KR20050026032A - Process for the production of l-lysine using coryneform bacteria - Google Patents

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

Method for producing L-lysine using coryneform bacteria {PROCESS FOR THE PRODUCTION OF L-LYSINE USING CORYNEFORM BACTERIA}

The present invention provides a method for producing L-lysine using coryneform bacteria that is resistant to diaminopimelic acid homologs, especially 4-hydroxy-diaminopimelic acid.

L-amino acids, in particular L-lysine, are used in human medicine and in the pharmaceutical industry, in the food industry and most particularly in animal nutrients.

Amino acids are known to be produced by fermentation of Coryneform bacteria, particularly Corynebacterium glutamicum strains. Due to their great importance, efforts have been made to improve the production methods. Improvements in the process may include, for example, fermentation technology means such as stirring and supply of oxygen, or the composition of nutrient media such as, for example, sugar concentrations during fermentation, or post-treatment of the product form, for example by ion exchange chromatography, Or inherent performance characteristics of the microorganism itself.

To improve the performance characteristics of these microorganisms, methods involving mutagenesis, selection and selection of mutants are used. In this way, a strain is obtained that is resistant to anti-metabolites such as, for example, lysine homologue S- (2-aminoethyl) -cysteine, or is nutritionally essential for regulatoryly important metabolites and produces L-amino acids. do.

For several years, recombinant DNA technology has also been used to improve L-amino acid producing strains of Corynebacterium glutamicum by amplifying individual amino acid biosynthetic genes and investigating their effects on L-amino acid production.

Purpose of the Invention

We have been keen to devise new principles for improved methods for the fermentative production of L-lysine using Coryneform bacteria.

Where L-lysine or lysine is mentioned below, this is understood to mean not only a base but also a salt such as, for example, lysine monohydrochloride or lysine sulfate.

The present invention provides a method for the fermentative production of L-lysine using Coryneform bacteria resistant to diaminopimelic acid homologs, particularly 4-hydroxydiaminopimelic acid. Homologs are generally used at concentrations of ≥ (greater / equivalent) 3 to ≤ (less / equivalent) 30 g / l.

The present invention also provides a method for the fermentative production of L-lysine using Coryneform bacteria that already produce L-lysine and are resistant to diaminopimelic acid homologs, especially 4-hydroxydiaminopimelic acid.

The invention also provides for a) fermenting L-lysine producing coryneform bacteria resistant to at least diaminopimelic acid homologs, especially 4-hydroxydiaminopimelic acid; b) enriching L-lysine in medium or bacterial cells; Optionally c) separating the L-lysine or L-lysine-containing feed additive from the fermentation broth such that ≧ 0-100% of the components from the fermentation broth and / or from the biomass are present. Provides a method for producing lysine

The present invention similarly provides a method for producing Coryneform bacteria resistant to diaminopimelic acid homologs, especially 4-hydroxydiaminopimelic acid.

The strain used preferably produces L-lysine before it is resistant to 4-hydroxydiaminopimelic acid.

Expression diaminopimelic acid homologs according to the invention include compounds such as 4-fluorodiaminopimelic acid, 4-hydroxydiaminopimelic acid, 4-oxodiaminopimelic acid or 2,4,6-triaminopimelic acid do.

The present invention also relates to one or more diamino selected from the group comprising 4-fluorodiaminopimelic acid, 4-hydroxydiaminopimelic acid, 4-oxodiaminopimelic acid and 2,4,6-triaminopimelic acid. Provided are mutant Coryneform bacteria producing L-lysine, resistant to pimelic homologues.

The invention also contains L-lysine produced according to the invention and contains no or only trace amounts of biomass and / or components derived from fermented broth formed during fermentation of L-lysine-producing microorganisms. To provide a feed additive based on fermented broth.

The term "traces" is understood to mean amounts of> 0% to 5%.

The present invention further provides a composition comprising 90% to 90% biomass and / or components derived from fermented broth formed during fermentation of L-lysine-producing microorganisms, a) containing L-lysine produced according to the present invention. It provides a feed additive based on fermented broth, characterized by containing in an amount of 100%.

