EP1084258A1

EP1084258A1 - Industrial method for producing heterologous proteins in e.coli and strains useful for said method

Info

Publication number: EP1084258A1
Application number: EP99923688A
Authority: EP
Inventors: Jérome Pierrard; Carole Guitton; Olivier Favre-Bulle
Original assignee: Aventis Animal Nutrition SA
Current assignee: Adisseo France SAS
Priority date: 1998-06-10
Filing date: 1999-06-08
Publication date: 2001-03-21
Also published as: IL140073A0; FR2780416B1; EA200100023A1; AU4046699A; WO1999064607A1; FR2780416A1; AU753879B2; JP2002517252A; CA2331150A1

Abstract

The invention concerns an industrial method for preparing heterologous proteins in E.coli, which consists in seeding and cultivating in an appropriate culture medium E.coli bacteria modified with an appropriate system for expressing heterologous proteins, characterised in that the E.coli strain is an E.coli W strain.

Description

INDUSTRIAL PROCESS FOR PRODUCING HETEROLOGOUS PROTEINS IN E. COLI AND STRAINS USEFUL FOR THE PROCESS

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.

The strains of E. coli most commonly used for molecular biology work are derived from the strain Kl 2 (Swartz, 1996, In Εscherichia coli and Salmonella, Cellular and Molecular Biology, 2 ^nd edition, ASM Press Washington, ppl693-1711). Derivatives of E. coli B, such as BL21, are also used for the production of proteins because of their physiological properties. 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. Εd, John Wiley & Sons, Inc, 5.0.1-5.0.3).

Numerous protein expression systems in bacterial hosts have been described (Makrides, 1996, Microbiol. Rev. 60: 512-538; Current Opinions in Biotechnology, 1996, 7). 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):

- it must be strong and lead to the accumulation of the protein of interest which can represent 10 to 50% of the total proteins of the host cell;

- it must be able to be regulated so as to be able as far as possible to decouple the biomass production phase from the protein production phase; - it must be inducible (transition from a level of low transcriptional activity to a maximum level of transcriptional activity) with simple and inexpensive process conditions.

Many promoters have been described for expression in E. coli (Makrides, 1996, supra; Weickert et al, 1996, Current Opinions in Biotechnology 7: 494-499). Among the 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. Among the heterologous promoters used for the production of proteins in E. coli, mention may be made of the promoters PL, PL-9G-50,? R-? L, T7, XPL- PT7, T3-lac, T5-lac, T4 gene 32, nprM-lac, VHb, Protein A. A number of drawbacks are linked to these promoters. We can cite for some of them the use of IPTG as an inducing molecule, the price of which can represent more than 14% of the cost of the medium. Others use temperature regulation, which is difficult to implement on the scale of an industrial fermenter of 100 m ³ .

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. This interaction is modulated by the ROP protein whose gene is present on pBR322 but not on certain derivatives such as plasmids of the pUC type (Lin-Chao and Cohen, 1991, Cell 65: 1233-1242). In terms of regulating the copy number of the expression plasmid in E. coli, several strategies are cited (Swartz, 1996, supra; Makrides, 1996, supra). j

In particular we note ^that 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. This locus has been characterized (Leung et ai, 1985, DNA 4: 351-355) and its insertion on multi-copy plasmids has been described as having a beneficial effect on the stability of these plasmids (Summers and Sherratt, 1984, Cell 36: 1097-1103).

If the above strains and expression systems make it possible to obtain good yields of production of heterologous proteins, their use remains limited to the production of heterologous proteins with very high added value for which the cost price of the production system (bacterial strain, medium and culture conditions, raw materials) is minimal compared to the value of the protein produced. As examples of such proteins with very high added value, there are more particularly heterologous proteins intended for pharmaceutical use such as, for example, human growth hormone, consensus human interferon alpha, human interleukins lβ, αl, 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).

However, for the production of mass chemical intermediates (Lee, 1997, Nature Biotech. 15: 17-18) or for the production of enzymes for industrial use, in particular the catalysts necessary for the production of chemical compounds, the price of returns from the production system becomes a dominant factor to be taken into account to assess the technical interest of said system.

