount of product expressed as biomass yield also depends on the culture system used.

In shake flasks, a host transformed with a vector of the invention can produce a culture expressing the product, typically in an amount of 0.5g/l medium. Using a fermenter, in batch and/or fed-batch mode, cultures expressing the product in a quantity generally greater than 0.5g/l of fermentation broth, preferably greater than 1g/l, in particular greater than 1.3g/l, are obtained.

Furthermore, in the fermentation method of the present invention using the host cell, at least 10 to 30OD can be obtained₆₀₀In particular at least 50OD₆₀₀，50OD₆₀₀And greater, more preferably at least 500OD₆₀₀And most preferably at least 1000OD₆₀₀High cell density. In particular, in the fed-batch process according to the invention, a 50OD can be obtained₆₀₀And greater than 1000OD₆₀₀Cell density in between.

For illustration, 1OD₆₀₀Corresponding to an average of about 0.322g dry mass/l. Thus, an OD of 100₆₀₀The values correspond to a dry mass/g of 32.2g and an OD of 500₆₀₀161g dry mass/l.

Fermentation focusing can maximize output changes by the specific induction protocol of the present invention, taking biomass, expression product and inducer consumption into account, respectively, as desired.

For example, the induction protocol combined with the 1 st impact induction and the additional replenishment of the inducer at an exponential rate results in high expression of the targeted polypeptide relative to the biomass concentration, which makes further processing, such as purification steps, more efficient, and, thus, saves time and cost.

Furthermore, the fermentation of the host cells of the invention allows a high replication speed without vector loss.

According to one embodiment of the fermentation process of the invention, a host cell, such as a Bacillus (Bacillus), is cultured comprising a vector carrying a promoter of the mannose operon, PmanR or PmanP, operably linked to a nucleic acid sequence encoding a targeting polypeptide. For the genus Bacillus (Bacillus), the preferred substrate is glucose. Furthermore, the inducer of the promoter of the mannose operon is mannose. Preferably, the fermentation is carried out in fed-batch mode. More preferably, as described above, the host cells are grown during the batch phase by addition of glucose only, up to an OD of about 20-30₆₀₀The cell density of (a). During the subsequent feeding period, a mixture of glucose and inducer mannose may be added.

As also described above, the ratio of glucose to mannose may vary, for example from 3: 1 to 1: 3, in the mixture. In addition, it is also possible to supply only mannose.

Preferably, the vector may also comprise, in addition to the mannose promoter, a complete or partial sequence regulating the gene manR.

Following fermentation and expression in the host cell, the expressed product, such as a polypeptide of interest, may then be recovered from the culture of the host cell. To obtain maximum yields of expressed product, the cells are usually harvested at the end of culture and lysed, such as by lysozyme treatment, sonication or French press lysis. Thus, the polypeptide is usually obtained for the first time as a crude lysate of the host cell. They can then be purified by standard protein purification procedures known in the art, which can include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity and immunoaffinity chromatography. Such well known and conventionally practiced methods are described, for example, in Ausubel et al, supra, and Wu et al (eds.), Academic Press inc, n.y.; immunochemical Methods In Cell And Molecular Biology. For example, purification of recombinantly produced immunoglobulins, which may be purified by immunoaffinity chromatography over a column containing a resin to which the expressed immunoglobulin binds specifically to a target molecule.

The invention also relates to methods and means for the intracellular heterologous expression of nucleic acids encoding, for example, polypeptides in prokaryotic hosts. In particular, the present invention relates to a vector and the use of the vector for the intracellular expression of heterologous polypeptides in a prokaryotic host using the vector of the present invention.

In intracellular expression, the polypeptide is expressed within the cytoplasm and is not transported from the cytoplasm to non-cytoplasmic locations. The polypeptide will be expressed within the cytoplasm either in an inclusion body form or in a soluble form. Methods for isolating and purifying polypeptides from cells, particularly from cell extracts, are also well known.

The mannose promoters of the invention are advantageous in that they can be tightly regulated, induced by common and non-toxic and thus industrially useful compounds.

Furthermore, the mannose promoter of the present invention and the vector comprising the mannose promoter are stable within cells and are not lost even after multiple cell replications. Thus, host cells transformed according to the invention can advantageously be grown to very high cell densities.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any 2 or more of said steps or features. The present disclosure is therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. Throughout this specification various references are cited, each of which is incorporated herein by reference in its entirety.

The above description will be more fully apparent with reference to the following examples. However, this example is merely illustrative of a method of carrying out the invention and is not intended to limit the scope of the invention.

[ examples ] A method for producing a compound

[ 1: isolation and characterization of the manR and manP promoter regions of the mannose operon

The following materials and methods were used if not otherwise stated:

[ bacterial strains and growth conditions ]

Escherichia coli (E.coli) JM109(Yanisch-Perron C.et al, Gene33, 1985, 103-containing strain 119) and Bacillus subtilis 3NA (Michel J.F.et al, J.appl.Bacteriol.33, 1970, 220-containing strain 227) were used as the main hosts for cloning and expression. Escherichia coli (E.coli) was grown in LB liquid medium (Luria S.E.et al, Virology 12, 1960, 348-. Bacillus subtilis was grown in LB liquid medium and C or S minimal medium at 37 ℃ (Martin-Verstraete I.et al, J.Mol.biol.214, 1990, 657-. The liquid medium and the agar plates were supplemented with 100. mu.g/ml spectinomycin, 10. mu.g/ml kanamycin or 5. mu.g/ml erythromycin, respectively. To induce the mannose promoter, sterile filtered or autoclaved D-mannose was added at a final concentration of 0.2% (w/v).

