CA2299105A1 - Process for the fermentative production of l-amino acids using coryneform bacteria - Google Patents

Abstract

This invention relates to a process for the production of L-amino acids using coryneform bacteria, in which the glutamate dehydrogenase gene is amplified.

Description

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Process for the fermentative production of L-amino acids using coryneform bacteria The present invention provides a process for the fermentative production of L-amino acids using coryneform bacteria, in which the glutamate dehydrogenase gene is amplified.
Prior art L-Amino acids are used in animal nutrition, human medicine and the pharmaceuticals industry.
L-Amino acids are produced by fermentation using strains of coryneform bacteria which produce L-amino acids, in particular using Corynebacterium glutamicum. Due to the significance of this group of products, efforts are constantly being made to improve the production process.
Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-(2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant amino acids and produce L-amino acids.
For some years, methods of recombinant DNA technology have also been used to improve strains of Corynebacterium glutamicum which produce L-amino acids by amplifying individual biosynthesis genes and investigating the effect on L-amino acid production. Review articles on this subject ' . 980182 BT

may be found inter alia in Kinoshita ("Glutamic 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 Sinskey (Critical Reviews in Biotechnology 15, 73-lb3 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
The enzyme glutamate dehydrogenase catalyses the reductive amination of oc-ketoglutaric acid to yield glutamic acid.
French published patent application 2 575 492 describes a DNA fragment from Corynebacterium melassecola 801 which bears a glutamate dehydrogenase gene. It is possibly used therein to increase glutamic acid production in the fermentation of Corynebacterium melassecola. The nucleotide sequence of the glutamate dehydrogenase gene of Corynebacterium glutamicum ATCC13032 has been described by Bormann et al. (Molecular Microbiology 6, 317-326 (1992)).
The nucleotide sequence of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus is stated in Snedecor et a1. (Journal of Bacteriology 193, 6162-6167 (1991) ) .
Object of the invention The inventors set themselves the object of providing novel measures for the improved fermentative production of other L-amino acids.
Description of the invention L-Amino acids are used in animal nutrition, human medicine and the pharmaceuticals industry. There is accordingly general interest in providing improved processes for the production of L-amino acids.
When L-amino acids are mentioned below, they are taken to mean the protein-forming amino acids L-lysine, L-threonine, L-isoleucine, L-valine, L-proline, L-tryptophan and . 980182 BT

