MXPA00001638A - Process for the fermentative production of l-amino acids using coryneform bacteria - Google Patents

Abstract

Preparation of L-amino acids comprises fermentation of coryneform bacteria in which the nucleotide sequence encoding glutamate dehydrogenase (GDH) is amplified, especially over-expressed.

Description

PROCESS FOR THE PREPARATION BY FERMENTATION OF L-AMINO ACIDS USING CORINEFORM BACTERIA Field of the invention The object of the invention is a process for the preparation by fermentation of L-amino acids using coryneform bacteria, in which the glutamate hydrogenase gene is reinforced. Background of the Invention L-amino acids are used in animal feed in human medicine and in the pharmaceutical industry. The L-amino acids are used by fermentation with strains that produce L-amino acids from coryneform bacteria, especially Corynebacterium glutamicum. Due to the great importance of this group of products, we continuously work to improve these preparation procedures. Process improvements may consist of technical fermentation measures such as agitation and oxygen feed, or the composition of nutritive media such as sugar concentration during fermentation, or post-processing to the form of a product, by means of ion exchange chromatography or by intrinsic properties of microorganisms. To improve the performance properties of these microorganisms, methods such as mutagenesis are used, REF .: 32803 selection and selection of mutants. In this way, strains are obtained which are resistant against antimetabolites, such as, for example, the lysine analogue S- (2-aminoethyl) -cysteine) or auxotrophs for the regulatory amino acids which produce L-amino acids. For several years, methods of DNA recombination technique have been used for the improvement of strains of strains that produce L-amino acids of Corynebacterium glutamicun, in which individual biosynthesis genes are amplified and their effect on the production of the L-amino acids. An article on this is among others that of Konoshita ("Glutamicum Acid Bacteria" in Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Jetten and Sunskey (Critical Reviews in Biotechnology 15, 73-.103 (1995)) and Sahm et al., (Annuals of the New York Academy of Science 782, 23-39 (1996)) . The glutamate dehydrogenase enzyme catalyzes the reductive amination of a.-ketoglutaric acid to qutamic acid In French publication 2675492 a DNA fragment of Corynebacterium melassecola 801 carrying a glutamate dehydrogenase gene is described. It is probably used there to increase the production of glutamine acid in the fermentation of Corynebacterium melassecola. The nucleotide sequence of the < glutamate dehydrogenase from Corynebacterium glutamicum ATCC13032 was described by Börmann et al. (Molecular Microbiology 6, 317-326 (1992)). The nucleotide sequence of the glutamate dehydroqenase gene of Peptostreptococcus asaccharolyticus is given by Snedecor et al. (Journal of bacteriology 173, 6162-6167 (1991)). Task of the Invention The inventors set themselves the task of obtaining new measures to improve the preparation by fermentation of other L-amino acids. Detailed Description of the Invention L-amino acids have application in animal feed, human medicine and the pharmaceutical industry. Therefore there is a general interest in a new and improved process for the production of L-amino acids. When L-amino acids are mentioned below, protein-forming amino acids are implicated, L-lysine, L-threonine, L-isoleucine, L-valine, L-proline, L-tryptophan and eventually its salts and also L-homoserine, especially L-lysine, L-threonine and L-tryptophan. The object of the invention is therefore a process for the preparation by fermentation of L-amino acids using coryneform bacteria which in particular already produce the corresponding L-amino acids and in which the nucleotide sequence encoding the enzyme glutamate hydrogenase is reinforced . Preferred embodiments are found