MXPA99000962A - Novel strains of escherichia coli - Google Patents

Abstract

The present invention relates to novel strains of Escherichia coli and fermentation processes involving these microorganisms. More specifically, the present invention relates to genetically-modified Escherichia coli strains and the use thereof for the production of the amino acids, particularly members of the aspartate family of amino acids such as threonine. The present invention also relates to methods of preparing E. coli strains for use in the fermentative production of amino acids.

Description

NEW CEPAS OF ESCHERIC ZEA COLI, METHODS FOR PREPARING THEM AND USING THESE IN FERMENTATION PROCESSES FOR THE PRODUCTION OF L-THREONINE FIELD OF THE INVENTION The present invention relates to new strains of Escherichia coli and to fermentation processes involving these microorganisms. More specifically, the present invention relates to genetically modified strains of Escherichia coli and to the use thereof for the production of amino acids, particularly members of the amino acid aspartate family, such as threonine. The present invention also relates to methods for preparing E. coli strains for use in the fermentative production of amino acids. BACKGROUND OF THE INVENTION In Escherichia coli, the amino acids L-threonine, L-isoleucine, L-lysine and L-methionine are derived in all or part of their carbon atoms from aspartate (aspartic acid) through the following common biosynthetic pathway (G. N. Cohen, "The common pathway to lysine, methionine and threonine", pp. 147-171 in Amino Acids: Biosynthesis and Genetic Regulation, K. M. Herrmann and R. L. Somerville, eds., Addison-elesley Publishing Co. , Inc., Reading, Mass. (1983)): REF. 29242 aspartate-? Aspartyl-phosphate- »aspartate semialdehyde? Homoserin? MET / THR / IE • i- 1YS The first reaction of this common route is catalyzed by one of three different aspartate kinases (AK, I, II or III), each of which is encoded by a separate gene and differs from the others in the way in which its activity and synthesis is regulated. Aspartate kinase I, for example, is encoded by the thrA gene, its activity is inhibited by threonine and its synthesis is repressed by threonine and isoleucine, in combination. However, AK II is encoded by the metL gene and its synthesis is repressed by methionine (although its activity is not inhibited by methionine or by paired combinations of methionine, lysine, threonine and isoleucine (F. Falcoz-Kelly et al. ., Eur. J. Biochem., 8: 146-152 (1969), JC Patte et al., Biochim, Biophys, Act 136: 245-251 (1967).) AK III is encoded by the lysC gene and its activity and syntheses are inhibited and repressed, respectively by lysine.Two of the AKs, I and II, are not different proteins, but rather they are a domain of complex enzymes that includes homoserine dehydrogenase I or II, respectively, each of which catalyzes the reduction of semialdehyde aspartate in homoserine (P. Truffa-Bachi et al., Eur. J. Biochem. 5: 73-80 (12968)). Homoserine dehydrogenase I, therefore, is also encoded by the thrA gene, its synthesis is repressed by threonine plus isoleucine and its activity is inhibited by threonine. II is similarly encoded by the metL gene and its synthesis is repressed by methionine. The threonine biosynthesis includes the following additional reactions: homoserin - > homoserine phosphate - > threonine. Phosphorylation of homoserine is catalyzed by homoserine kinase, which is a protein consisting of two identical subunits of 29 kDa encoded by the ti? R-3 gene and whose activity is inhibited by threonine (B. Burr et al., J. Bochem 51: 519-526 (1976)). The final step, the complex conversion of homoserine phosphate to L-threonine, is catalyzed by threonine synthetase, which is a 47 Da protein encoded by the thrC gene (C. Parsot et al., Nucleic Acids Res. 12: 7331 -7345 (1983)). The thrA, thrB and thrC genes, all belong to the thr operon, which is a simple operon located at 0 minutes in the genetic map of E. coli (J. Théze and I. Saint-Girons, J. Bacteriol, 228: 990 -998 (1974); J. Théze et al., J. Bacteriol. 227: 133-143 (1974)). These genes code, respectively, for aspartate kinase I-ho oserin dehydrogenase I, homoserine kinase and threonine synthetase. The biosynthesis of these enzymes is subject to multivalent repression by threonine and isoleucine (M. Freundlich, Biochem Biophys Res. Commun. 20: 277-282 (1963)). A regulatory region is located upstream of the first structural gene in the thr operon and its sequence has been determined (J. F. Gardner, Proc. Nati, Acad. Sci. USA 75: 1706-1710 (1979)). The thr attenuator, downstream of the transcription initiation site, contains a sequence encoding a leader peptide; this sequence includes eight threonine codons and four isoleucine codons. The thr attenuator also contains the classical mutually exclusive secondary structures that allow or prevent the transcription of RNA polymerase from the structural genes in the thr operon, depending on the levels of threonyl-AR? T and isoleucyl-tRNA. Due to the problems associated with obtaining high levels of amino acid production through natural biosynthesis (for example, repression of the thr operon by the desired product), bacterial strains having plasmids containing a thr operon with a thrA gene have been produced. encodes for a feedback-resistant enzyme. With such plasmids L-threonine has been produced on an industrial scale by fermentation processes employing a wide variety of microorganisms, such as Brevibacterium flavum, Serratia marcescens and Escherichia coli. For example, the E. coli strain BKIIM B-3996 (Debabov et al., US Patent No. 5,175,107) which contains the plasmid pVIC40, produces approximately 85 g / 1 in 36 hours. The host is a strain that requires threonine because its threonine synthetase enzyme is defective. In strain BKIIM B-3996, the recombinant plasmid pVIC40 is the one that provides the crucial enzymatic activities, that is, an AK I-HD-I resistant to feedback, homoserine kinase and threonine synthetase, necessary for threonine biosynthesis . This plasmid also complements the threonine auxotrophy of the host. The E. coli strain 29-4 (E. Shimizu et al., Biosci, Biotech, Biochem 5-9: 1095-1098 (1995)) is another example of a threonine-producing recombinant E. coli. Strain 29-4 was constructed by cloning the tíi-r operon of a threonine-producing mutant strain, strain E. coli K-12 (ßIM-4) (derived from the E. coli strain ATCC 21277), plasmid pBR322, which was subsequently introduced into the progenitor strain (K.