[ MATERIALS ] OF THE INVENTION

All chemicals were obtained from Sigma-Aldrich (Taufkrichen, Germany), Fluka (Buchs, Germany) or Merck (Darmstadt, Germany). Synthetic DNA oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany). Restriction enzymes and DNA modifying enzymes were purchased from Roche Applied Science (Mannheim, Germany) or New England Biolabs. (Frankfurt am Main, Germany). PCR was run with high fidelity-DNA polymerase from Fermentas (st. leon-Rot, germany) on MiniCycler from Biozym.

[ preparation and transformation of DNA ]

DNA-isolation from Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis) or from agarose gels was carried out using DNA preparation kits from Qiagen (Hilden, Gemrany) or Roche (Mannheim, Germany) as described by the manufacturer. Standard molecular techniques are used throughout the examples.

Escherichia coli (E.coli) was transformed with plasmid DNA as described by Chung C.T.et al, Proc.Natl.Acad.Sci.USA 86, 1989, 2172-2175. Bacillus subtilis (b.subtilis) was transformed with plasmid DNA according to the modified "paris method" (Harwood c.r. molecular Biological Methods for Bacillus, 1990, John Wiley & Sons ltd., England).

[ beta-galactosidase Activity measurement ]

0.1ml of cells to be examined in 0.9ml of Z-buffer are treated with 10. mu.l of toluene for 30min at 37 ℃. Beta-galactosidase activity was determined by the Miller method using o-nitrophenyl-beta-galactopyranoside at 22 ℃ (Miller J.H., 1972, experiments in molecular genetics, Cold Spring Harbor, NY).

[ oligonucleotides used ]

[ TABLE 1 ]

[ experiment 1: isolation of the promoter region of the DNA fragment carrying the mannose operon and determination of the transcription initiation sites of the manR promoter and manP promoter ]

Chromosomal DNA of Bacillus subtilis 168 was isolated by using DNeasy blood & tissue kit from Qiagen (Hilden, Germany).

From the obtained DNA an about 2.3kb DNA fragment with a complete manR containing the putative manR promoter and the intergenic region between manR and manP was amplified by PCR using primers s4693/s 4694.

Primer extension experiments using the obtained DNA fragment of about 2.3kb for determining the transcription initiation sites of manR promoter and manP promoter.

To isolate mRNA for primer extension, shuttle vectors were constructed from the Escherichia coli (E.coli) vector pIC20HE (Altenbuchner et al, 1992, Methods enzymol.216, 457-466) and Bacillus subtilis vector pUB110(MacKenzie et al, 1986, Plasmid 15, 93-103). Vectors containing the lys gene as a reporter gene encoding a mature form of lysostaphin from Staphylococcus haemolyticus (Staphylococcus simulans) (Recsai et al, 1987, Proc. Natl. Acad. Sci. USA84, 1127. Asca. 1131).

To this high copy pUB110 derivative, a 2.3kb DNA fragment was cloned upstream of the lysostaphin gene. The resulting plasmid was named pSUN178.4 and introduced into Bacillus subtilis 3 NA.

Bacillus subtilis 3NA with plasmid pSUN178.4 was grown in LB medium with kanamycin. In the exponential growth phase, the culture was induced with 0.2% mannose. After 1 hour of growth at 37 ℃, induced and non-induced cells were harvested. Total RNA was isolated using Qiagen-RNeasy Mini kit.

Primers s5006, s5007, s5097 and s5098 labeled with Cy5 at the 5' -end were used.

Primers s5006 and s5007 hybridize to +21 to +50 and +76 to +105, respectively, relative to the start codon of the lysostaphin gene. Primers s5097 and s5098 hybridize to +81 to +101 and +131 to +153, respectively, relative to the start codon of manR.

The same primers were used for the sequencing reaction of plasmid DNA of psun178.4 as a size standard. AMV-reverse transcriptase and T7-DNA polymerase from Roche were used for reverse transcription and DNA sequencing, respectively. Reverse transcription products and sequencing were analyzed on denaturing polyacrylamide sequencing gel (GE HEALTHCARE). All other reagents used were provided by the Amersham pharmacia Biotech AutoRead Sequencing kit.

The transcription start site of the manP-promoter was determined by using primer s 5006. The DNA sequence reaction of plasmid pSUN178.4 was prepared with the same primers and run on the same denaturing gel for comparison. FIG. 1 shows the DNA sequence around the manP promoter, which will be highlighted at the transcription initiation site of A (adenine nucleotide). The deduced cassettes-10 and-35 are in italics, the end of the manR gene and the start of the lys gene are marked by arrows, and the restriction sites for BglII, XbaI, AflII and NdeI are underlined.

The transcription start site of manR promoter was determined by RNA isolation and DNA sequencing as described above for manP promoter, except that primer s5098 incorporated in manR gene was used.

In FIGS. 2 and 3, the DNA sequence of the manR promoter region is shown, will be highlighted at the transcription start site of G (guanine nucleotide), the deduced-10 and-35 cassettes are indicated in italics, and the start of the lys gene and manR gene are indicated by arrows, respectively. The restriction site and the putative cre sequence are underlined.

When cells were induced with mannose as observed by the much stronger signal in the primer extension experiments, transcription from the manR promoter and especially from the manP promoter increased strongly.

The primers used are shown in table 1 above.

[ EXPERIMENT 2 ]

The primer extension experiment according to experiment 1 locates the transcription start site of manP promoter near the 3' -end of the intergenic region between manR and the beginning of manP. To determine the manP promoter region more precisely, the 2.3kb DNA fragment was shortened stepwise by PCR-amplification and the obtained sequence fragments of different lengths were cloned back into the same basic expression vector and expression studies.

[ construction of basic expression vector (a) ]

Constructing an expression vector containing promoterless lacZ as a reporter gene. The expression vector was designed as a shuttle vector that replicates in both bacillus subtilis (b. subtilis) and escherichia coli (e. coli) and was designated psun 272.1.