in the claims. The term "booster" describes in this respect the increase in intracellular activity of one or more enzymes in a microorganism, which are encoded by the corresponding DNA, for which, for example, the number of copies of the gene or genes is increased, a strong promoter or a gene that encodes the corresponding enzyme with high activity is used, and these are eventually combined measurements. The microorganisms that are the object of the present invention can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, melase, starches, cellulose or glycerin and ethanol. It may be a representative of the coryneform bacteria, especially of the genus Corynebacterium. In the genus Corynebacteirum should be mentioned in particular the type Corynebactyerium glutamicum, which are known in the enzyme for its ability to produce L-amino acids. Suitable the genus Corynebacterium especially the type Corynebacterium glutamicum strains are the native type known Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 strains and mutants or prepared strains such as, for example, the L-lysine producing strains Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 and Brevibacterium lactofermentum FERM-P 1712, or the strains producing L-threonine Corynebacterium glutamicum FERM-P 5835 Brevibacterium flavum FERM- P 4164 and Brevibacterium lactofermentum FERM-P 4180, or the strains producing L-isoleucine Corynebacterium glutamicum FERM-P 756 Brevibacterium flavum FERM-P 759 and Brevibacterium lactofermentum FERM-P 4192, or strains producing L-valine Brevibacteriu m flavum FERM-P 512 and Brevibacterium lactofermentum FERM-P 1845, or the strains producing L-tryptophan Corynebacterium glutamicum FERM-BP 478 Brevibacterium flavum FERM-P 475 and Brevibacterium lactofermentum FERM-P 7127. From this the inventors found as bacteria After the overexpression of the L-glutamate hydrogenase, they produce L-amino acids in an improved manner, and L-glutaminic acid is not claimed here. The glutamate hydrogenase gene of C. glutamicum described by Börmann et al. (Molecular Microbiology 6, 317-326 (1992)) can be used according to the invention. Furthermore, the glutamate dihydrogenase gene of other microorganisms, such as Peptostptococcus asaccharolytcus, which was described by Snededor et al. (Journal of Bacteriology 173, 6162-6167 (1991)). In addition, the alleles of the mentioned genes can be used, which is obtained by means of the degeneration of the genetic code by means of sense mutations (sense mutations) of neutral function. To obtain the over-expression the number of corresponding copies of the gene can be increased or the promoter and regulatory region, which is upstream of the structural gene, can be imitated. Similarly, the expression cartridges that are built upstream of the structural gene are used. By means of inducible promoters it is additionally possible to increase the expression in the course of pro-fermentation production of L-amino acids. By means of measures to lengthen the life of m-RNA expression is also improved. Furthermore, avoiding the reduction of the enzymatic protein reinforces the enzymatic activity. Genes or genetic constructs can be found in the plasmids with different numbers of copies or be integrated and amplified in the chromosome. Alternatively, an over-expression of the genes in question can be obtained by means of the modification of the composition of the medium and the performance of the culture. Data on this is found by the technician among others in the writings of Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsychiya and Morinaga (bio / technology 6, 428-430 (1988)) by Eikmanns et al. (Gene 102, 93-98 (1991)), in the European patent document EP 0 472 869, in the patent of US 4,601,893, by Schwarzer and Pühler (bio / technology 9, 84-87 (1991), by Reinscheid et al. Applied and Enviromental Microbiology 60, 126-132 (1994)), in LaBarre et al (Journal of Bacteriology 175, 