For the production of heterologous proteins in bacteria, the productivity of the culture system employed can be significantly increased using high cell density culture strategies (S. Makrides, 1996, supra; Wingfield, 1997, supra). Among these is the fed-batch strategy (Jung et ai, 1988, Ann. Inst. Pasteur / Micrbiol. 139: 129-146; Kleman et ai, 1996, Appl. Environ. Microbiol. 62: 3502-3507; Lee, 1996, supra; Bauer and White. 1976, Biotechnol. Bioeng. 18: 839-846). This strategy, combined with the use of a P _tr p promoter, made it possible to achieve significant productivities: 55 g of dry weight per liter and 2.2 g of heterologous protein per liter (Jung et ai, 1988, cited above ). Routine production of 35 to 50 g dry weight per liter is reported (Wingfield, 1997, cited above).

However, the above strains and systems do not make it possible to obtain sufficient culture densities for the industrial production of heterologous proteins whose value (cost) must be negligible with regard to their purpose (in particular for the preparation of catalysts organic). 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.

This strain W (ATCC 9637) is well known and described in numerous publications (Davies & Mingioli, 1950, J. Bact., 60: 17-28; Doy and Brown, 1965, Biochim. Biophys. Acta, 104: 377 -389; Brown and Doy, 1966, Biochim. Biophys. Acta, 118: 157-172; Wilson & Holden, 1969, J. Biol. Chem., 244: 2737-2742; Wilson & Holden, 1969, J. Biol. Chem., 244: 2743-2749; White, 1976, J. Gen. Microbiol., 96: 51-62; Shaw & Duncombe, 1963, Ananlyst 88: 694-701; Br. Pharmacopoeia, 1993, 2: A164-A169 ; Huang et al, US 3,088,880; Hamsher et al, US 3,905,868; Takahashi et a, US 3,945,888; Huang et al, US 3,239,427; Burkholder, 1951, Science, 114: 459-460; Prieto et al, 1996, J. Bact , 178: 11-120; Lee 1996, supra; Lee & Chang, 1995, Can. J. Microbiol. 41: 207-215; Lee et al., 1994, Biotechnol. Bioeng., 44: 1337-1347; Lee & Chang, 1993, Biotechnology Letters, 15: 971-974; Bauer and White, 1976, supra; Bauer and Shiloach, 1974, Biotechnol. Bioeng 16: 933-941; Gleiser and Bauer, 1981, Biotec hnol. Bioeng., 23: 1015-1021; Lee and Chang, 1995, Advances in Biochem. Εngine. / Biotech. 52: 27-58). 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 W strain has also been used in high cell density cultures (Bauer and White, 1976, supra; Bauer and Shiloach, 1974, supra; Gleiser and Bauer, 1981, supra; Lee and Chang, 1993, supra; Lee et al., 1997, Biotechnology Techniques 11: 59-62). Biomasses of 125 g dry weight per liter have thus been obtained (Lee and Chang, 1993, cited above) using sucrose as the carbon source.

However, this strain has never been described for the production of recombinant proteins. In addition, by combining a plasmid carrying the operon of Alcaligenes eutrophus encoding enzymes involved in the biosynthesis of PHA and a strategy of high cell density culture of the corresponding recombinant W strain, Lee and Chang (1993, cited above) obtained poorer PHA productivity than with an XL1-Blue strain derived from the Kl 2 strain (Lee and Chang, 1995, cited above; Lee, 1996, cited above).

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.

According to a particular embodiment of ^the invention, the strain W is a derivative of the strain deposited at the ATCC under number 9637 obtained by clonal selection or genetic manipulation. By 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 (defined, complex and semi-defined) 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

productivity of culture (Lee, 1996, supra) the development of such an environment, however, requires empirical optimization to take into account the economic constraints previously stated (Lee, 1996, supra).

According to a preferred embodiment of ^the invention, the culture medium contains sucrose as a main carbon source. By 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. According to a more preferential embodiment of the invention, 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.

Advantageously, 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.

According to an advantageous embodiment of the invention, 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. Preferably, 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. The expression “appropriate 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.

