USOO68584O6B1 (12) United States Patent (10) Patent No.: US 6,858,406 B1 Vrlijc et al. (45) Date of Patent: Feb. 22, 2005

(54) PROCESS FOR THE MICROBIAL (56) References Cited PRODUCTION OF AMINO ACIDS BY PUBLICATIONS BOOSTED ACTIVITY OF EXPORT CARRIERS Lodish et al. “Molecular Cell Biology', Fourth edition, Publisher: W.H. Freeman & company, pp. 255–256, 1999.* (75) Inventors: Marina Vrlijc, Jülich (DE); Lothar Vrljic et al. “Unbalance of L-Lysine flux in Corynebacte Eggeling, Jülich (DE); Hermann rium glutamicum and its use for the isolation of excretion Sahm, Jülich (DE) -defective mutants”, J. of Bacteriology, vol. 177 (4), Jul. 1995.* (73) ASSignee: Forschungszentrum Jilich GmbH, Vrljic et al., “A new type of transporter with a new type of Jülich (DE) cellular function: L-lysine export from Corynebacterium glutamicum”, 1996, Molecular Microbiology, vol. 22(5), pp. Notice: Subject to any disclaimer, the term of this 815-826. patent is extended or adjusted under 35 Ayala et al., “Modern Genetics”, second edition, 1984, U.S.C. 154(b) by 0 days. Glossary.* * cited by examiner (21) Appl. No.: 09/105,117 Primary Examiner Karen Cochrane Carlson ASSistant Examiner Rita Mitra (22) Filed: Jun. 17, 1998 (74) Attorney, Agent, or Firm-Klaus J. Bach Related U.S. Application Data (57) ABSTRACT The invention pertains to a proceSS for the microbial pro (63) Continuation-in-part of application No. PCT/DE96/02485, duction of amino acids. The process involves boosting the filed on Dec. 18, 1996. export carrier activity and/or export gene expression of a Foreign Application Priority Data microorganism which produces the desired amino acid. It (30) was found that a single specific gene is responsible for the Dec. 22, 1995 (DE) ...... 1954.8 222 export of a given amino acid, and on that basis, a process for 51) Int.nt. Cl.Cl." ...... C12P 21/06, C12N 1/20 the microbial production of amino acids, involving the controlled boosting of the export gene expression and/or C12N 5/00; CO7H 21/04; CO7K 14/00 export carrier activity of a microorganism was developed, (52) U.S. Cl...... 435/69.1; 435/252.32; which produces the amino acid. The boosted expression or 435/325; 435/320.1; 435/252.3; 536/23.7; activity of the export carrier resulting from this process 536/24.1; 530/350 increases the Secretion rate and thus increases transport of (58) Field of Search ...... 435/69.1, 252.32, the desired amino acid. 435/325, 320.1, 252.3, 106, 252.1; 536/23.7, 24.1; 530/350 17 Claims, 4 Drawing Sheets

B X YSINE EXCRETION

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0000\____0008_0009000;000Z 9S0S &HHIS;uJS88;X8 U.S. Patent Feb. 22, 2005 Sheet 2 of 4 US 6,858,406 B1

Cg Lys E MVIMEI FITGLLGASLILLSIGPONVLVIKOGIKREGLIAVLLVCLISDV 50 ; : ...... : : : : : EcYgga l ...... ------MILPLGPCNAFWMNOGIRRQYHIMIALLCAISDL 34

CalysF 5 l FLFIAGTLGVDLLSNAAPIVLDIMRWGGIAYLLWFAVMAAKDAMTNKVEi OO ...... : : : : : : ...... EcYgga 35 VLICAGIFGGSALLMQSPWLLALVTWGGVAFLLWYGFGAFKTAMss NIE, 83

cglys E 01 PQI IEETEPTVPDDTPLGGSAVATDT RNRVRVEVsV pKORVwVKPMLMA: 50 i ...... i : : ; ; ; ; ; ECYgga 84 ...... LASAEVMKOGRWK ...... I ATMLAW OM

C gLys E lS) VLTWLNPNAYLDAFVFIGGVGAQYGDIGRWIFAAGAFAASLIWFPLVGFG 200 ...... ; : : : I. : . . : : . . . . . : : : : : EcYgga 05 . . TWLNPHVYLDTFVVLGSLGGQLDVEPKRWFALGTISASFLWFFGLALL lS2

CLys E 201 AAALSRPLSSPKVWRW.INVVVAVVMTALAIKLMLM.G...... 236 ...... : : . : : . EcYgga 153 AAWLAPRLRTAKAQRIINLVVGCVMWFIALQL.ARDGIAHAQALFS 1.97

