AU4046699A

AU4046699A - Industrial method for producing heterologous proteins in E.coli and strains useful for said method

Info

Publication number: AU4046699A
Application number: AU40466/99A
Authority: AU
Inventors: Olivier Favre-Bulle; Carole Guitton; Jerome Pierrard
Original assignee: Rhone Poulenc Nutrition Animale SA
Current assignee: Adisseo France SAS
Priority date: 1998-06-10
Filing date: 1999-06-08
Publication date: 1999-12-30
Anticipated expiration: 2019-06-08
Also published as: EA200100023A1; EP1084258A1; AU753879B2; IL140073A0; CA2331150A1; FR2780416A1; FR2780416B1; JP2002517252A; WO1999064607A1

Description

WO 99/64607 PCT/FR99/01343 1 INDUSTRIAL PROCESS FOR PRODUCING HETEROLOGOUS PROTEINS IN E. COLI AND STRAINS USEFUL FOR SAID PROCESS The present invention relates to a novel 5 industrial process for producing heterologous proteins in E. coli. While for certain heterologous proteins with very high added value the cost price of the process for preparing them remains a factor which is negligible with compared to the purpose of the 10 heterologous protein (in the pharmaceutical domain in particular), the development of the industrial production of heterologous proteins of lower added value in E. coli involves taking into account production factors such as the necessity of having an 15 increased biomass and a very high content of heterologous proteins produced for the lowest possible cost, which cost should take account of the nature of the media, of the energetic and reagent yield and of the operating conditions. For industrial productions 20 using reaction volumes which can reach several dozens of m 3 , the simplest possible media and operating conditions will be sought. The present invention consists of the selection of an E. coli strain suitable for satisfying the conditions above, which are 25 essential for economically satisfactory industrial REPLACEMENT SHEET (RULE 26) 1 'rT 6~ WO 99/64607 PCT/FR99/01343 2 production of heterologous proteins, independently of the value of the protein produced. The strains of E. coli most commonly used for molecular biology studies derive from the strain K12 5 (Swartz. 1996, In Escherichia coli and Salmonella, Cellular and Molecular Biology, 2 nd edition, ASM Press Washington, pp. 1693-1711). Derivatives of E. coli B, such as BL21, are also used for producing proteins, because of their physiological properties. A table of 10 the strains most commonly used for producing recombinant proteins is given by Wingfield, 1997 (Current Protocols in Protein Science, Coligan et al. Ed. John Wiley & Sons, Inc. 5.0.1-5.0.3). Many systems for expressing proteins in 15 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, of its regulator, of a ribosome binding site followed by a restriction site which allows the 20 insertion of the gene of interest, of a structure which can be used as a transcription terminator, optionally of genes the coexpression of which increases the quality of the protein of interest overexpressed, and of one or more vectors which make it possible to 25 introduce these combinations into the host. ipAL REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 3 The promoter must have at least three properties in order to be used in a process for producing proteins (Makrides, 1996, mentioned above): - it must be strong and cause the accumulation of the 5 protein of interest, which can represent 10 to 50% of the total proteins of the host cell; - it must be capable of being regulated so as to be able, as far as possible, to uncouple the biomass production phase from the protein production phase; 10 - it must be inducible (passage from a level of low transcriptional activity to a maximum level of transcriptional activity) using simple and inexpensive process conditions. Many promoters have been described for expression in 15 E. coli (Makrides, 1996, mentioned above; Weickert et al., 1996, Current Opinions in Biotechnology 7 : 494 499). Among the homologous promoters used for producing proteins in E. coli, mention may be made of the lac, trp, lpp, phoA, recA, araBAD, proU, cst-1, tetA, cadA, 20 nar, tac, trc, lpp-lac, Psyn and cspA promotors. Among the heterologous promoters used for producing proteins in E. coli, mention may be made of the PL, PL-9G-50, PR-PL, T7, XPL-PT7, T3-lac, T5-lac, T4 gene 32, nprM lac, VHb and Protein A promoters. A certain number of 25 drawbacks are linked to these promoters. For some of REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 4 them, mention may be made of the use of IPTG as the inducer molecule, the price of which can represent more than 14% of the cost of the medium. Others use regulation by temperature, which is difficult to 5 implement on the scale of a 100 m 3 industrial fermenter. The vectors most commonly used for expressing proteins in E. coli derive from the plasmid pBR322 (Swartz, 1996, mentioned above; Makrides, 1996, mentioned above). They are present in cells at a 10 certain copy number, which is determined by the interaction of two RNAs encoded by the plasmid, RNAI and RAII (Polisky, 1988, Cell 55 : 929-932). The interaction of RNAI with RNAII inhibits the maturation of RNAII into a form required for the initiation of the 15 replication of the plasmid. This interaction is modulated by the protein ROP, the gene of which is present on pBR322 but not on certain derivatives, such as the pUC-type plasmids (Lin-Chao and Cohen, 1991, Cell 65 : 1233-1242). With regard to regulation of the 20 number of copies of the expression plasmid in E. coli, several strategies are mentioned (Swartz, 1996, mentioned above; Makrides, 1996, mentioned above). It will be appreciated in particular that a high number of copies of expression plasmid leads to a high level 25 of messenger RNAs of the desired protein, but can be 4AL REPLACEMENT SHEET (RULE 26) ujj Zc- WO 99/64607 PCT/FR99/01343 5 detrimental to the metabolism of the host cell (Bailey, 1993, Adv. Biochem. Eng. Biotechnol. 48 : 29-52). The stability of the expression plasmids is an important criterion, all the more so given that 5 industrial fermentations tend not to use antibiotics in the fermenters. Several strategies have been developed to stabilize expression plasmids, including the cloning of the cer locus of the natural plasmid ColEl. This locus has been characterized (Leung et al., 1985, 10 DNA 4 : 351-355) and its insertion into multicopy plasmids has been described as having a beneficial effect on the stability of these plasmids (Summers and Sherratt, 1984, Cell 36 : 1097-1103). While the strains and expression systems 15 above make it possible to obtain good heterologous protein production yields, 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, culture medium and 20 conditions, raw materials) is minimal compared with the value of the protein produced. As examples of such proteins with very high added value, there are more particularly the heterologous proteins intended for pharmaceutical use, such as for example human growth 25 factor, human alpha consensus interferon, human AL/4 REPLACEMENT SHEET (RULE 26) Wi

