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EP1525321A1 - Process for the production of l-lysine using coryneform bacteria - Google Patents

Process for the production of l-lysine using coryneform bacteria

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Publication number
EP1525321A1
EP1525321A1 EP03766147A EP03766147A EP1525321A1 EP 1525321 A1 EP1525321 A1 EP 1525321A1 EP 03766147 A EP03766147 A EP 03766147A EP 03766147 A EP03766147 A EP 03766147A EP 1525321 A1 EP1525321 A1 EP 1525321A1
Authority
EP
European Patent Office
Prior art keywords
lysine
gene
coding
acid
resistant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03766147A
Other languages
German (de)
French (fr)
Inventor
Brigitte Bathe
Stephan Hans
Walter Pfefferle
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evonik Operations GmbH
Original Assignee
Degussa GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Degussa GmbH filed Critical Degussa GmbH
Publication of EP1525321A1 publication Critical patent/EP1525321A1/en
Withdrawn legal-status Critical Current

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Classifications

    • 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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Abstract

The invention relates to a process for the production of L-lysine, in which the following steps are carried out: a) fermentation of the L-lysine producing coryneform bacteria that are at least resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid; b) enrichment of the L-lysine in the medium or in the bacterial cells; and optionally c) isolation of the L-lysine or L-lysine-containing feedstuffs additive from the fermentation broth, so that ≥ 0 to 100% of the constituents from the fermentation broth and/or from the biomass are present, and optionally bacteria are used in which in addition further genes of the biosynthesis pathway of L-lysine are enhanced, or bacteria are used in which the metabolic pathways that reduce the formation of L-lysine are at least partially switched off.