Microorganisms 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 of the genus Coryneform bacteria, in particular Corynebacterium. Among the genera Corynebacterium, mention is made of the species Corynebacterium glutamicum , whose ability to produce L-amino acids is known to those skilled in the art.

Suitable strains of the genus Corynebacterium, in particular Corynebacterium glutamicum species, in particular the following known wild-type strains, Corynebacterium glutamicum ATCC13032, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium acetonitrile know also pilrum (Corynebacterium acetoacidophilum) ATCC13870, Corynebacterium Melaka three-cola (Corynebacterium melassecola) ATCC17965, Corynebacterium gather more Mino jeneseu (Corynebacterium thermoaminogenes) FERM BP-1539 , Brevibacterium Playa boom Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020, and L-amino acid-producing mutants and / or strains produced from these strains , For example, Corynebacterium glutamicum FERM-P 1709, Brevibacteium plavum rium flavum ) FERM-P 1708, Brevibacterium lactofermentum FERM-P 1712, Corynebacterium glutamicum FERM-P 6463, Corynebacterium glutamicum FERM-P 6464, Coryne L such as Bacterium glutamicum ATCC 21513, Corynebacterium glutamicum ATCC 21544, Corynebacterium glutamicum ATCC 21543, Corynebacterium glutamicum DSM 4697 and Corynebacterium glutamicum DSM 5715 Lysine-producing strains.

Coryneform bacteria that are resistant to diaminopimelic acid homologs, particularly 4-hydroxydiaminopimelic acid, have been found to produce L-lysine in an improved manner.

The mutagenesis method described in the prior art is used to produce Coryneform bacteria according to the invention which are resistant to 4-hydroxydiaminopimelic acid.

For mutagenesis, conventional in vivo mutagenesis methods using mutagenic substances such as, for example, N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light can be used (Miller, JH: 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).

Coryneform bacteria resistant to 4-hydroxydiaminopimelic acid can be identified by plating on nutrient medium plates containing 4-hydroxydiaminopimelic acid. For this purpose a final concentration of 4-hydroxydiaminopimelic acid of about 5 to 15 g / l, for example about 10 g / l, in nutrient media is particularly suitable. Mutants that are resistant to 4-hydroxydiaminopimelic acid at this concentration can be distinguished from the parent strain unchanged by delayed growth. Mutants that are resistant to 4-hydroxydiaminopimelic acid after selection show improved L-lysine production.

In addition to this resistance to 4-hydroxydiaminopimelic acid, it also supports one or more of individual biosynthetic pathways, glycolysis, anaplerosis, citric acid cycles, pentose phosphate cycles, amino acid exports and optionally regulatory proteins. It may be advantageous to produce L-lysine by enhancing, in particular overexpressing. It is generally preferred to use endogenous genes.

The expression “endogenous gene” or “endogenous nucleotide sequence” is understood to mean a gene or nucleotide sequence present within a population of species.

The expressions "promoting" and "enhancing" refer to, for example, increasing the number of copies of a gene or genes, using genes encoding corresponding enzymes or proteins with strong promoters or high activity, and optionally combining these means. Thereby increasing the intracellular activity of one or more enzymes or proteins encoded by the corresponding DNA in the microorganism.

By using these enhancement, in particular overexpressing means, the activity or concentration of the corresponding protein is generally at least 10%, 25%, 50% with reference to the activity or concentration of the wild type protein and / or the activity or concentration of the protein in the starting microorganism. , Up to 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000%.

Thus, for the production of L-lysine, in addition to the resistance of the diaminopimelic acid homologues, one can in particular enhance, in particular overexpress, one or more genes selected from:

Gene lysC encoding feedback-resistant aspartate kinase (Accession No. P26512, EP-B-0387527; EP-A-0699759; WO 00/63388),

The gene dapA (EP-B 0 197 335), which encodes a dihydrodipicolinate synthase,

Gene gaps encoding glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992). Journal of Bacteriology 174: 6076-6086),

At the same time, the gene pyc (DE-A-198 31 609, EP-A-1108790), which encodes pyruvate carboxylase,

Gene zwf (JP-A-09224661, EP-A-1108790) encoding glucose-6-phosphate dehydrogenase,

At the same time, the gene lysE, which encodes a lysine releasing protein,

The gene zwa1 encoding the Zwal protein (DE: 19959328.0, DSM 13115),

The gene lysA encoding diaminopimelic acid decarboxylase (Accession No. X07563),

Gene sigC encoding sigma factor C (DE: 10043332.4, DSM14375),

The gene tpi encoding triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), and

Gene pgk encoding 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086).