Wiwa et al. , Agrie. Biol. Chem. 47: 2329-2334 (1983)). Strain 29-4 produces approximately 65 g / 1 of L-threonine in 72 hours Recombinant strains constructed in a similar manner using other organisms have been prepared. For example, the S. marcescens strain T2000 contains a plasmid that has a thr operon that codes for a thrA-resistant gene for feedback and produces approximately 100 g / 1 of threonine in 96 hours (M. Masuda et al., Applied Biochemistry and Biotechnology 37: 255-262 (1992) All of these strains contain plasmids that have multiple copies of the genes encoding threonine biosynthetic enzymes, which allows the overexpression of these enzymes This overexpression of genes of plasmid origin For thioonine biosynthetic enzymes, particularly the thrA gene encoding a feedback-resistant I-HD I AK, these strains can produce large amounts of threonine, other examples of microorganisms containing plasmids are described, for example, in US Patents Nos. 4,321,325, 4,347,318, 4,371,615, 4,601,983, 4,757,009, 4,945,058, 4,946,781, 4,980,285, 5,153,123 and 5,236.83. 1. However, strains containing plasmids such as these present problems that limit their usefulness for the commercial fermentative production of amino acids. For example, a significant problem with these strains is to ensure that the integrity of the strain containing the plasmid is maintained throughout the fermentation process, due to the potential loss of the plasmid during cell growth and division. To avoid this problem, it is necessary to selectively remove the plasmid-free cells during culture, for example using antibiotic resistance genes in the plasmid. However, this solution requires the addition of one or more antibiotics to the fermentation medium, which is not commercially practical for large scale fermentations. Another significant problem with strains containing plasmids is the stability of the plasmid. The high expression of enzymes whose genes are encoded in the plasmid, which is necessary for commercially practical fermentative processes, often causes instability of the plasmid (E. Shimizu et al., Biosci, Biotech, Biochem 59: 1095-1098 (nineteen ninety five)). The stability of the plasmid also depends on factors such as the culture temperature and the level of dissolved oxygen in the culture medium. For example, strain 29-4 containing plasmids was more stable at lower culture temperatures (30 ° C vs. 37 ° C) and higher levels of dissolved oxygen (E. Shimizu et al., Biosci, Biotech, Biochem. 59: 1095-1098 (1995)). Microorganisms that do not contain plasmids, while being less effective than those previously described, have also been used as threonine producers. E. coli strains such as H-8460, which is obtained by a series of conventional mutagenesis and selection of resistance to various metabolic analogues, produces approximately 75 g / l of L-threonine in 70 hours (Kino et al., U.S. Patent No. 5,474,918). Strain H-8460 is not a carrier of any recombinant plasmid and has a copy of the threonine biosynthesis genes on the chromosome. The lower productivity of this strain compared to plasmid-carrying strains, such as BKIIM B-3996, is believed to be due to lower enzyme activity (particularly those enzymes encoded by the thr operon) since these strains do not contain plasmids are carriers of only one copy of the biosynthetic threonine genes. Other examples of suitable microorganisms that do not contain plasmids are described, for example, in U.S. Patent Nos. 5,376,538; 5,342,766; 5,264,353; 5,217,883; 5,188,949; 5,164,307; 5,098,835; 5,087,566; 5,077,207; 5,019,503; 5,017,483; 4,996,147; 4,463,094; 4,452,890; 3,732,144; 3,711,375; 3,684,654; 3,684,653; 3,647,628; 3,622,453; 3,582,471; 3,580,810; 3,984; 830; and 3,375,173. Both in the -57 strains. coli carrier plasmids as in the non-plasmid carriers, the thr operon is controlled by the respective native threonine promoter of the particular strain. As described above, the expression of the native promoter is regulated by an attenuation mechanism controlled by a region of the DNA that codes for a leader peptide and contains a number of threonine and isoleucine codons. This region is translated by a ribosome that senses the levels of threonyl-tRNA and isoleucinyl-tRNA. When these levels are sufficient for the leader peptide to be translated, the transcription is terminated prematurely, but when the levels are insufficient for the leader peptide to be translated, the transcription does not end and the entire operon is transcribed, which, after the translation, results in increased production of threonine biosynthetic enzymes. Thus, when the levels of threonyl-tRNA and / or isoleucinyl-tRNA are low, the thr operon is transcribed to the maximum and the biosynthetic threonine enzymes are maximally produced. In the threonine-producing E. coli strain BKIIM B-3996, the threonine operon in the plasmid is controlled by its native promoter. As a result, the thr operon is only expressed to the maximum when the strain has a great lack of threonine and / or isoleucine. As the lack of threonine is not possible in a threonine-producing strain, these strains become auxotrophic for isoleucine, in order to obtain a higher level of enzymatic activity. Another way to resolve the attenuation control is to decrease the level or levels of threonyl-tRNA and / or isoleucinyl-AR? T in the cell. A thrS mutant, for example, having a threonyl-AR? T-synthetase exhibiting a 200-fold decrease in its apparent affinity for threonine, results in overexpression of the thr operon, presumably at the low level of treonyl-AR T (EJ Johnson et al., J. Bacteriol. 129: 66-10 (1977)). However, in fermentation processes using these strains, the cells must be supplemented with isoleucine in the growth stage, due to their deficient isoleucine biosynthesis. Subsequently, in the production stage, the cells are deprived of isoleucine to induce the expression of threonine biosynthetic enzymes. Therefore, one of the main disadvantages of using native threonine promoters to control the expression of threonine biosynthetic enzymes is that the cells must be supplemented with isoleucine. The antibiotic borrelidin is also known to reduce the enzymatic activity of the threonyl AR? T-synthetase and, thereby, inhibits the growth of E. coli (G.? Ass et al., Biochem. Biophys. Res. Commun. : 84 (1969)). In view of this reduced activity, certain borrelidin-sensitive strains of -... coli have been used to produce high levels of threonine (Japanese Published Patent Application No. 6752/76; US Patent No. 5,264,353). It was found that the addition of borrelidin to the culture increased the yield of L-threonine. Borrelidin sensitive strains of Brevibacterium and Corynebacterium have also been used to produce high levels of threonine (Japanese Patent No. 53-101591). Similarly, borrelidin-resistant E. coli mutants exhibit changes in the activity of the threonyl-tRNA synthetase. More specifically, it has been demonstrated that borrelidin-resistant E. coli strains exhibit one of the following characteristics: (i) constitutively increased levels of wild-type threonyl-tRNA synthetase; (ii) structurally altered threonyl-tRNA synthetase; or (iii) some unknown cellular alteration, probably due to a change in the membrane (G. Nass and J. Thomale, FEBS Lett 39: 182-186 (1974)). However, none of these mutants has been used for the fermentative production of L-threonine. In view of the previous discussion, there is still a need in the art for strains of microorganisms that efficiently produce amino acids such as threonine, but without the problems associated with the state of the art. BRIEF DESCRIPTION OF THE INVENTION Therefore, an objective of the present invention is to provide microorganisms that efficiently produce L-threonine with high yields, but which do not require any recombinant plasmid containing genes coding for threonine biosynthetic enzymes and preferably that do not have nutritional requirements of amino acids. Other objects, features and advantages of the present invention will be set forth in the detailed description of the preferred embodiments presented below and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the present invention will be realized and achieved by the methods particularly indicated in the written description and the claims thereof. These and other objects are achieved by the methods of the present invention, which, in a first embodiment, relates to a process for producing amino acids such as L-threonine, which comprises the steps of culturing an