Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
  • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to a new industrial process for the production of heterologous proteins in E. coli. If for certain heterologous proteins with very high added value the cost price of their preparation process remains a negligible factor compared to the purpose of the heterologous protein (in the pharmaceutical field in particular), the development of the industrial production of heterologous proteins of lower added value in E. coli requires taking into account production factors such as the need to have a high biomass and a very high content of heterologous proteins produced at the lowest possible cost, which cost must take into account the nature of the media, the energy and reagent yields, and the operating conditions. For industrial productions with reaction volumes of up to several tens of m 3 , the simplest possible environments and operating conditions will be sought.
• the present invention consists in the selection of an E. coli strain suitable for meeting the above conditions, essential for the economically satisfactory industrial production of heterologous proteins, independently of the value of the protein produced.
• E. coli most commonly used for molecular biology work are derived from the strain Kl 2 (Swartz, 1996, In ⁇ scherichia coli and Salmonella, Cellular and Molecular Biology, 2 nd edition, ASM Press Washington, ppl693-1711).
• a table of the strains most commonly used for the production of recombinant proteins is given by Wingfield, 1997 (Current Protocols in Protein Science, Coligan et al. ⁇ d, John Wiley & Sons, Inc, 5.0.1-5.0.3).
• An expression system consists of a promoter, its regulator, a ribosome binding site followed by a restriction site allowing the insertion of the gene of interest, a structure which can serve as a terminator for transcription, possibly of genes whose coexpression increases the quality of the protein of overexpressed interest and of one or more vectors making it possible to introduce these combinations into the host.
• the promoter must have at least three characteristics to be used in a protein production process (Makrides, 1996, supra):
• promoters have been described for expression in E. coli (Makrides, 1996, supra; Weickert et al, 1996, Current Opinions in Biotechnology 7: 494-499).
• homologous promoters used for the production of proteins in E. coli there may be mentioned the promoters lac, trp, lpp, phoA, recA, araBAD, proU, cst-1, tetA, cadA, nar, tac, trc, Ipp- lake, Ysyn, cspA.
• heterologous promoters used for the production of proteins in E. coli mention may be made of the promoters PL, PL-9G-50,? R-?
• RNAI and RNAII The most commonly used vectors for protein expression in E. coli are derived from the plasmid pBR322 (Swartz, 1996, supra; Makrides, 1996, supra). They are present in cells at a number of copies, determined by the interaction of two RNAs encoded by the plasmid, RNAI and RNAII (Polisky, 1988, Cell 55: 929-932). The interaction of RNAI with RNAII inhibits the maturation of AR II in a form necessary for the initiation of replication of the plasmid.
• a high copy number expression plasmid resulted in high levels of messenger RNA of the desired protein, but may be detrimental to the metabolism of the host strain (Bailey, 1993, Adv. Biochem. Eng Biotechnol. 48: 29-52).
• the stability of expression plasmids is an important criterion, especially since industrial fermentations tend not to use antibiotics in fermenters.
• Several strategies have been developed to stabilize the expression plasmids, including the cloning of the cer locus of the natural plasmid ColEl.
• heterologous proteins intended for pharmaceutical use such as, for example, human growth hormone, consensus human interferon alpha, human interleukins l ⁇ , ⁇ l, 2, l leukocyte interferon, human parathyroid hormone, human insulin, human serum albumin, human Proapolipoprotein Al (Lee, 1996, Trends in Biotechnol. 14: 98-105; Latta et ai, 1987, Bio / Technology 5: 1309-1313).
• the present invention resides in the selection of a particular E. coli strain, suitable for the industrial production of heterologous proteins.
• the strain useful for the process according to the invention is an E. coli W strain, more particularly the W strain referenced in the ATCC under the number 9637.