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WO 99/64607 PCT/FR99/01343 6 interleukins 10, al and 2, human leukocyte interferon, human parathyroid hormone, human insulin, human serum albumin or human proapolipoprotein A-1 (Lee, 1996, Trends in Biotechnol. 14:98-105; Latta et al., 1987, 5 Bio/Technology 5 : 1309-1313). However, for the mass production of chemical intermediates (Lee, 1997, Nature Biotech. 15 : 17-18) or for the production of enzymes for industrial use, in particular of the catalysts required for producing 10 chemical compounds, the cost price of the production system becomes a dominant factor to be taken into consideration in order to evaluate the technical advantage of said system. For the production of heterologous proteins 15 in bacteria, the productivity of the culture system employed can be significantly increased by using high cell density culturing strategies (S. Makrides, 1996, mentioned above; Wingfield, 1997, mentioned above). Among these is the fed-batch strategy (Jung et al., 20 1988, Ann. Inst. Pasteur/Microbiol. 139 : 129-146; Kleman et al., 1996, Appl. Environ. Microbiol. 62 3502-3507; Lee, 1996, mentioned above; Bauer and White, 1976, Biotechnol. Bioeng. 18 : 839-846). This strategy, combined with the use of a Ptrp promoter, has made it 25 possible to achieve significant productivities: 55 g of REPLACEMENT SHEET (RULE 26) r7 LU 0~rrO WO 99/64607 PCT/FR99/01343 7 dry weight per liter, and 2.2 g of heterologous protein per liter (Jung et al., 1988, mentioned above). Routine productions of 35 to 50 g of dry weight per liter are reported (Wingfield, 1997, mentioned above). 5 However, the strains and systems above do not make it possible to obtain culture densities which are sufficient for the industrial production of heterologous proteins for which the value (cost price) must be negligible compared to their purpose (in 10 particular for the preparation of biological catalysts). The present invention lies in the selection of a specific strain of E. coli, which is suitable for the industrial production of heterologous proteins. The 15 strain which is useful for the process according to the invention is an E. coli strain W, more particularly the strain W referenced at the ATCC under the number 9637. This strain W (ATCC 9637) is well known, and described in many publications (Davies & Mingioli, 20 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, 25 1976, J. Gen. Microbiol., 96: 51-62; Shaw & Duncombe, REPLACEMENT SHEET (RULE 26) 00 WO 99/64607 PCT/FR99/01343 8 1963, Analyst 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 al., US 3,945,888; Huang et al., US 3,239,427; Burkholder, 1951, Science, 114: 5 459-460; Prieto et al., 1996, J. Bact., 178: 11-120; Lee 1996, mentioned above; 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, 10 1976, mentioned above; Bauer and Shiloach, 1974, Biotechnol. Bioeng 16: 933-941; Gleiser and Bauer, 1981, Biotechnol. Bioeng., 23: 1015-1021; Lee and Chang, 1995, Advances in Biochem. Engine./Biotech. 52: 27-58). The strain W (ATCC9637) has thus been used 15 for the production of 3-polyhydroxybutyric acid (PHA) after introduction of a plasmid carrying the operon of Alcaligenes eutrophus encoding enzymes involved in the PHA biosynthesis (Lee and Chang, 1993, mentioned above; Lee and Chang, 1995, mentioned above; Lee et al., 20 1994). The strain W has also been used in high cell density cultures (Bauer and White, 1976, mentioned above; Bauer and Shiloach, 1974, mentioned above; Gleiser and Bauer, 1981, mentioned above; Lee and 25 Chang, 1993, mentioned above; Lee et al., 1997, REPLACEMENT SHEET (RULE 26) wJ WO 99/64607 PCT/FR99/01343 9 Biotechnology Techniques 11: 59-62). Biomasses of 125 g of dry weight per liter have thus been obtained (Lee and Chang, 1993, mentioned above) using sucrose as a carbon source. 5 However, this strain has never been described for the production of recombinant proteins. Furthermore, in combining a plasmid carrying the operon of Alcaligenes eutrophus encoding enzymes involved in PHA biosynthesis and a strategy of culturing the 10 corresponding recombinant strain W at high cell density, Lee and Chang (1993, mentioned above) obtained worse PHA productivity than with a strain XLl-Blue derived from the strain K12 (Lee and Chang, 1995, mentioned above; Lee, 1996, mentioned above). 15 The present invention relates, therefore, to an industrial process for preparing heterologous proteins in E. coli, in which E. coli bacteria modified with a suitable system for expressing heterologous proteins are seeded and cultured in a suitable culture 20 medium, characterized in that the strain of E. coli is an E. coli strain W. More preferably, the strain W is the strain W deposited at the ATCC under the number 9637. According to one particular embodiment of the 25 invention, the strain W is a derivative of the strain REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 10 deposited at the ATCC under the number 9637, obtained by clonal selection or genetic manipulation. According to the invention, the term "industrial process" is intended to mean any process in 5 which the bacterial culture volume is greater than the usual culture volume employed in research laboratories. Generally, the term "industrial process" is intended to mean any process for which the culture volume is greater than 2 liters, preferably greater than or equal 10 to 10 liters, more preferably greater than or equal to 20 liters, even more preferably greater than or equal to 50 liters. The process according to the invention is particularly suitable for culture volumes from several dozens of M 3 up to more than 100 M 3 . 15 The suitable culture medium is a culture medium which is suitable for the production of a high density of biomass and a high content of heterologous proteins produced. Several types of medium (defined, complex and semidefined) can be used for high cell 20 density culturing (Lee, 1996, mentioned above). While the known media of the prior art, and in particular semidefined media, make it possible to accumulate good reproducibility of the composition of the medium and good productivity of the culture (Lee, 1996, mentioned 25 above), the development of such a medium requires, ALQq, REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 11 however, empirical optimization for taking into account the economic constraints set out previously (Lee, 1996, mentioned above). According to one preferential embodiment of 5 the invention, the culture medium comprises sucrose as the main carbon source. According to the invention, the expression "main carbon source" is intended to mean that the sucrose represents at least 50% by weight of the total weight of the carbon sources of the culture 10 medium, more preferably at least 75% by weight, even more preferably at least 85% by 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 process 15 according to the invention, the culture medium can comprise suitable additives so as to increase the overall yield of the invention. These additives can have the ancillary function of behaving as a carbon source to the bacterial culture. However, these 20 additives will not be considered as a carbon source for the purpose 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 sole carbon source. REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 12 Advantageously, the amount of sucrose in the culture medium of the process according to the invention is between 0.1 and 300 g/l at the start of culturing, (before seeding), preferably between 0.5 and 5 200 g/l. It is understood that, since the sucrose constitutes the main carbon source of the medium according to the invention, the amount of sucrose will be decreasing during the process. In general, at the end of the reaction, the amount of sucrose in the 10 culture medium at the end of the reaction is between 0 and 10 g/l. According to one advantageous embodiment of the invention, the suitable culture medium also comprises a supplementary organic nitrogen source. This 15 supplementary organic nitrogen source can consist of all organic nitrogen sources known to a person skilled in the art. Preferably, the supplementary organic nitrogen source consists of protein extracts. These protein extracts have more preferably the following 20 composition: (in g amino acids per 100 g of product) alanine between 10 and 4, aspartic [lacuna] between 11 and 4, glycine between 22 and 2.5 and lysine between 7 and 4. Meat or potato peptones or proteins satisfy such a profile, is/are particularly preferred for the L/ REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 13 particularly the derivatives of potato proteins are preferred. According to the invention, the expression "suitable system for expressing heterologous proteins" 5 is intended to mean any expression system comprising regulation elements suitable for the expression of heterologous proteins in E. coli W. These regulation elements comprise in particular promoters, ribosome binding sites and transcription terminators. 10 Advantageously, the expression system comprises a Ptp promoter. The Ptrp promoter has been used in several examples (EP Application 0 198 745; CIP Application No. 08/194,588; Application WO 97/04083; Latta et al., 1987, Bio/Technology 5: 1309-1314; 15 Denefle et al., 1987, Gene 56: 61-70). In particular, Latta et al. (1990, DNA Cell. Biol. 9: 129-137) have conducted a detailed study on the influence of regulatory sequences upstream of the promoter, and of tandem-duplicated promoter sequences, and on the 20 influence of the coexpression of the TrpR repressor. Their reference construct, pXL534, was used as a basis for the construction of pXL642 (CIP Application No. 08/194,588), used in the examples which illustrate the present invention. Preferably, the Ptrp promoter c REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 14 comprises the nucleic acid sequence represented by sequence identifier No. 1 (SEQ ID NO 1). According to one embodiment of the invention, in order to improve the level of expression of the 5 heterologous protein, a coexpression of the molecular chaperones of E. coli GroESL (review by Makrides, 1996, mentioned above) is carried out. The increase in the intracellular concentration of the GroESL proteins makes it possible, in effect, to assist the folding of 10 the recombinant protein and thus improve 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 15 included in the expression system according to the invention. According to the invention, the term "heterologous protein" is intended to mean any protein produced by the process according to the invention 20 which is not naturally found in E. coli W, in the suitable expression system according to the invention. It can be a protein of nonbacterial origin, for example of animal, in particular human, or plant origin, or a protein of bacterial origin which is not naturally 25 produced by E. coli W, or a protein of bacterial origin REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 15 naturally produced by a bacterium other than E. coli W or a protein naturally produced by E. coli W, the expression of which is controlled by regulation elements different from those of the expression system 5 according to the invention, or finally, a protein which derives from the preceding ones after modification of certain elements of its primary structure. Of course, the process according to the invention applies to any protein of interest the 10 production of which requires a great accumulation of proteins before either extracting them and purifying them, totally or partially, or using them in a mixture with the biomass which will have made it possible to produce them. It is the case, for example, of enzymes 15 which are useful for the biocatalysis of chemical reactions, and which can be used without a prior isolation and purification procedure, or also of enzymes which are used in the host bacterium in the process of growing, for the biotransformation of 20 chemical compounds. Advantageously, the heterologous protein is an enzyme produced in industrial amounts for a subsequent use as a chemical reaction catalyst. According to one particular embodiment of the 25 invention, the enzyme is a nitrilase, advantageously a CO REPLACEMENT SHEET (RULE 26) LU