E. coli strain in a medium and recover the amino acid from the medium. The strain of E. coli used in this process has the following characteristics: (i) it contains a genetic determinant of amino acid biosynthesis, such as the threonine operon (which codes for the biosynthetic enzymes of threonine), in the chromosome under the control of a non-native promoter; and (ii) does not require any recombinant plasmid containing genes that code for threonine biosynthetic enzymes, to produce threonine. Another embodiment of the present invention relates to a biologically pure culture of an E. coli strain having the above characteristics. A further embodiment of the present invention relates to a process for producing amino acids such as L-threonine. Which includes the steps of growing a strain of J-J. coli in a medium and recover the amino acid from the medium, where the E. coli strain is resistant to borrelidin. Another embodiment of the present invention relates to a method for producing an E. coli strain useful for the fermentative production of amino acids such as threonine, which comprises the steps of (a) introducing genetic material from an amino acid producing microorganism to the chromosome of a strain of E. coli auxotroph, to convert that strain of E. coli into prototrophic; (b) inserting a non-native promoter into the chromosome prior to the chromosomal location of the biosynthetic genes of the - - amino acid, to control the expression thereof; and optionally (c) removing the amino acid nutritional requirements for and / or regulatory impediments to the amino acid biosynthesis of the chromosome. It should be understood that the above general description and the following detailed description are examples and only explanations and are intended to provide a further explanation of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the construction of plasmid pAD103 from Kohara 's lambda 676 and plasmid pUC19. Figure 2 illustrates the construction of plasmid pAD106 from plasmid pAD103 and plasmid pUC4k. Figure 3 illustrates the construction of plasmid pAD115 from plasmid pAD103 and plasmid pkk223-3. Figure 4 illustrates the construction of plasmid pAD123 from plasmid pAD115 and plasmid pAD106. Figure 5 illustrates the integration of the promoter region from plasmid pAD123 in the chromosome of E. coli. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In a first embodiment, the present invention relates to new bacterial strains that can be used in fermentation processes for the production of amino acids. The new bacterial strains of the present invention have the following characteristics: (i) the cells contain at least one thr operon, that is, at least one set of genes coding for threonine biosynthetic enzymes, in the chromosome under control from a non-native promoter; and (ii) the cells do not require any recombinant plasmid that codes for the biosynthetic enzymes of threonine, in order to produce threonine. Preferably, the strains of the present invention are capable of producing at least about 50 g / 1 L-threonine in about 30 hours, more preferably at least about 70 g / 1 in about 30 hours, still more preferably at least about 80 hours. g / 1 in about 30 hours and most preferably is at least at least 90 g / 1 in about 30 hours. Preferably, the strains of the present invention are capable of producing at least about 90 g / 1 in about 48 hours, preferably at least about 100 g / 1 in about 48 hours and more preferably at least about 110 g / 1 of threonine in about 48 hours. Preferably, the strains of the present invention are capable of producing L-threonine at an Index of at least 2 g / l / h, preferably at least about 2.5 g / l / h, even more preferably at least about 3 g / l. l / h and most preferably at least about 3.6 g / l / h. In a particularly preferred embodiment, the new bacterial strains also have no amino acid nutritional requirements for the fermentative production of threonine, that is, the cells do not require amino acid supplements to grow and produce threonine. In accordance with the present invention, the bacterial strain of the present invention does not require any recombinant plasmid containing one or more genes coding for threonine biosynthetic enzymes for the production of threonine, ie, the strain is capable of producing threonine without the need for one or more of the biosynthetic enzymes of threonine to be encoded by genes contained in a recombinant plasmid. Of course, the strains of the present invention optionally contain one or more recombinant plasmids, if desired. For example, while such plasmids are not required for the production of threonine, the strains of the present invention, however, may contain recombinant plasmids which code for threonine biosynthetic enzymes in order to increase threonine production. Similarly, the strains of the present invention may contain recombinant plasmids encoding other enzymes involved in threonine biosynthesis, such as aspartate semialdehyde dehydrogenase (asd). Preferably, the bacterial strains of the present invention are strains of Escherichia coli. More preferably, the bacterial strains of the present invention are E. coli strains that exhibit resistance to the macrolide antibiotic borrelidin. A particularly preferred example of the bacterial strains of the present invention is the strain of E. coli kat-13, which was deposited in the Cultivation Collection of the Agricultural Research Service (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA, on June 28, 1996 and to which the accession number NRRL B-21593 was assigned. The threonine operon (thr) in the chromosome of the cells of the bacterial strains of the present invention encodes the enzymes necessary for threonine biosynthesis. Preferably, the threonine operon consists of an AK-HD gene (thrA or metL), a homoserine kinase (thrB) gene and a threonine synthetase gene (thrC) More preferably, the thr operon consists of thrA (the AK I-HD I gene), thrB and thrC. Suitable thr operons can be obtained, for example, from the E. coli strain ATCC 21277 and the strain ATCC 21530 (ATCC = American Type Culture Collection, American Type Culture Collection). The thr operon of strain ATCC 21277 is particularly preferred. Multiple copies of the thr operon may be present on the chromosome. Preferably, the tii-r operon contains at least one non-attenuated gene, that is, the expression of the gene is not suppressed by the concentration (extracellular and / or intracellular) of one or more of the biosynthetic enzymes of threonine and / or of the products thereof (for example, threonine and isoleucine). The strain of the present invention may also contain a thr operon containing a defective thr attenuator (the regulatory region downstream of the transcription initiation site and upstream of the first structural gene) or a thr operon that lacks the thr attenuator. In a particularly preferred embodiment of the present invention, the thr operon encodes one or more biosynthetic threonine enzymes resistant to feedback, i.e., the activity of the enzymes is not inhibited by the extracellular and / or intracellular concentration of the intermediates and products of threonine biosynthesis. Preferably, the thr operon contains a gene that codes for a feedback-resistant AK-HD, such as an AK I-HD I that is resistant to feedback. The use of AK-HD resistant to feedback provides a higher level of enzymatic activity for the threonine biosynthesis, even in the presence of the L-threonine that is being produced. The expression of the threonine operon or operon in the strains of the present invention is controlled by a non-native promoter, i.e., a promoter that does not control the expression of the thr operon in the bacterial strains of E. coli normally found in nature. . Replacement of the native promoter of threonine biosynthetic enzymes by a strong non-native promoter to control the expression of the thr operon results in increased threonine production even with only one genomic copy of the thr operon. In addition, since a non-native promoter is used to control the expression of the threonine operon, it is not necessary to revert to the auxotrophic bacterial isoleucine strains in order to achieve a high threonine production. Illustrative examples of such promoters include, but are not limited to: the lac promoter; the trp promoter; the promoter P of the bacteriophage? the PR promoter; the lpp promoter; and the tac promoter. In the bacterial strains of the present invention it is particularly preferred to use the tac promoter.