optionally the salts thereof and also L-homoserine, in particular L-lysine, L-threonine and L-tryptophan.
The present invention provides a process for the fermentative production of L-amino acids using coryneform bacteria, which in particular already produce the corresponding L-amino acids and in which the nucleotide sequence coding for the enzyme glutamate dehydrogenase is amplified, in particular overexpressed.
Preferred embodiments are stated in the claims.
In this connection, the term "amplification" describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are coded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene which codes for a corresponding enzyme having elevated activity and optionally by combining these measures.
The microorganisms provided by the present invention are capable of producing L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are the known wild type strains Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC1406'7 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and mutants or strains produced therefrom, 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 L-threonine producing strains Corynebacterium glutamicum FERM-P 5835 Brevibacterium flavum FERM-P 4164 and Brevibacterium lactofermentum FERM-P 4180, or the L-isoleucine producing strains Corynebacterium glutamicum FERM-P 756 Brevibacterium flavum FERM-P 759 and Brevibacterium lactofermentum FERM-P 4192 or the L-valine producing strains Brevibacterium flavum FERM-P 512 and Brevibacterium lactofermentum FERM-P 1845, and the L-tryptophan producing strains Corynebacterium glutamicum FERM-BP 478 Brevibacterium flavum FERM-BP 475 and Brevibacterium lactofermentum FERM-P 7127.
The inventors discovered that, after overexpression of L-glutamate dehydrogenase, coryneform bacteria produce L-amino acids in an improved manner, wherein L-glutamic acid is not claimed here.
The glutamate dehydrogenase gene of C. glutamicum described by Bormann et al. (Molecular Microbiology 6, 317-326 5 (1992)) may be used according to the invention. The glutamate dehydrogenase gene from other microorganisms, such as for example that from Peptostreptococcus asaccharolyticus, which has been described by Snedecor et a1. (Journal of Bacteriology 173, 6162-6167 (1991)), is also suitable. Alleles of the stated genes arising from the degeneracy of the genetic code or from functionally neutral sense mutations may also be used.
Overexpression may be achieved by increasing the copy number of the corresponding genes, or the promoter and regulation region located upstream from the structural gene may be mutated. Expression cassettes incorporated upstream from the structural gene act in the same manner. It is additionally possible to increase expression during fermentative L-amino acid production by means of inducible promoters. Expression is also improved by measures to extend the lifetime of the mRNA. Enzyme activity is moreover amplified by preventing degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids in a variable copy number or be integrated in the chromosome and amplified. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the nutrient media and culture conditions.
The person skilled .in the art will find guidance in this connection inter alia in Martin et a1. (Bio/Technology 5, 137-146 (1987)), in Guerrero et a1. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et aI. (Gene 102, 93-98 (1991)), in European patent EPS 0 472 869, in US patent 4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in patent application WO 96/15246, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.
Examples of plasmids by means of which glutamate dehydrogenase may be overexpressed are pEKl.9gdh-1 and pEKExpgdh, which are present in strains ATCC13032/pEKl.9gdh-1 and DHSa/pEKExpgdh. Plasmid pEKl.9gdh-1 is a shuttle vector, which contains the NADP-dependent glutamate dehydrogenase gene of C. glutamicum.
Plasmid pEKExpgdh is a shuttle vector, which contains the NADP-dependent glutamate dehydrogenase [gene] of Peptostreptococcus asaccharolyticus.
It may additionally be advantageous for the production of the corresponding L-amino acids to overexpress one or more enzymes of the particular amino acid biosynthesis pathway as well as glutamate dehydrogenase. Thus, for example ~ the dapA gene which codes for dihydrodipicolinate synthase may additionally be overexpressed in order to improve L-lysine producing coryneform bacteria (EP-B
0197335), ~ the gene which codes for acetohydroxy acid synthase may additionally be overexpressed in order to improve L-valine producing coryneform bacteria (EP-B 0356739), ~ the gene which codes for anthranilic acid phosphoribosyl transferase may additionally be overexpressed in order to improve L-tryptophan producing coryneform bacteria (EP-B
0124048), ~ the gene which codes for homoserine dehydrogenase may additionally be overexpressed in order to improve coryneform bacteria which produce L-homoserine or L-threonine or L-isoleucine (EP-A 0131171).

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It may furthermore be advantageous for the production of the corresponding L-amino acid to switch off unwanted secondary reactions in addition to overexpressing glutamate dehydrogenase (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
For the purposes of L-amino acid production, the microorganisms according to the invention may be cultivated continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A
summary of known cultivation methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described in "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington D.C., USA, 1981). Carbon sources which may be used include sugars and carbohydrates, such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as for example soya oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as for example palmitic acid, stearic acid and linoleic acid, alcohols, such as for example glycerol and ethanol, and organic acids, such as for example acetic acid. These substances may be used individually or as a mixture. Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture.
Phosphorus sources which may be used are potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The ..culture medium must furthermore contain metal salts, such as for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium.
The stated feed substances may be added to the culture as a single batch or be fed appropriately during cultivation.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Antifoaming agents, such as for example fatty acid polyglycol esters, may be used to control foaming. Suitable selectively acting substances, such as for example antibiotics, may be added to the medium in order to maintain plasmid stability. Oxygen or gas mixtures containing oxygen, such as for example air, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20°C to 45°C and preferably from 25°C to 40°C. The culture is continued until a maximum quantity of the desired L-amino acid has been formed. This objective is normally achieved within 10 hours to 160 hours.
L-Amino acids may be analysed automatically using anion exchange chromatography with subsequent ninhydrin derivatisation, as described by Spackman et al. (Analytical Chemistry, 30, 1190 (1958)).
The following microorganisms have been deposited with Deutschen Sammlung fur Mikrorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the 3S Budapest Treaty:

Corynebacterium glutamicum strain ATCC13032/pEKl.9gdh-1 as DSM 12614.
Escherichia coli K12 strain DHSa/pEKExpgdh as DSM 12613.

