AU1760000A - 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

S&F Ref. 494670

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ORIGINAL

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Degussa-Huls Aktiengesellschaft DE-60287 Frankfurt am Main Germany Forschungszentr-um Julich GmbH D-52425 Julich Germany Actual Inventor(s): Address for Service: Invention Title: Achim Marx, Bettina Mockel, Walter Pfefferle, Hermann Sahmn, Albert De Graaf and Lothar Eggeling Spruson Ferguson St Martins Tower 3 1 Market Street Sydney NSW 2000 Process for the Fermentative Production of L-amino Acids using Coryneformn Bacteria The following statement is a full description of this invention, including the best method of performing it known to me/us:- D um e t r ,.ec 1 V o n~ 18 t-k6 2000

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Btch No: 5845c 980182 BT 1 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.

15 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 20 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 2 may be found inter alia in Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial Microorganisms, Demain and Solomon Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (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 a-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 15 sequence of the glutamate dehydrogenase gene of Corynebacterium glutamicum ATCC13032 has been described by B6rmann et al. (Molecular Microbiology 6, 317-326 (1992)) The nucleotide sequence of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus is stated in 20 Snedecor et al. (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 3 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 15 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 25 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 ATCC14067 980182 BT 4 Brevibacteriurn lactofe-rmen turn ATCC13869 and Brevibacteriurn divarica turn ATCC14020 and mutants or strains produced therefrom, such as for example the L-lysine producing strains Corynebacteriurn glutarnicum FERN-P 1709 Brevibacteriurn flavun FERN-P 1708 and Brevibacteriun lactoferrnenturn FERM-P 1712, or the L-threonine producing strains Corynebacteriurn glutanicun FERM-P 5835 Brevibacterium flavurn FERN-P 4164 and BDrevibacteriurn lactofermentun FERN-P 4180, or the L-isoleucine producing strains Corynebacterium glutanicun FERN-P 756 B3revibacteriurn flavun FERN-P 759 and Brevibacterium lactofermentum FERN-P 4192 20 or the L-valine producing strains Brevjbacterium flavun FERN-P 512 and Brevibacterium lactofermentum FERN-P 1845, and the L-tryptophan producing strains Corynebacterj ur glutanicun FERM-BP 478 Brevibacteriurn filavurn FERM-Bp 475 and Brevibacterium lactofernentun FERM-P 7127.

The inventors discovered that, after overexpression of L-glutamate dehydrogenase, coryneform bacteria produce 980182 BT L-amino acids in an improved manner, wherein L-glutamic acid is not claimed here.

The glutamate dehydrogenase gene of C. glutamicum described by B6rmann et al. (Molecular Microbiology 6, 317-326 (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 al. (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 15 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 20 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 25 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 al. (Bio/Technology 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (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 980182 BT 6 126-132 (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 pEK1.9gdh-1 and pEKExpgdh, which are present in strains ATCC13032/pEK1.9gdh-1 and DH5a/pEKExpgdh. Plasmid pEK1.9gdh-1 is a shuttle vector, which contains the NADPdependent glutamate dehydrogenase gene of C. glutamicum.

Plasmid pEKExpgdh is a shuttle vector, which contains the NADP-dependent glutamate dehydrogenase [gene] of 15 Peptostreptococcus asaccharolyticus.

a 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 Lvaline 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).

980182 BT 7 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 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, 15 1991)) or in the textbook by Storhas (Bioreaktoren und 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 20 various microorganisms are described in "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington USA, 1981). Carbon sources which may be used include sugars and carbohydrates, such as for example glucose, sucrose, lactose, fructose, maltose, 25 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 980182 BT 8 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 15 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 20 in order to maintain plasmid stability. Oxygen or gas oo 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 0 C to 45 0 C and preferably from 25 0 C to 40 0 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 und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty: 9 19.Corynebacterium glutamicum strain ATCC 13032/pEK.9gdh-1 as DSM 12614 on 8 January Escherichia coli Kl 2 strain DH5a/pEKExpgdh as DSM 12613 on 8 January 1999.

0 *0 G oo C08102 980182 BT Examples The present invention is illustrated in greater detail by the following practical examples.

To this end, testing was performed with amino acid 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 Brevibacterium flavum DSM5399 (EP-B- 0385 940) and c) the L-valine producing, isoleucine-requiring strain ~ATCC13032AilvA, which has been deposited as DSM12455 with Deutschen Sammlung fir Mikroorganismen und Zellkulturen in Braunschweig (Germany) in accordance 15 with the Budapest Treaty.

