MICROORGANISM STRAIN AND METHOD FOR ANTIBIOTIC-FREE, FERMENTATIVE PREPARATION OF LOW MOLECULAR WEIGHT SUBSTANCES AND PROTEINS

Abstract

The invention relates to a microorganism strain and a method for antibiotic-free fermentative preparation of low molecular weight substances and proteins. The microorganism strain for producing low molecular weight substances or proteins contains in its genome a mutation in a gene which brings about an auxotrophy in the strain. It further contains a production plasmid coding an enzyme for production of a low molecular weight substance or a recombinant protein and a functional copy of the gene whose chromosomal inactivation brings about the auxotrophy, wherein the auxotrophy is a non-feedable auxotrophy.

Claims

1. A microorganism strain for producing low-molecular-weight substances or proteins, said microorganism strain comprising: (a) a genome containing a mutation in a gene, which mutation brings about an auxotrophy in the microorganism strain, and (b) a production plasmid encoding at least one enzyme for producing: (i) a low-molecular-weight substance or at least one recombinant protein and (ii) a functional copy of the gene, the chromosomal inactivation of which brings about the auxotrophy, wherein the auxotrophy is a nonfeedable auxotrophy.

2-3. (canceled)

4. The microorganism strain as claimed in claim 1, wherein the mutation of the gene brings about the nonfeedable auxotrophy in the strain, and leads to inactivation of said gene.

5. The microorganism strain as claimed in claim 1, wherein the mutation of the gene brings about the nonfeedable auxotrophy in the strain, and leads to inactivation of an activity of a gene product coded by the gene.

6. A method for producing a strain as claimed in claim 1, wherein a temperature-sensitive plasmid is introduced into a microorganism strain which has a gene which can be converted by mutation or deletion into a modified gene which brings about a nonfeedable auxotrophy in the strain, which temperature-sensitive plasmid has a functional copy of the gene which is to be mutated or deleted, then the genome of the strain is mutated such that the strain has in said modified gene a mutation which leads to the nonfeedable auxotrophy in the strain and such that a functional copy of said modified gene is present on the temperature-sensitive plasmid, and then the temperature-sensitive plasmid is exchanged in said strain at a nonpermissive temperature for a production plasmid, wherein the production plasmid contains a gene encoding an enzyme for the production of the low-molecular-weight substance or the recombinant protein and also a functional copy of the gene which brings about the auxotrophy.

7. The method as claimed in claim 6, wherein the temperature-sensitive plasmid has a temperature-sensitive origin of replication.

8. The method as claimed in claim 7, wherein cells are exposed, immediately after the transformation with the production plasmid, to a temperature shock at 47-55 C. for 30-90 min and the further incubation is then carried out at the nonpermissive temperature of 37-45 C.

9. A method for producing low-molecular-weight substances or proteins comprising producing the low-molecular weight substances or proteins with a microorganism strain as claimed in claim 1 and an antibiotic-free fermentation medium.

10. The microorganism strain as claimed in claim 4, wherein the mutation of the gene brings about the nonfeedable auxotrophy in the strain, and leads to inactivation of an activity of a gene product coded by the gene.

11. A method for producing low-molecular-weight substances or proteins comprising producing the low-molecular-weight substances or proteins with a microorganism strain as claimed in claim 10 and an antibiotic-free fermentation medium.

Description

[0086] FIG. 1 shows a restriction and function map of plasmid pKD46 from Example 2.

[0087] FIG. 2 shows a restriction and function map of the plasmid pAF-ts-pyrH produced in Example 2.

[0088] FIG. 3 shows a restriction and function map the the plasmid pAF-ts-plsC produced in Example 2.

[0089] FIG. 4 shows a restriction and function map the the plasmid pMT1 used in Example 5.

[0090] FIG. 5 shows a restriction and function map of the expression plasmid pcysEX-GAPDH-ORF306_tetR produced in Example 5.

[0091] FIG. 6 shows a restriction and function map of the expression plasmid pCGT_tetR produced in Example 6.

[0092] FIG. 7 shows a restriction and function map of the expression plasmid pFab-anti-Lysozyme_tetR produced in Example 7.

[0093] FIG. 8 shows a restriction and function map of the expression plasmid pcysEX-GAPDH-ORF306 pyrH1_tetR produced in Example 8.

[0094] FIG. 9 shows a restriction and function map of the expression plasmid pCGT_pyrH1 tetR produced in Example 8.

[0095] FIG. 10 shows a restriction and function map of the expression plasmid pFab-anti-Lysozyme_pyrH1 tetR produced in Example 8.

[0096] FIG. 11 shows a restriction and function map of the expression plasmid pcysEX-GAPDH-ORF306_plsC1_tetR produced in Example 8.

[0097] FIG. 12 shows a restriction and function map of the expression plasmid pCGT_plsC1 tetR produced in Example 8.

[0098] FIG. 13 shows a restriction and function map of the expression plasmid pFab-anti-Lysozyme_plsC1 tetR produced in Example 8.

[0099] FIG. 14 shows a restriction and function map of the production plasmid pcysEX-GAPDH-ORF306_pyrH according to the invention produced in Example 9.