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Examples The present invention is illustrated in greater detail by the following practical examples.
To this end, testing was performed with amino acid 5 producing strains, in which the superiority of the claimed process is demonstrated:
a) the L-lysine producing strain Corynebacterium glutamicum DSM5715, (EP-B- 0435 132) and b) the L-threonine and L-isoleucine producing strain 10 Brevibacterium flavum DSM5399 (EP-B- 0385 940) and c) the L-valine producing, isoleucine-requiring strain ATCC130320i1vA, which has been deposited as DSM12455 with Deutschen Sammlung fur Mikroorganismen and Zellkulturen in Braunschweig (Germany) in accordance with the Budapest Treaty.
Example 1 Production of L-amino acid producers with amplified glutamate dehydrogenase Plasmid pEKl.9gdh-1 corresponds to the plasmid pEKl.9gdh described by Bormann et a1. (Molecular Microbiology 6, 317-326 (1992)). It was isolated from ATCC13032/pEKl.9gdh-1.
The known plasmid pEKExpgdh (Marx et al., Metabolic Engineering 1, 35-48 (1999)), which bears the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus (Snedecor et al., Journal of Bacteriology 173, 6162-6167 (1991)) was isolated in the same manner from E. coli strain DHSa/pEKExpgdh.
Strains DSM5715, DSM5399 and ATCC13032~i1vA were transformed with plasmid pEKl.9gdh-1 as described by Liebl et a1. (FEMS Microbiology Letters 65, 299-304 (1989)). The transformants were selected on brain/heart agar from Merck (Darmstadt, Germany) which had been supplemented with 50 mg/1 of kanamycin. In this manner, strains ' 980182 BT

DSM5715/pEKl.9gdh-1, DSM5399/pEKl.9gdh-1 and ATCC13032~i1vA/pEKl.9gdh-1 were obtained. Strain DSM5715 was transformed in the same manner with plasmid pEKExpgdh and strain DSM5715/pEKExpgdh obtained.
Example 2 Production of L-lysine Strain DSM5715/pEKl.9gdh-1 was precultivated in complex medium 2TY consisting of 16 g/1 of tryptone, 10 g/1 of yeast extract and 5 g/1 of NaCl. To this end, 60 ml of medium 2TY, contained in a 500 ml Erlenmeyer flask with 2 flow spoilers, were inoculated with an inoculating loop of the strain and the culture incubated for 12 hours at 150 rpm and 30°C.
In order to inoculate 60 ml of production medium, contained in a 500 ml Erlenmeyer flask with 2 flow spoilers, the preculture was centrifuged for 10 minutes at 5000 rpm in a Sepatech Minifuge RF (Heraeus, Hanau, Germany) centrifuge.
The supernatant was discarded and the pellet resuspended in 1 ml of production medium. An aliquot of this cell suspension was added to the production medium, such that an OD600 of approx. 2.0 was obtained. The production medium used was medium CGXII with a pH of 7.0 (Table 1) described by Keilhauer et a1. (Journal of Bacteriology 175, 5595-5603 (1993)) supplemented with 20 g/1 of glucose, 350 mg/1 of leucine and 50 mg/1 of kanamycin monosulfate. The cultures were incubated for 72 hours at 30°C and 150 rpm.