Advantageously, the expression system comprises an R ^ - _n promoter. The promoter P { _r p was used in several examples (Application ΕP 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). In particular, 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. Their reference construction, pXL534, served as the basis for the construction of pXL642 (CIP Application No. 08 / 194,588) used in the examples which illustrate the present invention. Preferably, the promoter P _tr p comprises the nucleic acid sequence represented by the sequence identifier No. 1 (SEQ ID NO 1).

According to one embodiment of the invention, to improve the level of expression of the heterologous protein, coexpression of the molecular chaperones of E. coli GroΕSL (reviewed by Makrides, 1996, cited above). Increasing the intracellular concentration of GroΕSL 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. By 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.

Of course, 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. Advantageously, the heterologous protein is an enzyme, produced in industrial quantities for later use as a catalyst for chemical reactions. According to a particular embodiment of the invention, ^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.

The examples below illustrate the present invention, without however seeking to limit its scope.

Figures 1 to 3 in the appendix represent maps of plasmids used in the various examples. 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;

Frey et al, 1992, Gene 113: 101-106); delta refers to the name of the deletion described in the text.

The techniques used are conventional techniques of molecular biology and microbiology, known to those skilled in the art and described for example by Ausubel et ai, 1987 (Current Protocols in Molecular Biology, John Willey and Sons, New York) , Maniatis et ai, 1982, (Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Sring Harbor, New York), Coligan et ai, 1997 (Current Protocols in Protein Science, John Wiley & Sons, Inc).

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 λ (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. The resulting plasmid, 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 1.2 kb EcoRI-Xbal fragment from pRPA-BCAT19 containing the P ^ p -: RBScII:: nitB fusion was then cloned into the vector pRPA-BCAT28 opened by the same enzymes to yield the plasmid pRPA-BCAT29 of 6.2 kb. 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 pHP45ΩTc (Fellay et ai, 1987, Gene 52: 147-154) to replace the ampicillin resistance marker with the tetracycline resistance marker. By destroying the Ndel site close to the origin of replication of the plasmid pRPA-BCAT29 by partial digestion Ndel and action of Polymerase I of E. coli (Klenow fragment), 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 (SΕQ ID NO 2) .

Example 2: Expression of nitrilase from A. faecalis ATCC 8750 in E. coli K12, BL21, W in "batch".

The plasmids pRPA-BCAT29 and pXL2035 (Levy-Schill et ai, 1995, Gene 161: 15-20) were introduced into the strains of E. coli DH5α (CLONΕCH, Product reference C1021-1), BL21 (Novagen. product reference 69386-1) and W (ATCC9637) by conventional electroporation. Expression cultures were carried out as described in Example 5 of application FR 96/13077 by reducing the preculture time to 8 hours and by setting the expression time to 16 hours. The biomasses after expression were estimated from the optical density of the cultures read at 660 nm (DO660) using the following relationship: biomass in grams of dry weight per liter of culture = DO660 x

0.35. The nitrilase activity measurements of the cultures were carried out as described in application FR 96/13077. For each strain, two clones were analyzed and for each clone, the experiment was repeated. Table 1 contains for each strain the average of the data obtained in the four experiments.

Table 1: Biomass and activities of the strains hosting the plasmids pRPA-BCAT29 and pXL2035

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 These data show that the strain of E. coli W (ATCC9637) is more efficient at expressing nitrilase NitB.

Example 3: Construction of pBCAT43.

The polyamide hydrolase gene from Comamonas acidovorans N12 described in application WO 97/04083 (pamll) was cloned into the vector pBCAT41. By introducing into the PCR primers the EcoRI and Ncol restriction sites in position

5 ′ of the gene and Xbal in position 3 ′, 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. After ligation of these two samples, the plasmid pRPA-BCAT43 was obtained: it 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 DH5α, BL21 and W in "batch". The plasmid pRPA-BCAT43 was introduced into the strains of E. coli DH5α,

L21 and W by conventional electroporation. Expression cultures were carried out as described in Example 2 above and varying the expression time from 14 to 24 hours. The biomasses after expression were estimated as in Example 2 above. The polyamide hydrolase activity measurements of the cultures were carried out as described in application WO 97/04083 with the following modifications: the cells were permeabilized with toluene by resuspending the cell pellets in a lOOmMtrs-HCl buffer, 5 mM EDTA pH8 , toluene 1% so as to have a dry cell concentration of approximately 5 g / 1; after vigorous stirring, the suspension is incubated for one hour at 4 ° C. then centrifuged and finally 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.