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WO 99/64607 PCT/FR99/01343 16 nitrilase of Alcaligenes faecalis (ATCC8750) described in patent application WO 98/18941 or a nitrilase of Comamonas testosteroni sp. described in CIP application No. 08/194,588, or an amidase such as those described 5 in applications WO 97/04083, EP 433 117 and EP 488 916, or a hydroxyphenylpyruvate dioxygenase described in application WO 96/38567. The present invention also relates to an E. coli strain W as defined above, characterized in that 10 it comprises a system for expressing heterologous proteins, in which the promoter is the Ptrp promoter defined above. The examples hereinbelow make it possible to illustrate the present invention without, however, 15 seeking to limit the scope thereof. The appended figures 1 to 3 represent maps of plasmids used in the various examples. Figure 1 represents the map of the plasmid pRPA-BCAT41. The sites in brackets are sites which were 20 eliminated during cloning. Ptrp: tryptophan promoter; nitB: nitrilase gene; TrrnB: transcription terminators; end ROP: end of the gene encoding the ROP protein (Chambers et al., 1988, Gene 68: 139-149); ORI: origin of replication; RNAI/II: RNAs involved in replication REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 17 (Chambers et al., mentioned above); Tc: tetracyclin resistance gene. Figure 2 reoresents 1 map of the plasmid pRPA-BCAT127. The sites between brackets have been 5 eliminated during 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: cer locus. 10 Figure 3 represents the map of the plasmid pRPA-BCAT103. The sites between brackets have been eliminated during cloning. Sm/Sp: streptomycin and spectinomycin resistance gene; parABCDE: par locus (Roberts and Helinski, 1992, J. Bacteriol. 174: 8119 15 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). Figure 4 represents the map of the plasmid 20 pRPA-BCAT126. Ptrp: tryptophan promoter; nitB: nitrilase gene; TrrnB: transcription terminators; ORI: origin of replication; RNAI*/II: mutated RNAs involved in replication; Tcr: tetracycline resistance gene; cer: cer locus. AL REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 18 Figure 5 represents the map of the plasmid pRPA-BCAT143. Sm/Sp: streptomycin and spectinomycin resistance gene; rep, mob, and ori: regions involved in the replication and transfer of the plasmid (Scholtz et 5 al., 1989, Gene 75: 271-288; Frey et al., 1992, Gene 113: 101-106); delta relates to the name of the deletion described in the text. The techniques used are conventional molecular biology and microbiology techniques known to 10 a person skilled in the art and described, for example, by Ausubel et al., 1987 (Current Protocols in Molecular Biology, John Wiley and Sons, New York), Maniatis et al., 1982, (Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New 15 York), Coligan et al., 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 conting the Ptrp 20 promoter, the ribosome binding site of the X phage cII gene (RBScII) and the nitrilase gene of Alcaligenes faecalis ATCC8750 (nitB) was extracted from the plasmid pRPA6BCAT6 (application FR 96/13077) using the EcoRI and XbaI restriction enzymes, so as to be cloned into 25 the vector pXL642 (described in CIP application AQ REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 19 No. 08/194,588) opened with the same restriction enzymes. The resulting plasmid, pRPA-BCAT15, was opened with the StuI and BsmI enzymes, and the 4.3 kb fragment was ligated with the purified 136 bp StuI-BsmI fragment 5 of pRPA-BCAT4 (application FR 96/13077) so as to produce the plasmid pRPA-BCAT19. The partial sequencing of pRPA-BCAT19 confirmed the replacement of the codon of the Asp279 residue of the nitrilase with the codon of an Asn279 residue. The 1.2 kb EcoRI-XbaI fragment of 10 pRPA-BCAT19 containing the Perp::RBScII:::nitB fusion was then cloned into the vector pRPA-BCAT28 opened with the same enzymes, so as to produce the 6.2 kb plasmid pRPA BCAT29. The vector pRPA-BCAT28 was obtained by ligating the 3.9 kb SspI-ScaI fragment of pXL642 (CIP 15 application No. 08/194,588) with the 2.1 kb SmaI fragment of pHP45QTc (Fellay et al., 1987, Gene 52: 147-154) in order to replace the ampicillin resistance marker with the tetracycline resistance marker. In destroying the NdeI site close to the origin of 20 replication of the plasmid pRPA-BCAT29 by partial NdeI digestion and the action of E. coli Polymerase I (Klenow Fragment), a plasmid pRPA-BCAT41 was obtained, the map of which is represented in Figure 1. The sequence of the expression cassette is represented by 25 sequence identifier No. 2 (SEQ ID NO 2). L REPLACEMENT SHEET (RULE 26) LU