• the strain W (ATCC9637) was thus used for the production of 3-polyhydroxybutyric acid (PHA) after introduction of a plasmid carrying the operon of Alcaligenes eutrophus coding for enzymes involved in the biosynthesis of PHA (Lee and Chang, 1993 , supra; Lee and Chang, 1995, supra; Lee et al., 1994).
• the present invention therefore relates to an industrial process for the preparation of heterologous proteins in E coli, in which is seeded and cultivated in an appropriate culture medium, modified E coli bacteria with an appropriate heterologous protein expression system, characterized in that the E. coli strain is an E. coli W. strain More preferably, the W strain is the W strain deposited at the ATCC under the number 9637.
• the strain W is a derivative of the strain deposited at the ATCC under number 9637 obtained by clonal selection or genetic manipulation.
• industrial process is meant according to the invention any process in which the culture volume of bacteria is greater than the usual culture volume used in research laboratories. In general, by industrial process is meant any process for which the culture volume is greater than 2 liters, preferably greater than or equal to 10 liters, more preferably greater than or equal to 20 liters, even more preferably greater than or equal to 50 liters. .
• the method according to the invention is particularly suitable for culture volumes of several ten m 3 , up to more than 100 m 3 .
• the appropriate culture medium is a culture medium suitable for obtaining a high density of biomass and a high content of heterologous proteins produced.
• Several types of media can be used for high cell density culture (Lee, 1996, cited above). If the media known from the state of the art and in particular semi-defined media make it possible to combine good reproducibility of the composition of the medium and good o
• the culture medium contains sucrose as a main carbon source.
• main source of carbon is meant according to the invention that the sucrose represents at least 50% by weight of the total weight of the carbon sources of the culture medium, more preferably at least 75% by weight, even more preferably at least 85% in weight.
• the culture medium comprises substantially only sucrose as a carbon source. It is understood that for the method according to the invention, the culture medium can comprise appropriate additives so as to increase the overall yield of the invention. These additives can have the additional function of behaving as a carbon source to the culture of bacteria. However, these additives will not be considered as a carbon source within the meaning of the present invention if the E coli W bacteria used in the process according to the invention cannot grow on said additives as the only carbon source.
• the amount of sucrose in the culture medium of the process according to the invention is between 0.1 and 300 g / 1 at the start of culture (before seeding), preferably between 0.5 and 200 g / 1. It is understood that the sucrose constituting the main source of carbon of the medium according to the invention, the amount of sucrose will decrease during the process. In general, at the end of the reaction, the amount of sucrose in the culture medium at the end of the reaction is between 0 and 10 g / l.
• the appropriate culture medium also comprises a source of additional organic nitrogen.
• This source of additional organic nitrogen can be constituted by all the sources of organic nitrogen known to those skilled in the art.
• the source of additional organic nitrogen consists of protein extracts. These protein extracts more preferably have the following composition: (in g amino acids per 100g of product) Alanine between 10 and 4, Aspartic between 11 and 4, Glycine between 22 and 2.5 and Lysine between 7 and 4.
• Peptones or proteins Meat or potato meet such a profile and are particularly preferred for the process according to the invention, more particularly the potato protein derivatives.
• heterologous protein expression system is understood to mean, according to the invention, any expression system comprising regulatory elements suitable for the expression of heterologous proteins in E. coli W. These regulatory elements include in particular promoters, ribosome binding sites, transcription terminators.
• the expression system comprises an R ⁇ - n promoter.
• the promoter P ⁇ r p was used in several examples (Application ⁇ P 0 198 745; Application CIP N ° 08 / 194,588; Application WO 97/04083; Latta et ai, 1987, Bio / Technology 5: 1309-1314; Denèfle et ai , 1987, Gene 56: 61-70).
• Latta et al (1990, DNA Cell. Biol. 9: 129-137) conducted a detailed study on the influence of regulatory sequences upstream of the promoter, of promoter sequences duplicated in tandem and on the influence of coexpression of the TrpR repressor.
• the promoter P tr p comprises the nucleic acid sequence represented by the sequence identifier No. 1 (SEQ ID NO 1).