The reporter gene lacZ was cleaved with NdeI and XmaI from pLA2 (hallimann a.et al, 2001, j.bacteriol.183, 6384-. To this plasmid, a pair of oligonucleotides s4956/4957 was inserted between the AflII/MunI restriction sites, in order to add the same tufA transcription terminator upstream of lacZ. Thus avoiding "read-through" from the plasmid promoter into lacZ and "read-through" out of lacZ into the flanking plasmid sequences by the terminator. The spectinomycin resistance Gene spc for both Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis) was amplified from plasmid pDG1730(Geurout-Fleury et al, 1996, Gene 180, 57-61) with oligonucleotide s4833/4835 and inserted into the plasmid obtained above. In addition, the Escherichia coli (E.coli) vector portion was shortened by deleting the BspHI/HindIII fragment. Subsequently, the EcoRI/SphI fragment containing the replication region of B.subtilis pMTLBS72(Lagodich et al, 2005, mol. biol. (Mosk)39, 345-348) was ligated to the plasmid.

The 2.3kb DNA fragment obtained in experiment 1 was inserted before lacZ of pSUN272.1 by digestion with AflII and NheI and ligation, thereby obtaining expression vector pSUN279.2, the plasmid map of which is shown in FIG. 4.

The primers used are shown in table 1 above.

[ B determination of expression efficiency of vector pSUN279.2 ]

Plasmids pSUN279.2 and pSUN272.1 obtained in (a) above were introduced into Bacillus subtilis 3 NA. The latter served as background control. Bacillus subtilis 3NA strain carrying one or the other plasmid is grown in LB medium containing spectinomycin and in exponential growth phase 0.2% mannose, 0.2% mannose plus 0.2% glucose or no sugar (uninduced control) is added to the culture for induction. After 1 hour induction, the cells were assayed for β -galactosidase activity by Miller's assay. The results are shown in FIGS. 5 and 7.

Non-induced cultures of bacillus subtilis (b.subtilis) containing psunf279.2 have shown a reasonably high basal level of β -galactosidase activity. The presence of mannose leads to a further 4-fold increase in β -galactosidase activity, whereas the activity with mannose and glucose is reduced, but still well above basal levels. The results clearly indicate that the promoter activity seen in pSUN279.2 can be derived from the region between manR and manP, from the region upstream of manR or from both.

Thus, the upstream region of manR and most of manR were deleted from pSUN279.2 by cleaving the 2.3kb DNA fragment of pSUN279.2 shown between SfoI and NruI in FIG. 4 to give plasmid pSUN284.1.

The resulting nucleic acid sequence of pSUN284.1 is shown in FIG. 6.

Bacillus subtilis 3NA was transformed with this plasmid, psun284.1, and expression efficiency was determined as described above. The results are shown in FIG. 5. As can be seen in fig. 5, this manR deleted vector psun284.1 in bacillus subtilis 3NA showed only about half of the basal level of β -galactosidase activity compared to psun279.2 in bacillus subtilis 3NA, an even stronger increase induced by mannose, and again a stronger decrease in the presence of glucose. These results demonstrate that the manP promoter is located between manR and manP and that the chromosomal copy of manR is sufficient to control the entire manP promoter copy on a low copy plasmid.

[ location of manP promoter region ]

To locate the promoter region of manP, as shown in FIG. 6, in addition to the shortened DNA fragment of pSUN284.1, a further shortened sequence fragment was prepared from the 2.3kb DNA fragment by restriction enzyme by cleavage at a restriction site at a different position upstream of the transcription start site of manP promoter.

Deletion of bp-81 and bp-80 upstream of the transcriptional start site of manP results in a DNA sequence comprising SEQ ID NO: 1, 2 nd deletion sequence.

Further deletions of bp-41 and bp-40 upstream of the transcription start site of manP were performed (deletion No. 3 sequence).

In a similar manner to plasmid pSUN284.1 in (2b) above, a plasmid comprising deletion No. 2, pSUN290, and deletion No. 3, pSUN297.5 was constructed by inserting the PCR product amplified with primers s4802/s5203 and s5262/s5203, respectively, into pSUN272.1 via restriction enzymes EcoRV and NheI.

The plasmid was inserted into Bacillus subtilis 3NA and cultured as described in (b) above. After 1 hour induction, the cells were assayed for β -galactosidase activity as described in (b) above. The results are shown in FIG. 7.

As shown in fig. 7, no strain containing pSUN290 and pSUN284.1 showed significant differences regarding lacZ induction by mannose. However, in B.subtilis 3NA of pSUN297.5 containing the deletion No. 3 sequence, the induction by mannose was completely eliminated and the basal expression level was almost 0. From these results, the ManR binding site of the manP mannose promoter region is located between bp-80 and-35 relative to the transcription start site of manP.

[ experiment 3: determination of the manR promoter

[ a) identification of cre sequence ]

Since most CCR are mediated by catabolite control protein a (ccpa), a search for the corresponding binding site (cre sequence) was performed in the whole mannose operon with the Clone Manager program using DNA alignment functions. For alignment, cre consensus 5 '-WWTGNAARCGNWWWCAWW-3' was used.

As shown in FIGS. 2 and 3, a putative cre sequence was found only in the promoter region of manR, downstream of the-10 cassette.

SEQ ID NO: 3 comprises a region starting from bp-122 to the start codon of lacZ, SEQ ID NO: 4 comprises the region starting from bp-122 to bp +7 (inclusive) of the sequence shown in figure 3, whereas SEQ ID NO: 5 includes the region beginning at bp-122 to bp-1 (inclusive) of the sequence shown in FIG. 3.