In addition, for the production of L-lysine, in addition to resistance to 4-hydroxydiaminopimelic acid, it may be advantageous to simultaneously attenuate, in particular reduce expression, one or more genes selected from the following groups:

The gene pck encoding phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047),

Gene pgi encoding glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969),

The gene poxB encoding pyruvate oxidase (DE: 1995 1975.7, DSM 13114),

Gene deaD (DE: 10047865.4, DSM14464), which encodes a DNA helicase

The gene citE (PCT / EP01 / 00797, DSM 13981), encoding the citrate lysase,

The gene menE encoding O-succinylbenzoic acid CoA-ligase (DE: 10046624.9, DSM 14080),

Gene mikE17 (DE: 10047867.0, DSM 14143), encoding the transcriptional regulator MikE17 and

Gene zwa2 encoding the Zwa2 protein (DE: 19959327.2, DSM 13113).

The term "attenuation" refers to, for example, using a gene or allele encoding a weak promoter or a corresponding enzyme with low activity, or inactivating a corresponding gene or enzyme (protein) and optionally Combinations are described in terms of reducing or stopping the intracellular activity of one or more enzymes (proteins) encoded by the corresponding DNA in a microorganism.

By using these weakening means the activity or concentration of the corresponding protein is generally 0-75%, 0-50%, 0-25%, of the activity or concentration of the wild-type protein and / or the activity or concentration of the protein in the initial microorganism, Reduced to 0-10%, or 0-5%.

Finally, for the production of L-lysine, in addition to resistance to 4-hydroxydiaminopimelic acid, it may also be advantageous to stop unwanted secondary reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academmic Press, London, UK, 1982).

Microorganisms produced according to the present invention are also included in the present invention and for the purpose of producing L-lysine, a batch method (batch culture) or a fed-batch method (addition method) or a repeated addition-batch method It can be cultured continuously or discontinuously in (repeated addition method). A summary of known culture methods can be found in the Chmiel textbook (Bioprozesstechnik 1. Eunfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbooks of Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag). , Brunswick / Wiesbaden, 1994).

The culture medium used must meet the requirements of the individual strain in a suitable manner. Descriptions of culture media for various microorganisms are included in the handbok ("Manual of Methods for General Bacteriology" of the American Society for Bacteriology, Washington D.C., USA, 1981).

Carbon sources include, for example, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, for example oils and fats such as soybean oil, sunflower oil, peanut oil and palm oil, Fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as, for example, acetic acid can be used. These materials can be used individually or in mixtures.

Nitrogen sources include organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy bean flour and urea, or sulfuric acid Inorganic compounds such as ammonium, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. Nitrogen sources can be used individually or in mixtures.

As the phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium phosphate or the corresponding sodium-containing salts can be used. The culture medium should also contain further salts of metals, such as magnesium sulfate or iron sulfate, for example necessary for growth. Finally, in addition to the substances mentioned above, essential growth promoters such as amino acids and vitamins can be used. Apart from these, suitable precursors may be added to the culture medium. The aforementioned starting materials may be added to the culture in the form of a single batch, or may be supplied in a suitable manner during the culture.

To adjust 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 necessary. To control foam formation, antifoaming agents such as, for example, fatty acid polyglycol esters can be used. In order to maintain the stability of the plasmid, a suitable substance which selectively acts, for example antibiotics, can be added to the medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as, for example, air, is fed into the culture. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until a maximum amount of desired product is formed. This goal is typically achieved within 10 to 160 hours.