• heterologous protein to improve the level of expression of the heterologous protein, coexpression of the molecular chaperones of E. coli Gro ⁇ SL (reviewed by Makrides, 1996, cited above). Increasing the intracellular concentration of Gro ⁇ SL proteins makes it possible to assist the folding of the recombinant protein and thus improves the level of active protein (Weicker et al., 1996, Curr. Opin. Biotechnol. 7: 494-499) .
• the genes whose coexpression promotes the expression of the heterologous protein according to the invention, and its quality, are included in the expression system according to the invention.
• heterologous protein is meant according to the invention any protein produced by the process according to the invention which is not naturally found in E.
• coli W in the appropriate expression system according to the invention. It may be a protein of non-bacterial origin, for example of animal origin, in particular human or vegetable, or also a protein of bacterial origin not produced naturally by E. coli W, or alternatively a protein of bacterial origin produced naturally by another bacterium than E. coli W or alternatively a protein produced naturally by E. coli W, the expression of which is controlled by regulatory elements distinct from those of the expression system according to the invention, or finally of a protein derived from the preceding after modifications of certain elements of its primary structure.
• the method according to the invention is applicable to any protein of interest whose production requires a high accumulation of proteins, before either the extract and purify the whole or in part or to use them mixed with the biomass that will have produced them.
• This is the case for example of enzymes useful for biocatalysis of chemical reactions, which can be used without prior transaction isolation and purification or as enzymes that are used in the host bacterium during growth for the biotransformation of chemical compounds.
• the heterologous protein is an enzyme, produced in industrial quantities for later use as a catalyst for chemical reactions.
• the enzyme is nitrilase, preferably a nitrilase!
• Alcaligenes faecalis (ATCC8750) described in patent application WO 98/18941 or a nitrilase from Comamonas testosteroni sp. described in application CIP n ° 08 / 194,588, or an amidase such as those described in applications WO 97/04083, EP 433 1 17, EP 488 916, or also a hydroxyphenylpyruvate dioxygenase described in application WO 96/38567.
• the present invention also relates to an E. coli W strain as defined above, characterized in that it comprises a system for expression of heterologous proteins, the promoter of which is the Ptrp promoter defined above.
• FIG. 1 represents the map of the plasmid pRPA-BCAT41.
• the sites in parenthesis are sites that were eliminated during cloning.
• Ptrp tryptophan promoter
• nitB nitrilase gene
• TrrnB transcription terminators
• end ROP end of the gene coding for the protein ROP (Chambers et al. 1988, Gene 68: 139-149);
• ORI origin of replication;
• RNAI / II AR involved in replication (Chambers et al., Cited above); Te: tetracycline resistance gene.
• FIG. 2 represents 1 map of the plasmid pRPA-BCAT127. The sites in parenthesis were eliminated during the cloning.
• Ptrp tryptophan promoter
• nitB nitrilase gene
• TrrnB transcription terminators
• ORI origin of replication
• RNAI * / II mutated RNAs involved in replication
• Cm chloramphenicol resistance gene
• cer locus cer.
• FIG. 3 represents the map of the plasmid pRPA-BCAT103. The sites in parenthesis were eliminated during the cloning.
• Sm / Sp gene for resistance to streptomycin and spectinomycin
• parABCDE locus by (Roberts and Helinski, 1992, J. Bacteriol. 174: 8119-8132); rep, mob, D20 and ori: regions involved in the replication and transfer of the plasmid (Scholtz et al., 1989, Gene 75: 271-288; Frey et al., 1992, Gene 113: 101-106).
• FIG. 4 represents the map of the plasmid pRPA-BCAT126.
• Ptrp tryptophan promoter
• nitB nitrilase gene
• TrrnB transcription terminators
• ORI origin of replication
• RNAI * / II mutated RNAs involved in replication
• Tc r tetracycline resistance gene
• cer locus cer.
