ZA200302409B

ZA200302409B - Fermentation process for the preparation of L-amino acids using strains of the family enterobacteriaceae.

Info

Publication number: ZA200302409B
Application number: ZA200302409A
Authority: ZA
Inventors: Mechthild Rieping; Thomas Hermann; Christine Bastuck; Georg Thierbach
Original assignee: Degussa
Priority date: 2000-09-30
Filing date: 2003-03-27
Publication date: 2004-02-13

Description

Fermentation Process for the Preparation of L-Amino Acids using Strains of the Family Enterobacteriaceae

Field of the Invention . The present invention relates to a fermentation process for the preparation of L-amino acids, especially L-threonine, using strains of the family Enterobacteriaceae in which at . least the pckA gene is attenuated.

Prior Art

L-Amino acids are used in animal nutrition, in human medicine and in the pharmaceutical industry.

It is known to prepare L-amino acids by the fermentation of strains of Enterobacteriaceae, especially Escherichia coli and Serratia marcescens. Because of their great importance, attempts are constantly being made to improve ~ 15 the preparative processes. Improvements to the processes may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself. . The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites, e.g. the threonine analogue o-amino-f- hydroxyvaleric acid (AHV) or auxotrophic for metabolites of regulatory significance, and produce L-amino acids, e.g. L- threonine.

Methods of recombinant DNA technology have also been used for some years to improve L-amino acid-producing strains of the family Enterobacteriaceae by amplifying individual amino acid biosynthesis genes and studying the effect on production.

Object of the Invention

The object which the inventors set themselves was to provide novel procedures for improving the preparation of

L-amino acids, especially L-threonine, by fermentation.

Summary of the Invention

The invention provides a fermentation process for the preparation of L-amino acids, especially L-threonine, using microorganisms of the family Enterobacteriaceae which, in particular, already produce L-threonine and in which the nucleotide sequence (pckA gene) coding for the enzyme phosphoenolpyruvate carboxykinase (PEP carboxykinase) (EC 4.1.1.49) is attenuated.

Detailed Description of the Invention Co

Where L-amino acids or amino acids are mentioned in the following, this means one or more amino acids, including their salts, chosen from the group consisting of L- asparagine, L-threonine, L-serine, L-glutamate, L-glycine,

L-alanine, L-cysteine, L-valine, L-methionine, L- isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L- histidine, L-lysine, L-tryptophan, L-homoserine and L- : arginine. L-Threonine is particularly preferred.

In this context the term “attenuation” describes the reduction or switching-off, in a microorganism, of the intracellular activity of one or more enzymes (proteins) which are coded for by the appropriate DNA, for example by using a weak promoter or a gene or allele which codes for an appropriate enzyme with low activity, or inactivating the appropriate enzyme (protein), and optionally: combining these measures.

By attenuation measures, the activity or concentration of the corresponding protein is in general 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 or of the activity or concentration of the protein in the starting microorganism.

The process is characterized in that the following steps are carried out: : a) fermentation of microorganisms of the family

Enterobacteriaceae in which at least the pckA gene is attenuated, b) enrichment of the appropriate L-amino acid in the medium or in the cells of the microorganisms of the family Enterobacteriaceae, and . c) isolation of the desired L-amino acid.

The microorganisms provided by the present invention can produce L-amino acids from glucose, sucrose, lactose, 20 . fructose, maltose, molasses, optionally starch or optionally cellulose, or from glycerol and ethanol. Said microorganisms are representatives of the family

Enterobacteriaceae selected from the genera Escherichia,

Erwinia, Providencia and Serratia. The genera Escherichia and Serratia are preferred. The species Escherichia coli and Serratia marcescens may be mentioned in particular among the genera Escherichia and Serratia respectively.

Examples of suitable strains, particularly L-threonine- producing strains, of the genus Escherichia, especially of the species Escherichia coli, are:

Escherichia coli TF427

Escherichia coli H4578

Escherichia coli KY10935

Escherichia coli VNIIgenetika MG442

Escherichia coli VNIIgenetika M1

Escherichia coli VNIIgenetika 472T23

Escherichia coli BKIIM B-3996

Escherichia coli kat 13 :

Escherichia coli KCCM-10132.

Examples of suitable L-threonine-producing strains of the genus Serratia, especially of the species Serratia marcescens, are:

Serratia marcescens HNr2l

Serratia marcescens TLrl56

Serratia marcescens T2000.

L-Threonine-producing strains of the family

Enterobacteriaceae preferably possess, inter alia, one or more genetic or phenotypic characteristics selected from the group comprising resistance to a-amino-f-hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to oa-methylserine, resistance to diaminosuccinic acid, resistance to a-aminobutyric acid, resistance to borrelidine, resistance to rifampicin, resistance to valine analogues such as valine hydroxamate, resistance to purine analogues such as 6-dimethylaminopurine, need for L- methionine, optionally partial and compensable need for L- isoleucine, need for meso~-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, resistance to L-homoserine, resistance to

L-lysine, resistance to L-methionine, resistance to L- glutamic acid, resistance to L-aspartate, resistance to L- leucine, resistance to L-phenylalanine, resistance to L- serine, resistance to L-cysteine, resistance to L-valine, sensitivity to fluoropyruvate, defective threonine dehydrogenase, optionally capability for sucrose utilization, amplification of the threonine operon, amplification of homoserine dehydrogenase I-aspartate kinase I, preferably of the feedback-resistant form, 5 amplification of homoserine kinase, amplification of threonine synthase, amplification of aspartate kinase, optionally of the feedback-resistant form, amplification of aspartate semialdehyde dehydrogenase, amplification of phosphoenolpyruvate carboxylase, optionally of the feedback-resistant form, amplification of phosphoenolpyruvate synthase, amplification of transhydrogenase, amplification of the RhtB gene product, amplification of the RhtC gene product, amplification of the YfiK gene product, amplification of malate quinone oxidoreductase and amplification of a pyruvate carboxylase and attenuation of acetic acid formation.

It has been found that the production of L-amino acids, especially L-threonine, by microorganisms of the family

Enterobacteriaceae is improved after attenuation and, in particular, switching-off of the pcka gene coding for PEP carboxykinase (EC 4.1.1.49).

The nucleotide sequence of the pckA gene of Escherichia coli has been published by Medina et al. (Journal of ‘Bacteriology 172, 7151-7156 (1990)) and can also be taken from the genome sequence of Escherichia coli published by

Blattner et al. (Science 277, 1453-1462 {1297)). The nucleotide sequence of the pckA gene of Escherichia coli is represented in SEQ ID No. 1 and the amino acid sequence of the corresponding gene product is represented in SEQ ID No. 2.

The pckA genes described in the above literature references can be used according to the invention. It is alsopossible to use alleles of the pckA gene which result from the degeneracy of the genetic code or from neutral sense mutations. :

Attenuation can be achieved for example by reducing or switching off the expression of the pckA gene or the 5S catalytic properties of the enzyme protein. Both measures may optionally be combined.