[0100] FIG. 15 shows a restriction and function map of the production plasmid pCGT_pyrH according to the invention produced in Example 9.

[0101] FIG. 16 shows a restriction and function map of the production plasmid pFab-anti-Lysozyme_pyrH according to the invention produced in Example 9.

[0102] FIG. 17 shows a restriction and function map of the production plasmid pcysEX-GAPDH-ORF306_plsC according to the invention produced in Example 9.

[0103] FIG. 18 shows a restriction and function map of the production plasmid pCGT_plsC according to the invention produced in Example 9.

[0104] FIG. 19 shows a restriction and function map of the production plasmid pFab-anti-Lysozyme_plsC according to the invention produced in Example 9.

[0105] The following examples serve to further elucidate the invention. All the molecular biology and microbiology methods used, such as polymerase chain reaction (PCR), gene synthesis, isolation and purification of DNA, modification of DNA by restriction enzymes, Klenow fragment and ligase, transformation, P1 transduction, etc., were carried out in the way which is known to a person skilled in the art, described in the literature or recommended by the respective manufacturers.

Example 1: Amplification of the Marker Gene (pyrH or plsC) Gene with its Own Promoter

[0106] A DNA fragment of about 1.0 kb in size, coding for the pyrH gene including native promoter region, was amplified using the primers pyrH-NcoI-fw (SEQ ID No. 5) and pyrH-NcoI-rev (SEQ ID No. 6). The template used for the PCR reaction was chromosomal DNA from the E. coli strain W3110 (ATCC 27325).

[0107] The PCR fragment of about 1.0 kb in size was purified via an agarose gel electrophoresis and isolated from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) according to the information from the manufacturer. Thereafter, the purified PCR fragment was digested using the restriction enzyme NcoI and stored at 20 C. In an analogous manner, a DNA fragment of about 1.0 kb in size, coding for the plsC gene including native promoter region, was amplified, purified, digested and stored. For the amplification, the primers plsC-NcoI-fw (SEQ ID No. 7) and plsC-NcoI-rev (SEQ ID No. 8) were used.

Example 2: Generation of the Plasmids pAF-Ts-pyrH and pAF-Ts-plsC with Temperature-Sensitive Origin of Replication

[0108] The starting plasmid used for the construction of the plasmids pAF-ts-pyrH and pAF-ts-plsC with temperature-sensitive origin of replication was the plasmid pKD46 (Datsenko and Wanner, 2000, P.N.A.S. 97: 6640-6645). A restriction and function map of plasmid pKD46 is shown in FIG. 1. The PCR products described in Example 1 and digested using NcoI, which PCR products code for the pyrH gene or plsC gene with its own promoter, were cloned into the NcoI restriction site of pKD46. The ligation preparations was transformed into DH5-T1R E. coli cells (Life Technologies GmbH), multiplied in said cells, and the DNA sequence of the isolated plasmids was verified by means of sequencing. Two of the altogether 4 possible constructs which were generated in this manner have the designations pAF-ts-pyrH and pAF-ts-plsC (see FIGS. 2 and 3).

Example 3: Transformation of Selected E. coli Strains Using pAF-Ts-pyrH or pAF-Ts-plsC

[0109] The plasmids pAF-ts-pyrH and pAF-ts-plsC with temperature-sensitive origin of replication that are described in Example 2 were transformed into the two E. coli strains W3110 (ATCC 27325) and W31101pp3 (described in US2008076158 A1 as leaky strain) using the CaCl.sub.2 method known to a person skilled in the art. The transformed cells were selected on LB agar plates containing 100 mg/l ampicillin. The strains generated in this manner have the designations W3110/pAF-ts-pyrH, W31101pp3/pAF-ts-pyrH, W3110/pAF-ts-plsC and W31101pp3/pAF-is-plsC.

Example 4: Deletion of the Gene pyrH (Inactivation of Uridylate Kinase) or of the Gene plSC (Inactivation of 1-Acylglycerol-3-Phosphate O-Acyltransferase) in E. coli

[0110] A) Deletion of the Gene pyrH

[0111] The gene pyrH, which codes for the enzyme uridylate kinase (PyrH) in E. coli, was deleted in the E. coli strains W3110/pAF-ts-pyrH and W31101pp3/pAF-ts-pyrH according to the red method developed by Datsenko and Wanner (Datsenko and Wanner, 2000, P.N.A.S. 97: 6640-6645). A DNA fragment coding for the kanamycin resistance marker gene (kanR) was amplified using the primers pyrH-fw (SEQ ID No. 9) and pyrH-rev (SEQ ID No. 10). The primer pyrH-fw codes for a sequence consisting of 30 nucleotides that is homologous to the 5-end of the pyrH gene and a sequence comprising 20 nucleotides that is complementary to a DNA sequence encoding one of the two FRT sites (FLP recognition target) on the plasmid pKD13 (Coli Genetic Stock Center (CGSC) No. 7633). The primer pyrH-rev codes for a sequence consisting of 30 nucleotides that is homologous to the 3-end of the pyrH gene and a sequence comprising 20 nucleotides that is complementary to a DNA sequence encoding the second FRT site on the plasmid pKD13.