For each strain, from 1 to 24 clones were analyzed and for each clone, one to seven independent experiments were carried out. Table 2 contains the average of the data obtained for each strain.

Table 2: Biomass and activities of the strains hosting the plasmid pRPA-BCAT43

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

These data show that the strain of E. coli W (ATCC9637) is more efficient in expressing Pamll polyamidase. Example 5: Construction and characterization of the plasmid pBCAT41-531.

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. After adding an identical volume of 50 mM sodium phosphate buffer pH6 containing 0.5 mM EDTA, 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. Among the transformants obtained, a clone was selected because of a productivity of the culture 3 times higher than that of a culture of the strain DH5α (pRPA-BCAT41, pXL2035). The plasmid pRPA-BCAT41-531 which it housed was extracted and reintroduced into a new DH5α host hosting the plasmid pXL2035. Three clones were then analyzed under the conditions described in Example 2 by comparing them to 3 DH5α clones (pRPA-BCAT41, pXL2035) and the results are presented in the table in Table 3.

Table 3: Biomass and activities of the strains hosting the plasmids pRPA-BCAT41, pRPA-BCAT41-531 and pXL2035

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.

These results indicate that the improvement in the productivity of the cultures is correlated with the presence of the plasmid pRPA-BCAT41-531.

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.

Table 4: Biomass and activities of the strains hosting the plasmids pRPA-BCAT41, pRPA-BCAT41-531, pRPA-BCAT86 and pXL2035

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 results show that the improvement in the productivity of the cultures harboring pRPA-BCAT41-531 is not due to an improvement in the specific activity of the strain and that this improvement is not caused by a mutation in the fragment carrying the Ptrp promoter and the nitB gene.

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 sequencing of the 994 bp region of pRPA-BCAT41-531 which extends from the Tthl ll site and which covers the origin of replication of the plasmid revealed two differences with respect to the sequence of the corresponding region of pBR322 (GeneBank # J01749, name: SYNPBR322). Referring to the numbering given in the sequence J01749 (0 is the middle of the single EcoRI site), we found that an insertion of an A had taken place after the base 2319 and that the C of position 3039 is replaced by a T at pRPA-BCAT41-531. 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). It is this latter mutation which is responsible for the lower number of copies of pRPA-BCAT41-531 in DH5 and which is responsible for the better nitrilase productivity of the cultures of the strain DH5α (pRPA-BCAT41-531, pXL2035).

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.

After 24 hours of fermentation, 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.

It is clear in this example that nitrilase is expressed much better in E. coli W than in E. coli BL21 and that the recombinant E. coli W BIOCAT 714 grows much better than the recombinant E. coli BL21 BIOCAT 594.

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:

To this medium, an organic nitrogen source of animal origin is added.

The use of increasing concentration of organic nitrogen of animal origin significantly increases the specific activity of cells.

Example 9 Influence of organic nitrogen of vegetable origin.

The culture conditions are identical to those of Example 8. In this example, organic nitrogen of vegetable origin is added.

The addition of vegetable organic nitrogen does not give identical results depending on the origin.

Surprisingly, the addition of potato protein gives as good a result as organic nitrogen of animal origin.

Example 10: influence of the carbon source.

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.

In this example, it is observed that the use of sucrose (zero syrup) as a carbon source significantly increases the specific activity of the cells.

Example 11 Construction of a TrpR Regulator Co-Expression Plasmid

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 DH5α (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.