Gene expression can be reduced by an appropriate culture technique, by genetic modification (mutation) of the signal structures of gene expression, or by means of antisense RNA technology. Examples of signal structures of gene expression are repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. Those skilled in the art will find relevant information inter alia in e.g. Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998) ), ’

Carrier and Keasling (Biotechnology Progress 15, 58-64 (1999)), Franch and Gerdes (Current Opinion in Microbiology - 3, 159-164 (2000)) and well-known textbooks on genetics and molecular biology, for example the textbook by Knippers . ("“Molekulare Genetik” ("Molecular Genetics”), 6th edition,

Georg Thieme Verlag, Stuttgart, Germany, 1995) or the textbook by Winnacker (“Gene und Klone” (“From Genes to

Mutations which cause a change or reduction in the catalytic properties of enzyme proteins are known from the state of the art. Examples which may be mentioned are the studies of Qiu and Goodman (Journal of Biological Chemistry 272, 8611-8617 (1997)), Yano et al. (Proceedings of the

National Academy of Sciences USA 95, 5511-5515 (1998) ) and

Wente and Schachmann (Journal of Biological Chemistry 266, 20833-20839 (1991)). Surveys can be found in well<=known textbooks on genetics and molecular biology, e.g. the textbook by Hagemann (“Allgemeine Genetik” (“General

Genetics”), Gustav Fischer Verlag, Stuttgart, 1986).

Mutations to be taken into consideration are transitions, transversions, insertions and deletions. Depending on the effect of amino acid exchange on the enzyme activity, the term missense mutations or nonsense mutations is used.

Insertions or deletions of at least one base pair in a gene cause frame shift mutations, the result of which is that false amino acids are incorporated or translation is terminated prematurely. Deletions of several codons typically lead to a complete loss of enzyme activity.

Instructions for the production of such mutations form part of the state of the art and can be found in well-known textbooks on genetics and molecular biology, e.g. the textbook by Knippers (“Molekulare Genetik” (“Molecular

Genetics”), 6th edition, Georg Thieme Verlag, Stuttgart,

Germany, 1995), the textbook by Winnacker (“Gene und Klone” (“From Genes to Clones”), VCH Verlagsgesellschaft,

Weinheim, Germany, 1990) or the textbook by Hagemann ("Allgemeine Genetik” (“General Genetics”), Gustav Fischer

Verlag, Stuttgart, 1986).

An example of a plasmid by means of which the pckA gene of

Escherichia coli can be attenuated and, in particular, switched off by position-specific mutagenesis is plasmid

PMAK705Apcka (Figure 1). It contains only part of the 5 region and part of the 3’ region of the pckA gene. A 349 bp segment of the coding region is missing (deletion).

The sequence of this DNA, which can be used for mutagenesis of the pckA gene, is represented in SEQ ID No. 3.

The deletion mutation of the pckA gene can be incorporated into suitable strains by gene or allele exchange.

A common method is the method of gene exchange using a conditionally replicating pSC10l derivative, pMAK705, as described by Hamilton et al. (Journal of Bacteriology 174, 4617-4622 (1989)). Other methods described in the state of the art, for example that of Martinez-Morales et al. (Journal of Bacteriology, 7143-7148 (1999)) or that of Boyd et al. (Journal of Bacteriology 182, 842-=847 (2000)), can also be used.

When exchange has been carried out, the form of the ApckA allele represented in SEQ ID No. 4, which is a further subject of the invention, is present in the strain in : question.

Mutations in the pckA gene or mutations involving expression of the pckA gene can also be transferred to different strains by conjugation or transduction. .

Furthermore, for the production of L-amino acids, especially L-threonine, with strains of the family

Enterobacteriaceae, it can be advantageous not only to attenuate the pckA gene but also to amplify one or more =~ } enzymes of the known threonine biosynthetic pathway, or enzymes of the anaplerotic metabolism, or enzymes for the production of reduced nicotinamide adenine dinucleotide ‘phosphate.

In this context the term “amplification” describes the increase in the intracellular activity, in a microorganism, of one or more enzymes or proteins which are coded for by the appropriate DNA, for example by increasing the copy number of the gene(s), using a strong promoter or using a gene coding for an appropriate enzyme or protein with a high activity, and optionally combining these measures.

By amplification measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by .at least 10%, 25%, 50%, 75%, . 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.

Thus, for example, one or more genes selected from the . group comprising: 5S eo the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase (US-2A-4,278,765), e the pyc gene coding for pyruvate carboxylase

DE-A-19 831 609), eo ‘the pps gene coding for phosphoenolpyruvate synthase (Molecular and General Genetics 231, 332 (1992), * the ppc gene coding for phosphoenolpyruvate carboxylase (Gene 31, 279-283 (1984)), ¢ the pntA and pntB genes coding for transhydrogenase - (European Journal of Biochemistry 158, 647-653 (1986)), e¢ the rhtB gene for homoserine resistance (EP-A-0994190), and e the rhtC gene for threonine resistance (EP-A-1013765), ¢ the gdhA gene coding for glutamate dehydrogenase (Gene 27:193-199 (1984) can be simultaneously amplified and, in particular, overexpressed.

Furthermore, for the production of L-amino acids, especially L-threonine, it can be advantageous not only to attenuate the pckA gene but also to attenuate and, in particular, switch off one or more genes selected from the group comprising: : : :

* the tdh gene coding for threonine dehydrogenase (Ravnikar and Somerville, Journal of Bacteriology 169, 4716-4721 (1987)), e the mdh gene coding for malate dehydrogenase (EC 1.1.1.37) (Vogel et al., Archives in Microbiology 149, 36-42 (1987)), * the gene product of the open reading frame (orf) yjfa (Accession Number AAC77180 of the National Center for

Biotechnology Information (NCBI, Bethesda, MD, USA) and

SEQ ID No. 5), and ¢ the gene product of the open reading frame (orf) ytfp (Accession Number AAC77179 of the National Center for

Biotechnology Information (NCBI, Bethesda, MD, USA) and

SEQ ID No. 5), : or to reduce the expression. i»

It is preferred to attenuate the open reading frame yjfA and/or the open reading frame ytfP. So

It is also possible according to the invention to attenuate the open reading frames yjfA and/or ytfP independently of the pckA gene, in order to achieve an improvement in the amino acids, in particular L-threonine production.

The invention accordingly also provides a process, characterized in that the following steps are carried out: d) fermentation of microorganisms of the

Enterobacteriaceae family in which at least the open reading frame yjfA and/or -ytfP is attenuated, e) enrichment of the L-amino acid in the medium or in the cells of the microorganisms of the

Enterobacteriaceae family, and

£) isolation of the L-threonine, constituents of the fermentation broth and the biomass in its. entirety or portions thereof optionally being isolated as a solid product together with the L-amino acid.

An example of a plasmid by means of which the open reading frames yjfA and ytfP of Escherichia coli can be attenuated and, in particular, switched off by position-specific mutagenesis is plasmid pMAK705AyjfA (Figure 2). It contains only the 5' and 3' flanks of the ytfP-yjfA region, ° including very short residues of the open reading frames

YJfA and ytfP. A 337 bp long part of the ytfP-yjfA region is missing (deletion). The sequence of this DNA, which can be used for mutagenesis of the ytfP-yjfA region, is represented in SEQ ID No. 6.