[ B) evaluation of expression efficiency of manR promoter ]

To evaluate the expression efficiency of the manR promoter, an expression vector such as pSUN284.1 was constructed as described above and designated pSUN 291. To this end, a DNA fragment of approximately 600bp upstream, comprising the putative manR-promoter and manR, was amplified with primers s5208/s5209, and the linearized plasmid DNA pSUN279.2 was used as template and inserted before the lacZ of plasmid pSUN272.1 by digestion with KpnI and AflIII and ligation.

The DNA sequence is shown in FIG. 3.

Plasmid pSUN291 was introduced into Bacillus subtilis 3NA and β -galactosidase activity was measured as described in experiment (2 b).

The results are shown in FIG. 5. In this context, basal expression has been relatively high and also increased by about 3-fold by the addition of 0.2% mannose. The addition of glucose results in repression of beta-galactosidase activity to nearly basal expression levels.

The results indicate that the manR promoter is not only a weakly constitutive promoter, but is also involved in mannose and CCR regulation.

[ c) location of the manR promoter region ]

To further locate the promoter region of the manR DNA-sequence as in experiment (2c), DNA-fragments of different lengths were prepared from the DNA-sequence as contained in pSUN291 by cleavage at restriction sites and by restriction enzymes at different positions upstream of the transcription start site of the manR promoter as shown in FIG. 3.

The 1 st deletion sequence was obtained by cleaving the sequence shown in FIG. 3 to bp-100 and bp-99 upstream of the transcription initiation site G, and the 2 nd deletion sequence was obtained by cleaving to bp-83 and bp-82 upstream of the transcription initiation site G.

In analogy to experiment (2c), the 1 st and 2 nd deletion sequences obtained were introduced into pSUN272.1 and the resulting plasmids were named pSUN384.1 and pSUN385.2, respectively.

Each plasmid was inserted into bacillus subtilis 3NA and cultured as shown in experiment 2 b. After 1 hour induction, the cells were assayed for β -galactosidase activity as shown in experiment 2 b. The results are shown in FIG. 8. No significant difference was noted in the lacZ induction of mannose by pSUN384.1 compared to pSUN 291. However, in bacillus subtilis 3NA containing psunl385.2 with deletion 2, the induction by mannose was completely eliminated and the basal expression level was almost 0. From this result, it was found that the ManR binding site of the manR promoter region is located between bp-99 and bp-35 relative to the transcription start site of manR.

[ II ] use of the promoter region of the mannose operon in high cell density fermentation

[ experiment 4: transformation (C)

The growth and expression capacity of model hosts carrying the promoter regions of the invention were tested. Plasmid pMW168.1 was constructed as shown in FIG. 6 using a nucleic acid sequence based on the manP promoter region as introduced into plasmid pSUN284.1, and as used in experiment 2c, and introduced into B.subtilis 3NA as a host by transformation.

[ A ] construction of plasmid pMW168.1 ]

Shuttle vectors replicable in escherichia coli (e.coli) and bacillus subtilis (b.subtilis) were designed as shown in experiment (2a), except that eGFP was used as a reporter gene instead of lacZ. The transcriptional initiation region of manP was also replaced with the transcriptional initiation region of the gene gsiB (stress protein; Jurgen et al, supra).

Furthermore, the start codon and 6 codons after the start codon of eGFP were substituted.

The schematic structure of the promoter-and transcription initiation regions obtained is as follows:

the arrangement of genes (arrows) and regions (cassettes) with associated restriction sites is shown.

In general, plasmid pmw168.1 was obtained as shown in the following scheme:

in the scheme, the names of vector-DNA, insert-DNA and complementary oligonucleotide used are shown in the cassette, and the restriction enzymes used are indicated at the corresponding sites with respect to PCR products, primers and template-DNA as in brackets.

The cloning procedure was performed with escherichia coli (e.coli) JM 109.

The plasmids used were: pUC18 positive selection and cloning vectors for PCR products with amp-resistance (Yanoch-Perron et al, supra); an amp-resistant vector for expression and cloning of pWA21 from escherichia coli (e.coli) (weger et al, 2008, BMC biotechnol.8, 2); pSUN202.4pUB 110 derivatives having manP promoter region and amp and kan resistance, shuttle vectors for Escherichia coli (E.coli) and Bacillus subtilis (B.subtilis); and pSUN266.1pUC18 derivatives with integration sites between the ter-sequences and spc and amp resistance.

The primer sequences used were as follows:

use of complementary oligonucleotides and a single restriction site Bg/II, AflII and BamHI were substituted for the transcription initiation region including the initiation codon and the codon following the initiation codon. Construction of the vector was initiated by replacement of the transcriptional initiation region of the T7 gene 10 of vector pWA21 with the translational initiation region of tufA from B.subtilis via complementary oligonucleotides s5019 and s5020, respectively (Weger et al, supra). In a further cloning step, this transcriptional initiation region is replaced by the gsiB transcriptional initiation region. The final plasmid pMW168.1 contains an ori included from pUB110⁺The rep gene of (1).

The plasmid map of pMW168.1 is shown in FIG. 9.

[ (b) determination of structural stability and separation ]

Bacillus subtilis 3NA was transformed with the vector pmw168.1 and the structural stability and stable propagation of the vector on cell division (split) was determined.

Bacillus subtilis 3NA transformed with pMW168.1 was used in LB_SpcPreculture in medium and subsequent transfer to LB without selective pressure₀-a culture medium.

Incubation was performed at 37 ℃. At the end of the exponential growth phase, each culture was inoculated into fresh LB₀-a culture medium. This procedure was repeated until 100 passages were obtained, during transfer to fresh medium, according to Harwood et al, 1990, Molecular biological Methods for Bacillus, John Wiley&Sons ltd. based on the obtained measured OD-values.