[0112] The amplified PCR product was introduced by means of electroporation into the E. coli strains W3110/pAFts-pyrH and W31101pp3/pAF-ts-pyrH (see Example 3). The selection for cells with chromosomal integration of the kanamycin resistance marker gene (kanR) was done on LB agar plates containing 50 mg/l kanamycin and 100 mg/l ampicillin. The removal of the chromosomally introduced kanamycin resistance marker gene (kanR) was achieved using the enzyme FLP recombinase, which is encoded on the plasmid pCP20 (CGSC No. 7629). The selection for pCP20-contained cells was done on LB agar plates containing 100 mg/l ampicillin (selection for pAF-ts-pyrH) and 34 mg/l chloramphenicol (selection for pCP20). Owing to a temperature-sensitive origin of replication (ori), the plasmid pCP20 can, after transformation has been carried out, be removed by cultivating the E. coli cells at a nonpermissive, i.e., elevated temperature, for example at 42 C.

[0113] A first selection for loss of the temperature-sensitive plasmid pCP20 with simultaneous maintenance of the temperature-sensitive plasmid pAF-ts-pyrH was done on LB agar plates containing 100 mg/l ampicillin (selection for pAF-ts-pyrH). Later on, the preselected ampicillin-resistant bacteria clones were checked for kanamycin sensitivity, i.e., the loss of the chromosomally introduced kanamycin marker gene, and for chloramphenicol sensitivity, i.e., the loss of the temperature-sensitive plasmid pCP20.

[0114] Only kanamycin- and chloramphenicol-sensitive but ampicillin-resistant clones were finally checked for the chromosomal deletion of the pyrH gene using the primers pyrH-check-for (SEQ ID No. 11) and pyrH-check-rev (SEQ ID No. 12). The template used for the checking of the chromosomal pyrH deletion by means of PCR was chromosomal DNA from the selected ampicillin-resistant, chloramphenicol- and kanamycin-sensitive clones. The thus generated and checked ampicillin-resistant E. coli strains with chromosomal pyrH deletion and plasmid-encoded pyrH expression have the designations W3110pyrH/pAF-ts-pyrH and W31101pp3pyrH/pAF-ts-pyrH.

B) Deletion of the plsC Gene

[0115] Analogously to the pyrH gene, the gene plsC, which codes for the enzyme 1-acylglycerol-3-phosphate O-acyltransferase (PlsC) in E. coli, was deleted in the E. coli strains W3110/pAF-ts-plsC and W31101pp3/pAF-is-plsC (Datsenko and Wanner, 2000, P.N.A.S. 97: 6640-6645). A DNA fragment coding for the kanamycin resistance marker gene (kanR) was amplified using the primers plsC-fw (SEQ ID No. 13) and plsC-rev (SEQ ID No. 14). The primer plsC-fw codes for a sequence consisting of 30 nucleotides that is homologous to the 5-end of the plsC gene and a sequence comprising 20 nucleotides that is complementary to a DNA sequence encoding one of the two FRT sites (FLP recognition target) on the plasmid pKD13 (Coli Genetic Stock Center (CGSC) No. 7633). The primer plsC-rev codes for a sequence consisting of 30 nucleotides that is homologous to the 3-end of the plsC gene and a sequence comprising 20 nucleotides that is complementary to a DNA sequence encoding the second FRT site on the plasmid pKD13.

[0116] The amplified PCR product was introduced by means of electroporation into the E. coli strains W3110/pAFts-plsC and W31101pp3/pAF-is-plsC (see Example 3). The removal of the chromosomally introduced kanamycin resistance marker gene (kanR) was again done using the enzyme FLP recombinase (encoded on plasmid pCP20). The selection for pCP20-contained cells was also done here on LB agar plates containing 100 mg/l ampicillin (selection for pAF-ts-pyrH) and 34 mg/l chloramphenicol (selection for pCP20). A first selection for loss of the temperature-sensitive plasmid pCP20 with simultaneous maintenance of the temperature-sensitive plasmid pAF-ts-plsC was done on LB agar plates containing 100 mg/l ampicillin (selection for pAF-ts-pyrH). Later on, the preselected ampicillin-resistant bacteria clones were checked for kanamycin sensitivity, i.e., the loss of the chromosomally introduced kanamycin marker gene, and for chloramphenicol sensitivity, i.e., the loss of the temperature-sensitive plasmid pCP20.

[0117] These clones were finally checked for the chromosomal deletion of the plsC gene using the primers plsC-check-for (SEQ ID No. 15) and plsC-check-rev (SEQ ID No. 16). The template used for the checking of the chromosomal plsC deletion by means of PCR was chromosomal DNA from the selected ampicillin-resistant, chloramphenicol- and kanamycin-sensitive clones.

[0118] The thus generated and checked ampicillin-resistant E. coli strains with chromosomal plsC deletion and plasmid-encoded plsC expression have the designations W3110plsC/pAF-ts-plsC and W31101pp3plsC/pAF-ts-plsC.