An further example of a plasmid by means of which the open reading frames yjfA and ytfP of Escherichia coli can be attenuated and, in particular, switched off by position- specific mutagenesis is the plasmid PMAKT705A90bp : (Figure 5). It also contains only the 5' and 3' flanks of the ytfP-yjfA region including very short residues of the open reading frames yjfA and ytfP. A 90 bp long part of the ytfP-yifA region is missing (deletion). The sequence of this DNA, which can be used for mutagenesis of the ytfpP-- yifA region, is represented in SEQ ID No. 7. -

This deletion mutation can be incorporated into suitable strains by gene or allele replacement. It is also possible to transfer mutations in the open reading frames yifA and/or ytfP or mutations affecting expression of these open - reading frames into various ‘strains by conjugation or transduction.

When replacement has been carried out, the form of the

AytfP and AyjfA allele represented in SEQ ID No. 6 or SEQ

ID No. 7, which are a further subject of the invention, is present in the strain in question.

Furthermore, for the production of L-amino acids, especially L-threonine, it can be advantageous, in addition 5S to the individual or joint attenuation of the pckA gene or of the open reading frames yjfA and/or ytfP, to switch off undesired 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 prepared according to the invention can ‘be cultivated by the batch process or the fed batch process. A summary of known cultivation methods is provided in the textbook by Chmiel (Bioprozesstechnik 1.

Einfiihrung in die Bioverfahrenstechnik (Bioprocess So

Technology 1. Introduction to Bioengineering) (Gustav

Fischer Verlag, Stuttgart, 1991)) or in the textbook by -

Storhas (Bioreaktoren und periphere Einrichtungen Co (Bioreactors and Peripheral Equipment) (Vieweg Verlag, =

Brunswick/Wiesbaden, 1994)).

The culture medium to be used must appropriately meet the demands of the particular strains. Descriptions of culture media for various microorganisms can be found in the - handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington DC, USA, 1981).

Carbon sources which can be used are sugars and carbohydrates, €.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and optionally cellulose, oils and fats, e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, e.g. palmitic acid, stearic acid and linoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids, e.g. acetic acid. These substances can be used individually or as a mixture.

Nitrogen sources which can be used are organic nitrogen- containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour ~ and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as a mixture.

Phosphorus sources which can be used are phosphoric acid, . potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium salts. The culture medium must also contain metal salts, e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins can be used in addition to the substances mentioned above. Suitable precursors can also be added to the culture medium. Said feed materials can be added to the culture all at once or fed in appropriately during cultivation.

The pH of the culture is controlled by the appropriate use of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled using antifoams such as fatty acid polyglycol esters. The stability of plasmids can be maintained by adding suitable selectively acting substances, e.g. antibiotics, to the medium. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gaseous mixtures, e.g. air, into the culture. The temperature of the culture is normally 25°C to 45°C and preferably 30°C to 40°C. The culture is continued until the formation of L-amino acids or L-threonine has reached a maximum. This objective is normally achieved within 10 hours to 160 hours.

L-Amino acids can be analyzed by means of anion exchange chromatography followed by ninhydrin derivation, as. described by Spackman et al. {Analytical Chemistry 30, 1190 (1958) ), or by reversed phase HPLC, as described by :

Lindroth et al. (Analytical Chemistry 51, 1167-1174 (1979)).

A pure culture of the Escherichia coli K-12 strain

DH5a/pMAK705 was deposited on 12th September 2000 at the

Deutsche Sammlung fir Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell

Cultures, Braunschweig, Germany) in accordance with the

Budapest Treaty as DSM 13720.

A pure culture of the Escherichia coli K-12 strain

MG442ApckA was deposited on 2nd October 2000 at the

Cultures, Braunschweig, Germany) in accordance with the

Budapest Treaty as DSM 13761.

A pure culture of the Escherichia coli K-12 strain B- 3996kurAtdhApckA/pVIC40 was deposited on 9th March 2001 at" the Deutsche Sammlung fir Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell

Cultures, Braunschweig, Germany) in accordance with the

Budapest Treaty as DSM 14150.

A pure culture of the Escherichia coli K-12 strain

MG442A90yJjfA was deposited on 9th May 2001 at the Deutsche

Sammlung fiir Mikroorganismen und Zellkulturen (DSMZ =

German Collection of Microorganisms and Cell Cultures,

Braunschweig, Germany) in accordance with the Budapest

Treaty as DSM 14289.

It is also possible according to the invention individually to attenuate the open reading frames ytfP and yjfA in order to improve the production of L-amino acids.

The process according to the invention is used for the preparation of L-amino acids, e.g. L-threonine, L- isoleucine, L-methionine, L-homoserine and L-lysine, especially L-threonine, by fermentation.

The present invention is illustrated in greater detail below with the aid of Examples. :

The isolation of plasmid DNA from Escherichia coli and all the techniques for restriction, Klenow treatment and alkaline phosphatase treatment were carried out as described by Sambrook et al. (Molecular cloning - A laboratory manual (1989), Cold Spring Harbor Laboratory

Press). Unless indicated otherwise, the transformation of

Escherichia coli was carried out as described by Chung et . al. (Proceedings of the National Academy of Sciences USA 86, 2172-2175 (1989)).

The incubation temperature for the preparation of strains and transformants was 37°C. Temperatures of 30°C and 44°C were used in the gene exchange process of Hamilton et al.

Example 1

Construction of the deletion mutation of the pckA gene

Parts of the 5 and 3' regions of the pcka gene of

Escherichia coli K12 were amplified using the polymerase chain reaction (PCR) and synthetic oligonucleotides. The nucleotide sequence of the pckA gene in E. coli K12 MG1655 (SEQ ID No. 1) was used to synthesize the following PCR primers (MWG Biotech, Ebersberg, Germany) : pckA'5'-1: 5’ - GATCCGAGCCTGACAGGTTA - 3" pckA’S'-2: 5 - GCATGCGCTCGGTCAGGTTA - 3’

PckA’3'-1: 5 - AGGCCTGAAGATGGCACTATCG - 3' pcka'3'-2: 5 - CCGGAGAAGCGTAGGTGTTA - 3’. .

The chromosomal E. coli K12 MG1655 DNA used for the PCR was isolated with “Qiagen Genomic-tips 100/G” (QIAGEN, Hilden,

Germany) according to the manufacturer’s instructions. An approx. 500 bp DNA fragment from the 5’ region of the pckA gene (denoted as pckl) and an approx. 600 bp DNA fragment from the 3’ region of the pckA gene (denoted as pck2) could be amplified with the specific primers under standard PCR conditions (Innis et al. (1990), PCR Protocols. A Guide to

Methods and Applications, Academic Press) using Taq DNA polymerase (Gibco-BRL, Eggenstein, Germany). The PCR products were each ligated with vector pCR2.1TOPO (TOPO TA

Cloning Kit, Invitrogen, Groningen, The Netherlands) according to the manufacturer’s instructions and . transformed into E. coli strain TOP10F'. Plasmid-carrying cells were selected on LB agar containing 50 pg/ml of ampicillin. After isolation of the plasmid DNA, vector :

PCR2.1TOPOpck2 was cleaved with the restriction enzymes

Stul and XbaI and, after separation in 0.8% agarose gel, the pck2 fragment was isolated with the aid of the QIAquick

Gel Extraction Kit (QIAGEN, Hilden, Germany). After isolation of the plasmid DNA, vector PCR2.1TOPOpckl was cleaved with the enzymes EcoRV and Xbal and ligated to the isolated pck2 fragment. The E. coli strain DH5a was transformed with the ligation mixture and plasmid-carrying cells were selected on LB agar containing 50 pg/ml of ampicillin. After isolation of the plasmid DNA, control cleavage with the enzymes Spel and Xbal was used to detect plasmids containing, in cloned form, the mutagenic DNA sequence represented in SEQ ID No. 3. One of the plasmids was denoted as pCR2.1TOPOApckA.