In addition to the threonine operon, the chromosome of the bacterial cells of the present invention preferably also contains at least one gene encoding the enzyme aspartate semialdehyde dehydrogenase (asd). Preferably, the chromosome of the cells of the present invention contains at least one asd gene, at least one thrA gene, at least one thrB gene and at least thrC. Of course, the chromosome can contain multiple copies of one or more of these genes. Threonine dehydrogenase (tdh) catalyzes the oxidation of L-threonine to a-amino-β-ketobutyrate. Accordingly, in a particularly preferred embodiment, the chromosome of the cells of the present invention further contains at least one defective threonine dehydrogenase (tdh) gene. The defective tdh gene can be a gene having a reduced level of threonine dehydrogenase expression or a gene encoding a threonine dehydrogenase mutant having a reduced enzyme activity compared to that of the native threonine dehydrogenase. Preferably, the defective tdh gene used in the strains of the present invention does not express threonine dehydrogenase. Illustrative examples of suitable tdh genes that do not express threonine dehydrogenase, include a tdh gene having a chloramphenicol acetyltransferase gene (cat) inserted therein, or a tdh gene having a Tn5 transposon inserted therein, as described in US Patent No. 5,175,107. The bacterial strains of the present invention can be prepared by any of the methods and techniques known and available to those skilled in the art. Illustrative examples of suitable methods for constructing the bacterial strains of the present invention include mutagenesis using suitable agents such as NTG; gene integration techniques mediated by linear DNA fragments transformants and homologous recombination; and transduction mediated by the bacteriophage Pl. These methods are well known in the art and are described, for example, in J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1992); M. Singer and P. Berg, Genes & Genomes, University Science Books, Mili Valley, California (1991); J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); P. B. Kaufman et al. , Hadbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton, Florida (1995); Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J. E. Thompson, eds., CRC Press, Boca Raton, Florida (1993); and P. F. Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press, New York, NY (1989). In a particularly preferred embodiment of the present invention, the strain of E. coli 472T23, which requires threonine to grow, can be transformed into a threonine producer using a Pl-mediated transduction to induce the threonine operon of the E strain. ATCC 21211 coli, which can be obtained from the North American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA. This fcfr-r operon consists of a feedback-resistant aspartate kinase-homoserine dehydrogenase (thrA) gene, a homoserine kinase (thrB) gene and a threonine synthetase (thrC) gene. To improve the production of threonine in the strains of the present invention, the threonine dehydrogenase gene defective of the E. coli strain CGSC6945 (relevant genotype: tdh-1:: catl212; obtained in the E. coli Genetic Stock Center, 355 Osborne Memorial Laboratory, Department of Biology, Yale University, New Haven, Connecticut 06520-8104, USA) can be introduced by transfusion Pl. The resulting threonine producer can be further enhanced by mutagenesis with NTG and / or by selecting borrelidin resistance. Plasmids carrying an antibiotic resistance marker gene can be constructed, such as the kan gene (which encodes for kanamycin resistance) and a strong promoter, such as PL or tac, preferably flanked by DNA upstream of the thrA and a few hundred base pairs of the wild-type thrA gene (ie, not the entire thrA gene), and can be used as a vehicle to deliver the desired DNA fragments on the chromosome. The fragment in the plasmid can be isolated by digestion with a suitable restriction enzyme and can be purified and subsequently introduced, through transformation or electroporation, into a strain to remove the control region of the threonine operon and replace it by homologous recombination. with the desired fragment, ie, a marker gene of antibiotic resistance and a strong promoter at the beginning of the thrA gene. This fragment can then be transferred to the borrelidin-resistant strain by a Pl-transduction. The isoleucine requirement of the preferred host strain, 472T23, can be eliminated, for example, by introducing a wild-type allele of the marker through a transduction Pl. The unwanted nutritional requirements of other hosts may be similarly removed or in accordance with other methods known and available to those skilled in the art. A second embodiment of the present invention relates to the use of the bacterial strains described above in fermentation processes for the production of amino acids of the aspartate family. L-threonine, for example, is obtained by culturing the bacterial strains of the present invention in a synthetic or natural medium containing at least one carbon source, at least one source of nitrogen and, as appropriate, inorganic salts, growth and the like. Illustrative examples of suitable carbon sources include, but are not limited to: carbohydrates, such glucose, fructose, sucrose, starch hydrolyzate, cellulose hydrolyzate and molasses; organic acids such as acetic acid, propionic acid, formic acid, malic acid, citric acid and fumaric acid; and alcohols such as glycerol. Illustrative examples of suitable nitrogen sources include, but are not limited to: ammonia, including gaseous ammonia and aqueous ammonia; ammonium salts of organic or inorganic acids such as ammonium chloride, ammonium phosphate, ammonium sulfate and ammonium acetate; and other substances containing nitrogen including meat extract, peptones, corn syrup, casein hydrolyzate, soy hydrolyzate and yeast extract. After cultivation, the L-threonine that has accumulated in the culture broth can be separated according to any of the known methods, for example, by the use of ion exchange resins such as described in US Pat. 5,342,766. This method involves first removing the microorganisms from the culture broth by centrifugation and then adjusting the pH of the broth to approximately 2 using hydrochloric acid. The acidified solution is then passed through a strongly acidic cation exchange resin and the adsorbent eluted using dilute aqueous ammonia. The ammonia is removed by evaporation in vacuo and the resulting solution condensed. The addition of alcohol and the subsequent cooling provides crystals of L-threonine. Other amino acids of the aspartate family can be produced by methods similar to that described in more detail below. Isoleucine, for example, can be prepared from the bacterial strains of the present invention containing, on the chromosome or, in a plasmid, an amplified gene ilvA or a tdc gene both of which code for threonine deaminase, which is the first enzyme involved in the biotransformation of threonine in isoleucine. The amplification of this gene, for example, by the use of an ilvA gene that codes for a feedback-resistant enzyme, produces an increase in the biosynthesis of isoleucine. Similarly, methionine can be prepared by microorganisms such as E. coli containing at least one meth operon on the chromosome, ie, the metL gene (which codes for a K II-HD II), the metA gene ( homoserin succinyl transferase), the metB gene (cystathionine? -sintetase), the mete gene (cystathionine ß-lyase) and the genes metE and metH (homocysteine methylase). These genes, including the variants resistant to feedback thereof and, optionally, a non-native promoter, can be introduced into the chromosome of the host microorganism in accordance with one or more of the general methods previously described and / or known to the technicians. in the matter. Similarly, lysine can be prepared by microorganisms containing a gene encoding lysine biosynthetic enzymes (preferably a biosynthetic enzyme of the lysine resistant to feedback encoded by the lysC and / or dapA genes) and, optionally, , a non-native promoter. A third embodiment of the present invention relates to the use of borrelidin-resistant bacterial strains in fermentation processes for L-threonine production. Preferably, the borrelidin-resistant strains are mutants of an E. coli strain. A particularly preferred embodiment of such mutants is the E. coli kat-13 strain, which was deposited at the Agricultural Research Service Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA, on June 28, 1996 and to which the access number NRRL B-21593 was assigned. The resistance to borrelidin can be determined by any of the accepted methods known to those skilled in the art. For example, strains resistant to borrelidin can be isolated by seeding the candidate strains in a minimal medium containing approximately 139 uM borrelidin., as described in G. Nass and J. Thomale, FEBS Lett. 39: 182-186 (1974). In addition, resistance to borrelidin in certain strains manifests itself as a change of one or more phenotypic characteristics of the cells, for example, mutants of the strain of E. coli 6-8 resistant to borrelidin and its derivatives, have a Round appearance, more than similar to a cane. In such cases, the evidence of a change in a phenotypic characteristic may be sufficient to adequately identify strains resistant to borrelidin.

