CA2301407A1

CA2301407A1 - Production of l-amino acids by fermentation using coryneform bacteria

Info

Publication number: CA2301407A1
Application number: CA002301407A
Authority: CA
Inventors: Douwe Molenaar; Michel Eduard Van Der Rest; Bettina Mockel
Original assignee: Degussa Huels AG
Current assignee: Evonik Operations GmbH
Priority date: 1999-03-19
Filing date: 2000-03-16
Publication date: 2000-09-19
Also published as: EP1038969A3; DE19912384A1; BR0001342A; ID29659A; AU2236900A; EP1038969A2; ZA200001354B; JP2000270888A; HUP0001176A2; SK3742000A3; KR20000076897A; HU0001176D0; CN1267734A

Abstract

The invention relates to a process for the production of L-amino acids by fermentation using coryneform bacteria in which the mqo gene is amplified.

Description

Production Of L-amino Acids By Fermentation Using Coryneform Bacteria The invention provides a process for the production of L-amino acids, especially L-lysine, by fermentation using coryneform bacteria in which the mqo gene is amplified.
L-amino acids, especially L-lysine, are used in the feeding of animals, in human medicine and in the pharmaceuticals industry.
It is known that those amino acids are produced by fermenting strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of their great importance, work is continually being done to improve the production processes. Improvements to the process may concern measures relating to the fermentation process, such as, for example, stirring and oxygen supply, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or working-up to the product form by, for example, ion-exchange chromatography, or the intrinsic performance properties of the microorganism itself.
To improve the performance properties of those micro-organisms, methods of mutagenesis, selection and mutant selection are used. In that manner there are obtained strains that are resistant to antimetabolites, such as, for example, the lysine analogue S-(2-aminoethyl)-cysteine, or are auxotrophic for amino acids which are important in terms of regulation, and produce L-amino acids.
For some years, methods of recombinant DNA technology have also been used to improve the L-amino-acid-producing strains of Corynebacterium gl_utamicum by ampii.fyi.ng individual genes of amino acid biosynthesis and studying the effect on the production of L-amino acids. General articles on that subject will 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)), Eggeling (Amino Acids 6, 261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et a1. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
L-amino acids, especially L-lysine, axe used in the feeding of animals, in human medicine and in the pharmaceuticals industry. There is, therefore, a general interest in making available new, improved processes for the production of those compounds.
The inventors have set themselves the task of making available new measures for the improved production of L-amino acids, especially L-lysine, by fermentation.
Any mention of L-lysine or lysine hereinbelow is to be understood as meaning not only the base but also the salts, such as, for example, lysine monohydrochloride or lysine sulfate.
The invention provides a process for the production by fermentation, of L-amino acids, especially L-lysine, using coryneform bacteria which, especially, already produce th.t~
desired amino acid and in which the nucleotide sequence coding for the enzyme malate:quinone oxidoreductase (mqo gene) is amplified, especially overexpressed.
The term "amplification" in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, by, for example, increasing the copy number of the gene or genes, using a strong promoter or using a gene that codes for a corresponding enzyme having a high degree of activity, and optionally combining those measures.
The microorganisms provided by the present invention can produce L-amino acids, especially L-lysine, from glucose, saccharose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They are representatives of coryneform bacteria, especially of the genus Corynebacterium. In the genus Corynebacterium, special mention may be made of the species Corynebacterium glutamicum, which is known to those skilled in the art for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, especially 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 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020 and L-amino-acid-producing, especially L-lysine-producing, mutants and strains produced therefrom, such as, for example, Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Brevibacterium flavum FERM-P 6463 and Brevibacterium flavum FERM-P 6964.
The inventors have found that coryneform bacteria produce L-amino acids, especially L-lysine, in an improved manner after overexpressi.on of malate:quinone oxidoreductase.

The mqo gene codes for the enzyme malate:quinone oxido-reductase (EC 1.1.99.16), which catalyses the oxidation of malate to oxalacetate with transfer of the electrons to ubiquinone-1 (Molenaar et al., European Journal of Bio-chemistry 254, 395-403 (1998)). The nucleotide sequence of the mqo gene of Corynebacterium glutamicum has likewise been determined by Molenaar et al. (European Journal of Biochemistry 254, 395-403 (1998)) and is generally available at the nucleotide sequence databank of the National Center for Biotechnology Information (NCBI, Behesda, MD, USA) under Accession Number AJ 22 4946.
The mqo gene of C. glutamicum described by Molenaar et al.
(European Journal of Biochemistry 254, 395-403 (1998)) can be used according to the invention. It is also possible to use alleles of the mqo gene which result from the degeneracy of the genetic code or by function-neutral sense mutations.
In order to achieve an overexpression, the copy number of the corresponding genes can be increased, or the promoter and regulation region, which is located in front of the structural gene, can be mutated. Expression cassettes, which are inserted in front of the structural gene, have the same effect. By means of inducible promoters it is additionally possible to increase the expression in the course of the production of L-lysine by fermentation. The expression is likewise improved by measures for lengthening the life of the m-RNA. The enzyme activity is also increased by preventing the degradation of the enzyme protein. The genes or gene constructs can either be present in plasmids with different copy numbers or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can also be achieved by changing the composition of the media and the manner in which culturing is carried out.

The person skilled in the art will find instructions there-for inter alia in Martin et a1. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), 5 in Eikmanns et a1. (Gene 102, 93-98 (1991)), in European Patent Specification EP-B 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 a1.
(Journal of Bacteriology 175, 1001-1007 (1993)), in Patent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)) (sic) ~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.
An example of a plasmid with the aid of which malate:
quinone oxidoreductase can be overexpressed is pRMl7 (Molenaar et al., 1998, European Journal of Biochemistry 254, 395-403). Plasmid pRMl7 is based on the shuttle vector pJCl, which is described in Cremer et al. (Molecular and General Genetics 220, 478-480).
In addition, it may be advantageous for the production of L-amino acids to overexpress one or more enzymes of the corresponding biosynthesis pathway as well as malate:
quinone oxidoreductase. Thus, for example, in the production of L-lysine ~ the dapA gene coding for dihydrodipicolinate synthase can be overexpressed at the same time (EP-B 0 197 335), or ~ a DNA fragment mediating S-(2-aminoethyl)-cysteine resistance can be amplified at the same time (EP-A 0 088 166).
It may also be advantageous for the production of I~-amino acids to exclude undesired secondary reactions in addition to the overexpression of malate:quinone oxidoreductase (Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
The microorganisms produced according to the invention may be cultivated continuously or discontinuously in the batch process or in the fed batch or repeated fed batch process for the purposes of the production of L-amino acids. A
summary of known cultivation methods is described 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 meet the requirements of the strains in question in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). There may be used as the carbon source sugars and carbohydrates, such as, for example, glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, 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. Those substances may be used individually or in the form of a mixture. There may be used as the nitrogen source organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. 'Ihe nitrogen sources may be used individually or in the form of a mixture. There may be used as the phosphorus source potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium must also contain salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above-mentioned substances. Moreover, suitable pre-stages may be added to the culture medium. The mentioned substances may be added to the culture in the form of a single batch or may be fed in in a suitable manner during the cultivation.
In order to control the pH of the culture, basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, are used in a suitable manner. For controlling the development of foam, antifoams, such as, for example, fatty acid polyglycol esters, can be used. In order to maintain the stability of plasmids, suitable substances having a selective action, for example antibiotics, may be added to the medium. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as, for example, air, are introduced into the culture. The temperature of .the culture is normally from 20°C to 45°C and preferably from 25°C to 40°C. Culturing is continued until a maximum of the desired L-amino acid has formed. That aim is normally achieved within a period of from 10 hours to 160 hours.
Analysis of L-amino acids may be carried out by anion-exchange chromatography with subsequent ninhydrin derivatisation, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190).
The Corynebacterium glutamicum strain DM22/pRMl7 was deposited at the Deutsche Sammlung von Mikroorgani_smen and Zellkulturen (Braunschweig, Germany) under number DSM12711 i.n accordance with the Budapest Treaty.

The process according to the invention is used for the production of L-amino acids, especially L-aspartic acid, L-asparagine, L-homoserine, L-threonine, L-isoleucine and L-methionine, by fermentation using coryneform bacteria, especially for the production of L-lysine.

Examples The present invention is explained in greater detail below by means of Examples.
To that end, tests were carried out using the L-lysine-producing Corynebacterium glutamicum strain DSM5715 (EP-B-0 435 132), in which the advantageousness of the claimed process becomes clear:
Example 1 Production of L-lysine producers containing amplified malate:quinone oxidoreductase The strain DSM5715 was transformed as in Liebl et a1. (FEMS
Microbiology Letters 65, 299-304 (1989)) with the plasmid pRMl7 (Molenaar et al., 1998, European Journal of Bio-chemistry 254 (395-403)). Selection of the transformants was carried out on LBHIS agar to which 25 mg/1 of kanamycin had been added. LBHIS agar consists of LB medium (Sambrook et al. (Molecular Cloning a Laboratory Manual (1989) Cold Spring Harbour Laboratories)) to which there have been added 37 g/1 of brain heart bouillon from Merck (Darmstadt, Germany), 0.5 M sorbitol and 15 g/1 of agar-agar. In that manner, the strain DSM5715/pRMl7 was formed. The strain DSM5715/pJCl was produced in the same manner.
Example 2 Production of L-lysine The strains DSM5715/pRMl7 and DSM5715/pJCl were first incubated on brain-heart agar, to which kanamycin (25 mg/1) had been added, for 24 hours at 33°C. For cultivation in liquid medium, CgIII medium (Kase & Nakayama, Agricultural and Biological Chemistry 36 (9) 1611-1621 (1972)), to which kanamycin (25 mg/1) had additionally been added, was used.
To that end, 10 ml of medium, which were contained in 100 ml Erlenmeyer flasks with 4 baffles, were inoculated with an inoculum of the strain and the culture was incubated for 16 hours at 240 rpm and 30°C. The culture was subsequently used further as a pre-culture.
The production or test medium used was MM medium, to which 5 kanamycin (25 mg/1) had additionally been added.
In the process using strain DSM5715, the corresponding media did not contain kanamycin.
The composition and preparation of the MM medium was as follows:

10 Corn Steep Liquor (CSL) 5 g/1 3-morpholino-propanesulfonic acid (MOPS) 20 g/1 glucose 50 g/1 (autoclaved separately) Salts:

(NH4)2504) 25 g/1 KH2P04 0.1 g/1 MgS04*7H20 1.0 g/1 CaCl2*2H20 10 mg/1 FeS04*7H20 10 mg/1 MnS04*H20 5.0 mg/1 biotin 0.3 mg/1 (sterilised by filtration) thiamine*HC1 0.2 mg/1 (sterilised by filtration) CaC03 25 g/1 leucine 0.1 g/1 CSL, MOPS and the salt solution were adjusted to pH 7 using ammonia water and autoclaved. The sterile substrate and vitamin solutions and the dry autoclaved CaC03 were then added.
Cultivation was carried out in 100 ml Erlenmeyer flasks with baffles, which had been charged with 10 ml of the above-described production medium. The cultures were so inoculated with the pre-cult=ure that the optical density at the start was 0.1. Cultivation was carried out at 33°C and 80o relative humidity.
After incubation for 72 hours, the optical density of the culture suspension and the concentration of L-lysine that had formed were determined. The optical density was determined using an LP2W photometer from Dr. Lange (Berlin, Germany) at a measuring wavelength of 660 nm (sic) L-lysine was determined using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by ion-exchange chromatography and post-column reaction with ninhydrin detection. The result of the test is shown in Table 1.
Table 1 --Strain OD L-lysine g/1 DSM5715 10.1 16.4 DSM5715/pJCl 9.9 16.5 DSM5715/pRMl7 10.2 17.8

Claims

1. A process for the production of L-amino acids by fermentation of coryneform bacteria, wherein bacteria are used in which the nucleotide sequence coding for malate:quinone oxidoreductase is amplified, especially overexpressed.

2. A process according to claim 1, wherein bacteria are used in which other genes of the biosynthesis pathway of the desired L-amino acid are additionally amplified.

3. A process according to claim 1, wherein bacteria are used in which at least some of the metabolic pathways that reduce the formation of the desired L-amino acid are eliminated.

4. A process according to claim 1, 2 or 3, wherein a strain transformed with a plasmid vector is used, and the plasmid vector carries the nucleotide sequence coding for malate:quinone oxidoreductase.

5. A process according to claim 4, wherein there are used bacteria transformed with plasmid vector pRM17 deposited in Corynebacterium glutamicum, under number DSM12711.

6. A process according to any one of claims 1 to 5, wherein coryneform bacteria which produce L-aspartic acid, L-asparagine, L-homoserine, L-threonine, L-isoleucine or L-methionine are used.

7. A process according to any one of claims 1 to 5, wherein coryneform bacteria which produce L-lysine are used.

8. A process according to claim 7, wherein the dapA
gene coding for dihydrodipicolinate synthase is overexpressed at the same time.

9. A process according to claim 7, wherein a DNA
fragment mediating S-(2-aminoethyl)-cysteine resistance is amplified at the same time.

10. A process for the production of L-amino acids by fermentation according to any one of claims 1 to 9, wherein the following steps are carried out:
(a) fermentation of the bacteria producing the desired L-amino acid, in which bacteria at least the malate:quinone oxidoreductase gene is amplified, (b) concentration of the L-amino acid in the medium or in the cells of the bacteria, and (c) isolation of the L-amino acid that has been produced