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WO 99/64607 PCT/FR99/01343 20 Example 2: Expression of the nitrilase of A. faecalis ATCC8750 in "batch" E. coli K12, BL21 and W. The plasmids pRPA-BCAT29 and pXL2035 (Levy 5 Schill et al., 1995, Gene 161: 15-20) were introduced into the strains DH5a (CLONECH. Product reference C1021-1), BL21 (Novagen, product reference 69386-1) and W (ATCC9637) of E. coli by conventional electroporation. Expression cultures were prepared as 10 described in Example 5 of application FR 96/13077, reducing the preculture time to 8 hours and fixing the expression time at 16 hours. The biomasses after expression were estimated according to the optical density of the cultures, read at 660 nm (OD660), using 15 the following equation: biomass in gram of dry weight per liter of culture = OD660 x 0.35. The measurements of nitrilase activity of the cultures were carried out as described in application FR 96/13077. For each strain, two clones were analyzed and for each clone, 20 the experiment was repeated. Table 1 contains, for each strain, the mean of the data obtained in the four experiments. Table 1: Biomass and activities of the strains harboring the plasmids pRPA-BCAT29 and pXL2035 ?AL REPLACEMENT SHEET (RULE 26) LUJ

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WO 99/64607 PCT/FR99/01343 21 STRAINS BIOMASS ACTIVITY (U) PRODUCTIVITY (g/l) (P) DH5a 0.15 10.4 1.6 BL21 0.37 6.3 2.4 w 0.65 7.00 4.5 ABBREVIATIONS: g/l: 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. 5 These data show that the strain W of E. coi (ATCC937) is more effective at expressing the nitrilase Nit. Example 3: Construction of pBCAT43. The polyamide hydrolase gene of Comamonas 10 acTdovorans N12 described in application WO 97/04083 (pamll) was cloned into the vector pBCAT4 . This polyamide hydrolase gene was amplified by PCR in the form of a 1.26 kb DNA fragment, while introducing, in the PCR primers, the EcoRI and NcoI restriction sites 15 in the 5' position of the gene and the XbaI restriction site in the 3' position. This fragment was then treated successively with the EcoRI enzyme and Mung Bean nuclease. After extraction of the proteins with phenol chloroform-isoamyl alcohol, the treatment was continued 20 with an XbaI digestion. Similarly, the vector pRPA REPLACEMENT SHEET (RULE 26) 0 PKrr D06 WO 99/64607 PCT/FR99/01343 22 BCAT41 was opened with the NdeI enzyme, and then treated with Mung Bean nuclease. After extraction of the proteins with phenol-chloroform-isoamyl alcohol, the treatment was continued with an XbaI digestion. 5 After ligation of these two samples, the plasmid pRPA BCAT43 was obtained: it contains the Ptp promoter and the RBScII binding site separated from the translation start codon of the pamll gene by the sequence: AATACTTACACC. 10 Example 4: Expression of the polyamidase PamII in "batch" E. coli DH5cL, BL21 and W. The plasmid pRPA-BCAT43 was introduced into the strains DH5a, L21 and W of E. coli by conventional electroporation. Expression cultures were prepared as 15 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 measurements of polyamide hydrolase activity of the cultures were carried out as described in application 20 WO 97/04083, with the following modifications: - the cells were permeabilized with toluene by resuspending the cell pellets in a 100 mM trs-HCl, 5 mM EDTA, pH8, 1% toluene buffer so as to have a dry cell concentration of approximately 5 g/l; after 25 vigorous shaking, the suspension is incubated for one REPLACEMENT SHEET (RULE 26) cg QAl, WO 99/64607 PCT/FR99/01343 23 hour at 4 0 C and then centrifuged, and finally, the pellets of permeabilized cells are taken up in a 100 mM, pH7, phosphate buffer. - the hydrolysis activity was measured on the AB 5 oligomer (one molecule of adipic acid condensed to one molecule of hexamethylenediamine) present at 2.5 g/l in the reaction medium containing 0.1 M potassium phosphate buffer at pH 7, and incubated at 30 0 C with stirring: 10 - 100 microliter samples are taken at regular intervals while adding to them the same volume of 0.2 N NaOH; - the samples are analyzed by HPLC after ten-fold dilution in a solution of 50 mM H 3

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4 . 15 For each strain, from 1 to 24 clones were analyzed and for each clone, one to seven independent experiments were conducted. Table 2 contains, for each strain, the mean of the data obtained. Table 2: Biomass and activities of the strains 20 harboring the plasmid pRPA-BCAT43 REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 24 STRAINS NB CULTURES ACTIVITY (U) PRODUCTIVITY (P) DH5a 11 0.77 0.3 BL21 3 1.4 1.8 W 24 2.1 2.6 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 W (ATCC9637) 5 of E. coli is more effective for expressing the PamII polyamidase. Example 5: Construction and characterization of the plasmid pBCAT41-531. The plasmid pRPA-BCAT41 underwent a 10 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, pp. 81-212) and 15 Humphreys et al., 1976 (Mol. Gen. Genet. 145: 101-108). Five micrograms of plasmid DNA purified on a cesium chloride gradient were incubated for 20 minutes at 800C in a 50 mM sodium phosphate buffer, pH 6, containing 0.5 mM EDTA and 0.4 M NH 2 OH. After the addition of an 20 identical volume of 50 mM sodium phosphate buffer, REPLACEMENT SHEET (RULE 26)