The borrelidin resistant mutants useful in this embodiment of the present invention are capable of producing threonine. The genes that code for threonine biosynthetic enzymes may be present on the chromosome or contained in plasmids or mixtures thereof. Multiple copies of these genes may also be present. Preferably, the genes encoding the biosynthetic threonine enzymes are resistant to attenuation control and / or encode feedback-resistant enzymes. As noted above, the borrelidin-resistant strains of the present invention may contain one or more recombinant plasmids, if desired. For example, the microorganisms of the present invention may contain recombinant plasmids that code for the biosynthetic enzymes of threonine. The bacterial strains of the present invention, similarly, may contain recombinant plasmids encoding other enzymes involved in threonine biosynthesis, such as aspartate semialdehyde dehydrogenase (asd) or growth-enhancing enzymes. Additionally, borrelidin-resistant strains can be modified if desired, for example, in order to increase threonine production, remove nutritional requirements and the like, using any of the methods and techniques known and available to those skilled in the art. . Illustrative examples of suitable methods for modifying the borrelidin-resistant E. coli mutants and their variants include, but are not limited to: mutagenesis by irradiation with ultraviolet light or X-rays, or by a treatment with a chemical mutagen such as nitrosoguanidine ( N-methyl-N '-nitro-N-nitrosoguanidine), methyl methanesulfonate, nitrogen mustard and the like; gene integration techniques such as those mediated by DNA fragments of linear transformation and homologous recombination and transduction mediated by bacteriophages such as Pl. These methods are well known in the art and are described, for example, in J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1992); M. Singer and P. Berg, Genes & Genomes, University Science Books, Mili Valley, California (1991); J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); P. B. Kaufman et al. , Hadbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton, Florida (1995); Methods in Plant Molecular Biology and Biotechnology, B. R. Glick and J. E. Thompson, eds., CRC Press, Boca Raton, Florida (1993); and P. F. Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press, New York, NY (1989). Preferably, the borrelidin-resistant mutants of the present invention are modified to include a non-native promoter upstream of, and in an operable relationship with, one or more of the genes encoding threonine biosynthetic enzymes, regardless of if these genes are in the chromosome and / or are contained in plasmids. According to a particularly preferred mode of this embodiment of the present invention, L-threonine is obtained by culturing at least one borrelidin-resistant bacterial strain in a natural or synthetic medium containing at least one carbon source, at least one source of nitrogen and, as appropriate, inorganic salts, growth factors and the like, as described above. The accumulated threonine can be recovered by any of the methods known to those skilled in the art. The following examples are illustrative only and are not intended to limit the scope of the present invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations of the methods of the present invention are possible., without departing from the spirit and the scope of it. Thus, it is intended that the present invention encompass the modifications and variations that fall within the scope of the appended claims and their equivalents. All patents and publications mentioned herein are incorporated by reference only. Example 1: Preparation of the strain of E. coli kat-13 A. Transfer of the threonine operon from the E. coli strain ATCC 21277 to the chromosome of the strain of E. coli 472T23. The strain of E. coli ATCC 21277 (Patent North American No. 3,580,810), available in the Collection American Type Culture, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA, is resistant to amino-β-hydroxyvaleric acid (AHV), but requires proline, thiamin, isoleucine, methionine to grow in a minimal medium. Strain ATCC 21277 is known to accumulate 6.20 g / 1 of threonine in a fermentation process. The threonine operon of strain ATCC 21277 consists of an aspartate kinase I-homoserine dehydrogenase I (thrA) gene encoding a feedback-resistant enzyme, a homoserine kinase (thrB) gene and a threonine synthetase gene (thrC). ). The strain of E. coli 472T23, which is deposited in the collection of commercial microorganisms of the former Soviet Union in the Research Institute of Antibiotics of the Former Soviet Union (USSR Antibiotics Research Institute) under the Registration Number BKIIM B-2307, is known to require threonine and isoleucine to grow in a minimum medium containing glucose, ammonia, vitamin B, and mineral salts. This strain can not produce threonine because it carries a defective thrC gene, which is an essential gene for threonine biosynthesis. Strain 472T23 also carries a defective threonine deaminase gene, ilvA, which codes for the first enzyme in the biosynthesis of isoleucine. A lysate of bacteriophage Pl was prepared by growing the phage in strain ATCC 21277. Next, strain 472T23 was infected with this PI lysate, in which a small number of phage were carrying the threonine operon of strain ATCC 21277. After infection, the threonine synthesizing bacteria were selected by dispersing them in a minimum medium E [glucose 0.05 g / l; MgSO4-7H2 0.2 g / 1; citric acid -H20 2.0 g / 1; K2HP04 10.0 g / 1; NaHNH4P04 • 4H20 3.5 g / 1; agar 15.0 g / 1] supplemented with 0.25 g / 1 isoleucine. Several threonine prototrophic transductants, which "were carriers of the threonine operon of strain ATCC 21277, were now able to grow in plates of minimal medium supplemented only with isoleucine." These transductants were selected by fermentation in shake flasks for their production of threonine, as will be described later in the Example 2. One of them, the G9 strain, producer of threonine, was selected for subsequent manipulations of the strain. B. Transfer of a defective threonine dehydrogenase (tdk) gene inserted with a chloramphenicol acetyltransferase gene (cat) into the chromosome of the E. coli strain G9. The strain CGSC6945, carrier of a defective threonine dehydrogenase (tdh) gene, was obtained from the JSJ. co-li Genetic Stock Center, 355, Osborne Memorial Laboratory, Department of Biology, Yale University, New Haven, Connecticut 06520-8104, USA. The threonine dehydrogenase gene is defective because the gene for chloramphenicol acetyltransferase is inserted into it (cat). To transfer this defective gene to strain G9, Pl phages were grown on strain CSCG6945 and the lysate was used to infect the G9 strain. Several transducers resistant to chloramphenicol were selected and threonine production was determined by shake flask fermentation as described in Example 2. One of them, the G909 strain, with a threonine titer higher than the G9 strain, was selected for subsequent manipulations. C. Insertion of a non-native promoter into the chromosome of the E. coli strain G909. With, in order to introduce the tac promoter into the chromosome of strain G909, a recombination j? homologous between a fragment of linear DNA and the chromosome of an exonucleasease V minus (recD). The linear DNA fragment contained 1.5 kb of the upstream sequence (5 'end) of the threonine operon, or kanamycin resistant marker, sequence of the tac promoter and approximately 480 bp of the thrA gene. This fragment, which provided a homology of the 5 'end, a selection marker (resistance to kanamycin), a strong and controllable promoter for the threonine operon (tac) and a homology of the 3' end, respectively, was generated from the Following way. The threonine operon of the W3110 wild-type E. coli strain was cloned into the Sphl restriction enzyme site of plasmid pUC19, using DNA from the clone lambda 676 of Dr. Yuji Kohara, Department.