Description

Process for the production of L-lysine using Coryneform
Bacteria
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.
Prior Art
-amino acids, in particular L-lysine, are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry and most particularly in animal nutrition.
It is known to produce amino acids by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. On account of their great importance efforts are constantly being made to improve the production processes . 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.
In order to improve the performance properties of these microorganisms methods involving mutagenesis, selection and choice of mutants are employed. In this way 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.
For some years recombinant DNA technology methods have also been employed to improve L-amino acid producing strains of Corynebacterium glutamicum, by amplifying individual amino acid biosynthesis genes and investigating the effect on L- a ino acid production.
Object of the Invention
The inventors have been involved in devising new principles for improved processes for the fermentative production of L-lysine using coryneform bacteria.
Description of the Invention
Where 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:
a) fermentation of the L-lysine producing coryneform bacteria that are at least resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid;
b) enrichment of the L-lysine in the medium or in the bacterial cells; and optionally c) isolation of the L-lysine or L-lysine-containing feedstuffs additive from the fermentation broth, so that > 0 to 100% of the constituents from the fermentation broth and/or from the biomass are present.
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.
The 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
• 4-fluorodiaminopimelic acid,
• 4-hydroxydiaminopimelic acid,
• 4-oxodiaminopimelic acid, or
• 2, 4, 6-triaminopimelic acid. /
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 .
The term "traces" is understood to mean amounts of > 0% to 5%.
The invention additionally provides feedstuffs additives based on fermentation broth, characterised in that
a) they contain L-lysine produced according to the invention, and
b) they contain the biomass and/or constituents from the fermentation broth in an amount of 90% to
100% that are formed during the fermentation of the L-lysine-producing microorganisms .
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. Among the genus 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
Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FER BP-1539
Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020
and L-amino acid-producing mutants and/or strains produced therefrom,
such as for example the L-lysine-producing strains
Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM-P 6464 Corynebacterium glutamicum ATCC 21513 Corynebacterium glutamicum ATCC 21544 Corynebacterium glutamicum ATCC 21543 Corynebacterium glutamicum DSM 4697 und Corynebacterium glutamicum DSM 5715.
It has been found that coryneform bacteria that are resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid, produce L-lysine in an improved manner.
In order to produce the coryneform bacteria according to the invention that are resistant to 4-hydroxydiaminopimelic acid, mutagenesis methods described in the prior art are used.
For the 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) .
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.
In addition it may be advantageous for the production of 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 . The use of endogenous genes is in general preferred.
The expressions "endogenous genes" or "endogenous nucleotide sequences" are understood to mean the genes or nucleotide sequences present in the population of a species .
The expressions "enhancement" and "to enhance" describe in this connection the increase of the intracellular activity of one or more enzymes or proteins in a microorganism that are coded by the corresponding DNA, by for example increasing the number of copies of the gene or genes, employing a strong promoter or a gene that codes for a corresponding enzyme or protein having a high activity, and optionally combining these measures.
By means of these enhancement, in particular overexpression measures, 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.
Thus, for the production of L-lysine, in addition to the resistance to diaminopimelic acid analogues, in particular one or more of the genes selected from the following group may be enhanced, in particular overexpressed:
• the gene lysC coding for a feedback-resistant aspartate kinase (Accession No. P26512, EP-B-0387527; EP-A-0699759; WO 00/63388) ,
• the gene dapA coding for dihydrodipicolinate synthase (EP-B 0 197 335) ,
• the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992) . Journal of Bacteriology 174:6076-6086),
• simultaneously the gene pyc coding for pyruvate carboxylase (DE-A-198 31 609, EP-A-1108790) ,
• the gene zwf coding for glucose-6-phosphate dehydrogenase (JP-A-09224661, EP-A-1108790),
• simultaneously the gene lysE coding for the lysine export protein (DE-A-195 48 222),
• the gene zwal coding for the Zwal protein (DE: 19959328.0, DSM 13115),
• the gene lysA coding for diaminopimelic acid decarboxylase (Accession No. X07563),
• the gene sigC coding for the sigma factor C (DE: 10043332.4, DSM14375) , • the gene tpi coding for triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086) and
• the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086).
Furthermore it may be advantageous for the production of 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 gene pck coding for phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047),
• the gene pgi coding for glucose-6-phosphate isomerase (US 09/396,478, DSM 12969),
• the gene poxB coding for pyruvate oxidase (DE:1995 1975.7, DSM 13114) ,
• the gene deaD coding for DNA helicase (DE: 10047865.4, DSM14464) ,
• the gene citE coding for citrate lysase (PCT/EP01/00797, DSM13981),
• the gene menE coding for O-succinylbenzoic acid CoA- ligase (DE: 10046624.9, DSM14080) ,
• the gene mikEl7 coding for the transcription regulator MikEl7 (DE: 10047867.0, DSM14143) and
• the gene zwa2 coding for the Zwa2 protein (DE: 19959327.2, DSM 13113) .
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 .
By means of these attenuation 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.
Finally it may be advantageous for the production of L- lysine, in addition to the resistance to 4-hydroxydiaminopimelic acid, also to switch off undesirable secondary reactions (Nakayama: "Breeding of Amino Acid
Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .
The 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. 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 und periphere Einrichtungen (Vieweg Verlag, Brunswick/ Wiesbaden, 1994)).
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).
As carbon source there may be used 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. These substances may be used individually or as a mixture.
As 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. The nitrogen sources may be used individually or as a mixture.
As 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. Finally, essential growth promoters such as amino acids and vitamins may be used in addition to the aforementioned substances . Apart from these, 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.
In order to regulate the pH of the culture 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. In order to control foam formation antifoaming agents such as for example fatty acid polyglycol esters may be used. In order to maintain the stability of plasmids, suitable selectively acting substances, 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 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.
Methods for the determination of L-lysine are known from the prior art. The analysis may be carried out as described by Spackman et al. (Analytical Chemistry, 30, (1958) , 1190) by anion exchange chromatography followed by ninhydrin derivatisation, or by reversed phase HPLC as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174) .
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.
By means of the described processes it is possible to isolate coryneform bacteria that are resistant to diaminopimelic acid analogues, in particular
4-hydroxy-diaminopimelic acid, and to produce L-lysine in an improved manner according to the described fermentation processes. Example 1
Screening for clones resistant to 4-hydroxydiaminopimelic acid.
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.
A pure culture of the strain DM1725 was deposited as DSM 15662 on 6 June 2003 at the German Collection for Microorganisms and Cell Cultures (DSM Brunswick) according to the Budapest Convention.
For screening on colonies that are resistant to 4-hydroxydiaminopimelic acid, the strain DSM 15662 after UV mutagenesis (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2nd 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.
Example 2
Production of lysine
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.
For this purpose the strains are first of all incubated on agar plates for 24 hours at 33°C. Using this agar plate culture 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. Using this preculture a main culture is inoculated so that the initial optical density (OD - 660 nm) of the main culture is
0.1 OD. The medium MM is also used for the main culture.
Medium MM
CSL 5 g/1
MOPS 20 g/1
Glucose (separately autoclaved) 50 g/1
Salts :
(NH4)2S04 25 g/1
KH2P04 0.1 g/1
MgS04 x 7 H20 1.0 g/1
CaCl2 x 2 H20 10 mg/1
FeS04 x 7 H20 10 mg/1
MnS04 x H20 5.0 mg/1
Biotin (sterile filtered) 0.3 mg/1
Thiamine x HCl (sterile filtered) 0.2 mg/1
CaC03 25g/l
CSL (Corn Steep Liquor) , MOPS (morpholinopropanesulfonic acid) and the salt solution are adjusted with ammonia water to pH 7 and autoclaved. The sterile substrate and vitamin solutions as well as the dry autoclaved CaC03 are then added.
Culturing is carried out in a 10 ml volume in a 100 ml
Erlenmeyer flask equipped with baffles. The culturing is carried out at 33 °C and 80% atmospheric humidity.
After 72 hours the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 instrument (Beckmann Instruments GmbH, Munich) . 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 result of the experiment is shown in Table 1
Table 1
BUDAPEST TREATY ON THE I TERNAΗONAL ECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNAΗONAL FORM
Degussa AG
Kantstr.2
33790 Halle/Westf. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNAΗONAL DEPOSITARY AUTHORITY identified at the bottom of this page
L IDENT CAΗON OF THE MICROORGANISM
Identification reference given by the DEPOSITOR: Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: DM1725
DSM 15662
IL SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under L above was accompanied by:
( x ) a scientific description
( x) a proposed taxonomic designation
(Mark with a cross where applicable).
HL RECEIPT AND ACCEPTANCE
This International Depositary Authority accepts the microorganism identified τmder L above, which was received by it on 2003-06-06 (Date of the original deposit)1. T vv . YY. v
IV. RECEIPT OF REQUEST FOR CONVERSION
The 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).
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signatures) of person(s) having the power to represent the MKROORGANBMEN UND ZELL ULTUREN GmbH International Depositary Authority or of authorized officials):
Address: MascheroderWeg lb D-38124 Braunschweig
Date: 2003-06-10
1 Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired.
Form DSMZ-BP/4 sole age) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Degussa AG
Kantstr. 2
33790 Halle/Westf.
VIABILΠΎ STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page
L DEPOSITOR π. IDENTIFICATION OF THE MICROORGANISM
Name: Degussa AG Accession number given by the
Kantstr. 2 INTERNAΗONAL DEPOSITARY AUTHORITY:
Address: 33790 Halle/Westf.
DSM 15662 Date of the deposit or the transfer1:
2003-06-06 . VIABILΠΎ STATEMENT
The viability of the microorganism identified under II above was tested on 2003-06-06 On that date, the said microorganism was
(χ)3 viable
( )3 no longer viable
IV. CONDITIONS UNDER WHICH THE VIABnJTY TEST HAS BEEN PERFORMED4
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature^) of person(s) having the power to represent the
MKROORGANISMEN UND 2ELLKULTUREN GmbH International Depositary Authority or of authorized officials):
Address: MascheroderWeg lb D-38124 Braunschweig
Date: 2003-06-10
1 Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).
2 In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test
3 Mark with a cross the applicable box.
4 Fill in if the information has been requested and if the results of the test were negative.
Form DSMZ-BP/9 (sole page) 12/2001