The results are shown in FIG. 10.

After about 15 passages, more than 99, 9% of the cells, and even after 20 passages, about 90% of the cells still carried the vector. From only about passage 25, more and more cells lose the vector.

To determine the structural stability of the plasmids, the plasmids were isolated from 20 colonies after 15 generations. Approximately 0.5. mu.g of each isolated plasmid was compared with pMW168.1 isolated from Escherichia coli (E.coli) as a control by agarose gel electrophoresis. No difference in the runs of the plasmid and control was observed, indicating no structural variation.

These results show that plasmid pmw168.1 not only has high structural stability, but also has stable segregation as expected in fermentation.

[ experiment 5: fermentation (c)

Bacillus subtilis 3NA transformed with plasmid pmw168.1 containing the reporter gene eGFP was used for 6 fermentation runs with different induction protocols and monitored on-line by observing the fluorescent signal of eGFP.

As the fermentation medium, a known medium for high cell density fermentation of escherichia coli (e.coli) disclosed in Wilms et al, 2001, biotechnol.bioeng.73, 95-103 and shown below was used.

Materials and methods

In general, also for fermentation experiments, standard molecular techniques are used, if not stated otherwise.

[ optical Density ]

For the determination of the Optical Density (OD), a spectrophotometer Ultrospec 1100pro from Amersham biosciences was used at 600nm according to the manufacturer's protocol.

[ determination of Dry Biomass concentration ]

To determine the dry biomass concentration c_xA hygrometer MB 835Halogen from Ohaus was used.

[ spectrophotometric measurement of fluorescence ]

The expression and fluorescence of eGFP were analyzed by means of a multifunctional reader GENios from TECAN using the reader software XFluor 4 (version V4.11) with the following measurement parameters, respectively:

measuring parameters	Value taking
		Photosensitive filter	485nm
Emission filter	535nm
		Acquisition (Manual)	60
Integration time	20μs
		Number of flashes	3
Read mode	Top part

[ Online fluorescence measurement in fermentation tank ]

During fermentation, eGFP expression was monitored on-line using a fluorescent probe (MicropackHPX-2000, high power xenon light source from Ocean Optics, Inc.; S2000 fiber optic spectrometer).

The measurement parameters were as follows: 485nm of photosensitive filter, 535nm of emission filter and 0.6 of filter. For recording and storage, Ocean Optics SpectraSuite software was used.

As a Relative Fluorescence Unit (RFU) indicates fluorescence. Shortly before 4.000 RFUs were obtained, the integration time of 50ms was changed to 25ms and then to 10 ms. In these cases, the measured values are multiplied by 2 and 5, respectively.

[ culture of preculture ]

Single colonies were placed on LB agar plates and cultured overnight in 5ml Spizizens Minimal Medium (SMM) containing 0.02% (w/v) Casamino Acid (CA) and antibiotics. 1ml of the overnight culture was added to 20ml SMM containing 0.02% (w/v) CA and antibiotics and incubated at 37 ℃ for 5-6 h in 250ml Erlenmeyer flasks (preculture 1).

10ml of preculture 1 were added to 200ml of batch medium comprising 5g/l glucose and incubated at 37 ℃ for 8h in a 1l Erlenmeyer flask (preculture 2). For the inoculation of the fermenter, use was made of a seed having an OD of between 1.2 and 2.2₆₀₀Preculture 2 of (1).

【】

In general, the fermentation is carried out according to the principle of Wilms et al, 2001, Biotechnol.Bioeng.73, 95-103.

Once glucose, carbon source, was completely consumed, batch mode was switched to fed-batch mode.

By adding the make-up solution exponentially in the fed-batch phase, 0.10h can be obtained^-1And at the same time avoid being repressed by glucose catabolites, since glucose is consumed immediately by the cell.

[ protein analysis ]

The crude protein extract of the harvested cells was analyzed by SDS-polyacrylamide gel electrophoresis with a polyacrylamide gel consisting of 3% concentrated gel and 12% separation gel having the following composition:

components	Concentrated gum (3%)	Separating glue (12%)
			Deionized H2O	3.00ml	6.70ml
TRIS 0.5M pH6.8	1.25ml	-
			TRIS 1.5M pH8.8	-	5.00ml
SDS 10％(w/v)	0.05ml	0.20ml
			Acrylamide 30% (w/v)	0.67ml	8.00ml
APS 10％(w/v)	0.05ml	0.10ml
			TEMED	0.005ml	0.01ml

A Twin Mini gel chamber from Biometra was used.

For denaturation, 12. mu.l of the crude extract of the protein mixture were mixed with 3. mu.l of 5 XSDS-applied buffer and incubated for 5 minutes at 95 ℃ in Thermomixer 5438 from Eppendorf. After cooling to room temperature, the sample was separated by centrifugation and placed completely on the gel.

During separation in the concentrate, the current was 10mA and increased to 20mA after the bromophenol front reached the concentrate. Using 1 XSDS-electrophoresis buffer and Roth's length standardFor separation. Once the bromophenol front completely exited the gel, the electrophoresis was terminated. To detect different protein bands, the gel was incubated with Coomassie staining solution for 30 minutes at room temperature, and then treated with de-staining solution for 30 minutes at room temperature. To remove the remaining blue background from the gel, the gel was incubated in 7.5% acetic acid for several hours.