• FIG. 5 represents the map of the plasmid pRPA-BCAT143.
• Sm / Sp gene for resistance to streptomycin and spectinomycin; rep, mob, and ori: regions involved in the replication and transfer of the plasmid (Scholtz et ai, 1989, Gene 75: 271-288;
• delta refers to the name of the deletion described in the text.
• Example 1 Construction of the expression plasmids pBCAT29 and pBCAT41.
• the 1.27 kb fragment containing the promoter Ptrp, the binding site of the ribosome of the eyelash gene of phage ⁇ (RBScII) and the nitrilase & Alcaligenes faecalis ATCC8750 (nitB) gene was extracted from the plasmid pRPA6BCAT6 (Request FR 96/13077 ) using the restriction enzymes EcoRI and Xbal to be cloned into the vector pXL642 (described in application CIP N ° 08 / l 94.588) opened by the same restriction enzymes.
• pRPA-BCAT15 was opened by the enzymes StuI and Bsml and the 4.3 kb fragment was ligated with the Stul-Bsml fragment of 136 bp purified from pRPA-BCAT4 (Application FR 96/13077) to yield the plasmid pRPA-BCAT19.
• the partial sequencing of pRPA-BCAT19 confirmed the replacement of the codon of the residue Asp279 of nitrilase by the codon of a residue Asn279.
• the vector pRPA-BCAT28 was obtained by ligating the 3.9 kb Sspl-Scal fragment of pXL642 (CIP application N ° 08 / 194.588) with the 2.1 kb Smal fragment of pHP45 ⁇ Tc (Fellay et ai, 1987, Gene 52: 147-154) to replace the ampicillin resistance marker with the tetracycline resistance marker.
• the plasmid pRPA-BCAT41 was obtained, a map of which is shown in FIG. 1.
• the sequence of the expression cassette is represented by the sequence identifier No. 2 (S ⁇ Q ID NO 2) .
• Example 2 Expression of nitrilase from A. faecalis ATCC 8750 in E. coli K12, BL21, W in "batch".
• ABBREVIATIONS g / 1: gram of dry weight per liter of culture; U: kg of HMTBA formed per hour and per kg of dry weight; P: kg of HMTBA formed per hour and per liter of culture
• the polyamide hydrolase gene from Comamonas acidovorans N12 described in application WO 97/04083 was cloned into the vector pBCAT41. By introducing into the PCR primers the EcoRI and Ncol restriction sites in position
• the gene for this polyamide hydrolase was amplified by PCR in the form of a DNA fragment of 1.26 kb. This fragment was then treated successively with the EcoRI enzyme and the Mung Bean nuclease. After extraction of the proteins with phenol-chloroform-isoamilic alcohol, the treatment continued with Xbal digestion. Similarly, the vector pRPA-BCAT41 was opened with the enzyme NdeI and then treated with Mung Bean nuclease. After extraction of the proteins with phenol-chloroform-isoamilic alcohol, the treatment continued with Xbal digestion.
• the plasmid pRPA-BCAT43 contains the Ptrp promoter, I e binding site RBScII separated from the translation initiation codon of the gene pamll by the sequence: AATACTTACACC.
• Example 4 Expression of the Pamll polyamidase in E. coli DH5 ⁇ , BL21 and W in "batch".
• the plasmid pRPA-BCAT43 was introduced into the strains of E. coli DH5 ⁇ ,
• the pellets of permeabilized cells are taken up in a 100 mM phosphate buffer, pH7.
• the hydrolysis activity was measured on the oligomer AB (a molecule of adipic acid condensed with a molecule of hexamethylene diamine) present at 2.5 g / 1 in the reaction medium containing potassium phosphate buffer 0, 1 M at pH 7 and incubated at 30 ° C with shaking; samples of 100 microliters are taken at regular intervals by adding to them the same volume of 0.2 N NaOH; the samples are analyzed by HPLC after dilution to the tenth in a 50 mM H 3 P0 4 solution.