Example 2

Construction of exchange vector PMAK705ApckA

After restriction with the enzymes Spel and Xbal and separation in 0.8% agarose gel, the pckA allele described in Example 1 was isolated from vector PCR2.1TOPOApckA and ligated to plasmid pMAK705 (Hamilton et al., Journal of

Bacteriology 174, 4617-4622 (1989)) which had been digested with the enzyme XbaI. DH5a was transformed with the ligation mixture and plasmid-carrying cells were selected on LB agar containing 20 pg/ml of chloramphenicol. After isolation of the plasmid DNA and cleavage with the enzymes

Hpal, KpnI, HindIII, Sall and PstI, successful cloning was detected. The exchange vector formed, PMAK705ApckA (= pMAK705deltapckA), is shown in Figure 1.

Example 3 :

Position-specific mutagenesis of the pcka gene in the E. : coli strain MG442

The L-threonine-producing E. coli strain MG442 is described in patent US-A-4,278,765 and deposited in the Russian 20 .National Collection of Industrial Microorganisms (VKPM,

Moscow, Russia) as CMIM B-1628.

The strain MG442 has a resistance to a-amino-f- hydroxyvaleric acid and has an optionally partial and compensable need for L-isoleucine.

For exchange of the chromosomal pckA gene for the plasmid- coded deletion construct, MG442 was transformed with SE plasmid pMAK705ApckA. The gene exchange was carried out by : the selection method described by Hamilton et al. (Journal of Bacteriology 174, 4617-4622 (1989)) and was verified by standard PCR methods (Innis et al. (1990), PCR Protocols. A

Guide to Methods and Applications, Academic Press) using the following oligonucleotide primers: pckA’S'-1: 5 - GATCCGAGCCTGACAGGTTA - 3’ pckA’3'-2: 5' ~- CCGGAGAAGCGTAGGTGTTA - 3’

The strain obtained was denoted as MG442ApckA.

Example 4 :

Preparation of L-threonine with the strain MG442ApckA

MG442ApckA was cultivated on minimum medium of the following composition: 3.5 g/1 of Na,HPO4-2H,0, 1.5 g/l of

KH2PO4, 1 g/1 of NH, Cl, 0.1 g/l of MgS04-7H20, 2. g/1 of glucose and 20 g/1 of agar. The formation of L-threonine was checked in 10 ml batch cultures contained in 100 ml :

Erlenmeyer flasks. These were inoculated with 10 ml of a preculture medium of the following composition: 2 g/l of yeast extract, 10 g/l of (NHy);S0,, 1 g/l of KHPO4, 0.5 g/l of MgS04-7H;0, 15 g/l of CaCO; and 20 g/l of glucose, and incubated for 16 hours at 37°C and 180 rpm on an ESR incubator from Kiithner AG (Birsfelden, Switzerland). 250 pl of this preculture were transferred to 10 ml of a production medium (25 g/l of (NH4),SO4, 2 g/l of KH,POy, 1 g/1 of MgSO4-7H;0, 0.03 g/l of FeSO47H,0, 0.018 g/l of

MnS04-1H,0, 30 g/l of CaCos, 20 g/1 of glucose) and incubated for 48 hours at 37°C. After incubation, the optical density (OD) of the culture suspension was determined with an LP2W photometer from Dr. Lange (Berlin,

Germany) at a measurement wavelength of 660 nm.

The concentration of L-threonine formed was then determined in the sterile-filtered culture supernatant with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by means of ion exchange chromatography and postcolumn reaction with ninhydrin detection.

The result of the experiment is shown in Table 1.

Table 1

Strain oD L-Threonine (660 nm) g/l } Example 5 - :

Preparation of L-threonine with the strain

MG442ApckA/pMW218gdhA 5.1 Amplification and cloning of the gdhA gene

The glutamate dehydrogenase gene from Escherichia coli K12 is amplified using the polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence for the gdhA gene in E. coli K12 MG1655 (gene library: Accession No. AE000270 and No. AEQ00271) PCR primers are synthesized (MWG Biotech, Ebersberg, Germany):

Gdhl: 5° - TGARACACTTCTGGCGGTACG - 3°

Gdh2: 5‘ ~ CCTCGGCGAAGCTAATATGG - 3°

The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolated according to the manufacturers instructions with "QIAGEN Genomic-tips 100/G" (QIAGEN, Hilden, Germany).

A DNA fragment approx. 2150 bp in size, which comprises the ~~ 20 gdhA coding region and approx. 350 bp 5'-flanking and approx. 450 bp 3'-flanking sequences, can be amplified with the specific primers under standard PCR conditions (Innis et al.: PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with the Pfu-DNA polymerase (Promega

Corporation, Madison, USA). The PCR product is cloned in the plasmid pCR2.1TOPO and transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands, Product

Description TOPO TA Cloning Kit, Cat. No. K4500-01).

Successful cloning is demonstrated by cleavage of the plasmid pCR2.1TOPOgdhA with the restriction enzymes EcoRI and ECORV. For this, the plasmid DNA is isolated by means of the "QIAprep Spin Plasmid Kits" (QIAGEN, Hilden,

Germany) and, after cleavage, separated in a 0.8 % agarose gel. 5.2 Cloning of the gdhA gene in the plasmid vector pMW218

The plasmid pCR2.1TOPOgdhA is cleaved with the enzyme

EcoRI, the cleavage batch is separated on 0.8% agarose gel and the gdhA fragment 2.1 kbp in size is isolated with the aid of the "QIAquick Gel Extraction Kit“ (QIAGEN, Hilden,

Germany). The plasmid pMW218 (Nippon Gene, Toyama, Japan) is cleaved with the enzyme EcoRI and ligated with the gdhA fragment. The E. coli strain DH5a is transformed with the ligation batch and pMW218-carrying cells are selected by plating out on LB agar (Lennox, Virology 1955, 1: 190), to which 20 pg/ml kanamycin are added.

Successful cloning of the gdhA gene can be demonstrated after plasmid DNA isolation and control cleavage with EcoRI and EcoRV. The plasmid is called pMW218gdhA (Figure 3). 5.3 Preparation of the strain MG442ApckA/pMW218gdhA

The strain MG442ApckA obtained in Example 3 and the strain

MG442 are transformed with the plasmid pMW218gdhA and transformants are selected on LB agar, which is supplemented with 20 pg/ml kanamycin. The strains

MG442ApckA/pMW218gdhA and MG442/pMW218gdhA are formed in this manner.