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Table 1 Component Concentration per litre (NHa) ZISOQ 20 g Urea 5 g KH2 P04 1 g KZHP04 1 g MgS04 ~ 7Hz0 0.25 g 3-Morpholinopropane- 42 g sulfonic acid FeS04 ~ 7H20 10 mg MnS04 ~ H20 10 mg ZnS04 ~ 7Hz0 1 mg CuS04 0.2 mg NiCl2 ~ 6H20 0.02 mg CaCl2 10 mg Protocatechuic acid 0.03 mg Biotin 200 ~,g Optical density (OD) (Biochrom Novaspec 4049, LKB
Instrument GmbH, Grafelfing, Germany) was then determined S at a measuring wavelength of 600 nm, as was the concentration of L-lysine formed using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column reaction with ninhydrin detection. Table 2 shows the result of the test.
Table 2 Strain OD L-lysine g/1 DSM5715 16.5 4.5 DSM5715/pEKl.9gdh-1 19.4 6.2 Example 3 Production of L-threonine and L-isoleucine Strain DSM5399/pEKl.9gdh-1 was precultivated in complete medium CgIII (Kase & Nakayama, Agricultural and Biological Chemistry 36 (9) 1611- 1621 (1972)) with 50~g/ml of kanamycin. To this end, 10 ml of medium CgIII, contained in a 100 ml Erlenmeyer flask with 4 flow spoilers, were inoculated with an inoculating loop of the strain and the culture incubated for 16 hours at 240 rpm and 30°C.
In order to inoculate 10 ml of production medium, contained in a 100m1 Erlenmeyer flask with 4 flow spoilers, the OD
(660 nm) of the preculture was determined. The main culture was inoculated to an OD of 0.1. The production medium used was the medium CgXII described by Keilhauer et al. (Journal of Bacteriology 1993, 175: 5595 - 5603). The composition of the medium is shown in Example 2. 4% of glucose and 50 mg/1 of kanamycin sulfate were added. The cells were incubated for 48 hours at 33°C, 250 rpm and 80% atmospheric humidity.
Optical density at 660 nm was then determined, as was the concentration of the L-threonine and L--isoleucine formed as stated in Example 2. Table 3 shows the result of the test.

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Table 3 Strain OD L-threonine L-g/1 isoleucine DSM5399 10.5 1.77 1.05 DSM5399/pEKl,9gdh-1 11.0 2.26 1.44 Example 4 Production of L-valine Strain ATCC130320i1vA/pEKl.9gdh-1 was precultivated in complete medium CgIII (Kase & Nakayama, Agricultural and Biological Chemistry 36 (9) 1611- 1621 (1972)) with 50~g/ml of kanamycin. To this end, 50 ml of medium CgIII, contained in a 500 ml Erlenmeyer flask with 4 flow spoilers, were inoculated with an inoculating loop of the strain and the culture incubated for 16 hours at 140 rpm and 30°C.
In order to inoculate 60 ml of production medium, contained in a 500 ml Erlenmeyer flask with 4 flow spoilers, the OD
(660 nm) of the preculture was determined. The main culture was centrifuged and the supernatant discarded. The pellet was resuspended in 5 ml of production medium and the main culture inoculated to an OD of 0.3. The production medium used was medium CgXII (Keilhauer et al., Journal of Bacteriology 1993 175: 5595 - 5603) as described in Example 3 (with 4% glucose). The cells were incubated for 48 hours at 30°C, 150 rpm.
Optical density at 660 nm was then determined, as was the concentration of L-valine formed as described as stated in Example 2. Table 4 shows the result of the test.