Molecular Biology, School of Sciences, Nagoya University, Chikusa-ku, Nagoya, Japan. The DNA of clone lambda 676 and pUC19 were subsequently digested with the enzyme Sphl. The pUC19 fragment was subsequently dephosphorylated with shrimp alkaline phosphatase (SAP) and purified on agarose gel. The 6.9 kb fragment of the threonine operon of the lambda clone was also purified. Subsequently, these two fragments were ligated by the T4 DNA ligase to generate plasmid pAD103. An upstream flanking region was then constructed for the homologous recombination and the kanamycin resistance marker. PAD103 was digested with the restriction enzymes BstEII, Bbal and blunt ended by treatment with the Klenow fragment. The 1.5 kb fragment containing only the 5 'end (upstream) of the threonine operon (but not the thr operon itself or its control region) was isolated and ligated to the kanamycin resistance gene fragment of pUC4K (Pharmacia) , which was digested with the SalI restriction enzyme and treated with the Klenow fragment to conform to the 3 'end overhangs, to generate the intermediate plasmid pAD106. PAD103 was also digested with the restriction enzyme Taql and blunt-ended by treatment with the Klenow fragment. The fragment containing the native ribosome binding site and approximately 480 bp of the coding sequence of the thrA gene was isolated and then ligated to a fragment of pKK233-3 (Pharmacia), which had previously been digested with the restriction enzyme Smal and dephosphorylated with SAP, to obtain the plasmid pAD115, which contained the DNA sequence of the tac promoter, the ribosome binding sites and a few hundred bases of the thrA gene. The pAD115 was subsequently digested with the restriction enzyme BamHI and 0.75 kb of the DNA fragment containing the desired DNA sequences were isolated. PAD106 was also digested with Bait-HI and then dephosphorylated with SAP. Subsequently, the two fragments were ligated to obtain the plasmid pAD123, which contained the DNA sequence upstream of the threonine operon, a marker gene of kanamycin resistance, the tac promoter and approximately 480 bp of the start of the thrA gene. Then, pAD123 was digested with Spel, Bgl and the fragment containing the desired DNA sequences was isolated. The exonuclease strain V minus (recD) was prepared by growing phage PI in E. coli strain KW251 (relevant genotype: argAßl:: Tnl 0, recD1014, obtained in Pharmacia), which contained a recD gene with a cotransfusible transposon insertion TnlO in the argA gene. The lysate prepared from the phage was subsequently used to infect the G9 strain and the tetracycline resistant transductant G9T7 was isolated. The DNA fragment of plasmid pAD123 was introduced into E. coli strain G09T7 by electroporation. The strain resistant to kanamycin G9T7 was isolated and a lysate of phage Pl was prepared by growing the phage in this strain. Then, the phage Pl lysate was used to transduce strain G909. One of the kanamycin-resistant transductants of strain G909, tac3, which showed a higher threonine titre in the presence of IPTG, was isolated in a shake flask study. Then, a lysate of phage Pl was prepared with strain tac3 and subsequently used to infect strain 6-8 (to be described later) Transductants resistant to kanamycin were selected and one of them was isolated, strain 6 8tac3, which produced an even greater titre than the tac3 strain in a shake flask study D. NTG mutagenesis and isolation of borrelidin resistant mutants from E. coli strains G909 and 6-8. strain G909 were mutagenized by treatment with N-methyl-N '-nitro-N-nitrosoguanidine (NTG) (50 mg / l, 30 min at 36 ° C) using conventional methods.The resulting cells were dispersed in a plate of medium minimum agar E containing 0.25 g / 1 of L-isoleucine and 0.1% v / v of borrelidin After incubation for 3-5 days at 36 ° C, the large colonies that formed on the plate, which included strain 6-8, were selected by a borrelidin resistance test and L-threonine production. To test the resistance to borrelidin, each strain was cultured in 20 ml of the seed medium SM [32.5 g / 1 glucose; 1 g / 1 MgSO4-7H20; 24.36 g / 1 of K2HP04; 9.52 g / 1 of KH2P04; 5 g / 1 of (NH4) 2S? 4; 15 g / 1 yeast extract; pH 7.2] at 36 ° C for 17 hours with stirring.