Example 5: Generation of a Production Plasmid Containing Antibiotic Resistance Gene for the Production of Cysteine

[0119] The starting plasmids used for the cloning and expression of the genes cysEX (codes for feedback-resistant variants of serine acyltransferase; CysE) and orf306 (codes for 0-acetylserine/cysteine exporter; EamA) was the base plasmid pMT1 and the production plasmid pACYC184-LH-cysEX-orf306 described in EP0885962B1.

[0120] pMT1 contains not only the tetracycline resistance gene (tetR), but also the tac promoter, which is repressed by the LacIq gene product, the gene of which is likewise present on the plasmid, and which can be turned on by an inducer such as, for example, D-lactose or isopropyl--D-thiogalactopyranoside (IPTG). A restriction and function map of plasmid pMT1 is shown in FIG. 4. The sequence of the plasmid pMT1 is deposited in the sequence listing (SEQ ID No. 17).

[0121] For the generation of a new production plasmid for the production of cysteine, based on pMT1, a NcoI-BsaBI fragment from the plasmid pACYC184-LH-cysEX-orf306 (described in EP0885962 B1), which codes for the genes cysEX and orf306, was ligated with a 2458 bp NcoI-PvuII fragment (codes for ColE1 ori and tetracycline resistance, tetR) from the plasmid pMT1. The ligation preparation was transformed into DH5-T1R E. coli cells (Life Technologies GmbH), multiplied in said cells, and the DNA sequence of the isolated plasmids was verified by means of sequencing. The resulting expression plasmid has the designation pcysEX-GAPDH-ORF306_tetR (see FIG. 5).

Example 6: Generation of a Production Plasmid Containing Antibiotic Resistance Gene for the Production of -CGTase

[0122] The starting plasmids used for the cloning and expression of the cyclodextrin glycosyltransferase (CGTase) gene from Klebsiella pneumoniae M5a1 (Genebank No. M15264) was again the plasmid pMT1 and the plasmid pCGT described in US2008076158 A1.

[0123] For the generation of a new production plasmid for the production of CGTase, based on pMT1, a MauBI-BsaI fragment from the plasmid pCGT, which codes for the CGTase gene from Klebsiella pneumoniae M5a1, was ligated with a 4004 bp MauBI-BsaI fragment from the plasmid pMT1. Said 4004 bp fragment from the plasmid pMT1 codes for the ColE1 ori, the lac/tac operator and the tetracyline resistance gene (tetR).

[0124] The ligation preparation was transformed into DH5-T1R E. coli cells (Life Technologies GmbH), multiplied in said cells, and the DNA sequence of the isolated plasmids was verified by means of sequencing. The resulting expression plasmid has the designation pCGT_tetR (see FIG. 6).

Example 7: Generation of a Production Plasmid Containing Antibiotic Resistance Gene for the Production of Fab-Anti-Lysozyme

[0125] The starting plasmids used for the cloning and expression of the genes for the anti-lysozyme Fab fragment was again the plasmid pMT1 and the pFab-anti-lysozyme described in US20080076158 A1.

[0126] For the generation of a new production plasmid for the production of the antibody fragment Fab-anti-lysozyme, based on pMT1, a MauBI-BsaI fragment from the plasmid Fab-anti-lysozyme, which codes for the two chains, i.e., the heavy chain (V.sub.H-C.sub.H1 domains) and the light chain (V.sub.L-C.sub.L domains) of the anti-lysozyme Fab fragment, was ligated with a 4004 bp MauBI-BsaI fragment from the plasmid pMT1. Said 4004 bp fragment from the plasmid pMT1 codes for the ColE1 ori, the lac/tac operator and the tetracyline resistance gene (tetR).

[0127] The ligation preparation was transformed into DH5-T1R E. coli cells (Life Technologies GmbH), multiplied in said cells, and the DNA sequence of the isolated plasmids was verified by means of sequencing. The resulting expression plasmid has the designation pFab-anti-Lysozyme_tetR (see FIG. 7).

Example 8: Generation of Production Plasmids Containing the Marker Gene pyrH or plsC as Basis for the Creation of Production Plasmids According to the Invention without Antibiotic Resistance Gene

[0128] The starting plasmids used for the generation of production plasmids containing pyrH or plsC as marker gene were the plasmids pcysEX-GAPDH-ORF306_tetR, pCGT_tetR and pFab-anti-Lysozyme_tetR, as described in Examples 5 to 7. All these plasmids have an individual NcoI restriction site (see FIGS. 5 to 7). This universal NcoI restriction site was used for the clonning or integration of the genes pyrH or plsC.