5.4 Preparation of L-threonine

The preparation of L-threonine by the strains

MG442ApckA/pMW218gdhA and MG442/pMW218gdhA is tested as described in Example 4. The minimal medium and the 5S preculture medium are additionally supplemented with 20 pg/ml kanamycin.

The result of the experiment is summarized in Table 2.

Table 2

Strain oD L-Threonine oT (660 nm) g/l

MG442 /pMi218gdhA 5.6

Example 6

Preparation of L-threonine with the strain

MG442ApckA/pMW219rhtC 6.1 Amplification of the rhtC gene

The rhtC gene from Escherichia coli K12 is amplified using the polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence for the rhtC gene in E. coli K12 MG1655 (gene library:

Accession No. AE000458, Zakataeva et al. (FEBS Letters 452, 228-232 (1999)), PCR primers are synthesized (MWG Biotech,

Ebersberg, Germany):

RhtCl: 5‘ - CTGTTAGCATCGGCGAGGCA - 3°

RhtC2: 5‘ - GCATGTTGATGGCGATGACG — 3°

A DNA fragment approx. 800 bp in size can be amplified with the specific primers under standard PCR conditions (Innis et al.: PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with Pfu-DNA polymerase (Promega

Corporation, Madison, USA). 6.2 Cloning of the rhtC gene in the plasmid vector pMW219

The plasmid pMW219 (Nippon Gene, Toyama, Japan) is cleaved with the enzyme Saml and ligated with the rhtC-PCR fragment. The E. coli strain DH5a is transformed with the ligation batch and pMW219-carrying cells are selected on LB agar, which is supplemented with 20 pg/ml kanamycin.

Successful cloning can be demonstrated after plasmid DNA isolation and control cleavage with KpnI, HindIII and Ncol.

The plasmid pMW219rhtC is shown in Figure 4. 6.3 Preparation of the strain MG442ApckA/pMW219rhtC

The strain MG442ApckA obtained in Example 3 and the strain

MG442 are transformed with the plasmid pMW219rhtC and transformants are selected on LB agar, which is supplemented with 20 pg/ml kanamycin. The strains

MG442ApckA/pMW219rhtC and MG442/pMW219rhtC are formed in this manner. 6.4 Preparation of L-threonine

The preparation of L-threonine by the strains

MG442ApckA/pMW219rhtC and MG442/pMW219rhtC is tested as described in Example 4. The minimal medium and the preculture medium are additionally supplemented with 20 pg/ml kanamycin.

The result of the experiment is summarized in Table 3.

Table 3

Strain - OD L~-Threonine (660 nm) g/l c : .

Example 7

Preparation of L-threonine with the strain

B-3996kurAtdhApckA/pVIC40

The L-threonine-producing E. coli strain B-3996 is described in US-A- 5,175,107 and deposited at the Russian

National Collection for Industrial Microorganisms (VKPM,

Moscow, Russia).

The strain B-3996 has, inter alia, a resistance to a-amino-

B-hydroxyvaleric acid, has an attenuated, in particular switched-off, or defective threonine dehydrogenase, has an enhanced homoserine dehydrogenase I aspartate kinase I in the feed back resistant form, has an optionally partial and compensable need for L-isoleucine and has the ability to utilize sucrose.

7.1 Preparation of the strain B-3996kurAtdhApckA/pVIC40

After culture in antibiotic-free complete medium for approximately ten generations, a derivative of strain B- ~ 3996 which no longer contains the plasmid pVIC40 is isolated. The strain formed is streptomycin-sensitive and is designated B-3996kur.

The method described by Hamilton et al. (Journal of :

Bacteriology (1989) 171: 4617-4622), which is based on the use of the plasmid pMAK705 with a temperature-sensitive replicon, was used for incorporation of a deletion into the tdh gene. The plasmid pDR121 (Ravnikar and Somerville,

Journal of Bacteriology (1987) 169:4716-4721) contains a

DNA fragment from E. coli 3.7 kilo-base pairs (kbp) - in : size, on which the tdh gene is coded. To generate a deletion of the tdh gene region, pDR121 is cleaved with the restriction enzymes ClaI and EcoRV and the DNA fragment 5 kbp in size isolated is ligated, after treatment with

Klenow enzyme. The ligation batch is transformed in the E. coli strain DH5a and plasmid-carrying cells are selected on

LB agar, to which 50 pg/ml ampicillin are added.

Successful deletion of the tdh gene can be demonstrated after plasmid DNA isolation and control cleavage with :

EcoRI. The EcoRI fragment 1.7 kbp in size is isolated, and ligated with the plasmid pMAK705, which is partly digested with EcoRI. The ligation batch is transformed in DHS5¢. and plasmid-carrying cells are selected on IB agar, to which 20 pg/ml chloramphenicol are added. Successful cloning is demonstrated after isolation of the plasmid DNA and - cleavage with EcoRI. The pMAK705 derivative formed is designated pDM32.

For the gene replacement, B-3996kur is transformed with the plasmid pDM32. The replacement of the chromosomal tdh gene with the plasmid-coded deletion construct is carried out by the selection process described by Hamilton et al. and is verified by standard PCR methods (Innis et al. (1990), PCR

Protocols. A Guide to Methods and Applications, Academic

Press) with the following oligonucleotide primers:

Tdhl: 5‘'-TCGCGACCTATAAGTTTGGG-3"

Tdh2: 5‘-AATACCAGCCCTTGTTCGTG-3"'.

The strain formed is tested for kanamycin sensitivity and is designated B-3996kurAtdh.

For the position-specific mutagenesis of the pckA gene, B- 3996kurAtdh is transformed with the replacement vector

PMAK705ApckA described in Example 2. The replacement of the chromosomal pckA gene by the plasmid-coded deletion construct is carried out as described in Example 3. The strain obtained is called B-3996kurAtdhApckA.

B-3996kurAtdh and B-3996kurAtdhApckA are transformed with the plasmid pVIC40 isolated from B-3996 and plasmid- carrying cells are selected on LB agar with 20 pg/ml - streptomycin. In each case a selected individual colony is called B-3996kurAtdh/pVIC40 and B-3996kurAtdhApckA/pVIC40. 7.2 Preparation of L-threonine

The preparation of L-threonine by the strains

B-3996kurAtdh/pVIC40 and B-3996kurAtdhApckA/pVIC40 is tested as described in Example 4. The minimal medium, the preculture medium and the production medium are additionally supplemented with 20 pg/ml streptomycin.

The result of the experiment is summarized in Table 4.

Table 4

Strain OD L-Threonine (660 nm) |. g/l oe pepe

Example 8

Preparation of L-lysine with the strain TOC21RApckA

The L-lysine-producing E. coli strain pDA1/TOC21R is described in the patent application F-A-2511032 and deposited at the Collection Nationale de Culture de

Microorganisme (CNCM = National Microorganism Culture ..

Collection, Pasteur Institute, Paris, France) under number

I-167. The strain and the plasmid-free host are also described by Dauce-Le Reverend et al. (European Journal of ~ Applied Microbiology and Biotechnology 15:227-231 (1982)) under the name TOCR21/pDAl. 8.1 Position-specific mutagenesis of the pckA gene in the

E. coli strain TOC21R

After culture in antibiotic-free LB medium for approximately six generations, a derivative of strain . pPDA1/TOC21R which no longer contains the plasmid pDAl is isolated. The strain formed is tetracycline-sensitive and is called TOC21R.