Methods of measuring L-lysine are known from the prior art. Assays can be subjected to anion exchange chromatography as described in Spackman et al., Analytical Chemistry, 30, (1958), 1190, followed by ninhydrin derivatization, or by Lindroth et al., Analytical Chemistry (1979). 51: 1167-1174).

The process according to the invention is used for the fermentative production of L-lysine.

The concentration of L-lysine may optionally be adjusted to the desired value by adding L-lysine.

By using the described method, it is possible to isolate Coryneform bacteria resistant to diaminopimelic acid homologs, especially 4-hydroxy-diaminopimelic acid, and to produce L-lysine in an improved manner according to the described fermentation method. Can be.

Example 1

Screening for clones resistant to 4-hydroxydiaminopimelic acid

Corynebacterium glutamicum strain DM1725 was produced by multiple nontargeting and targeting mutations including genetic engineering, selection and mutant selection from Corynebacterium glutamicum ATCC13032. This strain is resistant to the lysine homologue S- (2-aminoethyl) -L-cysteine and has two identical complete copies of the LysC gene encoding a feedback-resistant aspartate kinase. Two copies are located at the LysC gene site on the chromosome. Feedback-resistant aspartate kinase is not sensitive to inhibition by a mixture of lysine (or lysine homolog S- (2-aminoethyl) -L-cysteine, 100 mM) and threonine (10 mM), in contrast to wild type The activity of aspartate kinase is inhibited up to 10% residual activity. The strain is streptomycin resistant.

Pure cultures of strain DM1725 were deposited with DSM 15662 at the German Collection for Microorganisms and Cell Cultures (DSM Brunswick) on June 6, 2003 in accordance with the Budapest Convention.

4-hydroxy-for diaminopimelic acid to the screening of the resistant colonies, UV mutagenesis: after (Sambrook et al, Molecular Cloning A Laboratory Manual 2 nd Edition, Cold Spring Harbor, New York, 1989..) Strain DSM 15662 is placed on an LB agar plate containing 4-hydroxydiaminopimelic acid. Agar plates are supplemented with 10 g / l 4-hydroxydiaminopimelic acid. Colony growth is observed over 48 hours. Mutants that are resistant to 4-hydroxydiaminopimelic acid at this concentration can be distinguished from unmodified parent strains by improved growth. In this way, clones are identified that show much better growth compared to DSM 15662. The strain is identified as DSM 15662_Hdap_r.

Example 2

Production of lysine

Example 1 culturing the Corynebacterium glutamicum (C. glutamicum) strain DSM 15662_Hdap_r obtained in in a suitable nutrient medium for producing lysine and the lysine content in the culture supernatant is measured.

For this purpose the strains are first incubated at 33 ° C. for 24 hours on agar plates. This agar plate culture is used to inoculate the preculture (10 ml of medium in a 100 ml Erlenmeyer flask). Medium MM is used as medium for preculture. The preculture is incubated for 24 hours at 33 ° C. at 240 rpm on a vibrator. Using this preculture, the main culture is inoculated such that the initial absorbance of the main culture (OD-660 nm) is 0.1 OD. Medium MM is also used for main culture.

Badge MM

CSL 5 g / ℓ

MOPS 20 g / ℓ

Glucose (separately autoclaved) 50 g / ℓ

salts:

(NH ₄ ) ₂ SO ₄ 25 g / ℓ

KH ₂ PO ₄ 0.1 g / ℓ

MgSO ₄ × 7 H ₂ O 1.0 g / ℓ

CaCl ₂ × 2 H ₂ O 10 mg / l

FeSO ₄ × 7 H ₂ O 10 mg / l

MnSO ₄ x H ₂ O 5.0 mg / l

Biotin (sterile filtered) 0.3 mg / l

Thiamine × HCl (sterile filtered) 0.2 mg / l

CaCO ₃ 25 g / ℓ

CSL (corn immersion), MOPS (morpholinopropanesulfonic acid) and salt solution are adjusted to pH 7 with ammonia water and autoclaved. Then, sterile substrate and vitamin solution and dry autoclaved CaCO ₃ are added.

Cultivation is carried out in a 10 ml volume in a 100 ml Erlenmeyer flask equipped with baffles. Cultivation is performed at 33 ° C. and 80% atmospheric humidity.