[0129] For this purpose, the PCR products described in Example 1 and cut using the restriction enzyme NcoI were ligated with the plasmids pcysEX-GAPDH-ORF306_tetR, pCGT_tetR and pFab-anti-Lysozyme_tetR, after they were likewise cut using NcoI. The individual ligation preparations were transformed into DH5-T1R E. coli cells (Life Technologies GmbH), multiplied in said cells, and the DNA sequence of the isolated plasmids was verified by means of sequencing. The resulting constructs have, depending on the orientation and on the marker gene used, the following designations: [0130] pcysEX-GAPDH-ORF306_pyrH1_tetR (see FIG. 8) and pcysEX-GAPDH-ORF306_pyrH2_tetR [0131] pCGT_pyrH1_tetR (see FIG. 9) and pCGT_pyrH2_tetR [0132] pFab-anti-Lysozyme_pyrH1_tetR (see FIG. 10) and pFab-anti-Lysozyme_pyrH2_tetR [0133] pcysEX-GAPDH-ORF306_plsC1_tetR (see FIG. 11) and pcysEX-GAPDH-ORF306_plsC2_tetR [0134] pCGT_plsC1_tetR (see FIG. 12) and pCGT_plsC2_tetR [0135] pFab-anti-Lysozyme_plsC1_tetR (see FIG. 13) and pFab-anti-Lysozyme_plsC2_tetR

[0136] For the final removal of the tetR gene, the procedure was continued with the variants pcysEX-GAPDH-ORF306_pyrH1_tetR, pCGT_pyrH1_tetR, pFab-anti-Lysozyme_pyrH1_tetR, pcysEX-GAPDH-ORF306_plsC1_tetR, pCGT_plsC1_tetR and pFab-anti-Lysozyme_plsC1_tetR (see FIGS. 8 to 13).

Example 9: Removal of the Antibiotc Resitance Gene tetR and Transformation of the Production Plasmids Containing pyrH or plsC as Remaining Marker Gene into E. coli Strains with Chromosomal pyrH or plsC Deletion

[0137] The starting plasmids used for the generation of production plasmids without the antibiotic resistance gene tetR and containing pyrH or plsC as marker gene were the plasmids pcysEX-GAPDH-ORF306_pyrH1_tetR, pCGT_pyrH1_tetR, pFab-anti-Lysozyme_pyrH1_tetR, pcysEX-GAPDH-ORF306_plsC1_tetR, pCGT plsC1_tetR and pFab-anti-Lysozyme_plsC1_tetR, as described in Example 8. The removal of the antibiotic resistance gene tetR from the plasmids pFab-anti-Lysozyme_pyrH1_tetR and pFab-anti-Lysozyme_plsC1_tetR, as described in Example 8, was achieved via a digestion using the restriction enzyme ClaI and subsequent religation.

[0138] For the plasmids pcysEX-GAPDH-ORF306_pyrH1_tetR and pcysEX-GAPDH-ORF306_plsC1_tetR, a similar procedure was carried out, except that the gene tetR was removed via a partial digestion using ClaI, since two further ClaI restriction sites are situated in the structural genes cysEX and orf306 (see FIGS. 8 and 11).

[0139] In the case of pCGT_plsC1_tetR, the tetR gene was removed from the plasmid via a digestion using the enzymes StuI (cuts to leave blunt end) and FspI (cuts to leave blunt end). In the case of pCGT_pyrH1_tetR, the tetR gene was likewise removed via a partial digestion using the enzymes StuI (cuts to leave blunt end) and FspI (cuts to leave blunt end), since a further FspI restriction site is situated in the pyrH gene (see FIG. 9).

[0140] After the restriction digest, the respective linear vector fragments without tetR were purified via an agarose gel electrophoresis and isolated from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) according to the information from the manufacturer. Thereafter, the tetR-free vector fragment in question was religated. Using a modified CaCl.sub.2 method, the corresponding ligation preparations were transformed into the strains W3110pyrH/pAF-ts-pyrH and W31101pp3pyrH/pAF-ts-pyrH or W3110pyrH/pAF-ts-plsC and W31101pp3pyrH/pAF-ts-plsC, which strains are described in Example 4. For the transformation, i.e., the introduction of the antibiotic resistance-free production plasmids containing pyrH or plsC as marker gene into E. coli strains with corresponding chromosomal deletion (pyrH or plsC), the following procedure was carried out:

[0141] After the addition of 5 to 20 l of the ligation preparation in question to 100 l of CaCl.sub.2-competent cells of the strains W3110pyrH/pAF-ts-pyrH and W31101pp3pyrH/pAF-ts-pyrH or W3110plsC/pAF-is-plsC and W31101pp3plsC/pAF-ts-plsC, the cells were incubated on ice for a further 30 minutes. After a brief heat shock at 42 C. for 45 seconds, the cells were cooled on ice for 2 min. Thereafter, 900 l of LB medium were added to the transformation preparation, and the cells were incubated/regenerated, not at 37 C. as customary, but at 47 C. to 55 C. for 30 to 90 min. The further incubation at elevated nonpermissive temperature was then carried out on LB agar plates or in liquid LB medium without antibiotic (e.g., without tetracycline) at 40-45 C. for 15 to 24 h.

[0142] The 30-90 minute regeneration phase at 47 C. to 55 C. and the cultivation for 15 to 24 h at elevated, nonpermissive temperature facilitates the exchange of the temperature-sensitive plasmids pAF-ts-pyrH or pAF-ts-plsC for the final, antibiotic resistance-free production plasmid containing pyrH or plsC as new selection marker gene.

[0143] The best transformation results were achieved when the transformed cells were regenerated at 52 C. for 60 min and then incubated on LB agar plates at 42 C. for 20 h.