For replacement of the chromosomal pckA gene by the plasmid-coded deletion construct, TOC21R is transformed with the plasmid pMAK705ApckA (Example 2). The gene replacement is carried out by the selection method described by Hamilton et al. (1989) Journal of Bacteriology

174, 4617 — 4622) and is verified by standard PCR methods (Innis et al. (1990) PCR Protocols. A Guide to Methods and

Applications, Academic Press) with the following oligonucleotide primers: : pckA'5'-1; 5' —- GATCCGAGCCTGACAGGTTA - 3© pckA3'-2: 5' - CCGGAGAAGCGTAGGTGTTA - 31

The strain obtained is called TOC21RApckA. 8.2 Preparation of L-lysine with the strain TOC21RApckA

The formation of L-lysine by the strains TOC21RA pckA and

TOC21R is checked in batch cultures of 10 ml contained in 100 ml conical flasks. For this, 10 ml of preculture medium of the following composition: 2 g/l yeast extract, 10 g/1 (NH4) 2804, 1 g/1 KHzPO4, 0.5 g/l MgS02*7H,0, 15 g/l CaCOs, 20 g/1 glucose are inoculated and the batch is incubated for 16 hours at 37°C and 180 rpm on an ESR incubator from

Kihner AG (Birsfelden, Switzerland). 250 pl of this preculture are transinoculated into 10 ml of production medium (25 g/l (NH) 2S04, 2 g/l KH,PO4, 1 g/l MgS0,*7H20, 0.03 g/1 FeS04*7H20, 0.018 g/l MnSO4*1H,0, 30 g/l CaCOs;, 20 g/l glucose, 25 mg/l L-isoleucine and 5 mg/l thiamine) and the batch is incubated for 72 hours at 37°C. After the incubation the optical density (OD) of the culture suspension is determined with an LP2W photometer from Dr.

Lange (Berlin, Germany) at a measurement wavelength of 660 nm.

The concentration of L-lysine formed is then determined in the sterile-filtered culture supernatant with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column reaction with : ninhydrin detection.

The result of the experiment is shown in Table 5.

Table 5

Strain OD L-Lysine (660 nm) g/l

Example 9

Preparation of L-isoleucine with the strain

B-3996kurAtdhilvA*ApckA/pVIC40 9.1 Preparation of the strain B- 3996kurAtdhilvA*ApckA/pVIC40

The strain B-3996kurAtdh, which is in need of L-isoleucin, obtained in Example 7.1 is transduced with the aid of the : phage Plkc (Lennox, Virology 1, 190-206 (1955); Miller,

Experiments in Molecular Genetics, Cold Spring Harbor

Laboratory 1972) and L-isoleucine-prototrophic transductants are isolated.

For this, the phage Plkc is multiplied on the strain MG1655 15, (Guyer et al., Cold Spring Harbor Symposium of Quantitative

Biology 45, 135-140 (1981) and Blattner et al., Science. 277, 1453-1462 (1997))and the phage lysate is employed for the transduction of the strain B-3996kurAtdh. The multiplicity of the infection is approximately 0.2.

Selection for L-isoleucine-prototrophic transductants is carried out on minimal agar, which contains 2 g/1 glucose and 10 mg/l L-threonine. An L-isoleucine-prototrophic transductant is isolated, smeared on to LB agar for purification or isolation and called B-3996kurAtdhilva‘.

The pckA gene of the strain B-3996kurAtdhilvA* is then replaced, as described in Example 3, by the ApckA allele prepared in Example 1 and 2. The strain obtained is called

B-3996kurAtdhilvA*ApckA.

The strains B-3996kurAtdhilvA* and B-3996kurAtdhilvA*Apcka .are transformed with the plasmid pVIC40 isolated from strain B-3996 and plasmid-carrying cells are selected on LB agar, which is supplemented with 20 pg/ml streptomycin. In each case a selected individual colony is called B- 3996kurAtdhilvA*ApckA/pVIC40 and B-3996kurAtdhilvA*/pvVIC40. 9.2 Preparation of L-isoleucine

The preparation of L-isoleucine by the strains "B~3996kurAtdhilvA*/pvIC40 and B- 3996kurAtdhilvA*ApckA/pVIC40 is tested under the test conditions as described in Example 4. The minimal medium, the preculture medium and the production medium are additionally supplemented with 20 pg/ml streptomycin.

The result of the experiment is shown in Table 6.

Table 6

Strain Co oD L-Isoleucine (660 nm) mg/l

B-3996kurAtdhilvA*ApckA/pvICA0

Example 10

Preparation of L-valine with the strain B-12288ApckA

The L-valine-producing E. coli strain AJ 11502 is described in the patent specification US-A-4391907 and deposited at the National Center for Agricultural Utilization Research (Peoria, Illinois, USA) as NRRL B-12288. 10.1 Position-specific mutagenesis of the pckA gene in the

E. coli strain B+~1288

After culture in antibiotic-free LB medium for approximately six generations, a plasmid-free derivative of strain AJ 11502 is isolated. The strain formed is ampicillin-sensitive and is called AJ11502kur. ~ For replacement of the chromosomal pckA gene by the plasmid-coded deletion construct, AJ11502kur is transformed with the plasmid pMAK705ApckA (see Example 2). The gene replacement is carried out by the selection method described by Hamilton et al. (1989) Journal of Bacteriology 174, 4617 — 4622) and is verified by standard PCR methods (Innis et al. (1990) PCR Protocols. A Guide to Methods and

Applications, Academic Press) with the following oligonucleotide primers: pckA'5'-1: 5‘ - GATCCGAGCCTGACAGGTTA - 3° pckA3'-2: 5' — CCGGAGAAGCGTAGGTGTTA — 3°

The strain obtained is called AJ11502kurApckA. The plasmid described in the patent specification US-A-4391907, which carries the genetic information in respect of valine production, is isolated from strain NRRL B-12288. The strain AJ11502kurApckA is transformed with this plasmid.

One of the transformants obtained is called B-12288ApckaA.

10.2 Preparation of L-valine with the strain B-12288ApckA

The formation of L-valine by the strains B-12288ApckA and

NRRL B-12288 is checked in batch cultures of 10 ml contained in 100 ml conical flasks. For this, 10 ml of preculture medium of the following composition: 2 g/l yeast extract, 10 g/l (NH4).SO0s, 1 g/1 KH;PO4, 0.5 g/1 MgSO4*7H,0, 15 g/1 CaCOs;, 20 g/l glucose and 50 mg/l ampicillin are inoculated and the batch is incubated for 16 hours at 37°C and 180 rpm on an ESR incubator from Kithner AG (Birsfelden, switzerland). 250 pl of this preculture are transinoculated into 10 ml of production medium (25 g/l (NH4),80s, 2 g/l

KH>POy4, 1 g/l MgSO, *7H,0, 0.03 g/l FeS04*7H,0, 0.018 g/l

MnSO4*1H,0, 30 g/1 CaCO;, 20 g/l glucose, 5 mg/l thiamine and 50 mg/l ampicillin) and the batch is incubated for 72 hours at 37°C. After the incubation the optical density (OD) of the culture suspension is determined with an LP2W photometer from Dr. Lange (Berlin, Germany) at a measurement wavelength of 660 nm.