FIG 2 U.S. Patent Feb. 22, 2005 Sheet 3 of 4 US 6,858,406 B1

COMPLEMENTATION OF EXPORT DEFECTS

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14

12 US 6,858,406 B1 1 2 PROCESS FOR THE MICROBIAL connection, it is known that the overexpression of the PRODUCTION OF AMNO ACDS BY transketolase achieved by recombinant techniques improve BOOSTED ACTIVITY OF EXPORT the product generation of L-tryptophan, L-tyrosine or CARRIERS L-phenalanine (EP 0600 463 A2). Furthermore, the reduc tion of the phosphenol pyruvate carboxylase activity in This application is a continuation in part of PCT/DE96/ Corynebacterium provides for an improvement in the gen 02485 filed on Dec. 18, 1996, which claims benefit of a eration of aromatic amino acids (EPO 331 145). foreign (German) application 19548 222.0 filed on Dec. 22, All these attempts to increase the productivity have the 1995. aim to overcome the limitation of the cytosolic Synthesis of the amino acids. However, as a further limitation basically BACKGROUND OF THE INVENTION also the export of the amino acids formed in the interior of a cell into the culture medium should be taken into consid The invention relates to a process for the microbial eration. As a result, it has been tried to improve this export production of amino acids, to export genes, to regulator and, consequently, the efficiency of the amino acid produc genes, to vectors, to transformed cells, to membrane tion. For example, the cell permeability of the Corynebac proteins, and to uses. 15 terium has been increased by biotin deficiency, detergence or Amino acids are of high economical interest and there are penicillin treatment. However, these treatments were effec many applications for the amino acids: for example, tive eXclusively in the production of glutamate, whereas the L-lysine as well as L-threonine and L-tryptophan are needed Synthesis of other amino acids could not be improved in this as feed additives, L-glutamate as Seasoning additive, manner. Also, bacteria Strains have been developed in which L-isoleucine, and L-tryosine in the pharmaceutical industry, the activity of the Secretion System is increased by chemical L-arginine and L-isoleucine as medicine or L-glutamate and or physical mutations. In this way, for example, a Coryne L-phenylalanine as a starting Substance for the Synthesis of bacterium glutamicum Strain has been obtained which has an fine chemicals. improved Secretion activity and is therefore especially Suit A preferred method for the manufacture of these different able for the L-Lysine production. (DE 0203320). amino acids is the biotechnological manufacture by means 25 Altogether, the attempts to increase the Secretion of of micro-organisms, Since, in this way, the biologically amino-acids formed within the cell have all in common that effective and optically active form of the respective amino an increase efflux of amino acids on the basis of the Selected acid is directly obtained and Simple and inexpensive raw non-directed and non-specific methods could be achieved materials can be used. AS microorganisms, for example, only accidentally. Corynebacterium glutamicum and its relatives S.Sp. flavum and ssp lactofermentum (Lieblet al; Int. J-System Bacteriol Solely in the German patent application No. 195 23 (1991) 41:255-260) as well as Escherichia coli and related 279.8-41, a process is described which provides for a bacteria can be used. well-defined increase of the secretion of amino acids formed However, these bacteria produce the amino acids only in internally in a cell by increasing the expression of genes the amounts needed for their growth Such that no exceSS coding for the import of amino acids. The understanding on amino acids are generated and are available. The reason for 35 which this proceSS was based, that is, the cell utilizes import this is that in the cell the biosynthesis of the amino acids is proteins for the export of amino acids as well as the fact that controlled in various ways. As a result, different methods of by nature microorganisms do not generate and release increasing the formation of products by overcoming the exceSS amino acids lets one assume that export genes or control mechanisms are already known. In these processes, proteins Specific for the amino acid transport do not exist, for example, amino acid analogs are utilized to render the 40 but that the amino acids are excreted by way of other export control of the biosynthesis ineffective. A method is Systems. described, for example, wherein Corynebacterium Strains The export Systems known So far export poisonous metal are used which are resistant to L-tyrosine and ions, toxic antibiotica and higher molecular toxins. These L-phenylalanine analogs (JP 19037/1976 and 39517/1978). export Systems are relatively complex in their structure. Also methods have been described in which bacteria resis 45 Generally, membrane proteins of the cytoplasmic membrane tant to L-lysine and also to L-threonine analogs are used in are involved which however cause only a partial reaction of order to overcome the control mechanisms (EPO 205 849 the export So that presumably additional extra cytoplasmic B1, UK patent application GB 2 152 509 A). Support proteins are needed for the transport (Dink, T. et al., A family of large molecules across the Outer membranes of Furthermore, microorganisms constructed by recombi gram-negative bacteria., J. Bacteriol. 1994, 176: nant DNA techniques are known wherein the control of the 50 biosynthesis has also been eliminated by cloning and 3825-3831). Furthermore, it is known that, with the sec expressing the genes which code for the key enzymes which dependent export System for extra-cellular proteins, at least cannot be feed-back inhibited any more. For example, a Six different protein components are essential for the export. recombinant L-lysinee producing bacterium with plasmid This State-of-the-art Suggests that also the Systems, which coded feedback-resistant aspartate kinase is known (EP are responsible for the export of amino acids, but which are 0381527). Also, a recombinant L-phenylalanine producing 55 not known So far comprise Several protein components or bacterium with feedback resistant prephenate dehyrodge respectively, Several genes are responsible for the export of nase has been described (JP 124375/1986; EPO 488 424). In amino acids. A hint in this direction could be the various addition, increased amino acid yields have been obtained by mutants which are defective in the lysine export as described overexpression of genes which do not code for feedback by Vrylic et al.