[0144] A preliminary selection for loss of the temperature-sensitive plasmid pAF-ts-pyrH or pAF-ts-plsC with simultaneous exchange for the respective pyrH- or plsC-containing production plasmid without tetracycline resistance gene tetR was done first of all on LB agar plates without antibiotic.

[0145] Thereafter, the preselected bacteria clones were checked for ampicillin sensitivity, i.e., the loss of the temperature-sensitive plasmid pAF-ts-pyrH or pAF-ts-plsC.

[0146] The pyrH- or plsC-coding production plasmids from ampicillin-sensitive clones were finally checked by means of restriction digest. Restriction maps of the pyrH-coding production plasmids pcysEX-GAPDH-ORF306_pyrH, pCGT_pyrH and pFab-anti-Lysozyme_pyrH, each without the antibiotic resistance gene tetR, are depicted in FIGS. 14 to 16. Restriction maps of the plsC-coding production plasmids pcysEX-GAPDH-ORF306_plsC, pCGT_plsC and pFab-anti-Lysozyme_plsC, each without the antibiotic resistance gene tetR, are depicted in FIGS. 17 to 19.

[0147] The thus generated and checked antibiotic resistance-free E. coli strains containing pyrH- or plsC-coding production plasmid and with chromosomal pyrH or plsC deletion have the designations: [0148] W3110pyrH/pcysEX-GAPDH-ORF306_pyrH [0149] W31101pp3pyrH/pCGT_pyrH [0150] W31101pp3pyrH/pFab-anti-Lysozyme_pyrH [0151] W3110plsC/pcysEX-GAPDH-ORF306_plsC [0152] W31101pp3plsC/pCGT_plsC [0153] W31101pp3plsC/pFab-anti-Lysozyme_plsC

Example 10: Cysteine Fermentation

[0154] Preliminary Culture 1:

[0155] In an Erlenmeyer flask (100 ml), 20 ml of LB medium were inoculated with the particular E. coli strain W3110/pcysEX-GAPDH-ORF306_tetR, W3110pyrH/pcysEX-GAPDH-ORF306_pyrH or W3110plsC/pcysEX-GAPDH-ORF306_plsC and incubated on a shaker (150 rpm, 30 C.) for seven hours. For a cultivation of construct W3110/pcysEX-GAPDH-ORF306_tetR in the context of the prior art, i.e., with antibiotic as selection agent, the medium was supplemented with 15 mg/L teracyclineteracycline.

[0156] Preliminary Culture 2:

[0157] Thereafter, the preliminary culture 1 was transferred in full to 100 ml of SM1 medium (12 g/l K.sub.2HPO.sub.4, 3 g/l KH.sub.2PO.sub.4, 5 g/l (NH.sub.4).sub.2SO.sub.4, 0.3 g/l MgSO.sub.47H.sub.2O, 0.015 g/l CaCl.sub.22H.sub.2O, 0.002 g/l FeSO.sub.47H.sub.2O, 1 g/l Na.sub.3 citrate2H.sub.2O, 0.1 g/l NaCl, 1 ml/l trace element solution, consisting of 0.15 g/l Na.sub.2MoO.sub.42H.sub.2O, 2.5 g/l H.sub.3BO.sub.3, 0.7 g/l CoCl.sub.26H.sub.2O, 0.25 g/l CuSO.sub.45H.sub.2O, 1.6 g/l MnCl.sub.24H.sub.2O, 0.3 g/l ZnSO.sub.47H.sub.2O), which was supplemented with 5 g/l glucose and 5 mg/l vitamin B1. The cultures were shaken in Erlenmeyer flasks (1 l) at 30 C. for 17 h at 150 rpm. After this incubation, the optical density at 600 nm (OD.sub.600) was between 3 and 5. For the cultivation of W3110/pcysEX-GAPDH-ORF306_tetR in the context of the prior art, i.e., with antibiotic as selection agent, the medium was supplemented with 15 mg/L teracyclineteracycline.

[0158] Main Culture:

[0159] The fermentation was carried out in fermenters, BIOSTAT B model, from Sartorius Stedim. A culture vessel with 2 l total volume was used. The fermentation medium (900 ml) contains 15 g/l glucose, 10 g/l tryptone (Difco), 5 g/l yeast extract (Difco), 5 g/l (NH.sub.4).sub.2SO.sub.4, 1.5 g/l KH.sub.2PO.sub.4, 0.5 g/l NaCl, 0.3 g/l MgSO.sub.47H.sub.2O, 0.015 g/l CaCl.sub.22H.sub.2O, 0.075 g/l FeSO.sub.47H.sub.2O, 1 g/l Na.sub.3 citrate2H.sub.2O and 1 ml of trace element solution (see above) and 0.005 g/l vitamin B1. The pH in the fermenter was initially adjusted to 6.5 by pumping in a 25% NH.sub.4OH solution.