The concentration of L-valine formed is then determined in the sterile-filtered culture supernatant with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column reaction with ninhydrin detection.

The result of the experiment is shown in Table 7.

Table 7 :

Strain oD L-Valine (660 nm) g/l

Example 11

Construction of deletion mutations of the ytfP-yjfA gene region

The ytfP-yjfA gene region is amplified from Escherichia coli K12 using the polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence of the ytfP-yjfA gene region in E. coli K12 MG1655 (SEQ ID No. 5), the following PCR primers are synthesized (MWG Biotech, Ebersberg, Germany): oo ytfP-1: 5‘ - GGCGATGTCGCAACAAGCTG — 3° ) : ytfP-2: 5‘ - CTGTTCATGGCCGCTTGCTG —- 3°

The chromosomal E. coli K12 MG1655 DNA employed for the PCR is isolated according to the manufacturers instructions : with "Qiagen Genomic-tips 100/G" (QIAGEN, Hilden, Germany). :

A DNA fragment approx...1300 bp in size can be amplified © with the specific primers under standard PCR conditions (Innis et al. (1990) PCR Protocols. A Guide to Methods and

Applications, Academic Press) with Tag-DNA polymerase (Gibco-BRL, Eggenstein, Germany). The PCR product is ligated with the vector pCR2.1TOPO (TOPO TA Cloning Kit,

Invitrogen, Groningen, The Netherlands) in accordance with the manufacturers instructions and transformed into the E. coli strain TOP10F'. Selection of plasmid-carrying cells takes place on LB agar, to which 50 pg/ml ampicillin are added. After isolation of the plasmid DNA, successful cloning of the PCR product is checked with the restriction enzymes EcoRI and NsiI.

To generate a 337 bp deletion in the yftP-yjfA region, the vector pCR2.1TOPOytfP-yjfA is cleaved with the restriction enzymes NdeI and Sspl and the DNA fragment 4.8 kbp in size is ligated, after treatment with Klenow enzyme.

To generate a 90 bp deletion, the vector pCR2.1TOPOytfP- yJfA is cleaved with the enzymes NdeI and SplI and the DNA fragment 5 kbp in size is ligated, after treatment with

Klenow enzyme.

The E. coli strain DHSa is transformed with the ligation batches and plasmid-carrying cells are selected on LB agar, to which 50 pg/ml ampicillin is added. After isolation of the plasmid DNA those plasmids in which the mutagenic DNA sequence shown in SEQ ID No. 6 and SEQ ID No. 7 is cloned are detected by control cleavage with the enzyme EcoRI. The plasmids are called pCR2.1TOPOAyjfA and pCR2.1TOPOA90Dbp.

Example 12

Construction of the replacement vectors pMAK705AyjfA and

PMAK705A%0bp

The ytfP-yijfA alleles described in Example 11 are isolated from the vectors pCR2.1TOPOAyjfA and PCR2.1TOPOA9Obp after restriction with the enzymes SacI and Xbal and separation in 0.8% agarose gel, and ligated with the plasmid PMAK705 (Hamilton et al. (1989) Journal of Bacteriology 174, 4617 - 4622), which is digested with the enzymes SacI and XbaI.

The ligation batches are transformed in DHS5a and plasmid-

TT Cartying cells” ate selected on LB agar, to which 20 pg/ml } chloramphenicol are added. Successful cloning is demonstrated after isolation of the plasmid DNA and cleavage with the enzymes SacI and XbaI. The replacement vectors formed, pMAK705AyjfA (= pMAK705deltayjfA) and

PMAK705A90bp ( = pMAK705delta90bp), are shown in Figure 2 and in Figure 5. :

Example 13

Position-specific mutagenesis of the ytfP-yjfA gene region in the E. coli strain MG442

For replacement of the chromosomal ytfP-yjfA gene region with the plasmid-coded 90 bp deletion construct, MG442 is transformed with the plasmid pMAK705A90bp, The gene replacement is carried out by the selection method described by Hamilton et al. (1989) Journal of Bacteriology 174, 4617 - 4622) and is verified by standard PCR methods (Innis et al. (1990) PCR Protocols. A Guide to Methods and

Applications, Academic Press) with the following oligonucleotide primers: ytfP-1l: 5' - GGCGATGTCGCRAACAAGCTG - 3‘ ytfP-2: 5‘ - CTGTTCATGGCCGCTTGCTG — 3°

The strain obtained is called MG442A90yjfA.

Example 14

Preparation of L-threonine with the strain MG442A90yj fA

The preparation of L-threonine by the strain MG442A90yjfA is tested as described in Example 4. The result of the experiment is summarized in Table 8.

Table 8 .Strain oD L-Threonine : (660 nm) g/l

Example 15

Preparation of L-threonine with the strain :

MG442A90yj fAApckA 15.1 Preparation of the strain MG442A90yjfAApckA

The pckA gene of the strain MG442A90yjfA is replaced, as described in Example 3, by the ApckA allele (see Example 1 and 2). The strain obtained is called MG442A90yj fAApckA. 15.2 Preparation of L-threonine

The preparation of L-threonine with the strain

MG442A90yjfRAApckA is carried out as described in Example 4. The result is shown in Table 9. :

. Table 9

Strain OD | L-Threonine (660 nm) g/1

Brief Description of the Figures: e Figure 1: pMAK705ApckA ( = PMAK705deltapcka) .5 e Figure 2: PMAK7054yjfA ( = PMAK705deltayj fA) e Figure 3: pMW218gdhA ® Figure 4: pMW219rhtC ~~ ® Figure 5: PMAKT705A%90bp ( = PMAK705delta%90bp)

The length data are to be understood as approx. data. The abbreviations and designations used have the following meaning: e cat: Chloramphenicol resistance gene ® rep-ts: Temperature-sensitive replication region of the plasmid pSC101 ee pckl: Part of the 5' region of the pcka gene e pck2: Part of the 3' region of the pcka gene e ytfP‘-yjfA‘: DNA sequence containing truncated coding regions of ytfP and yjfA ® kan: Kanamycin resistance gene e gdhA: Glutamate dehydrogenase gene se rhtC: Threonine resistance-imparting gene

The abbreviations for the restriction enzymes have the following meaning ¢ BamHI: restriction endonuclease from Bacillus amyloliquefaciens ® BglII: restriction endonuclease from Bacillus globigii e Clal: restriction endonuclease from Caryphanon latum ®¢ ECORI: restriction endonuclease from Escherichia coli e EcoRV: restriction endonuclease from Escherichia coli ® HindIII: restriction endonuclease from Haemophilus influenzae e KpnI: restriction endonuclease from Klebsiella pneumoniae eo PstI: restriction endonuclease from Providencia stuartii eo Pvul: restriction endonuclease from Proteus vulgaris ® SacI: restriction endonuclease from Streptomyces achromogenes eo Sall: restriction endonuclease from Streptomyces albus e Smal: restriction endonuclease from Serratia © marcescens eo Xbal: restriction endonuclease from Xanthomonas badrii e XhoI: restriction endonuclease from Xanthomonas holcicola

Claims

What is claimed is:

1. Fermentation process for the preparation of L-amino acids, especially L-threonine, wherein the following steps are carried out: : a) fermentation of the microorganisms of the family Enterobacteriaceae producing the desired L-amino acid, in which microorganisms at least the pckA gene or nucleotide sequences coding therefor are attenuated and, in particular, switched off, b) enrichment of the L-amino acid in the medium or in the bacterial cells, and Cc) isolation of the L-amino acid, constituents of the fermentation broth and the biomass in its entirety or portions thereof optionally being isolated as a solid product together with the L-amino acid.