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WO 99/64607 PCT/FR99/01343 25 pH 6, containing 0.5 M EDTA, the reaction mixture was dialyzed against a large excess of 10 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA and 100 nM NaCl. The plasmid DNA was then recovered by precipitation and 5 approximately 20 ng of DNA was introduced by electroporation into the strain DH5a harboring the plasmid pXL2035. Among the transformants obtained, one clone was selected because the productivity of the culture was 3 times higher than that of a culture of 10 the strain DH5a (pRPA-BCAT41, pXL2035). The plasmid pRPA-BCAT41-531 that it was harboring was extracted and reintroduced into a new DH5a host harboring the plasmid pXL2035. Three clones were then analyzed under the conditions described in example 2, comparing them with 15 3 DH5O clones (pRPA-BCAT41, pXL2035), and the results are given in table in Table 3. Table 3: Biomass and activities of the strains harboring the plasmids pRPA-BCAT41, pRPA-BCAT41-531 and pXL2035 REPLACEMENT SHEET (RULE 26) 'A of WO 99/64607 PCT/FR99/01343 26 Strains Biomass Activity (U) Productivity (g/1) (P) DH5CC(pRPA-BCAT41, 0.21 12 2.5 pXL2035) DH5a(pRPA-BCAT41- 0.63 12 7.5 531, pXL2035) ABBREVIATIONS: g/l: 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. 5 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-XbaI fragment containing the Prp::nitB fusion was extracted from the plasmid 10 pRPA-BCAT41 in order to be cloned in place of the one 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 15 results are given in Table 4. Table 4: Biomass and activities of the strains harboring the plasmids pRPA-BCAT41, pRPA-BCAT41-531, pRPA-BCAT86 and pXL2035 REPLACEMENT SHEET (RULE 26) 4U' WO 99/64607 PCT/FR99/01343 27 Strains Biomass Activity (U) Productivity (g/1) (P) DH5cX(pRPA-BCAT41, 0.20 13.8 2.7 pXL2035) DH5ox(pRPA-BCAT41- 0.68 11.0 7.4 531, pXL2035) DH5U(pRPA-BCAT86, 0.69 11.9 8.1 pXL2035) ABBREVIATIONS: g/l: 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. 5 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 10 and the nitB gene. Example 6: Characterization of a mutation carried by the plasmid pBCAT41-531 responsible for the improvement in productivity of the cultures of strains 15 expressing nitrilase. The analysis of the amount of protein produced by the strains of example 5, by polyacrylamide R A N H(RAEE -T REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 28 gel electrophoresis in the presence of SDS, showed that all these constructs led to levels of nitrilase polypeptide synthesis which were comparable among the strains described in this example. On the other hand, 5 preparations of plasmid DNA of pRPA-bCAT41 and pRPA BCAT41-531 prepared from equivalent amounts of biomass demonstrated that the plasmid pRPA-BCAT41-531 is present at lower number of copies than its parent pRPA-BCAT41. The sequencing of the 994 bp region of 10 pRPA-BCAT41-531, which stretches from the Tth111I site and 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). By referring to the 15 numbering given in the sequence J01749 (0 is the middle of the unique EcoRI site), we found that an insertion of an A had taken place after base 2319, and that the C of position 3039 is replaced with a T, in pRPA-BCAT41 531. The first difference can be attributed to an error 20 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 al., 1988, Gene 68: 139-149). The second error 25 corresponds to a transition, a characteristic effect of RARN( I~ REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 29 hydroxylamine on DNA (Drake and Baltz, 1976, Annu. Rev. Biochem. 45: 11-37), and is located at the second nucleotide of the region transcribed into RNA I involved in the replication of pBR322 (Chambers et al., 5 mentioned above). It is the latter mutation which is responsible for the lower number of copies of pRPA BCAT41-531 in DH5a and which is responsible for the better nitrilase productivity of the cultures of the strain DH5L (pRPA-BCAT41-531, pXL2035). 10 Example 7: Expression of the nitrilase of A. faecalis ATCC 8750 in "fed-batch" (semicontinuous culture) E.coli BL21 and E. coli W. The plasmids pRPa-BCAT41-531 and pXL2035 were 15 introduced by electroporation into the strains BL21 (reference mentioned above) and W (ATCC9637) so as to give the RPA-BIOCAT594 [BL21 (pRPA-BCAT41-531, pXL2035)] and RPA-BIOCAT714 [W (pRPA-BCAT41-531, pXL2035)] strains, respectively. The recombinants 20 E. coli BIOCAT 594 and E. coli BIOCAT 714 were cultured in 3.5 liter fermenters containing 2 liters of medium with the following composition: T RA REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 30 Compound Concentration in g/1 KH2PO4 8 K2HPO4 6. 3

(NH

4 ) 2

SO

4 0.75 MgSO 4 7H 2 0 2.5 Iron sulfate 0.04 CaCl2.2H20 0. 05 Manganese sulfate 0.01 Cobalt chloride 0.004 Zinc sulfate 0.002 Sodium molybdate 0.002 Copper chloride 0.002 Boric acid 0.0005 Citrate [lacuna].H20 1.7 Glucose monohydrate 95 L-tryptophan 0.1 Meat peptone 5 Yeast extract 3 The pH is maintained at 7.0 by adding aqueous ammonia. The oxygen saturation is maintained at 20% by adding air in a proportion of 1 volume/volume of 5 medium/minute and by stirring. The glucose is introduced at the start at a final concentration of 2 g/l. After having been totally consumed, it is p REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 31 introduced continuously from a stock solution with the following composition: 700 g/l glucose; 19.6 g/l MgSO 4 .7H 2 0. The rate of addition is 2.2 g of glucose/h.1 of medium. 5 After fermentation for 24 hours, the medium is recovered and centrifuged, and the dry weight is estimated in g/l. The enzymatic activity is measured following a protocol given in patent WO 96/09403. It is expressed in kilos of ammonium 3-hydroybutanoate formed 10 per hour and per kilo of dry cells. Strain Final Final Yield on biomass activity glucose BIOCAT 594 (BL21) 27 g/l 13 23% BIOCAT 714 (W) 40 g/l 17 40% In this example, it appears clearly that the nitrilase is expressed much better in E. coli W than in 15 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 20 source of animal origin. REPLACEMENT SHEET (RULE 26) )4j WO 99/64607 PCT/FR99/01343 32 The E. coli strain W BIOCAT 714 is cultured in a 3.5 liter fermenter containing 2 liters of medium with the following composition: Compound Concentration in the medium in g/l