The composition of the buffer solution and the dyeing and de-dyeing solutions was as follows:

buffer/solution	Components	Concentration of
			Coomassie	Coomassie R250	2.0g
Dyeing solution	Coomassie G250	0.5g
				EtOH	425ml
	MeOH	50ml
				Glacial acetic acid	100ml
	Deionized H2O	ad 1.0l
De-staining solution	EtOH	450ml
				Glacial acetic acid	100ml
	Deionized H2O	450ml
5 × application of buffer	TRIS/HCl(2M，pH6.8)	6.25ml
				EDTA	0.146g
	SDS(40％(w/v))	6.25ml
				Beta-mercaptoethanol (pure)	2.50ml
	Glycerol (86% (v/v))	29.00ml
				Bromophenol blue	0.05g
	Deionized H2O	ad 50ml
10 Xelectrophoresis buffer	TRIS	30g
				Glycine	144g
	SDS(20％(w/v))	50ml
				Deionized H2O	ad 1.0l

Note:

TRIS: tris (hydroxymethyl) aminomethane

SDS (sodium dodecyl sulfate): sodium dodecyl sulfate

APS: ammonium persulfate

TEMED: n, N, N ', N' -tetramethylethylenediamine,

EDTA: ethylene diamine tetraacetic acid.

[ Induction of Gene expression ]

For induction of gene expression, different patterns of addition of inducer solution were evaluated (induction protocol):

1. addition in single portions at given time points (impact induction)

2. At intervals, impact induction is combined with further induction, wherein

■ is further added in stepwise increasing amounts at a constant rate, or

■ is further added at an exponentially increasing rate,

3. addition of the inducer solution was initiated after a given cell density was reached.

Table 2: culture medium used

Culture medium	Components	Concentration of
LB0Culture medium (pH7.2)	Tryptone	10.0g
				Yeast extract	5.0g
	NaCl	5.0g
				H2O, de-ionized	ad 1.0l
Spizizens Minimalmedium(SMM)	(NH4)2SO4	2.0g
				KH2PO4	6.0g
	K2HPO4	14.0g
				Na3Citric acid salt	1.0g
	MgSO4	0.2g
				D-glucose＊	5.0g
	H2O, de-ionized	ad 1.0l
			Batch-culture medium for fermentation of Bacillus subtilis	(NH4)2H-citrate salt	1.00g/l
	Na2SO4	2.68g/l
				(NH4)2SO4	0.50g/l
	NH4Cl	0.50g/l
				K2HPO4	14.60g/l
	NaH2PO4xH2O	4.00g/l
				D-glucose＊	25.00g/l
	MgSO4(1m)＊	2.00ml/l
				TES＊(As follows)	3.00ml/l

^＊To be autoclaved separately

Microelement solution (TES)	CaCl2×2H2O	0.50g/l
				FeCl3×6H2O	16.70g/l
	Na2-EDTA	20.10g/l
				ZnSO4×7H2O	0.18g/l
	MnSO4×H2O	0.10g/l
				CuSO4×5H2O	0.16g/l
	CoCl2×6H2O	0.18g/l

The pH was adjusted with 2M NaOH and 1M HCl solutions, respectively. For the agar plates, 15g/l Euroagar from BD company was additionally added.

All cultures were autoclaved at 121 ℃ for about 30 min.

[ fermentation run 1: induction of impact

Fermentation run 1 was carried out in a 30l reactor (Bioengineering D598 and D596). The batch volume was 8 l. Dependent on OD₆₀₀Inoculating 200-400 ml of preculture 2 to initiate OD₆₀₀Adjusted to 0.1.

During the batch phase, the temperature was 30 ℃ overnight and after 12h, increased to 37 ℃. By adding 24% (v/v) NH during the whole fermentation₄OH, adjusting the pH to about 7.0. The aeration speed can be adjusted to 30 l/min. At the start of the batch phase, the aeration rate was 10 l/min.

The compositions of feed media I and II are shown in Table 3 below.

Table 3: composition of feed media I and II

^＊Microelement solution pH7.0

Media I and II were added in proportion to their total volume, i.e., 4.2: 1.0 (corresponding to 80.8% media I and 19.2% media II for total make-up F).

To start the induction in the fed-batch phase, one portion of 0.2% (w/v) D-mannose solution was added.

The dry biomass concentration and the monitored fluorescence signal are shown in fig. 11a and 11b, respectively.

In the figure, dry biomass c is_xThe concentration of (d) is plotted logarithmically for the duration of the incubation. The batch and fed-batch phases are separated by a vertical line.

The monitored fluorescent signal at emission wavelength 535nm is plotted against the incubation period. Arrows indicate points of induction.

From FIG. 11a, a maximum dry biomass (DM) concentration of 82.75g DM/l is obtained, which corresponds to about 970g DM based on a reaction volume of 11.7 l.

In the first addition, a total of 71.5g of inducer D-mannose were consumed with 16 g.

During the whole fed-batch phase, the specific growth rate μ was 0.10h^-1。

As shown in FIG. 11b, after the first addition of D-mannose, the fluorescence signal strongly increased to a maximum of about 2,200RFU within the first 5 hours of the fed-batch phase. Then, the signal continuously decreases. This decrease in expression rate is presumed to be due to consumption of the inducer and/or sequestration effects by increased cell mass. Addition of a further 0.5% (w/v) mannose solution after 37 hours resulted in a new increase in the value of the fluorescence signal up to 2, 100 RFU.

(fermentation run 2: combined induction with constant velocity ]

The same procedure as in run 1 was repeated except for the pattern change of the addition of the inducer. As in run 1, 0.2% (w/v) D-mannose solution was added in a single portion at the start of the fed-batch phase. Once RFU reaches the turning point of the curve for the fluorescence signal of run 1, 1,500, addition of the inducer of fraction 2 is initiated.

During the 2 nd addition step, 20% (w/v) mannose solution was added in stepwise increments at a constant rate at an average rate of 0.39g/min until the entire 2 nd serving had been added.

The results are shown in fig. 12a and 12b, showing dry biomass concentration and fluorescence signal curves, with the same designations as in fig. 11a and 11 b. In fig. 12b, the addition point of the 1 st portion and the start and end of the addition of the 2 nd portion are indicated by arrows.