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Table 4 Strain OD L-valine g/1 ATCC130320i1vA 18.5 0.29 ATCC130320i1vA /pEKl.9gdh-1 17.6 0.45 Example 5 Production of L-lysine, L-valine and L-alanine 5 Strain DSM5715/pEKExpgdh was precultivated in complex medium 2TY consisting of 16 g/1 of tryptone, 10 g/1 of yeast extract and 5 g/1 of NaCl. To this end, 60 ml of medium 2TY, contained in a 500 ml Erlenmeyer flask with 2 flow spoilers, were inoculated with an inoculating loop of 10 the strain and the culture incubated for 12 hours at 150 rpm and 30°C.
In order to inoculate 60 ml of production medium, contained in a 500 ml Erlenmeyer flask with 2 flow spoilers, the preculture was centrifuged for 10 minutes at 5000 rpm in a' 15 Sepatech Minifuge RF (Heraeus, Hanau, Germany) centrifuge.
The supernatant was discarded and the pellet resuspended in 1 ml of production medium. An aliquot of this cell suspension was added to the production medium such that an OD600 of approx. 0.4 was obtained. The production medium used was medium CGC (table 5) described by Schrumpf et a1.
(Journal of Bacteriology 173, 4510-4516 (1991)), supplemented with 25 g/1 of glucose, 350 mg/1 of leucine, 42 g/1 of 3-morpholinopropanesulfonic acid and 50 mg/1 of kanamycin monosulfate at pH 7. The cultures were incubated for 30 hours at 30°C and 150 rpm.

Table 5 Component Concentration per litre (NH4) 5 g zS04 Urea 5 g KH2 0 . 5 g K2HP04 0.5 g MgS04 7H20 0.25 g ~

FeS04 7H20 10 mg ~

MnS04 H20 10 mg ~

ZnS04 7H20 1 mg ~

CuS04 0.2 mg NiCl2 6H20 0.02 mg ~

CaClz 2H20 10 mg ~

Biotin 200 ~.g Optical density (OD) ( Biochrom Novaspec 4049, LKB
Instrument GmbH, Grafelfing, Germany) was then determined at a measuring wavelength of 600 nm, as was the concentration of L-alanine, L-lysine and L-valine formed using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column reaction with ninhydrin detection. Table 6 shows the result of the test.

Table 6 Strain OD L- L-lysine L-valine Alanine g/1 g/1 DSM5715 29.4 Traces 1.6 0.1 DSM5715/pEKExpgdh 19.4 0.6 2.1 0.6

Claims (10)

1. Process for the production of L-amino acids by fermentation of coryneform bacteria, characterised in that, bacteria are used in which the nucleotide sequence coding for glutamate dehydrogenase is amplified, in particular overexpressed.

2. Process according to claim 1, characterised in that, the glutamate dehydrogenase produced in the bacteria used is NADP dependent.

3. Process according to claim 1, characterised in that, the glutamate dehydrogenase produced in the bacteria used is NAD dependent.

4. Process according to claim 1, characterised in that, bacteria are used in which the remaining genes in the metabolic pathway for the formation of the desired L-amino acids are additionally amplified.

5. Process according to claim 1, characterised in that, bacteria are used in which the metabolic pathways which reduce the formation of the desired L-amino acid(s) are at least partially switched off.

6. Process according to one of more of the preceding claims, characterised in that, a strain transformed with a plasmid vector ( ) is used and the plasmid vector bears the nucleotide sequence coding for glutamate dehydrogenase.

7. Process according to claim 6, characterised in that, a bacterium transformed with the plasmid vector pEK1.9gdh-1, deposited in Corynebacterium glutamicum under number DSM 12614, is used.

8. Process according to claim 6, characterised in that, bacteria transformed with the plasmid vector pEKExpgdh, deposited in E. coli under the number DSM 12613, are used.

9. Process for the fermentative production of L-amino acids according to one or more of the preceding claims, characterised in that the following steps are performed:
a) fermentation of the bacteria producing L-amino acid(s), in which at least the glutamate dehydrogenase gene is amplified, b) accumulation of the desired L-amino acid(s) in the medium or in the cells of the bacteria and c) isolation of the L-amino acid(s).

10. Process according to one or more of the preceding claims, characterised in that coryneform bacteria producing one or more of the amino acids L-lysine, L-threonine, L-isoleucine, L-valine, L-proline, L-tryptophan and L-homoserine are used.