After 72 hours OD is measured at a measurement wavelength of 660 nm using a Biomek 1000 instrument (Beckmann Instruments GmbH, Munich). The amount of lysine formed is measured by post-column derivatives by ion exchange chromatography and ninhydrin detection using an amino acid analyzer provided from Eppendorf-BioTronik, Hamburg, Germany.

The results of the experiments are shown in Table 1.

Strain OD (660 nm) Lysine, HClg / ml DSM 15662 11.6 16.2 DSM 15662_Hdap_r 11.9 18.9

Claims (11)

a) fermenting L-lysine producing coryneform bacteria resistant to at least diaminopimelic acid homologs, especially 4-hydroxydiaminopimelic acid; b) enriching L-lysine in medium or bacterial cells; Randomly c) separating the L-lysine or L-lysine-containing feed additive from the fermentation broth such that ≧ 0-100% of the components from the fermentation broth and / or from the biomass are present. How to produce lysine. The method of claim 1, wherein a bacterium in which additional genes of the biosynthetic pathway of L-lysine is also enhanced. The method of claim 1, wherein a bacterium is used which at least partially disrupts metabolic pathways that reduce the formation of L-lysine. Method according to claim 1, characterized in that for the production of L-lysine, the fermentation of Coryneform microorganisms simultaneously enhanced, in particular overexpressed, one or more genes selected from the group: Gene lysC, which encodes a feedback-resistant aspartate kinase, The gene dapA, which encodes a dihydrodipicolinate synthase, Gene gap encoding glyceraldehyde-3-phosphate dehydrogenase, Gene pyc, which encodes pyruvate carboxylase, Gene zwf, which encodes glucose-6-phosphate dehydrogenase, At the same time, the gene lysE, which encodes a lysine releasing protein, The gene zwa1, which encodes a Zwal protein Gene lysA, encoding diaminopimelic acid decarboxylase, The gene sigC encoding sigma factor C, Gene tpi encoding triose phosphate isomerase, or Gene pgk encoding 3-phosphoglycerate kinase. The method according to claim 1, characterized by expressing Coryneform microorganisms simultaneously attenuating at least one gene selected from the group for the production of L-lysine: Pck gene encoding phosphoenol pyruvate carboxykinase, Pgi gene encoding glucose-6-phosphate isomerase, Gene deaD, encoding a DNA helicase, The gene citE, which encodes citrate lyase, The gene menE encoding O-succinylbenzoic acid CoA-ligase, The gene mikE17, which encodes the transcriptional regulator MikE17, The gene poxB encoding pyruvate oxidase, or The gene zwa2, which encodes a Zwa2 protein. The method according to any one of claims 1 to 5, characterized in that microorganisms of Corynebacterium glutamicum species are used. The method according to any one of claims 1 to 6, characterized in that microorganisms of Corynebacterium glutamicum species are used that are resistant to 4-hydroxydiaminopimelic acid. One or more diaminopimelic acid homologs selected from the group comprising 4-fluorodiamino-pimelic acid, 4-hydroxydiaminopimelic acid, 4-oxo-diaminopimelic acid and 2,4,6-triaminopimelic acid A mutant of Coryneform bacteria producing L-lysine, resistant to. The process according to any one of claims 1 to 7, which produces L-lysine and is 4-fluorodiamino-pimelic acid, 4-hydroxydiaminopimelic acid, 4-oxo-diaminopimelic acid and 2,4, And a mutant of Coryneform bacteria that is resistant to one or more diaminopimelic acid homologs selected from the group comprising 6-triaminopimelic acid. a) contains L-lysine produced according to any one of claims 1 to 7 and 9, b) a feed additive based on fermented broth, characterized in that it contains from 0% to 5% biomass and / or components derived from fermented broth formed during fermentation of L-lysine-producing microorganisms. a) contains L-lysine produced according to any one of claims 1 to 7 and 9, A feed additive based on fermented broth, characterized by containing from 90% to 100% biomass and / or components derived from fermented broth formed during fermentation of L-lysine-producing microorganisms.