2. Process according to claim 1, wherein microorganisms are used in which other genes of the biosynthetic pathway of the desired L-amino acid are additionally amplified.

3. Process according to claim 1, wherein microorganisms are used in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partially switched off. :

4. Process according to claim 1, wherein the expression of the polynucleotide(s) coding for the pckA gene is attenuated and, in particular, switched off.

5. Process according to claim 1, wherein the regulatory and/or catalytic properties of the polypeptide (enzyme protein) coded for by the polynucleotide pckA are reduced.

6. Process according to claim 1, wherein microorganisms of the family Enterobacteriaceae in which one or more genes selected from the group comprising:

6.1 the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase,

6.2 the pyc gene coding for pyruvate carboxylase,

6.3 the pps gene coding for phosphoenolpyruvate synthase,

6.4 the ppc gene coding for phosphoenolpyruvate carboxylase,

6.5 the pntA and pntB genes coding for transhydrogenase,

6.6 the rhtB gene for homoserine resistance, and

6.7 the rhtC gene for threonine resistance,

6.8 the gdhA gene coding for glutamate . dehydrogenase are simultaneously amplified and, in particular, overexpressed are fermented for the preparation of L- " amino acids.

7. Process according to claim 1, wherein microorganisms of the family Enterobacteriaceae in which one or more genes selected from the group comprising:

7.1 the tdh gene coding for threonine dehydrogenase,

7.2 the mdh gene coding for malate dehydrogenase,

7.3 the gene product of the open reading frame (orf) yjfA, and

7.4 the gene product of the open reading frame (orf) ytfp, are attenuated and, in particular, switched off, or the expression is reduced, are fermented for the preparation of L-amino acids.

8. Fermentation process for the preparation of L-amino acids, especially L-threonine, wherein the following steps are carried out: a) fermentation of the microorganisms of the family Enterobacteriaceae producing the desired L-amino acid, in which microorganisms at least the open reading frames yjfA and/or ytfP or nucleotide sequences coding therefor are attenuated and, in particular, switched off, : b) enrichment of the L-amino acid in the medium or in the bacterial cells, and c) isolation of the L-amino acid, constituents of the fermentation broth and the biomass in its entirety or portions thereof optionally being isolated as a solid product together with the L-amino acid.

8. Process according to claim 1 or 8, wherein L- isoleucine, L-valine, L-lysine or L-threonine is prepared.

10. L-Amino acid-producing microorganisms of the family Enterobacteriaceae in which at least the pckA gene or : nucleotide sequences coding therefor are attenuated and, in particular, switched off.

11. L-Amino acid-producing microorganisms of the family Enterobacteriaceae according to claim 10, which additionally have one or more features selected from the group comprising: a resistance to o—amino-B- hydroxyvaleric acid, an amplified homoserine dehydrogenase I-aspartate kinase I in the feed back resistant form, an optionally compensable partial need for L-isoleucine, an attenuated threonine dehydrogenase and the ability to utilize sucrose.

12. L-Amino acid-producing microorganisms of the family Enterobacteriaceae, in which at least the open reading frame yjfA and/or ytfP or nucleotide sequences coding therefor are attenuated and, in particular, switched off.

13. L-Amino acid-producing microorganisms of the family Enterobacteriaceae according to claim 12, which additionally have one or more features selected from B the group comprising: a resistance to a-amino-B- hydroxyvaleric acid, an amplified homoserine dehydrogenase I-aspartate kinase I in the feed back resistant form, an optionally compensable partial need for L-isoleucine, an attenuated threonine dehydrogenase and the ability to utilize sucrose.

14. Plasmid pMAK705ApckA, shown in Figure 1, containing parts of the 5 and 3' regions of the pckA gene, corresponding to SEQ ID No. 3. .

15. Plasmid pMAK705AyjfA, shown in Figure 2, containing the 5' and 3’ flanks of the ytfP-yjfA region, including very short residues of the open reading frames yjfA- and ytfP, corresponding to SEQ ID No. 6.

16. Plasmid pMAK705A90bp, shown in Figure 5, containing the 5' and 3' flanks of the ytfP-yjfA region, including very short residues of the open reading frames yjfA- and ytfP, corresponding to SEQ ID No. 7.

17. Isolated polynucleotide from microorganisms of the family Enterobacteriaceae containing a polynucleotide sequence coding for the 5 and 3' regions of the pckA gene, shown in SEQ ID No. 4, which is particularly suitable as a constituent of plasmids for the position- specific mutagenesis of the pckA gene.

18. Isolated polynucleotide from microorganisms of the family Enterobacteriaceae containing the 5' and 3' flanks of the ytfP-yjfA region, shown in SEQ ID No. 6, which is particularly suitable as a constituent of plasmids for the position~specific mutagenesis of the open reading frames ytfP and/or yjfA.

19. L-Threonine-producing strains of the family Enterobacteriaceae containing a deletion mutation in the pckA gene, corresponding to SEQ ID No. 4.

20. L-Threonine-producing strains of the family Enterobacteriaceae containing a deletion mutation in the open reading frame ytfP, corresponding to SEQ ID

No. 6 or 7.

21. L-Threonine-producing strains of the family Enterobacteriaceae containing a deletion mutation in the open reading frame yjfA, corresponding to SEQ ID

No. 6 or 7.

22. L-Threonine-producing strains of the family Enterobacteriaceae according to claim 19, additionally containing a deletion mutation in the open reading frame ytfP, corresponding to SEQ ID No. 6 or 7.

23. L-Threonine-producing strains of the family Enterobacteriaceae according to claim 19, additionally containing a deletion mutation in the open reading frame yjfA, corresponding to SEQ ID No. 6 or 7.

24. L-Threonine-producing strains of the family Enterobacteriaceae according to claims 19, 20 or 21, wherein they have one or more features selected from the group comprising: a resistance to o-amino-p- hydroxyvaleric acid, an amplified homoserine dehdrogenase I-aspartate kinase I in the feed back resistant form, an optionally compensable partial need for L-isoleucine, an attenuated threonine dehydrogenase and the ability to utilize sucrose.

25. Escherichia coli K-12 strain MG442ApckA deposited under number DSM 13761 at the Deutsche Sammlung flr Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures). :

26. Escherichia coli K-12 strain MG442A90yjfA deposited under number DSM 14289 at the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures).

27. Escherichia coli K-12 strain B3996kurAtdhpckA/PVIC40, deposited under number DSM 14150 at the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures).