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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor

Definitions

• the invention provides a process for the production of L- lysine using coryneform bacteria that are resistant to diaminopimelic acid analogues, in particular 4-hydroxy- diaminopimelic acid.
• -amino acids in particular L-lysine
• L-lysine are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry and most particularly in animal nutrition.
• Process improvements may relate to fermentation technology measures, such as for example stirring and provision of oxygen, or the composition of the nutrient media, such as for example the sugar concentration during the fermentation, or the working-up to the product form by for example ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.
• strains are obtained that are resistant to antimetabolites such as for example the lysine analogue S- (2-aminoethyl) -cysteine, or that are auxotrophic for regulatorily important metabolites and that produce L-amino acids.
• antimetabolites such as for example the lysine analogue S- (2-aminoethyl) -cysteine, or that are auxotrophic for regulatorily important metabolites and that produce L-amino acids.
• the inventors have been involved in devising new principles for improved processes for the fermentative production of L-lysine using coryneform bacteria.
• L-lysine or lysine are mentioned hereinafter, this is understood to mean not only the bases, but also the salts such as for example lysine monohydrochloride or lysine sulfate.
• the invention provides a process for the fermentative production of L-lysine using coryneform bacteria that are resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid.
• the analogues are generally used in concentrations of > (greater than/equal to) 3 to ⁇ (less than/equal to) 30 g/1.
• the invention also provides a process for the fermentative production of L-lysine using coryneform bacteria that already produce L-lysine and that are resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid.
• This invention furthermore provides a process for the production of L-lysine in which the following steps are carried out:
• the invention similarly provides a process for the production of coryneform bacteria that are resistant to diaminopimelic acid analogues, in particular 4-hydroxy- diaminopimelic acid.
• strains that are used produce L-lysine preferably already before the resistance to 4-hydroxydiaminopimelic acid.
• the expression diaminopimelic acid analogues according to the present invention includes compounds such as
• the present invention also provides mutant coryneform bacteria producing L-lysine that are resistant to one or . more of the diaminopimelic acid analogues selected from the group comprising 4-fluorodiaminopimelic acid, 4-hydroxydiaminopimelic acid, 4-oxodiaminopimelic acid or 2,4,6- triaminopimelic acid.
• the invention moreover provides feedstuffs additives based on fermentation broth that contain L-lysine produced according to the invention and.no or only traces of biomass and/or constituents from the fermentation broth formed during the fermentation of the L-lysine-producing microorganisms .
• traces is understood to mean amounts of > 0% to 5%.
• the invention additionally provides feedstuffs additives based on fermentation broth, characterised in that
• the microorganisms that are provided by the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
• These microorganisms may be representatives of coryneform bacteria, in particular of the genus Corynebacterium.
• Corynebacterium there should in particular be mentioned the species Corynebacterium glutamicum, which is known to the specialists in this field for its ability to produce L- amino acids.
• Suitable strains of the genus Corynebacterium in particular of the species Corynebacterium glutamicum, are in particular the following known wild type strains
• coryneform bacteria that are resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid, produce L-lysine in an improved manner.
• mutagenesis there may be employed conventional, in vivo mutagenesis processes using mutagenic substances such as for example N-methyl-N' -nitro-N-nitrosoguanidine or ultraviolet light (Miller, J. H. : A Short Course in Bacterial Genetics . A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992) .
• mutagenic substances such as for example N-methyl-N' -nitro-N-nitrosoguanidine or ultraviolet light (Miller, J. H. : A Short Course in Bacterial Genetics . A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992) .
• the coryneform bacteria that are resistant to 4-hydroxydiaminopimelic acid may be identified by plating out on nutrient media plates containing 4-hydroxydiaminopimelic acid. End concentrations of ca. 5 to 15 g/1, for example ca. 10 g/1 of 4-hydroxydiamino-pimelic acid in the nutrient medium are particularly suitable for this purpose. At this concentration mutants resistant to 4-hydroxydiaminopimelic acid may be distinguished from the unchanged parent strains by a delayed growth. After selection the mutants resistant to 4-hydroxydiaminopimelic acid exhibit an improved L- lysine production.
• L- lysine in addition to the resistance to 4-hydroxydiaminopimelic acid to enhance, in particular overexpress, one or more enzymes of the respective biosynthesis pathway, glycolysis, anaplerosis, citric acid cycle, pentose phosphate cycle, amino acid export and optionally regulatory proteins .
• endogenous genes is in general preferred.
• endogenous genes or “endogenous nucleotide sequences” are understood to mean the genes or nucleotide sequences present in the population of a species .
• the activity or concentration of the corresponding protein is generally raised by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000%, referred to the activity or concentration of the wild type protein and/or the activity or concentration of the protein in the starting microorganism.
• L- lysine in addition to the resistance to 4-hydroxydiaminopimelic acid, simultaneously to attenuate, in particular reduce the expression, of one or more of the genes selected from the following group:
• the term "attenuation” describes in this connection the reduction or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism that are coded by the corresponding DNA, by using for example a weak promoter or a gene or allele that codes for a corresponding enzyme with a low activity or inactivating the corresponding gene or enzyme (protein) , and optionally combining these measures .
• the activity or concentration of the corresponding protein is generally reduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild type protein, and/or the activity or concentration of the protein in the initial microorganism.
• microorganisms produced according to the invention are also covered by the invention and may be cultivated continuously or discontinuously in a batch process (batch cultivation) or in a fed-batch process (feed process) or repeated fed-batch process (repetitive feed process) for the purposes of producing L-lysine.
• batch cultivation or in a fed-batch process (feed process) or repeated fed-batch process (repetitive feed process) for the purposes of producing L-lysine.
• feed process fed-batch process
• repetitive feed process for the purposes of producing L-lysine.
• the culture medium to be used must satisfy in a suitable manner the requirements of the respective strains. 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).
• sugars and carbohydrates such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as for example soy bean oil, sunflower oil, groundnut 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.
• sugars and carbohydrates such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
• oils and fats such as for example soy bean oil, sunflower oil, groundnut 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
• organic acids such as for example acetic acid.
• nitrogen source there may be used organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
• organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea
• 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 source there may be used phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
• the culture medium must furthermore contain salts of metals, such as for example magnesium sulfate or iron sulfate, that are necessary for growth.
• essential growth promoters such as amino acids and vitamins may be used in addition to the aforementioned substances .
• suitable precursors may be added to the culture medium.
• the aforementioned starting substances may be added to the culture in the form of a single batch or may be fed in in an appropriate manner during the cultivation.
• basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid are used as appropriate.
• acidic compounds such as phosphoric acid or sulfuric acid are used.
• antifoaming agents such as for example fatty acid polyglycol esters may be used.
• suitable selectively acting substances for example antibiotics, may be added to the medium.
• oxygen or oxygen-containing gas mixtures such as for example air are fed into the culture.
• the temperature of the culture is normally 20°C to 45°C, and preferably 25°C to 40°C. Cultivation is continued until a maximum amount of desired product has been formed. This target is normally achieved within 10 hours to 160 hours.
• the process according to the invention serves for the fermentative production of L-lysine.
• the concentration of L-lysine may optionally be adjusted to the desired value by the addition of L-lysine.
• Example 1 4-hydroxy-diaminopimelic acid, and to produce L-lysine in an improved manner according to the described fermentation processes.
• Example 1 4-hydroxy-diaminopimelic acid, and to produce L-lysine in an improved manner according to the described fermentation processes.
• the Corynebacterium glutamicum strain DM1725 was produced by multiple untargeted and targeted mutagenesis including genetic engineering methods, selection and mutant selection from C. glutamicum ATCC13032.
• the strain is resistant to the lysine analogue S- (2-aminoethyl) -L-cysteine and has two identical complete copies of the LysC gene that code for a feedback-resistant aspartate kinase. The two copies are located at the LysC gene site on the chromosome.
• the feedback-resistant aspartate kinase is insensitive to inhibition by mixtures of lysine (or the lysine analogue S- (2-aminoethyl) -L-cysteine, lOOmM) and threonine (lOmM) , but in contrast to this the activity of aspartate kinase in the wild type is inhibited up to 10% residual activity.
• the strain is streptomycin resistant.
• the strain DSM 15662 after UV mutagenesis (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2 nd Edition, Cold Spring Harbor, New York, 1989) is plated out on LB agar plates containing 4- hydroxydiaminopimelic acid. The agar plates are supplemented with 10 g/1 of 4-hydroxydiaminopimelic acid. The growth of the colonies is observed over 48 hours. At this concentration mutants that are resistant to 4-hydroxydiaminopimelic acid can be distinguished from the unaltered parent strain by an improved growth. In this way a clone is identified that exhibits a much better growth compared to DSM 15662.
• the strain is identified as DSM 15662_Hdap_r.
• the C. glutamicum strain DSM 15662_Hdap_r obtained in Example 1 is cultured in a nutrient medium suitable for the production of lysine and the lysine content in the culture supernatant is determined.
• the strains are first of all incubated on agar plates for 24 hours at 33°C.
• a preculture is inoculated (10 ml of medium in a 100 ml Erlenmeyer flask) .
• the medium MM is used as medium for the preculture.
• the preculture is incubated for 24 hours at 33°C at 240 rpm on a vibrator.
• a main culture is inoculated so that the initial optical density (OD - 660 nm) of the main culture is
• the medium MM is also used for the main culture.
• CSL Corn Steep Liquor
• MOPS morpholinopropanesulfonic acid
• the salt solution is adjusted with ammonia water to pH 7 and autoclaved.
• the sterile substrate and vitamin solutions as well as the dry autoclaved CaC0 3 are then added.
• Culturing is carried out in a 10 ml volume in a 100 ml
• the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 instrument (Beckmann Instruments GmbH, Kunststoff) .
• the amount of lysine formed is determined by ion exchange chromatography and post-column derivatisation with ninhydrin detection, using an amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) .
• the microorganism identified under L above was accompanied by:
• microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

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• 2002
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