The cells were harvested and washed with minimal E. The cell suspension was inoculated into a sterile tube containing 3 ml of minimal medium E and O, 0.1, 0.5 or 1 μM borrelidin. After 24 hours of cultivation at 36 ° C with shaking, the growth was determined by measuring the optical density at 660 nm. The results in relation to growth in the absence of borrelidin are shown below.

E. Removal of the requirement of isoleucine and the repressor gene of lactose (lacl). By introducing a non-native tac promoter and a thrA-resistant gene for feedback, the expression of the thr (thrA, thrB, thrC) operon is no longer controlled by the attenuation mechanism. As a result, isoleucine deprivation and / or the presence of an ilotrophic auxotrophic marker is no longer necessary for the production of threonine. Accordingly, the ilvA wild-type marker was introduced by transduction to strain 6- 8tac3 to set the isoleucine requirement to the strain, ie, eliminate the need for a medium supplemented with isoleucine for cell growth. The phage lysate Pl prepared from strain CGSC7334 (relevant genotype: lacI42:: Tnl O, lacZUl l ß; obtained in E. coli Genetic Stock Center, 355 Osborne Memorial Laboratory, Department of Biology, Yale University, New Haven, Connecticut 06520-8104, USA) was used to infect strain 6-8tac3 and positive transductants for isoleucine biosynthesis were selected. These transductants produced approximately the same amount of L-threonine as strain 6-8tac3 in a study in shake flasks. One of these transductants, strain 6-8tac3ile + was selected for subsequent manipulations. Since the threonine operon of strain 6-8tac3ile is under the control of the tac promoter, it was necessary to use isopropyl-β-D-thiogalactoside (IPTG) to induce the cells to fully express the th-r operon. The use of IPTG to induce the expression of the thr operon, however, is less preferred in accordance with the methods of the present invention. Accordingly, to eliminate this unnecessary regulatory impediment, a defective lac (lacl) repressor gene was introduced by infecting strain 6-8tac3ile + with phage Pl prepared in strain CGSC7334. The resulting transductants (6-8tac31acI-) were tested for their resistance to tetracycline and the tetracycline-resistant colonies were selected.

Example 2: Study of fermentation in shake flasks for the production of threonine. A comparison of threonine production between several strains of E. coli was determined by its fermentation performance in shake flasks. The tested strains were grown on LB agar medium [10 g / 1 tryptone, 5 g / 1 extract, 15 g / 1 agar]. After one to two days of growth, the cells were suspended in 5 ml of seed medium [dextrose 32.5 g / 1; K2HPO4 24.5 g / 1; KH2PO4 9.5 g / 1; Yeast extract 15 g / 1; (NH4) S? 4 5 g / 1; MgS? 4'7H20 1 g / 1] at pH 7.2. The strains were grown for 24 hours with a stirring speed of 250 rpm at 37 ° C. Then, 15 ml of the fermentation medium [dextrose 40 g / 1; yeast extract 2 g / 1; citric acid 2 g / 1; (NH4) 2S04 25 g / 1; MgSO4 • 7H20 2.8 g / 1; CaCO3 20 g / 1; trace metal solution 2 ml] at pH 7.2 was added to the seed medium and the fermentation process was carried out at 37 ° C with a stirring speed of 250 rpm. After cultivation, the amount of L-threonine that had accumulated in the medium was analyzed by HPLC (ISCO Model 2353 pump, Rainin Refractive Index Model RI-1 detector and aminex column Hp87-CA). The amount of L-threonine produced by each of the strains tested is presented below.

Example 3: Fermentation study. The E. coli strains of the present invention and their precursors were tested for the production of L-threonine by fermentation. The G909 strain was tested under the following conditions. 0.5 1 of the aqueous culture medium containing 30 g / 1 of trypticase soy broth and 5 g / 1 of yeast extract was inoculated in a 2 1 shake flask., with 1.5 ml of G909 and incubated in agitation at 35 ° C at 200 rpm, for 8.5 hours. 0.9 ml (0.03%) of the mature culture was inoculated in a glass fermenter containing 3.0 1 of the seed fermenting medium [10 g / 1 d.s. of corn liquor, 0.4 g / l of L-isoleucine, 2.5 g / l of KH2P04, 2.0 g / l of MgSO4-7H20, 0.5 g / l of (NH4) 2 S04, 0.192 g / l of anhydrous citric acid, 0.03 g / 1 of FeS04-7H20, 0.021 g / 1 of MnS04H20 and 80 g / 1 of dextrose]. The incubation was carried out under the following conditions: temperature of 39 ° C during the first 18 hours and then 37 ° C for the rest of the process; pH 6.9 (maintained by the addition of NH 4 OH); 3.5 LPM air flow; initial agitation of 500 rpm, which was then increased to maintain the optical density (OD) at 20%; and back pressure of 1-2 psi.