Claims

Patent Claims
1. Process for the production of L-lysine, characterised in that the following steps are carried out:
a) fermentation of the L-lysine producing coryneform bacteria that are at least resistant to diaminopimelic acid analogues, in particular 4-hydroxydiaminopimelic acid;
b) enrichment of the L-lysine in the medium or in the bacterial cells; and optionally
c) isolation of the L-lysine or L-lysine-containing feedstuffs additive from the fermentation broth, so that > 0 to 100% of the constituents from the fermentation broth and/or from the biomass are present.
2. Process according to claim 1, characterised in that bacteria are used in which in addition further genes of the biosynthesis pathway of L-lysine are enhanced.
3. Process according to claim 1, characterised in that bacteria are used in which the metabolic pathways that reduce the formation of L-lysine are at least partially switched off.
4. Process according to claim 1, characterised in that for the production of L-lysine coryneform microorganisms are fermented in which at the same time one or more of the genes selected from the following group is/are enhanced, in particular overexpressed:
4.1 the gene lysC coding for a feedback-resistant aspartate kinase,
4.2 the gene dapA coding for dihydrodipicolinate synthase,
4.3 the gene gap coding for glyceraldehyde-3- phosphate dehydrogenase,
4.4 the gene pyc coding for pyruvate carboxylase,
4.5 the gene zwf coding for glucose-6-phosphate dehydrogenase,
4.6 simultaneously the gene lysE coding for the lysine export protein,
4.7 the gene zwal coding for the Zwal protein,
4.8 the gene lysA coding for diaminopimelic acid decarboxylase,
4.9 the gene sigC coding for the sigma factor C,
4.10 the gene tpi coding for triose phosphate isomerase, or
4.11 the gene pgk coding for 3-phosphoglycerate kinase.
5. Process according to claim 1, characterised in that for the production of L-lysine coryneform microorganisms are fermented in which at the same time one or more of the genes selected from the following group is/are attenuated:
5.1 the pck gene coding for phosphoenol pyruvate carboxykinase,
5.2 the pgi gene coding for glucose-6-phosphate- isomerase,
5.3 the gene deaD coding for DNA helicase,
5.4 the gene citE coding for citrate lysase,
5.5 the gene menE coding for O-succinylbenzoic acid CoA-ligase,
5.6 the gene mikEl7 coding for the transcription regulator MikEl7,
5.7 the gene poxB coding for pyruvate oxidase, or
5.8 the gene zwa2 coding for the Zwa2 protein.
6. Process according to one or more of the preceding claims, characterised in that microorganisms of the species Corynebacterium glutamicum are used.
7. Process according to one or more of the preceding claims, characterised in that microorganisms of the species Corynebacterium glutamicum that are resistant to 4-hydroxydiaminopimelic acid are used.
8. Mutants of coryneform bacteria producing L-lysine and that are resistant to one or more of the diaminopimelic acid analogues selected from the group comprising 4-fluorodiamino-pimelic acid, 4- hydroxydiaminopimelic acid, 4-oxo-diaminopimelic acid or 2, 4, 6-triaminopimelic acid.
9. Process according to claims 1 to 7, characterised in that mutants of coryneform bacteria are used that produce L-lysine and that are resistant to one or more of the diaminopimelic acid analogues selected from the group comprising 4-fluorodiaminopimelic acid, 4- hydroxydiaminopimelic acid, 4-oxo-diaminopimelic acid or 2,4, 6-triaminopimelic acid.
10. Feedstuffs additives based on fermentation broth, characterised in that a) they contain L-lysine produced according to claims 1 to 7 or 9 , and
b) they contain the biomass and/or constituents from the fermentation broth formed during the fermentation of the L-lysine-producing microorganisms in an amount of 0% to 5%.
11. Feedstuffs additives based on fermentation broth, characterised in that
a) they contain L-lysine produced according to claims 1 to 7 or 9, and
they contain the biomass and/or constituents from the fermentation broth formed during the fermentation of the L-lysine-producing microorganisms in an amount of 90% to 100%.
EP03766147A 2002-07-31 2003-07-10 Process for the production of l-lysine using coryneform bacteria Withdrawn EP1525321A1 (en)

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US8404465B2 (en) 2009-03-11 2013-03-26 Celexion, Llc Biological synthesis of 6-aminocaproic acid from carbohydrate feedstocks
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US9890405B2 (en) 2012-08-23 2018-02-13 Bioamber Inc. Recombinant bacterial cells producing (S)-2-amino-6-hydroxypimelate
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