K

2

HPO

4 8

(NH

4

)

2

SO

4 0.75 MgSO 4 7H 2 0 2.5 Iron sulfate 0.04 CaCl 2 .2H 2 0 0.04 Manganese sulfate 0.026 Cobalt chloride 0.004 Zinc sulfate 0.013 Sodium molybdate 0.001 Copper chloride 0.001 Boric acid 0.00025 AlCl 3 0.00125 Citrate [lacuna] .H 2 0 1.7 Glucose monohydrate 95 L-tryptophan 0.1 Yeast extract 3 5 The pH is maintained at 7.0 by adding aqueous ammonia. The oxygen saturation is maintained at 20% by REPLACEMENT SHEET (RULE 26) 44/ 773, WO 99/64607 PCT/FR99/01343 33 adding air in a proportion of 1 volume/volume of medium/minute and by stirring. The glucose is introduced at the start at a final concentration of 2 g/l. After having been totally consumed, it is 5 introduced continuously from a stock solution with the following composition: 700 g/l glucose; 19.6 g/l MgS0 4 .7H 2 0. The rate of addition is 2.2 g of glucose/h.1 of medium. An organic nitrogen source of animal origin 10 is added to this medium. Organic nitrogen Final Final Yield on source of animal biomass activity glucose origin None 30 2 40% 2.5 g/l of meat 33 12 40% peptone 5 g/l of meat 40 25 45% peptone 5 g/l of casein 35 20 43% The use of an increasing concentration of organic nitrogen of animal origin significantly 15 increases the specific activity of the cells. REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 34 Example 9: Influence of the organic nitrogen of plant origin. The culture conditions are identical to those of example 8. In this example, organic nitrogen of 5 plant origin is added. Organic nitrogen Final Final Yield on source of animal biomass activity glucose origin None 30 2 40% 5 g/l of soybean 31 4 40% peptone 5 g/l of wheat 32 5 40% peptone 7.5 g/l of sodium 35 17 43% hydrolysate of potato protein (Alburex SP; Roquette) The addition of plant organic nitrogen does not give identical results depending on the origin. 10 Surprisingly, the addition of potato protein gives as good a result as the organic nitrogen of animal origin. REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 35 Example 10: Influence of the carbon source. The E. coli strain W BIOCAT 714 is cultured in a 3.5 liter fermenter containing 2 liters of medium 5 with the following composition: Compound Concentration in the medium in g/l Corn-steep 40 LAB2218 (Roquette) Yeast extract 3 MgSO 4 7H 2 0 2.5 The pH is maintained at 7.0 by adding aqueous ammonia. The oxygen saturation is maintained at 20% by 10 adding air in a proportion 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/l. After having been totally consumed, it is introduced continuously from a stock solution with the 15 following composition: 700 g/1 carbon source; 19.6 g/l MgSO 4 .7H 2 0. The rate of addition is 2.2 g of glucose or of sucrose/h.1 of medium. The carbon source is varied in this example. REPLACEMENT SHEET (RULE 26)

C'

WO 99/64607 PCT/FR99/01343 36 Carbon source Final Final Yield on biomass activity carbon Glucose monohydrate 38 11 45% 90 g/l "Syrup zero" 38 17 45% (EUROSUCRE) 90 g/l In this example, it is observed that the use of sucrose ("syrup zero") as a carbon source significantly increases the specific activity of the 5 cells. Example 11: Construction of a plasmid for coexpression of the TrpR regulator A 434 bp DNA fragment which carries the trpR 10 gene and its promoter was extracted from the plasmid pRPG9 (Gunsalus and Yanofsky, 1980, Proc. Natl. Aca. Sci. USA 77: 7117-7121) using the AatII and StuI restriction enzymes. This fragment was cloned into the plasmid pSL301 (Brosius, 1989, DNA 8: 759-777) by 15 ligating it to the approximately 3.1 kb AatII-StuI fragment, so as 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 EcoRI-NotI fragment in order to be cloned into the plasmid pXL2035 in place (RA4Z REPLACEMENT SHEET (RULE 26) 4'1 WO 99/64607 PCT/FR99/01343 37 of a 240 bp EcoRI-NotI fragment. The resulting plasmid, pRPA-BCAT34, is therefore a derivative of pKT230 which allows the expression of the GroESL chaperones and of the TrpR regulator. 5 Example 12: Influence of the coexpression of GroESL and of TrpR. The plasmid pRPA-BCAT34 was introduced by electroporation into the strains DH5a (pRPA-BCAT29), 10 BL21 (pRPA-BCAT29) and W (pRPA-BCAT29). Expression cultures of various strains were prepared as described in example 2, and the results are given in Table 5. Table 5: Biomass and activities of the strains 15 harboring combinations the plasmids pRPA-BCAT29, pXL2035 and pRPA-BCAT34 Combinations pRPA-BCAT29 pRPA-BCAT29 pRPA-BCAT29 pXL2035 pRPA-BCAT34 HOST U P U P U P DH5-alpha 0.37 0.16 10.4 1.6 2.0 0.7 BL21 0 0.0 6.4 2.4 5.6 2.0 W 1.7 0.96 7.0 4.5 8.9 6.5 REPLACEMENT SHEET (RULE 26) 4u4 WO 99/64607 PCT/FR99/01343 38 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. 5 The results show that the coexpression of GroESL makes it possible to increase the productivity of the cultures whatever the strain under consideration, by improving the specific activity of the cultures. This effect is correlated with an 10 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 15 makes it possible, in W, to improve the productivity of the cultures. Example 13: Influence of the presence of a cer locus on pRPA-BCAT41 20 The 382 bp HpaII fragment containing the cer locus of the plasmid ColEl (Leung et al., 1985, DNA 4: 351-355) was cloned into the replicative form of the Ml3mp7 phage at one of the 2 AccI sites. The construct obtained then made it possible to extract, with the 25 EcoRI enzyme, an approximately 430 bp fragment REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 39 containing the cer locus, which was cloned into pRPA-BCAT41 at the EcoRI site, thereby producing the plasmid pRPA-BCAT66. This plasmid was introduced by electroporation into the strain W harboring the plasmid 5 pRPA-BCAT34. Expression cultures of various strains were prepared as described in example 2, extending the duration of the expression cultures to 24 hours and studying three clones of each strain in a sole experiment. The mean results are given in Table 6. 10 Table 6: Biomass and activities of the strains harboring the plasmids pRPA-BCAT41, pRPA-BCAT66 and pRPA-BCAT34 Strains Biomass Activity Productivity (g/l) (U) (P) W (pRPA-BCAT41, pRPA-BCAT34) 2.1 6.9 14.5 DH5a (pRPA-BCAT66, pRPA-BCAT34) 1.8 10.0 18.0 15 ABBREVIATIONS: g/l: 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 [lacuna] REPLACEMENT SHEET (RULE 26) -'p 44 WO 99/64607 PCT/FR99/01343 40 These results show that adding the cer locus to the plasmid for expression of the nitrilase leads to an improvement in the productivity of the cultures. 5 Example 14: Construction of the plasmid pRPA-BCAT127 After elimination of the unique NdeI site of the plasmid pRPA-BCAT30 by digestion and formation of blunt ends with polymerase I (Klenow fragment), the 10 trpR gene was extracted from this latter plasmid in the form of an approximately 300 bp fragment prepared by treatment with the AatII enzyme followed by the action of polymerase I (Klenow fragment), and then, after inactivation of the reaction mixture, by digestion with 15 the SacII enzyme. This fragment was cloned into the pRPA-BCAT66 plasmid after opening this plasmid with Tthlll followed by treatment with polymerase I Klenow fragment) and, after inactivation, with SacII. The plasmid pRPA-BCAT82 was thus obtained. Its origin of 20 replication was replaced with that of the plasmid pRPA-BCAT41-531 by replacing the approximately 1.12 kb Bstll07I-EamllO5I fragment. The construct selected during this cloning, the plasmid pRPA-BCAT99, has an artefact which is in the form of a deletion of one 25 nucleotide at the Eaml105I site, transforming this site REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 41 into a unique PshAI site. The resistance marker of the plasmid pRPA-BCAT99 was then changed by cloning, between the AatII and PshAI sites, an approximately 1.07 kb AatII-PshAI fragment prepared after PCR 5 amplification of the gene encoding chloramphenicol resistance from the matrix pACYC184 (New England Biolabs #401-M), using the primers Cml and Cm2, the sequence of which is: Cml : 5'-CCCCCCGACAGCTGTCTTGCTTTCGAATTTCTGCC 10 Cm2 5'-TTGACGTCAGTAGCTGAACAGGAGGG The plasmid thus obtained was called pRPA-BCAT123. It was then modified by eliminating the trpR gene in the form of an approximately 0.525 kb SacI-Bst1107I fragment, and reclosing the plasmid after forming blunt 15 ends with the Pfu polymerase (15 minutes at 75 0 C in the buffer recommended by the manufacturer Stratagene, and in the presence of 0.2 mM of deoxynucleotides). The plasmid thus obtained is the plasmid pRPA-BCAT127, the map of which is represented schematically in Figure 2. 20 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 25 approximately 3.2 kb SfiI-ScaI fragment with the REPLACEMENT SHEET (RULE 26) 4/1 \),q WO 99/64607 PCT/FR99/01343 42 approximately 2.42 kb SfiI-ScaI fragment of the plasmid RSF101D20 (Frey et al., 1992, Gene 113: 101-106). This fragment contains a deletion in the 5' portion of the gene encoding the RepB primase, and reduces the 5 frequency of transfer of the plasmid by 6 logs (Frey et al., mentioned above). The plasmid thus obtained, pRPA-BCAT98, has several advantages: the loss of its mobilization functions makes it comply with the rules of industrial biosafety while at the same time 10 retaining its properties of replication in Gram negative bacteria. The par locus (Gerlitz et al., 1990, J. Bacteriol 172: 6194-6203) was then cloned on pRPA BCAT98 as follows. The approximately 2.3 kb SphI-BamHI 15 fragment of pGMA28 (Gerlitz et al., mentioned above) was first cloned into the vector pUC18, thereby allowing its extraction in the form of a HindIII-EcoRI fragment so as to clone it into the vector pMTL22 (Chambers et al., 1988, Gene 68: 139-49). The HindIII 20 site was then destroyed by HindIII digestion and Klenow treatment. An approximately 2.38 kb fragment was then extracted with the PstI and BglII enzymes so as to be cloned into the vector pXL2426 at the PstI and BamHI sites and to produce the vector pXL2572. The vector 25 pXL2426 originates from the replacement of the 2.38 kb REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 43 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 NdeI and BamHI sites, of an approximately 0.960 bp 5 NdeI-BamHI fragment of pRR71 (Weinstein et al., 1992, J. Bacteriol. 174: 7486-7489) made it possible to reconstitute the par locus as a whole on the plasmid pXL2573. This locus was then extracted from pXL2573 in the form of a 2.6 kb EcoRI-blunt end (after treatment 10 with PstI and Klenow) fragment in order to be cloned on the plasmid pRPA-BCAT98 opened with EcoRI and SacI, the latter end having been treated with the Pfu polymerase. The resulting plasmid was called pRPA-BCAT103 and its map is represented schematically in Figure 3. 15 Example 16: Use of the plasmids pRPA-BCAT98, pRPA-BCAT103 and pRPA-BCAT127 for expressing the nitrilase in W. The plasmids pRPA-BCAT127, pRPA-BCAT98, pRPA 20 BCAT103, pXL2035 and pXL2231 (application FR 96/13077) were introduced into the strain W of E. coli by electroporation, and expression cultures were prepared under the conditions described in example 2, using the following antibiotics: 12 pg/ml tetracycline for 25 pXL2231, 50 sg/ml kanamycin for pXL2035, 100 gg/ml REPLACEMENT SHEET (RULE 26) 4/ WO 99/64607 PCT/FR99/01343 44 streptomycin for pRPA-BCAT98 and pRPA-BCAT103, and 20 pg/ml chloramphenicol for pRPA-BCAT127. For each combination of plasmids, two to three clones were analyzed, and the mean results are given in Table 7. 5 Table 7: Biomass and activities of the strains harboring the plasmids pRPA-BCAT41-531, pRPA-BCAT127, pRPA-BCAT98, pRPA-BCAT103, pXL2035 and pXL2231 Combination Biomass Activity Productivity (g/1) (U) (P) pBCAT127/pXL2231 1.43 4.9 7 pBCAT127/pBCAT103 1.75 7 12 pBCAT127/pBCAT98 1.72 11.2 19 pBCAT127/pXL2035 1.70 7.2 12 pBCAT41- 1.36 5.9 8 531/pXL2035 10 ABBREVIATIONS: g/l: 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 pRPA-BCAT127/pRPA-BCAT98 and 15 pRPA-BCAT127/pRPA-BCAT103 allow an at least equivalent productivity to be obtained, using plasmids which are in conformity with the European criteria for biosafety. REPLACEMENT SHEET (RULE 26) 0o WO 99/64607 PCT/FR99/01343 45 Example 17: Construction of the plasmid pRPA-BCAT126 The resistance marker of the plasmid pRPA-BCAT99 described in example 14 was changed as 5 follows. The vector was opened with the PshAI and AatII enzymes and then treated with the Pfu polymerase (5 min at 750C in the buffer recommended by the manufacturer Stratagene, and in the presence of 0.2 mM of deoxynucleotides), and the approximately 3.95 kb 10 fragment was extracted from an agarose gel using the Quiaex kit (Quiagen) [other systems for recovering DNA can also be used, in particular those of chromatographic type]. It was ligated according to a conventional process with the 1.32 kb HindIII-BsmI 15 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 tetracyclin resistance gene oriented in the same direction of 20 transcription as the cassette for expressing the nitrilase was named pRPA-BCATlll. This plasmid was then opened with the NsiI and BstZl7I enzymes and then treated with the Pfu polymerase, and religated in order to eliminate the 0.47 kb fragment carrying the trpR REPLACEMENT SHEET (RULE 26) 7i7- WO 99/64607 PCT/FR99/01343 46 gene. The plasmid obtained was named pRPA-BCAT126, the map of which is represented in Figure 4, Example 18: Construction of the plasmid 5 pRPA-BCAT143 The plasmid pRPA-BCAT98 described in example 15 was opened with the SfiI and ScaI enzymes in order to replace the 2.42 kb fragment carrying the deletion in the 5' portion of the gene encoding the RepB primase 10 with the 2.96 kb SfiI-ScaI fragment extracted from the plasmid RSF1010A18 carrying a 267 bp in-frame deletion in the 5' portion of the repB gene (Frey et al., 1992, Gene 113: 101-106). The deletion introduced on pRPA-BCAT143 decreases the frequency of transfer of the 15 plasmid to 10-6 (Frey et al., 1992, Gene 113: 101-106) and makes it comply with the demands of the rules of biosafety. Unlike the plasmid pRPA6BCAT98 described above, this novel plasmid conserves a copy number close to the unmodified plasmid pXL2035 (Ldvy-Schill et al., 20 1995, Gene 161: 15-20). It is represented in Figure 2. Example 19: Use of the plasmids pRPA-BCAT126 and pRPA-BCAT143 for expressing the nitrilase in W The plasmids pRPA-BCAT126, pRPA-BCAT127 25 (described above), pRPA-BCAT143, pRPA-BCAT98 and RAg, REPLACEMENT SHEET (RULE 26) 7R4 WO 99/64607 PCT/FR99/01343 47 pXL2035 were introduced into the strain W of E. coli by electroporation, and expression cultures were prepared under the conditions described in example 2, using the following antibiotics: 12 pg/ml tetracycline for 5 pRPA-BCAT41-531 and pRPA-BCAT126, 50 jg/ml kanamycin for pXL2035, 100 ptg/ml streptomycin for pRPA-BCAT98 and pRPA-BCAT143, and 20 pg/ml chloramphenicol for pRPA BCAT127. For each combination of plasmids, two to three clones were analyzed, and the mean results are given in 10 Table 8. Table 8: Biomass and activities of the strains harboring the plasmids pRPA-BCAT41-531, pRPA-BCAT126, pRPA-BCAT127, pRPA-BCAT98, pRPA-BCAT143 and pXL2035 15 Combination Biomass Activity Productivity (g/1) (U) (P) pBCAT41- 2.3 9.5 22 531/pXL2035 pBCAT41- 2.5 8.9 22 531/pBCAT143 pBCAT126/pXL2035 2.2 9.8 21 pBCAT126/pBCAT98 1.3 3.5 4.5 pBCAT126/pBCAT143 2.5 8.1 20 pBCAT127/pBCAT143 2.8 7.1 20 -A /REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 48 ABBREVIATIONS: g/l: 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 5 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 conserve the productivity of the cultures prepared with the strains harboring the plasmid 10 pXL2035. REPLACEMENT SHEET (RULE 26)