1001-1007 (1993)), in patent application WO 96/15246, by Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), by Makrides (Microbiological Revies 60: 512-538 (1996)) and in the known texts of Genetics and Molecular Biology Examples of plasmids that can be overexpressed with the help of their glutamate dehydrogenase are pEKl, 0gdh-l and pEKExpgdh, which are found in strains ATCC13032 / pEKl, 9gfdh-l and DH5a / pEKExpgd.The plasmid pEKl, 9gdh-l is a pendulum vector, which contains the glutamate-dependent dihydrogenase gene of glutamate. NADP of C-glutamicum The pELExpgdh plasmid is a pendulum vector containing the NAD-dependent glutamate dehydrogenase gene of Peptostreptoc occus asaccharolyticus. Additionally it may be advantageous for the production of the corresponding L-amino acids, in addition to over-expressing the glutamate dehydrogenase also one or more enzymes of the amino acid biosynthetic pathway in question. For example, • to improve the coryneproducer bacteria producing L-lysine, the coding gene can be overexpressed to dehydrodipicolinate synthase (EP-B 0197335), • to improve the coryneproducer bacteria producing L-valine additionally over-express the coding gene to acetohydroxy acid synthase (EP-B-0356739), • to improve the coryneproducer bacteria producing L-tryptophan additionally over-expressing the coding gene to anthranilic phosphoribosyl transferase (EP-B 0234048), • to improve the L-homoserin or L-threonine-producing coryneform bacteria or L-isoleucine additionally over-express the gene encoding homoserine dehydrogenase (EP-A 0131171). Furthermore, it may be advantageous for the production of the corresponding L-amino acids in addition to the overexpression of glutamate dehydrogenase to avoid unwanted side reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overpoduction of Microbial Products, Krumphanzl, Sikytam Vanek (eds.); Academic Press, London, UK, 1982), The microorganisms produced according to the invention can be grown continuously or discontinuously in batch processes or in the fed batch or repetitive feeding method. (repeated fed batch) in order to produce L-amio acids. A summary of known cultivation methods can be found in Chmiel's text (Bioprozestechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer verlag, Stuttgart, 1991)) or in the text of Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium used must suitably be sufficient for the requirements of the microorganisms in question. The description of culture media of different microorganisms can be found in the manual "Manual of Methods for General Bacteriology" of the American Society of Bacteriology (Washington D.C., US, 1981). As a carbon source, sugars and carbohydrates can be used, such as glucose, sucrose, lactose, fructose, maltose, melase, starches and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil. fatty acids such as palmitic acid, stearinic acid and linoleic acid, alcohols such as for example glycerin and ethanol and organic acids such as acetic acid. These substances can be used individually or in mixtures. Nitrogen-containing organic compounds such as peptone, yeast extract, meat extract, malt extract, corn steep water, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, phosphate can be used as nitrogen source. of ammonium, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used alone or as mixtures. Phosphoric acid, potassium dihydrophosphate or dipotassium hydrophosphate or the corresponding sodium-containing salts can be used as phosphorus sources. The culture medium must also contain salts of metals such as magnesium sulfate or iron, which are necessary for growth. Finally, growth promoters such as amino acids and vitamins can be used in addition to the mentioned substances. Suitable pre-stages can be added to the culture medium. The additives mentioned can be added to the culture in the form of a single charge or in a suitable form added during cultivation.