Table S: Biomass and activities of the strains hosting combinations of the plasmids pRPA-BCAT29, pXL2035 and pRPA-BCAT34

ABBREVIATIONS: U: Activity, kg of HMTBA formed per hour and per kg of dry weight; P: Productivity, kg of HMTBA formed per hour and per liter of culture

The results show that the coexpression of GroESL makes it possible to increase the productivity of the cultures whatever the strain considered by improving the specific activity of the cultures. This effect is correlated with an increase in the solubility of the nitrilase polypeptide, as shown by an analysis of the proteins by electrophoresis as described in application FR 96/13077. The effect of the coexpression of the TrpR regulator is variable according to the strains but allows W to improve the productivity of the cultures. Example 13 Influence of the presence of a cer locus on pRPA-BCAT41

The 382 bp Hpall fragment containing the cer locus of the plasmid ColEl (Leung et ai, 1985, DNA 4: 351-355) was cloned in the replicative form of phage M13mp7 at one of the 2 Accl sites. The construction obtained then made it possible to extract with the enzyme EcoRI a fragment of approximately 430 bp containing the cer locus which was cloned in pRPA-BCAT41 at the EcoRI site, which led to the plasmid pRPA-BCAT66. This plasmid was introduced by electroporation into the strain W hosting the plasmid pRPA-BCAT34. Expression cultures of different strains were carried out as described in Example 2 by extending the duration of the expression cultures to 24 hours and by studying three clones of each strain in a single experiment. The average results are presented in Table 6.

Table 6: Biomass and activities of the strains hosting the plasmids pRPA-BCAT41, pRPA-BCAT66 and pRPA-BCAT34

Biomass strains Productivity Activity

(g / 1) (U) (P)

W (pRPA-BCAT41, pRPA-BCAT 34) 2.1 6.9 14.5

DH5α (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

These results show that the addition of the cer locus to the nitrilase expression plasmid leads to improving the productivity of the cultures.

Example 14 Construction of the Plasmid pRPA-BCAT127

After elimination of the unique NdeI site from the plasmid pRPA-BCAT30 by digestion and blunt ends with polymerase I (Klenow fragment), 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. 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). The plasmid thus obtained, 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. to clone it into the vector pMTL22 (Chambers et ai, 1988, Gene 68: 139-49). The HindIII site was then destroyed by HindIII digestion and Klenow treatment. A fragment of about 2.38 kb was then extracted with the enzymes PstI and BglII to be cloned in the vector pXL2426 at the PstI and BamHI sites and lead to the vector pXL2572. 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. The cloning on the plasmid pXL2572 at the Ndel and BamHI sites of a fragment of approximately 0.960 bp Ndel-BamHI from pRR71 (Weinstein et al. 1992, J. Bacteriol. 174: 7486-7489) made it possible to reconstitute the locus by whole on plasmid pXL2573. This locus was then extracted from pXL2573 in the form of a 2.6 kb EcoRI-blunt end fragment (after treatment with PstI and Klenow) in order to be cloned on the plasmid pRPA-BCAT98 opened by EcoRI and Sacl, this last end having been treated with Pfu polymerase. The resulting plasmid was called pRPA-BCAT103 and its map is shown schematically in Figure 3.

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 (application FR 96/13077) 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 μg / ml for pXL2231, kanamycin 50 μg / ml for pXL2035, Streptomycin 100 μg / ml for pRPA- BCAT98 and pRPA-BCAT103, Chloramphenicol 20 μg / ml for pRPA-BCAT127. For each combination of plasmids, two to three clones were analyzed and the average results are presented in Table 7. Table 7: Biomass and activities of the strains hosting the plasmids pRPA-BCAT41-531, pRPA-BCAT127, pRPA-BCAT98, pRPA-BCAT103, pXL2035 and pXL2231

The combinations pRPA-BCAT127 / pRPA-BCAT98 and pRPA-BCAT127 / pRPA-

BCAT103 allow at least equivalent productivity by using plasmids that meet European biosecurity criteria.