Example 1 Production of L-amino acid producers with amplified glutamate dehydrogenase 20 Plasmid pEK1.9gdh-1 corresponds to the plasmid pEKl.9gdh described by B6rmann et al. (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 Strains DSM5715, DSM5399 and ATCC13032AilvA were transformed with plasmid pEK1.9gdh-l as described by Liebl et al. (FEMS Microbiology Letters 65, 299-304 (1989)). The transformants were selected on brain/heart agar from Merck (Darmstadt, Germany) which had been supplemented with mg/l of kanamycin. In this manner, strains 980182 BT 11 DSM5715/pEK1.9gdh-l, DSM5399/pEK1.9gdh-l and ATCC13032AilvA/pEK1.9gdh-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/pEK1.9gdh-1 was precultivated in complex medium 2TY consisting of 16 g/1 of tryptone, 10 g/l of yeast extract and 5 g/l of NaC1. 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 0

C.

9**9 In order to inoculate 60 ml of production medium, contained 15 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 al. (Journal of Bacteriology 175, 5595-5603 (1993)) supplemented with 20 g/l of glucose, 350 mg/l of S*os.: 25 leucine and 50 mg/l of kanamycin monosulfate. The cultures were incubated for 72 hours at 30 0 C and 150 rpm.

980182 BT 12 Table 1 Component

A

S.

A

A

A A (NHu2OS 4 NgSl4 -6H 2 0 Prlooaeic acid BiSon H2 Concentration per litre g ig ig 0.25 g 42 g Mg 10 Mg 1 mg 0.2 mg 0. 03 mg 200 g Optical density (OD) (Biochrom Novaspec 4049, LKB Instrument GmbH, Grdfelfing, Germany) was then determined at a measuring wavelength of 600 nm, as was the concentration of L-lysine formed using an amino acid analyser from Eppendorf-Bio'pronik (Hamburg, Germany) by ion 980182 BT 13 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 DSM5715/pEK1.9gdh-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 O:P 10 Chemistry 36 1611- 1621 (1972)) with 50pg/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 0

C.

15 In order to inoculate 10 ml of production medium, contained in a 100ml 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/l of kanamycin sulfate were added. The cells were incubated for 48 hours at 33 0 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.

980182 BT 14 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 ATCC13032AilvA/pEK1.9gdh-l was precultivated in complete medium CgIII (Kase Nakayama, Agricultural and Biological Chemistry 36 1611- 1621 (1972)) with of kanamycin. To this end, 50 ml of medium CgIII, contained in a 500 ml Erlenmeyer flask with 4 flow spoilers, were o 10 inoculated with an inoculating loop of the strain and the culture incubated for 16 hours at 140 rpm and 30 0

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 15 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 0 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.

980182 BT Table 4 Strain OD L-valine g/1 ATCC13032AilvA 18.5 0.29 ATCC13032AilvA /pEK1.9gdh-l 17.6 0.45 Example Production of L-lysine, L-valine and L-alanine Strain DSM5715/pEKExpgdh was precultivated in complex medium 2TY consisting of 16 g/l of tryptone, 10 g/l of yeast extract and 5 g/l of NaC1. 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 0

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 al.

(Journal of Bacteriology 173, 4510-4516 (1991)), supplemented with 25 g/l of glucose, 350 mg/l of leucine, 42 g/l of 3 -morpholinopropanesulfonic acid and 50 mg/l of kanamycin monosulfate at pH 7. The cultures were incubated for 30 hours at 30 0 C and 150 rpm.

980182 BT 16 Table Component Concentration litre

(NH

4 2

SO

4 5 g Urea 5 g

KH

2

PO

4 0.5 9

K

2

HPO

4 MgSO 4 7H 2 0 0.25 g FeSO, 7H 2 0 10 mg MnSO 4

H

2 0 10 mg ZnSO 4 7H 2 0 1 mg CUS0 4 0.2 mg NiCl 2 6H 2 0 0.02 mg CaC1 2 2H 2 0 10 Mg Biotin 200 /ig Optical density (OD) Biochrom Novaspec 4049, LKB Instrument GmbH, Gr~felf ing, 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-Biorronik (Hamburg, Germany) by ion exchange chromatography and postcolumn reaction with ninhydrin detection. Table 6 shows the result of the test.

980182 BT 17 Table G Strain OD

L

Al anin DSM5715 29.4 Traces DSM571S/pEKExpgdh 19.4 0.6

Claims (14)

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.

2. Process according to claim 1, characterised in that the nucleotide sequence coding for glutamate dehydrogenase is overexpressed.

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

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

Process according to claim 1 or claim 2, 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.

6. Process according to claim 1 or claim 2, 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.

7. Process according to any one 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. 20

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

9. Process according to claim 7, characterised in that, bacteria transformed with the plasmid vector pEKExpgdh, deposited in E. coli under the number DSM 12613, are used. 25

10. Process for the fermentative production of L-amino acids according to any one 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) 30

11. Process according to any one 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.

12. Process for the production of L-amino acids by fermentation of coryneform bacteria, said process being substantially as hereinbefore described with reference to any one of the examples.

13. L-amino acids produced by fermentation of coryneform bacteria in accordance with the process of any one of the preceding claims. Dated

14 February 2000 DEGUSSA-HULS AKTIENGESELLSCHAFT FORSCHUNGSZENTRUM JOLICH GMBH Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON C08102