From the results of fig. 12a, the maximum concentration of dry biomass was 67.6g DM/l, corresponding to about 804g DM based on 11.9l reaction volume. In total (1 st and 2 nd additions) 70g D-mannose was added.

The biomass yield was reduced by 17% compared to run 1. This and the low specific growth rate during the fed-batch phase, mu, 0.09h^-1In connection, however, the specific growth rate during the batch-phase was 0.43h^-1。

From the results of FIG. 12b, the fluorescence signal reached a maximum of about 4,900RFU and continued to decrease to about 2,500RFU after 25 hours.

In run 2, the expression rate can increase by 120% with a slight decrease in biomass concentration compared to run 1.

[ fermentation runs 3 and 4: combined induction with exponential velocity ]

In runs 3 and 4, a 3.7l small laboratory fermenter (Kleinlab fermentor from Bioengineering) was used. The batch volume (batch medium plus inoculum) was 1.5l in total. Dependent on OD₆₀₀Inoculating 100-200 ml of preculture 2 to initiate OD₆₀₀Adjusted to about 0.1. The temperature was 37 ℃ in the batch and fed-batch phases. During the fermentation, 24% (v/v) NH was used₄OH adjusted the pH to 7.0. The aeration rate was constant at 2l/min during the fermentation. The oxygen input is adjusted by the rotational speed of the stirrer. The fermentation pressure was initially 1.3 bar and then increased to 1.5 bar to enhance oxygen input as required. After complete consumption of the carbon source glucose, the batch operation was switched to a fed-batch operation.

Unlike run 2, in runs 3 and 4, the inducer solution was replenished at an exponentially increasing rate. Furthermore, medium I containing glucose was co-fed with medium II containing the inducer. The composition of the feed media I and II is shown in table 4 below:

table 4: composition of feed media I and II

All components of media I and II were autoclaved independently.

In both media, the pH was adjusted to 85% (v/v) H for solubility of the components₃PO₄Adjust to 3.3.

The total feed F at time t was calculated by the following formula:

wherein m is a maintenance factor (0.04g g)^-1h^-1)

Y_x/sCoefficient of specific yield of biomass relative to substrate (0.5 for glucose)

C_x0Initial biomass concentration in fed-batch phase

V₀Reactor volume starting in the fed-batch phase (═ batch volume)

C_s0Glucose concentration in the replenisher solution

To calculate, the D-mannose consumption of Bacillus subtilis (B.subtilis) was estimated with a yield coefficient Y_X/sThe glucose is equivalent.

In KLF, media I and II can be supplied independently, and the ratio of the ratios can be varied.

[ A ] fermentation run 3

The biomass concentration and the monitored fluorescence signal are shown in figures 13a and 13b, where the symbols are the same as in run 1.

At the start of the fed-batch phase, one portion of a 0.2% (w/v) mannose solution (16 g mannose in total) was added, and media I and II were fed exponentially starting at a ratio of 50: 50 (interval I). As the slope decreased, medium II containing the serving mannose increased to 60%, and total feed F (media I and II) to 125% for maintaining glucose-based growth (interval II). After about 2h, the slope decrease was again monitored and the portion of medium II increased to 66.6% while the total feed F increased to 150% (interval III). After the total consumption of medium II, fermentation was continued with 100% of total make-up medium I (not shown in fig. 10).

The procedure and data for run 3 are summarized in table 5 below:

table 5:

interval [ h ]]	Culture medium I: II [% ]]	F[％]	μ[h-1-]	RFU	Dry Biomass [ gDM/l]
						0-12			0.52	-
I 12-17	50∶50	100	0.09	7000
						II 17-19	50∶75	125	0.08	9000
III 19-22	50∶100	150	0.09	11000	22

A total of 50g mannose was added.

At each of 12h, 20h and 24h, samples were taken for analysis based on a total of 10OD₆₀₀Expression on SDS-gel of soluble protein fraction of cells.

The resulting SDS PAGE is shown below:

display from the left: (M) Length standards (M)) (1) after 12h, no induction; (2) after 20h, 8 h-induction; (3) after 24 hours, 12h induction.

After 20h and 24h, clear lanes appear at about 27kDa indicating expression of eGFP (arrows).

[ B ] fermentation run 4 ]

The same procedure as in run 3 was repeated except that the feed volume of medium II (mannose) was increased to 1.0l in total.

Dry biomass concentration and fluorescence signal are shown in fig. 14a and 12b, with the same designations as in run 1.

The procedure and data are summarized in table 6 below:

table 6:

interval [ h ]]	Culture medium I: II [% ]]	F[％]	μ[h-1-]	RFU	Dry Biomass [ gDM/l]
						0-15			0.40
I 15-22	50∶50	100		3500
						II 22-38	50∶75	120		10000-7800
III 38-39	50∶100	150		8900	40.4

A total of 200g mannose was added.

To compensate for the observed nitrogen deficiency, an additional feed (NH) was made at a constant rate after about 15 hours duration of fermentation₄)₂HPO₄。

In interval II, the maximum RFU of 10000 was reached, decreasing to 7800RFU during interval II.

[ a) fermentation runs 5 and 6: non-impact induction

Both fermentations were carried out in a 30l fermenter.

In both runs, cells were grown up to high cell density, and after reaching high cell density, exponentially increasing glucose replenishment rates were replaced with constant replenishment of mannose.

The feed media I, II and III used are shown in table 7 below:

table 7:

[ fermentation run 5 ]

Media I and II were added proportionally to their total volume at an exponentially increasing rate of 4.2: 1.0. After reaching a high cell density, the feed consisting of media I and II was replaced with a constant volume of feed consisting of media II and III in a ratio of 20: 80 corresponding to the volume of the last exponential feed rate of media I and II.