To control the pH of the culture, basic compounds are used, such as sodium hydroxide, potassium hydroxide, ammonia or acidic compounds such as phosphoric acid or sulfuric acid in a suitable form. To control foaming, anti-foaming agents such as fatty acid polyglycol ester or silicone oils can be used. To maintain the stability of the plasmids, selective substances, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or gaseous mixtures containing oxygen, such as air, are introduced into the culture. The culture temperature is usually at 20 ° C to 45 ° C and preferably at 25 ° C to 40 ° C. The culture is continued until a maximum amount of L-amino acid has been formed. This objective is normally reached in the course of 10 to 160 hours. The analysis of L-amino acids can be performed automatically based on anion exchange chromatography with the subsequent derivation of ninhydrin as described by Spackman et al. (Analytical Chemistry, 30, 1190 (1958)). The following microroganisms were deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty: Strain ATCC13032 (pEKI, 9gdh-l of Corynebacterum glutamicum, as DSM 12614 Strain DH5a / pEKExpgdh of Escherichia coli K12 as DSM 12613. EXAMPLES The present invention will be explained in more detail with the help of the exemplary embodiments. with amino acid producing strains, in which the superiority of the presented procedure is demonstrated: a) The strain of Corynebacterium glutamicum producing L-lysine DSM515, (EP-B-0435 132) and b) the strain of Brevibacterium flavum producing DSM5399 (EP-B-0385940) and c) The strain ATCC13032? IlvA producing L-valine that requires isoleucine, which was deposited as DSM12455 in the Collection German Microorganisms and Cell Cultures in Braunschweig (Germany) in accordance with the Budapest Treaty. Example 1 Preparation of producers of L-amino acids with reinforced glutamate dehydroaenase The plasmid pEK1, 9gfdh-l corresponds to the plasmid described by Börmann et al. (Molecular Microbiology 6, 317-326 (1992)). It was isolated from ATCC13032 / pEK1, Ogdh-1. Similarly, the known plasmid pEKExpgdh (Marx et al., Metabolic Engineering 1, 35-48 (1999)), which carries the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus (Snedecor et al., Journal of Bacteriology 173, 6162-6167). (1991)), was isolated from the E. coli strain DH5a / pEKExpgdh. Strains DSM5715, DSM5399 and ATCC13032? IlvA were transformed as described by Leibl et al. (Fems Microbiology letters 65, 299-304 (1989)) with the plasmid pekl, 9gdh-l. The selection of the transformants was carried out on a brain-heart agar from Merck (Darmstadt, Germany) with was supplemented with 50 mg / l. In this way the strains DSM5715 / Pekl, 9gdh-l were formed, DSM5399 / pEKl, 9gdh-l and ATCC13032? LLVa / Pekl, 9gdh-l. In the same way, strain DSM5715 was transformed with plasmid pEKExpgdh and strain DSM5715 / pEKExpgdh was obtained. Example 2 Preparation of L-lysine The strain DSM5715 / Pekl, 9gdh-1 was precultured in a 2TY complex medium consisting of 16 g / 1 Trypton, 10 g / 1 yeast extract and 5 g / 1 NaCl. For this, 60 ml of 2TY medium were added, which were obtained in a 500 ml Erlenmeyer flask with 2 mouths, inoculated with one unit of the strain and the culture was cultured for 12 hours at 150 rpm, and 30 ° C. To inoculate 60 ml of the production medium, which was obtained in the 500 ml Erlenmeyer flask with 2 nozzles, the preculture was centrifuged at 5000 rpm in a Sepatech Minifuge RF centrifuge (Heraeus, Hanau, Germany) for 10 minutes. The cream was discarded and the pellet was resuspended in 1 ml of production medium. An aliquot of this cell suspension was added to the production medium, in such a way that an OD600 of approx. 2.0. The CGXII medium described by Keilhauer et al. Was used as the production medium. (Journal of Bacteriology 175, 5595-5603 (1993)) with pH 7.0 (Table 1) supplemented with 20 g / 1 glucose, 350 mg / l leucine and 50 mg / l kanamycin monosulfate. The cultures were incubated for 72 hours at 30 ° C and 150 rpm. Table 1 The optical densities (OD) (Biochrom Novaspec 4049, LKB Instrument GmbH, Gräfing, Germany) were then determined at a measured wavelength of 600 nm and the concentration of L-lysine formed with an amino acid analyzer from Eppenmdorf. -BioTronik (Hamburg, Germany) by means of ion exchange chromatography and post-column reaction with detection of hybridine. Table 2 shows the results of this test. Table 2 Example 3 Preparation of L-threonine and L-isoleucine Strain DSM5399 / pEK1, 9gdh-1 was pre-cultured in complete medium CglII (Kase &Nakayama, Agricultural and Biological Chemistry 36 (9) 1611-1621 (1972) with 50 μg / ml kanamycin 10 ml of CglII medium was inoculated, which was obtained in the 100 ml Erlenmeyer flask with 4 nozzles with one unit of the strain and the culture was incubated for 16 hours at 240 rpm and 30 ° C. To inoculate 10 ml of production medium that was obtained in the 100 ml Erlenmeyer flask with 4 nozzles, the OD (660 n) of the preculture was determined.The main culture was inoculated at an OD of 0.1. used the CgXII medium described by Keilhauer et al., (Journal of Bacteriology 1993, 175: 5595-5603). The composition of the medium is represented in example 2. 4% glucose and 50 mg / l of kanamycin sulfate were added.