• Table 2 contains the average of the data obtained for each strain.
• ABBREVIATIONS NB: number; U: g of AB hydrolyzed per hour and per g of dry weight; P: g of AB hydrolyzed per hour and per liter of culture
• the plasmid pRPA-BCAT41 underwent a mutagenesis step carried out with hydroxylamine as described in Miller 1992 (Mutagenesis. A short course in bacterial genetics. "A laboratory manual and handbook for E. coli and related bacteria", Cold Spring Harbor Laboratory Press, Unit 4, pp81-212) and Humphreys et al, 1976 (Mol. Gen. Genêt. 145: 101-108).
• Five micrograms of plasmid DNA purified on a cesium chloride gradient were incubated for 20 minutes at 80 ° C. in a 50 mM sodium phosphate buffer pH6 containing 0.5 mM EDTA and 0.4 M NH 2 OH.
• the reaction mixture was dialyzed against a large excess of 10 mM Tris-HCl buffer pH7.5 containing 1 mM EDTA and 100 mM NaCl .
• the plasmid DNA was then recovered by precipitation and approximately 20 ng of DNA was introduced by electroporation into the strain DH5 harboring the plasmid pXL2035.
• a clone was selected because of a productivity of the culture 3 times higher than that of a culture of the strain DH5 ⁇ (pRPA-BCAT41, pXL2035).
• the plasmid pRPA-BCAT41-531 which it housed was extracted and reintroduced into a new DH5 ⁇ host hosting the plasmid pXL2035. Three clones were then analyzed under the conditions described in Example 2 by comparing them to 3 DH5 ⁇ clones (pRPA-BCAT41, pXL2035) and the results are presented in the table in Table 3.
• ABBREVIATIONS g / 1: gram of dry weight per liter of culture; U: kg of HMTBA formed per hour and per kg of dry weight; P: kg of HMTBA formed per hour and per liter of culture.
• the 1.27 kb EcoRI -Xbal fragment containing the fusion P f rp - '-nitB was extracted from the plasmid pRPA-BCAT41 to be cloned in place of that contained in pRPA-BCAT41-531.
• the resulting plasmid, pRPA-BCAT86 was introduced into the strain DH5a (pXL2035) and 3 transformants were studied under conditions similar to those described above. The results are collated in Table 4.
• ABBREVIATIONS g / 1: gram of dry weight per liter of culture; U: kg of HMTBA formed per hour and per kg of dry weight; P: kg of HMTBA formed per hour and per liter of culture
• Example 6 Characterization of a mutation carried by the plasmid pBCAT41-531 responsible for improving the productivity of cultures of strains expressing nitrilase. Analysis of the amount of protein produced by the strains of Example 5 by polyacrylamide gel electrophoresis in the presence of SDS showed that all these constructions lead to rates of synthesis of nitrilase polypeptide comparable between the strains described in this example . On the other hand, plasmid DNA preparations of pRPA-BCAT41 and pRPA-BCAT41-531 made from equivalent amounts of biomass have demonstrated that the plasmid pRPA-BCAT41-531 is present with a lower copy number than its parent pRPA-BCAT41.
• the first difference is due to an error during the action of the Klenow polymerase which was used to destroy one of the NdeI sites of pRPA-BCAT29 and is located in a region which is not described as playing a role in the replication of pBR322 (Chambers et ai, 1988, Gene 68: 139-149).
• the second error corresponds to a transition, a characteristic effect of hydroxyamine on DNA (Drake and Baltz, 1976, Annu. Rev. Biochem. 45: 11-37), and is located at the level of the second nucleotide of the region transcribed into RNA I involved in the replication of pBR322 (Chambers et al, cited above).
• Example 7 Expression of A. faecalis ATCC 8750 nitrilase in E. coli BL21 and E. coli W in "fed-batch" (semi-continuous culture).