The fluorescence signal is shown in FIG. 15, with the same designations as in run 1.

It is assumed that the minimal increase in fluorescence signal in fig. 15 after about 17h fermentation duration is due to short term leakage of medium III.

The procedure and data for run 5 are summarized in table 8 below:

table 8:

[ B ] fermentation run 6

The same procedure as in run 5 was repeated, except that after reaching a high cell density, a total of 600g of mannose was added with a 0.2(w/v) mannose solution shock induced (16 g mannose total) prior to constant addition of the supplement consisting of media II and III.

The fluorescence signal is shown in FIG. 16, with the same designations as in run 1.

The procedure and data for run 6 are summarized in table 9 below:

table 9:

[ evaluation of runs 1-6 ]

For evaluation at maximum fluorescence time, the dry biomass concentration c is determined_xVolume of reactor V_RThe duration of consumed mannose and of the initiation of self-induction and are summarized in table 10 below:

table 10:

based on the processing data for each run shown in table 10, the yield and hours at maximum fluorescence RFU/l were calculated. Moreover, expression efficiency is expressed as relative fluorescence calculated from the maximum fluorescence based on absolute biomass (gDM) and sensor concentration (gman/L).

The results are shown in table 11 below:

table 11:

these results clearly show that the present invention using a plasmid carrying the mannose promoter can be successfully used in high cell density fermentation processes and under-controlled expression by the addition of the inducer D-mannose.

Furthermore, by selecting an induction protocol, which takes biomass, expression products and inducer consumption into account, respectively, the fermenter can maximize the output change as required.

The combined shock induction and exponential replenishment protocol according to run 3 is particularly advantageous in view of the secondary downstream process inducers.

In this context, the high expression products produced with respect to biomass make further processing, such as purification steps, more efficient and, thus, time and cost saving.

Claims (22)

1. A method of culturing a prokaryotic host cell, wherein the prokaryotic host cell is transformed with an expression vector comprising an inducible promoter of the mannose operon of Bacillus subtilis operably linked to a transcription unit comprising a nucleic acid sequence encoding a polypeptide,

wherein the inducer of the inducible promoter is mannose or a derivative thereof capable of inducing the promoter,

wherein the content of the first and second substances,

in step 1, the prokaryotic host cell is cultured in the presence of a1 st carbon source different from the inducer, and

in step 2, the prokaryotic host cell is cultured in the presence of the inducer.

2. The method of claim 1, wherein the addition of the inducer is initiated after the culture reaches the assay parameter.

3. The method of claim 1 or 2, wherein the measured parameter is at least one selected from the group consisting of Optical Density (OD) and carbon source concentration.

4. The method of claim 3, wherein the measured parameter is optical density at 600 nm.

5. The process of any of the preceding claims, wherein the process is a fed-batch process.

6. The method of any one of the preceding claims, wherein all or part of the inducer is added continuously over time.

7. A process according to any one of claims 1 to 6 wherein at least part of the inducer is added in one portion.

8. The method of any one of the preceding claims, wherein at least a portion of the inducer is added at a rate that increases exponentially over time.

9. The method of any one of the preceding claims, wherein after the first addition of the inducer, at least one more addition of the inducer occurs after the culture reaches the 2 nd assay parameter.

10. The method of claim 9, wherein the 2 nd assay parameter is selected from the group consisting of optical density, OD, concentration of inducer in solution, concentration of polypeptide produced or signal intensity of reporter gene contained in the host cell.

11. The method according to any one of claims 1 to 10, wherein the promoter is selected from the group consisting of manP promoter and manR promoter

12. The method of any one of claims 1 to 11, wherein the fermentation medium is supplemented with a mixture of a1 st carbon source and an inducer, wherein the ratio of the 1 st carbon source to the inducer is from 3: 1 to 1: 3.

13. The method according to any of the preceding claims, wherein the vector comprises the complete or partial sequence regulating the gene manR.

14. The method of any one of the preceding claims, wherein the vector comprises a nucleic acid sequence selected from the group consisting of SEQ id nos: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, a sequence complementary thereto or a variant thereof.

15. The method of any one of the preceding claims, wherein the carbon source other than the inducer is glucose.

16. The method of any one of the preceding claims, wherein the nucleic acid sequence encoding the polypeptide is a heterologous nucleic acid sequence.

17. A method for producing a polypeptide in a prokaryotic host cell, comprising the steps of:

constructing a vector expressible in a prokaryotic host cell comprising an inducible promoter of the mannose operon of Bacillus subtilis operably linked to a transcriptional unit comprising a nucleic acid sequence encoding a polypeptide,

transforming a prokaryotic host cell with said vector, and

expressing the polypeptide by culturing the transformed host cell under the culture conditions of any one of claims 1-16.

18. The method of claim 15, further comprising the steps of: recovering the polypeptide from the cell or from the cell culture.

19. A recombinant prokaryotic host cell comprising an expression vector comprising:

a mannose-inducible promoter of the mannose operon of Bacillus subtilis, or

A purified and isolated nucleic acid sequence of the inducible promoter of the mannose operon,

wherein the purified and isolated nucleic acid sequence of the vector or the inducible promoter of the mannose operon is operably linked to a transcription unit comprising a nucleic acid sequence encoding a polypeptide.

20. The recombinant prokaryotic host cell of claim 19,

wherein the inducible promoter of the mannose operon is one according to any one of claims 11 to 13, or

Wherein the isolated and purified nucleic acid is one according to claim 14.

21. A recombinant prokaryotic host cell, wherein the host cell is selected from the group consisting of Firmicutes.

22. Use of the recombinant prokaryotic host cell of any one of claims 19-21 in the method of any one of claims 1-18.