Claims

1. Industrial process for preparing heterologous proteins in E. coli, in which E. coli 5 bacteria modified with a suitable system for expressing heterologous proteins are seeded and cultured in a suitable culture medium, characterized in that the strain of E. coli is an E. coli strain W.

2. Process according to claim 1, 10 characterized in that the strain W is the strain W deposited at the ATCC under the number 9637.

3. Process according to claim 1, characterized in that the strain W is a derivative of the strain deposited at the ATCC under the number 9637, 15 obtained by clonal selection or genetic manipulation.

4. Process according to one of claims 1 to 3, characterized in that the suitable culture medium is a culture medium which is suitable for the production of a high density of biomass and a high content of 20 heterologous proteins produced.

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

6. Process according to claim 5, 25 characterized in that the amount of L-tryptophan in the REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 50 culture medium is between 0.05 and 0.5 g/l, preferably between 0.1 and 0.3 g/l.

7. -- Process according to one of claims 1 to 6, characterized in that the culture medium comprises 5 sucrose as the main carbon source.

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

9. Process according to either of claims 7 10 and 8, characterized in that the amount of sucrose in the culture medium is between 0.1 and 300 g/l at the start of culturing, preferably between 0.5 and 200 g/l.

10. Process according to one of claims 1 to 9, characterized in that the suitable culture medium 15 also comprises a supplementary organic nitrogen source.

11. Process according to claim 10, characterized in that the supplementary organic nitrogen source consists of protein extracts.

12. Process according to either of claims 9 20 and 10, characterized in that the protein extract has the following composition: (in g amino acids per 100 g of product) alanine between 10 and 4, aspartic [lacuna] between 11 and 4, glycine between 22 and 2.5 and lysine between 7 and 4. REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 51

13. Process according to one of claims 10 to 12, characterized in that the supplementary organic nitrogen source consists of meat or potato peptones or proteins, more particularly the derivatives of potato 5 proteins.

14. Process according to one of claims 1 to 13, characterized in that the suitable system for expressing heterologous proteins comprises a Ptrp promoter. 10

15. Process according to claim 14, characterized in that the Ptrp promoter comprises the nucleic acid sequence represented by sequence identifier no. 1 (SEQ ID NO 1).

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

17. Process according to claim 16, characterized in that the enzyme is useful for the biocatalysis of chemical reactions. 20

18. Process according to claim 17, characterized in that the enzyme is a nitrilase.

19. E. coli strain W, characterized in that it comprises a system for expressing heterologous proteins, in which the promoter is the Ptrp promoter. REPLACEMENT SHEET (RULE 26) WO 99/64607 PCT/FR99/01343 52

20. Strain according to claim 19, characterized in that the Ptrp promoter comprises the nucleic acid sequence represented by sequence identifier no. 1 (SEQ ID NO 1). REPLACEMENT SHEET (RULE 26)