The cells were incubated at 33 ° C, 250 rpm and 80% humidity in the air for 48 hours. The optical densities were then determined at 660 nm and the concentration of L-threonine and L-isoleucine formed as described in Example 2. The results of these tests are given in Table 3.

Table 3 EXAMPLE 4 Preparation of L-valine Strain ATCC13032? IlvA / pEKI, 9gdh-1 was precultured in complete medium CglII (Kase &Nakayama, Agricultural and Biological Chemistry 36 (9) 1611-1621 (1972)) with 50 μg / ml Kanamycin For this, 50 ml of CglII medium was inoculated, which was obtained in the 500 ml Erlenmeyer flask with 4 nozzles with one unit of the strain and the culture was incubated for 16 hours at 140 rpm and 30 ° C. To inoculate 60 ml of production medium that was obtained in the 500 ml Erlenmeyer flask with 4 mouths, the OD (660 n) of the preculture was determined. The main culture was centrifuged and the residue was discarded. The pellet was taken in 5 ml of production medium and the main culture was inoculated to an OD of 0.3. The CgXII medium described by Keilhauer et al. Was used as the production medium, (Journal of Bacteriology 1993, 175: 5595-5603) as described in example 3 (with 4% glucose). The composition of the medium is represented in example 2. The cells were incubated at 30 ° C, 150 rpm for 48 hours. Next, the optical densities at 660 nm and the concentration of L-valine formed as described in example 2 were determined. Table 4 gives the results of those tests. Table 4 E p e 4 Preparation of L-lysine, L-valine and L-alanine Strain DSM5715 / pEKExpgdh was precultured in a complex 2TY medium consisting of 16 g / 1 Trypton, 10 g / 1 yeast extract and 5 g / 1 of NaCl. For this, 60 ml of 2TY medium were added, which were obtained in a 500 ml Erlenmeyer flask with 2 mouths, inoculated with one unit of the strain and the culture was cultured for 12 hours at 150 rpm, and 30 ° C. To inoculate 60 ml of the production medium, which was obtained in the 500 ml Erlenmeyer flask with 2 nozzles, the preculture was centrifuged at 5000 rpm in a Sepatech Minifuge RF centrifuge (Heraeus, Hanau, Germany) for 10 minutes. The cream was discarded and the pellet was resuspended "in 1 ml of production medium, an aliquot of this cell suspension was added to the production medium, in such a way that an OD600 of 0.4 was obtained. the CGC medium described by Schrumpf et al (Journal of Bacteriology 173, 4510-4516 (1991)) (Table 5) supplemented with 25 g / 1 glucose, 350 mg / l leucine and 52 g / 1 3-morpholinopropanesulfonic acid and 50 mg / l of kanamycin sulfate at a pH of 7. The cultures were incubated for 30 hours at 30 ° and 150 rpm.

The optical densities (OD) were then determined (Biochrom Novaspec 4049, LKB Instrument GmbH, Gráfeling, Germany) at a measured wavelength of 600 nm and the concentration of L-alanine, L-lysine and L-valine formed with a amino acid analyzer from the firm Eppenmdorf-BioTronik (Hamburg, Germany) by means of ion exchange chromatography and post-column reaction with detection of hybridine. Table 6 gives the results of this test. Table 6 It is clear that 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.

Claims (6)

1. CLAIMS Having described the invention as above, property is claimed as contained in the following:
2. Procedure for the preparation of L-amino acids by means of the fermentation of coryneform bacteria, characterized in that bacteria are used in which the nucleotide sequence coding for glutamate dehydrogenase is reinforced, in particular it is over-expressed. 2. Process according to claim 1, characterized in that the glutamate hydrogenase produced in the bacteria used depends on NADP.
3. 3. Method according to claim 1, characterized in that the glutamate hydrogenase produced in the bacteria used depends on NAD.
4. 4. Method according to claim 1, characterized in that bacteria are used in which the other genes of the metabolic pathway of the formation of the desired L-amino acids are additionally strengthened.
5. 5. Process according to claim 1, characterized in that bacteria are used in which the metabolic pathways that reduce the formation of the desired L-amino acids are at least partially deactivated.
6. 6. - Method according to one or more of the preceding claims, characterized in that a strain transformed with a plasmid vector is used and the plasmid vector carries the nucleotide sequence coding for glutamate dihydrogenase. 1 . - Process according to claim 6, characterized in that bacteria transformed with the plasmid vector pEK1, 9gdh-1 deposited in Corynebacterium glutamicum under the number DSM 12614 are used. 8. Method according to claim 6, characterized in that they are used bacteria transformed with the plasmid vector pEKExpgdh deposited in E. coli under the number DSM 12613. 9. - Process for the preparation by fermentation of L-amino acids according to one or more of the preceding claims, characterized in that the following steps are carried out a) fermentation of the L-amino acid producing bacteria in which at least the glutamate dihydrogenase gene is reinforced, b) increase of the desired L-amino acids in the medium or in the cells of the bacteria and c) isolated from the L-amino acids. 10. Method according to one or more of the preceding claims, characterized by using coryneform bacteria that produce one or more of the amino acids L-lysine, L-threonine, L-isoleucine, L-valine, L-proline, L -triptofano and L-homoserina.