Example 17 Construction of the Plasmid pRPA-BCAT126

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]. It was ligated according to a conventional method with the 1.32 kb HindIII-Bsml fragment extracted from the plasmid pBR322 (New England Biolabs, ref 303-3S) and then treated as above with the Pfu polymerase. Among the plasmids obtained, the plasmid containing the insert carrying the tetracycline resistance gene oriented in the same direction of transcription as the expression cassette of the nitrilase 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 RSF1010Δ18 carrying a phase deletion of 267 bp in the 5 'part of the repB gene (Frey et ai, 1992, Gene 113: 101-106). The deletion introduced on pRPA-BCAT143 reduces the frequency of transfer of the plasmid to 10 ^"6 (Frey et ai, 1992, Gene 113: 101-106) and makes it comply with the requirements of the biosafety rules. Unlike the plasmid pRPA6BCAT98 described above, this new plasmid retains a copy number close to the unmodified plasmid pXL2035 (Lévy-Schill et al., 1995, Gene 161: 15-20), and is shown in FIG. 2.

Example 19: Use of the plasmids pRPA-BCAT126 and pRPA-BCAT143 for the expression of nitrilase in W The plasmids pRPA-BCAT126, pRPA-BCAT127 (described above), pRPA-

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 μg / ml for pRPA-BCAT41-531 and pRPA-BCAT126, kanamycin 50 μg ml for pXL2035 , Streptomycin 100 μg / ml for pRPA-BCAT98 and pRPA-BCAT143, Chloramphenicol 20 μg / 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-

BCAT98, pRPA-BCAT143, pXL2035.

Unlike the plasmid pBCAT98, 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.

Claims

1. Industrial process for the preparation of heterologous proteins in E coli, in which seeds and cultivated in an appropriate culture medium are cultivated E coli bacteria modified with an appropriate heterologous protein expression system, characterized in that the strain of E . coli is an E. coli W. strain.

2. Method according to claim 1, characterized in that the strain W is the strain W deposited at the ATCC under the number 9637.

3. Method according to claim 1, characterized in that the strain W is a derivative of the strain deposited at the ATCC under the number 9637 obtained by clonal selection or genetic manipulation.

4. Method according ^to one of claims 1 to 3, characterized in that the suitable culture medium is a culture medium suitable for obtaining a high density of biomass and a high content produced heterologous proteins.

5. Method according to one of claims 1 to 4, characterized in that the culture medium comprises L-tryptophan.

6. Method according to claim 5 characterized in that the amount of L-tryptophan in the culture medium is between 0.05 and 0.5 g / 1, preferably between 0.1 and 0.3 g / 1

7. Method according to one of claims 1 to 6, characterized in that the culture medium comprises sucrose as the main source of carbon.

8. Method according to claim 7, characterized in that the culture medium comprises substantially only sucrose as carbon source.

9. Method according to one of claims 7 or 8, characterized in that the amount of sucrose in the culture medium is between 0.1 and 300 g / 1 at the start of culture, preferably between 0.5 and 200 g / 1.

10. Method according to one of claims 1 to 9, characterized in that the appropriate culture medium further comprises a source of additional organic nitrogen.

1 1. Method according to claim 10, characterized in that the source of additional organic nitrogen consists of protein extracts.

12. Method according to one of claims 9 or 10, characterized in that the protein extract has the following composition: (in g amino acids per 100g of product) Alanine between 10 and 4, Aspartic between 1 1 and 4, Glycine between 22 and 2.5 and Lysine between 7 and 4

13. Method according to one of claims 10 to 12, characterized in that the source of complementary organic nitrogen consists of peptones or proteins of meat or potato, more particularly derivatives of protein from apple, potato .

14. Method according to one of claims 1 to 13, characterized in that the appropriate heterologous protein expression system comprises a promoter tr -

15. The method of claim 14, characterized in that the trp promoter comprises the nucleic acid sequence represented by the sequence identifier No. 1 (SEQ ID NO 1).

16. Method according to one of claims 1 to 15, characterized in that the heterologous protein is an enzyme.

17. The method of claim 16, characterized in that the enzyme is useful for the biocatalysis of chemical reactions.

18. The method of claim 17, characterized in that the enzyme is a nitrilase.

19. E. coli W strain, characterized in that it comprises a system of expression of heterologous proteins whose promoter is the promoter

Ptrp- 20. A strain according to claim 19, characterized in that the tr promoter comprises the nucleic acid sequence represented by the sequence identifier No. 1 (SEQ ID NO 1).