• the plasmids pRPA-BCAT41-531 and pXL2035 were introduced by electroporation into the strains BL21 (reference cited above) and W (ATCC9637) to give respectively the strains RPA-BIOCAT594 [BL21 (pRPA-BCAT41-531, pXL2035)] and RPA- BIOCAT714 [W (pRPA-BCAT41-531, pXL2035)].
• the E. coli BIOCAT 594 and E. coli BIOCAT 714 recombinants were cultivated in 3.5 liter fermenters containing 2 liters of medium, the composition of which is as follows:
• the pH is maintained at 7.0 by adding ammonia.
• the oxygen saturation is maintained at 20% by adding air at the rate of 1 volume / volume of medium / minute and by stirring.
• Glucose is introduced at the start at a final concentration of 2 g / 1. After being completely consumed, it is introduced continuously from a stock solution whose composition is as follows: glucose 700 g / 1; MgSO 4 .7H 2 O 19.6 g / 1.
• the addition rate is 2.2 g of glucose / h. 1 in the middle.
• the medium is recovered, centrifuged and the dry weight is estimated in g / 1.
• the enzymatic activity is measured according to a protocol given in patent WO96 / 09403. It is expressed in kilos of ammonium 3-hydroybutanoate formed per hour and per kilo of dry cells.
• Example 8 Influence of the organic nitrogen source of animal origin.
• the E. coli W BIOCAT 714 strain is cultivated in a 3.5 liter fermenter containing 2 liters of medium, the composition of which is as follows:
• the pH is maintained at 7.0 by adding ammonia.
• the oxygen saturation is maintained at 20% by adding air at the rate of 1 volume / volume of medium / minute and by stirring.
• Glucose is introduced at the start at a final concentration of 2 g / 1. After being completely consumed, it is introduced continuously from a stock solution whose composition is as follows: glucose 700 g / 1; MgSO 4 .7H 2 O 19.6 g / 1.
• the addition rate is 2.2 g of glucose / h. 1 in the middle.
• the culture conditions are identical to those of Example 8. In this example, organic nitrogen of vegetable origin is added.
• potato protein gives as good a result as organic nitrogen of animal origin.
• Example 10 influence of the carbon source.
• the E. coli W BIOCAT 714 strain is cultivated in a 3.5 liter fermenter containing 2 liters of medium, the composition of which is as follows:
• the pH is maintained at 7.0 by adding ammonia.
• the oxygen saturation is maintained at 20% by adding air at the rate of 1 volume / volume of medium / minute and by stirring.
• the carbon source is introduced at the start at a final concentration of 2 g / 1. After being completely consumed, it is introduced continuously from a stock solution whose composition is as follows: carbon source 700 g / 1 ; MgSO 4 .7H 2 O 19.6 g / 1.
• the rate of addition is 2.2 g of glucose or sucrose / h. 1 in the middle.
• the carbon source is varied in this example.
• sucrose zero syrup
• a 434 bp DNA fragment carrying the trpR gene and its promoter was extracted from the plasmid pRPG9 (Gunsalus and Yanofsky, 1980, Proc. Natl. Aca. Sca. USA 77: 7117-7121) using the enzymes restriction Aatll and Stul. This fragment was cloned into the plasmid pSL301 (Brosius, 1989, DNA 8: 159-111) by ligating it to the approximately 3.1 kb AatlI-StuI fragment to give the plasmid pRPA-BCAT30.
• the trpR gene and its promoter were then extracted from pRPA-BCAT30 in the form of a 475 bp dEcoRI-NotI fragment to be cloned in the plasmid pXL2035 instead of a 240 bp EcoRI-NotI fragment.
• the resulting plasmid, pRPA-BCAT34 is therefore a derivative of pKT230 allowing the expression of the GroESL chaperones and of the TrpR regulator.
• Example 12 Influence of the co-expression of GroESL and TrpR.