The conclusion of the seed fermentor stage was determined by the exhaustion of dextrose. They added 315 ml (15%) of the mature inoculum from the seed fermenter to a glass fermenter containing the same medium (main medium fermentor) previously listed with the following exceptions: the volume was 2.1 1 and 0.34 g / 1 of L-isoleucine was added. Incubation was carried out for 48 hours under the following conditions: temperature of 37 ° C; pH 6.9 (maintained with NHOH); air flow of 3.5 LPM until 20 hours and then increased to 4.0 LPM; initial agitation of 500 rpm, which was subsequently increased to maintain the DO at 20%; back pressure of 1-2 psi; and dextrose level of 10 g / 1 (maintained by feeding with a solution of 50% w / w dextrose). The fermentation was finished after 48 hours. The G909 strain produced the following results. A final titre of 62.3 g / 1 of threonine with a total productivity of 274 g and a yield of 23. 2 ts • The tac3 strain was tested under the same conditions as the G909 described above, with the following exception: 1 g / 1 of IPTG was added at the start of the main fermentation stage. With the addition of IPTG, the tac3 strain produced a final titer of 85.7 g / 1 of threonine with a total productivity of 355 g and a yield of 28.8-s. Strain 6-8 was tested under the same conditions as G909 described above. Strain 6-8 produced the following results: a final titre of 74.1 g / 1 of threonine with a total productivity of 290 g and a yield of 28.3%. Strain 6-8tac3 was tested under the same conditions as the previous tac3 strain, including the addition of IPTG. Strain 6-8tac3 produced the following results: A final titre of 99.3 g / 1 of threonine with a total productivity of 421 g and a yield of 35.1%. Strain 6-8tac3ile + was tested under the same conditions as strain 6-8tac3 previously described, with the following exception: L-isoleucine was not required in the seed fermentor stage or in the main fermentation stage. Due to a failure of agitation at 22.5 hours, only the title was recorded at 22 hours (62 g / 1 threonine). Strain kat-13 was tested under the same conditions as strain 6.8tac3 previously described, with the following exception: it was not necessary to add IPTG.

Under these conditions, strain kat-13 produced a final titer of 102 g / 1 of threonine with a total productivity of 445 g and a yield of 33.1%. The relevant genotypes of the strains constructed, the supplements required for the fermentative production of threonine and the registered titles, are presented in the following Table: Bor-R: Borrelidin resistance MR: Not performed ND: Not available ptacthrABC: are the thrA, thrB and thrC genes under the control of the tac promoter.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as an antecedent, what is contained in the following is claimed as property.

Claims (24)

1. CLAIMS 1. A process for the production of L-threonine, characterized in that it comprises the steps of: (a) cultivating an E. coli strain in a medium; and (b) recovering the L-threonine that is produced by said E. coli strain; where the strain of E. coli: (i) contains in the chromosome at least one threonine (thr) operon operably linked with at least one non-native promoter; and (ii) does not require any recombinant plasmid containing one or more genes encoding one or more of the biosynthetic threonine enzymes in order to produce threonine.
2. 2. The process according to claim 1, characterized in that the E. coli strain is capable of producing at least about 50 g / 1 L-threonine in about 30 hours.
3. 3. The process according to claim 1, characterized in that the non-native promoter is selected from the group consisting of the tac promoter, the lac promoter, the trp promoter, the tp promoter, the PL promoter and the PR promoter.
4. 4. The process according to claim 1, characterized in that the threonine operon contains a gene that codes for an aspartate kinase-homoserine dehydrogenase resistant to feedback.
5. 5. The process according to claim 1, characterized in that the E. coli strain is the kat-13 strain.
6. 6. The process according to claim 3, characterized in that the non-native promoter is the tac promoter.
7. The process according to claim 1, characterized in that the E. coli strain contains a defective threonine dehydrogenase gene in the chromosome.
8. 8. The process according to claim 1, characterized in that the threonine operon is obtained from strain ATCC 21277.
9. 9. The process according to claim 1, characterized in that the E. coli strain is resistant to borrelidin.
10. 10. A method for producing an amino acid-producing E. coli strain that is not a carrier of recombinant plasmids encoding one or more of the biosynthetic enzymes of said amino acid, characterized in that it comprises the steps of: (a) introducing genetic material from an amino acid producing microorganism, to the chromosome of an E. coli strain. (b) inserting a non-native promoter into the chromosome prior to the chromosomal location of, and operably linked to, the amino acid biosynthetic genes, to control the expression thereof; and (c) optionally, removing regulatory impediments and / or nutritional requirements for the amino acid biosynthesis of said chromosome.
11. 11. A strain of the E. coli microorganism characterized by the following: (i) its chromosome contains at least one threonine operon (thr) operably linked with at least one non-native promoter; and (ii) does not require any recombinant plasmid containing one or more of the genes encoding one or more of the biosynthetic threonine enzymes in order to produce threonine.
12. 12. The strain according to claim 11, characterized in that the threonine operon consists of a feedback-resistant aspartate kinase I-homoserine dehydrogenase I gene (thrA), a homoserine kinase (r-3) gene, a gene of threonine synthetase (thrC)
13. 13. The strain according to claim 11, characterized in that the E. coli strain is the kat-13 strain.
14. 14. The strain according to claim 11, characterized in that the non-native promoter is the tac promoter.
15. 15. The strain according to claim 11, characterized in that the E. coli strain contains a defective threonine dehydrogenase gene in the chromosome.
16. 16. The strain according to claim 11, characterized in that the threonine operon is obtained from strain ATCC 21277.
17. 17. The strain according to claim 11, characterized in that the E. coli strain is resistant to borrelidin.
18. 18. The strain in accordance with the claim 11, characterized in that the E. coli strain has the characteristics of strain NRRL B-21593.
19. 19. A process for producing L-threonine, characterized in that it comprises the steps of: (a) cultivating a strain of E. coli resistant to borrelidin in a medium; and (b) recovering the L-threonine that is produced by the E. coli strain.
20. 20. The process according to claim 19, characterized in that the E. coli strain is capable of producing at least about 50 g / 1 of L-threonine in about 30 hours.
21. 21. The process according to claim 19, characterized in that the E. coli strain contains at least one thononine operon (thr) operably linked with at least one non-native promoter.
22. 22. The process according to claim 21, characterized in that the threonine operon contains a gene coding for an aspartate kinase-ho oserin dehydrogenase resistant to feedback.
23. 23. The process according to claim 19, characterized in that the E. coli strain is the kat-13 strain.
24. 24. The process according to claim 21, characterized in that the non-native promoter is the tac promoter.