[0160] During the fermentation, the pH was maintained by automatic correction at a level of 6.5 using 25% NH.sub.4OH. For the inoculation, 100 ml of the preliminary culture 2 were pumped into the fermenter vessel. The starting volume was therefore about 1 l. The cultures were initially stirred at 400 rpm and aerated with 2 vvm of a compressed air sterilized via a sterile filter. Under these starting conditions, the oxygen probe had been calibrated to 100% saturation prior to the inoculation. The nominal value for the O.sub.2 saturation during the fermentation was set to 50%. After the O.sub.2 saturation fell below the nominal value, a regulation cascade was started in order to bring the O.sub.2 saturation back to the nominal value. In this connection, the gas supply was first continuously increased (to max. 5 vvm) and then the stirring rate was continuously raised (to max. 1500 rpm).

[0161] The fermentation was carried out at a temperature of 30 C. After a fermentation time of 2 h, a sulfur source was fed in in the form of a sterile 60% sodium thiosulfate5H.sub.2O stock solution at a rate of 1.5 ml per hour. Once the glucose content in the fermenter of initially 15 g/l had dropped to approximately 2 g/l, a 56% glucose solution was continuously metered in. The feeding rate was set such that the glucose concentration in the fermenter, from then on, no longer exceeded 2 g/l.

[0162] Glucose was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA). For the fermentation of construct W3110/pcysEX-GAPDH-ORF306_tetR in the context of the prior art, i.e., with antibiotic as selection agent, the medium was supplemented with 15 mg/L teracyclineteracycline.

[0163] The fermentation period was 48 hours. Thereafter, samples were collected and the content of L-cysteine and the derivatives derived therefrom in the culture supernatant (especially L-cysteine and thiazolidine) and in the precipitate (L-cystine) were determined separately from one another. For this purpose, the colorimetric assay by Gaitonde (Gaitonde, M. K. (1967), Biochem. J. 104, 627-633) was used in each case. The L-cystine present in the precipitate first had to be dissolved in 8% hydrochloric acid before it could be quantified in the same way. The values listed in Table 1 for total cysteine correspond to the sum of the L-cysteine in the culture supernatant and L-cystine in the precipitate. In this connection, each molecule of L-cystine corresponds to two molecules of L-cysteine.

TABLE-US-00001 TABLE 1 Content of total cysteine (L-cysteine.sub.culture supernatant + L-cystine.sub.precipitate) in the culture broth after 48 h and stability of the production plasmids Total cysteine Plasmid Strain (g/L) stability W3110/pcysEX-GAPDH- 19.0 0.4 95% 5% ORF306_tet.sup.R (cultivated with tetracycline)* W3110/pcysEX-GAPDH- 10.0 4.1 60% 19% ORF306_tet.sup.R (cultivated without tetracycline) W3110pyrH 20.4 0.3 97% 3% pcysEX-GAPDH- ORF306 pyrH1** W3110plsC/pcysEX-GAPDH- 21.1 + 0.3 95% 5% ORF306_plsC** *Construct in the context of the prior art (comparative example) **Construct according to the invention

Example 11: Secretory Production of a Cyclodextrin Glycosyltransferase on the 10 l Scale (Fermentation)

[0164] With the aid of an lpp mutant of E. coli, biotechnologically relevant enzymes such as, for example, CGTases can be produced and secreted into the medium (US2008076158 A1).

[0165] The secretory production of the CGTase was carried out in 10 L stirred tank fermenters using the strains W31101pp3/pCGT_tetR (control), W31101pp3pyrH/pCGT_pyrH and W31101pp3pyrH/pCGT_plsC.

[0166] The fermenter filled with 6 l of the fermentation medium FM4 (1.5 g/l KH.sub.2PO.sub.4; 5 g/l (NH.sub.4).sub.2SO.sub.4; 0.5 g/l MgSO.sub.47H.sub.2O; 0.15 g/l CaCl.sub.22H.sub.2O, 0.075 g/l FeSO.sub.47H.sub.2O; 1 g/l Na.sub.3 citrate2H.sub.2O; 0.5 g/l NaCl; 1 ml/l trace element solution (0.15 g/l Na.sub.2MoO.sub.42H.sub.2O; 2.5 g/l Na.sub.3BO.sub.3; 0.7 g/l CoCl.sub.26H.sub.2O; 0.25 g/l CuSO.sub.45H.sub.2O; 1.6 g/l MnCl.sub.24H.sub.2O; 0.3 g/l ZnSO.sub.47H.sub.2O); 5 mg/l vitamin B.sub.1; 3 g/l Phytone; 1.5 g/l yeast extract; 10 g/l glucose) was inoculated in the ratio of 1:10 with a preliminary culture, which was cultivated overnight in the same medium. During the fermentation, a temperature of 30 C. was set and the pH was maintained constantly at a level of 7.0 by metering in NH.sub.4OH or H.sub.3PO.sub.4. Glucose was metered in throughout the fermentation, with a maximum glucose concentration in the fermentation medium of <10 g/l being striven for. Expression was induced by addition of isopropyl--D-thiogalactopyranoside (IPTG) to 0.1 mM at the end of the logarithmic growth phase.

[0167] For the fermentation of the construct W31101pp3/pCGT_tetR in the context of the prior art, i.e., with antibiotic as selection agent, the medium was supplemented with 15 mg/L teracyclineteracycline.