• the plasmid pRPA-BCAT34 was introduced by electroporation into the strains DH5 ⁇ (pRPA-BCAT29), BL21 (pRPA-BCAT29) and W (pRPA-BCAT29). Expression cultures of different strains were carried out as described in Example 2 and the results are presented in Table 5.
• DH5 ⁇ (pRPA-BCAT66, pRPA-BCAT34) 1, 10.0 18.0
• ABBREVIATIONS g / 1: gram of dry weight per liter of culture; U: kg of HMTBA formed per hour and per kg of dry weight; P: kg of HMTBA formed by
• the trpR gene was extracted from this latter plasmid in the form of a fragment of approximately 300 bp prepared by treatment with the enzyme Aatll followed by the action of polymerase I (Klenow fragment), then, after inactivation of the reaction mixture, by digestion with the enzyme SacII.
• This fragment was cloned into the plasmid pRPA-BCAT66 after opening it with Tthl ll followed by treatment with polymerase I Fragment by Klenow) and, after inactivation, with SacII.
• the plasmid pRPA-BCAT82 was thus obtained.
• the plasmid pRPA-BCAT41-531 Its origin of replication has been replaced by that of the plasmid pRPA-BCAT41-531 by replacing the Bstl l07I-Eaml l05I fragment of approximately 1.12 kb.
• the construction selected during this cloning, the plasmid pRPA-BCAT99 exhibits an artifact which manifests itself in the form of a deletion of a nucleotide at the Eaml 105I site, transforming this site into a single PshAI site.
• the resistance marker of the plasmid pRPA-BCAT99 was then changed by cloning between the Aatll and PshAI sites an AatlI-PshAI fragment of approximately 1.07 kb prepared after PCR amplification of the gene coding for resistance to chloramphenicol from the template.
• pACYC184 New England Biolabs # 401 -M using the primers Cml and Cm2 whose sequence is: Cml: 5'-CCCCCCGACAGCTGTCTTGCTTTCGAATTTCTGCC Cm2: 5'-TTGACGTCAGTAGCTGAACAGGAGGG
• the plasmid thus obtained was called pRPA-BCAT123. It was then modified by eliminating the trpR gene in the form of a SacI-Bstl l07I fragment of approximately 0.525 kb, and reclosing of the plasmid after blunt ends formed with Pfu polymerase (15 minutes at 75 ° C. in the buffer recommended by the supplier Stratagene and in the presence of 0.2 mM deoxynucleotides).
• the plasmid thus obtained is the plasmid pRPA-BCAT127, the map of which is shown diagrammatically in FIG. 2.
• Example 15 Construction of the plasmids pRPA-BCAT98 and pRPA-BCAT103.
• the plasmid pRPA-BCAT37 described in application FR 96/13077, was modified by replacing the Sfil-Scal fragment of approximately 3.2 kb with the Sfil-Scal fragment of approximately 2.42 kb of the plasmid RSF1010D20 (Frey et ai, 1992, Gene 113: 101-106).
• This fragment contains a deletion in the 5 ′ part of the gene coding for the RepB primase and reduces the transfer frequency of the plasmid by 6 log (Frey et ai, cited above).
• pRPA-BCAT98 has several advantages: the loss of its mobilization functions makes it conform to industrial biosecurity rules while retaining its replication characteristics in Gram-negative bacteria.
• the locus by (Gerlitz et al, 1990, J. Bacteriol 172: 6194-6203) was then cloned on pRPA-BCAT98 as follows.
• the approximately 2.3 kb Sphl-BamHI fragment of pGMA28 (Gerlitz et al, cited above) was first cloned into the vector pUC18, which made it possible to extract it in the form of a HindIII-EcoRI fragment.
• the vector pXL2426 comes from the replacement of the 2.38 kb SfiI-EcoRV fragment of pXL2391 (application FR 96/13077) with the 1.47 kb SfiI-EcoRV fragment of RSF1010D20.
• Example 16 Use of the plasmids pRPA-BCAT98, pRPA-BCAT103 and pRPA-BCAT127 for the expression of nitrilase in W.
• the plasmids pRPA-BCAT127, pRPA-BCAT98, pRPA-BCAT103, pXL2035 and pXL2231 were introduced into the strain of E. coli W by electroporation and expression cultures were carried out under the conditions described in Example 2 using the following antibiotics: Tetracycline 12 ⁇ g / ml for pXL2231, kanamycin 50 ⁇ g / ml for pXL2035, Streptomycin 100 ⁇ g / ml for pRPA- BCAT98 and pRPA-BCAT103, Chloramphenicol 20 ⁇ g / ml for pRPA-BCAT127.
• Table 7 Biomass and activities of the strains hosting the plasmids pRPA-BCAT41-531, pRPA-BCAT127, pRPA-BCAT98, pRPA-BCAT103, pXL2035 and pXL2231
• ABBREVIATIONS g / 1: gram of dry weight per liter of culture; U: kg of HMTBA formed per hour and per kg of dry weight; P: kg of HMTBA formed per hour and per liter of culture
• BCAT103 allow at least equivalent productivity by using plasmids that meet European biosecurity criteria.
• the resistance marker of the plasmid pRPA-BCAT99 described in Example 14 was changed as follows.
• the vector was opened with the enzymes PshAI and Aatll and then treated with Pfu polymerase (5 min at 75 ° C. in the buffer recommended by the supplier Stratagene and in the presence of 0.2 mM deoxynucleotides) and the fragment of approximately 3.95 kb was extracted from an agarose gel using the Quiaex kit (Quiagen) [other DNA recovery systems can also be used, in particular those of the chromatographic type].
• the plasmid obtained was named pRPA-BCATl 11. This plasmid was then opened by the enzymes Nsil and BstZ17I then treated with Pfu polymerase and religated in order to eliminate the 0.47 kb fragment carrying the trpR gene.
• the plasmid obtained was named pRPA-BCAT126, a map of which is shown in FIG. 4.
• Example 18 Construction of the Plasmid pRPA-BCAT143 The Plasmid pRPA-BCAT98 described in Example 15 was opened by the enzymes Sfil and Seal to replace the 2.42 kb fragment carrying the deletion in the 5 'part of the coding gene for the RepB primase by the 2.96 kb Sfil-Scal fragment extracted from the plasmid RSF1010 ⁇ 18 carrying a phase deletion of 267 bp in the 5 'part of the repB gene (Frey et ai, 1992, Gene 113: 101-106).
• Example 19 Use of the plasmids pRPA-BCAT126 and pRPA-BCAT143 for the expression of nitrilase in W
• BCAT143, pRPA-BCAT98, pXL2035 were introduced into the strain of E. coli W by electroporation and expression cultures were carried out under the conditions described in Example 2 using the following antibiotics: Tetracycline 12 ⁇ g / ml for pRPA-BCAT41-531 and pRPA-BCAT126, kanamycin 50 ⁇ g ml for pXL2035 , Streptomycin 100 ⁇ g / ml for pRPA-BCAT98 and pRPA-BCAT143, Chloramphenicol 20 ⁇ g / ml for pRPA-BCAT127. For each combination of plasmids, two to three clones were analyzed and the average results are presented in Table 8.
• Table 8 Biomass and activities of the strains hosting the plasmids pRPA-BCAT41-531, pRPA-BCAT126, pRPA-BCAT127, pRPA-
• ABBREVIATIONS g / 1: gram of dry weight per liter of culture; U: kg of HMTBA formed per hour and per kg of dry weight; P: kg of HMTBA formed per hour and per liter of culture
• the combinations of the plasmid pRPA-BCAT143 with one of the plasmids pRPA-BCAT41-531, pRPA-BCAT127 or pRPA-BCAT126 make it possible to maintain the productivity of the cultures produced with the strains hosting the plasmid pXL2035.

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