[0168] After 72 h of fermentation, samples were collected, the cells were removed from the fermentation medium by centrifugation and the CGTase content in the fermentation supernatant was determined, as described in Example 4 of US2008076158A1.

[0169] Table 2 lists the yields of functional CGTase and also the activities in the fermentation supernatant.

TABLE-US-00002 TABLE 2 Cyclodextrin glycosyltransferase yields in the fermentation supernatant after 72 hours of fermentation CGTase CGTase Plasmid Strain (U/ml) (mg/l) stability W3110lpp3/pCGT_tetR 555 15 3750 145 97% 3% (cultivated with tetracycline)* W3110lpp3/pCGT_tetR 340 65 2210 425 55% 21% (cultivated without tetracycline) W3110lpp3pyrH/ 560 25 3795 160 98% 2% pCGT_pyrH** W3110lpp3plsC/ 570 30 3810 190 98% 2% pCGT_plsC** *Construct in the context of the prior art (comparative example) **Construct according to the invention

Example 12: Secretory, Fermentative Production of the Fab Antibody Fragment Anti-Lysozyme-Fab on the 10 l Scale

[0170] With the aid of an lpp mutant of E. coli, functional Fab antibody fragments can also be produced extracellularly (US2008076158A1). In this case, the cell must simultaneously synthesize the corresponding fragments of the light chain, which comprises the domains V.sub.L and C.sub.L, and of the heavy chain, which comprises the domains V.sub.H and CH1, and then secrete them into the periplasm and ultimately into the fermentation medium. Outside the cytoplasm, the two chains then assemble to form the functional Fab fragment.

[0171] The present example describes the production of a Fab fragment of the well characterized anti-lysozyme antibody D1.3. The plasmids pFab-anti-Lysozyme_tetR, pFab-anti-Lysozyme_pyrH and pFab-anti-Lysozyme_plsC contain not only the marker genes tetR, pyrH and plsC, respectively, but also, inter alia, the structural genes for the HC and the LC of the Fab fragment in the form of an operon. In this case, the HC is fused in frame to the 3-end of the ompA signal sequence (ompA.sup.SS) and the LC is fused in frame to the 3-end of a CGTase signal sequence (cgt.sup.SS). The expression of the ompA.sup.SS-HC-cgt.sup.SS-LC operon is under the control of the tac promoter.

[0172] The production of the anti-lysozyme Fab fragment on the 10 l scale was carried out analogously to the method described in Example 11 on the basis of CGTase, using the strains W31101pp3/pFab-anti-Lysozyme_tetR, W31101pp3pyrH/pFab-anti-Lysozyme_pyrH and W31101pp3plsC/pFab-anti-Lysozyme_plsC. For the fermentation of E. coli W31101pp3/pFab-anti-Lysozyme_tetR in the context of the prior art, i.e., with antibiotic as selection agent, the medium was supplemented with 15 mg/L teracyclineteracycline.

[0173] After 72 h of fermentation, samples were collected and then the cells were removed from the fermentation medium by centrifugation.

[0174] The anti-lysozyme Fab fragment was purified from the fermentation supernatants by means of affinity chromatography, as described in Skerra (1994, Gene 141, 79-84).

[0175] The purified anti-lysozyme Fab fragment was quantified and its activity determined via an ELISA assay with lysozyme as antigen (Skerra, 1994, Gene 141, 79-84).

[0176] Table 2 lists the projected yields of functional anti-lysozyme Fab fragment in the fermentation supernatant, on the basis of isolated amounts of, in each case, 20 ml of fermentation supernatant after 72 h of fermentation.

TABLE-US-00003 TABLE 3 Anti-lysozyme Fab fragment yields in the fermentation supernatant after 72 h of fermentation Anti-lysozyme Fab fragment yields [mg/l] in the fermentation Plasmid Strain supernatant (projected) stability W3110lpp3/pFab-anti- 1440 110 97% 3% Lysozyme_tetR (cultivated with tetracycline)* W3110lpp3/pFab-anti- 625 250 55% 20% Lysozyme_tetR (cultivated without tetracycline) W3110lpp3pyrH/pFab- 1580 115 98% 2% anti-Lysozyme_pyrH** W3110lpp3plsC/pFab- 1610 130 98% 2% anti-Lysozyme_plsC** *Construct in the context of the prior art (comparative example) **Construct according to the invention

Example 13: Determination of Plasmid Stability

[0177] Plasmid stability was checked by means of plasmid preparation with subsequent restriction digest. For this purpose, completion of the cultivation of the production strains (e.g., after 72 h of fermentation) was followed by plating out various dilutions of the cultures on LB agar plates. For the subsequent ascertainment of plasmid stability, i.e., the identification of plasmid-bearing cells (colonies), only LB plates with individual colonies were used for the evaluation.

[0178] Altogether, 50 individual colonies were cultivated in liquid LB medium for 15 to 20 hours and then plasmid DNA was isolated from said cultures. Characteristic restriction patterns for the individual production plasmids were used as a basis to verify the correctness of the isolated plasmids.