(J. Bacteriol (1995) 177:4021-4027). Sensitive enzymes of the amino acids Synthesis. For 60 example, the lysine formation is improved by increased SUMMARY OF THE INVENTION synthesis of the dihydrodipicolinate synthase (EP 0 197 It has now been found Surprisingly that only a single 335). Also, the threonine formation is improved by increased Specific gene is responsible for the export of amino acids So synthesis of threonine dehydratase (EP 0436 886 Al). that, in accordance with the invention, for the first time a Further experiments for increasing the amino acid pro 65 method for the microbial manufacture of amino acids is duction aim at an improved generation of the cellular provided wherein clearly the export gene expression and/or primary metabolites of the central metabolism. In this the export carrier activity of a microorganism producing US 6,858,406 B1 3 4 amino acids is increased. The increased export expression or respective transport gene occurs according to the usual respectively, activity of the export carrier resulting from this methods. If a transport gene is isolated and cloned from proceSS leads to an increased Secretion rate So that the export Corynebacterium then for example, the method of homo of the respective amino acid is increased. The microorgan loguous complementation of an export defective mutant is isms. So modified also accumulate an increased part of the suitable (J.Bacteriol. (1995)177: 4021-4027). If a direct respective amino acid in the culture medium. cloning of the Structure gene is not possible vector Sequences may first be inserted into the transport gene For an increase in the export carrier activity especially the whereupon it is isolated by way of “plasmid rescue” in the endogenic activity of an amino acid producing microorgan form of inactive fragments. For the process according to the ism is increased. An increase of the enzyme activity can be invention genes from the C. glutamicum ATCC 13032 or C. obtained for example by an increased Substrate consumption glutamicum S.Sp. flavum 14067 or also, C. glutamicum S.Sp. achieved by changing the catalytic center or by eliminating lacto fermentum ATCC 13869 are particularly suitable. The the effects of enzyme inhibitors. An increased enzyme isolation of the genes and their invitro recombination with activity can also be caused by an increased enzyme Synthesis known vectors (Appl. Env. Microbial (1989)55: 684–688; for example by gene amplification or by eliminating factors Gene 102(1991)93–98) is followed by the transformation which inhibit the enzyme biosynthesis. The endogene export into the amino acid producing Strains by electroporation activity is increased preferably by mutation of the endogenic 15 (Lieblet al. (1989)FEMS Microbiol Lett. 65; 299-304) or export gene. Such mutations can be generated either in an conjugation (Schafer et al. (1990) J. Bacteriol. uncontrolled manner in accordance with classic methods as 172:1663–1666). For the transfer, preferably vectors with for example by UV irradiation or by mutation causing low numbers of copies are used. AS host cells, preferably chemicals or in a controlled manner by gene-technological Such amino acid producers are used which are deregulated in methods Such as deletion(s) insertion(s) and/or nucleotide the Synthesis of the respective amino acids and/or which exchange(s). have an increased availability of central metabolism metabo The export gene expression is increased by increasing the lites. number of gene copies and/or by increasing regulatory After isolation, export genes with nucleotide Sequences factors which positively affect the export gene expression. can be obtained which code for the amino acid Sequences For example, a strengthening of regulatory elements takes 25 given in SEQ ID No.(A)2 and shown in table 3 or for their place preferably on the transcription level by increasing allele Variations or, respectively, which include the nucle particularly the transcription signals. This can be accom otide sequence of 1016 to 1726 according to SEQID No. (A) plished for example in that, by changing the promoter 1 and table 2 or a DNAy sequence which is effective Sequence arranged before the Structure gene, the effective essentially in the same way. Also here, allele Variations or neSS of the promoter is increased or by completely replacing equally effective DNA sequences include particularly func the promoter by more effective promoters. An amplification tional derivatives in the sense indicated above for the of the transcription can also be achieved by accordingly regulatory sequences. These export genes are preferably influencing a regulator gene assigned to the export gene as used in the process according to the invention. will be explained further below. On the other hand, an One or Several DNA sequences can be connected to the amplification of the translation is also possible, for example, export gene with or without attached promoter or by improving the stability of the m-RNA. 35 respectively, with or without associated regulator gene, So To increase the number of gene copies the export gene is that the gene is included in a gene Structure. installed in a gene construct or, respectively, in a vector, By cloning of export genes, plasmids or, respectively, preferably, a vector with a Small number of copies. The gene vectors can be obtained which contain the export gene and construct includes regulatory gene Sequences, which are which, as already mentioned, are Suitable for the transfor Specifically assigned to the export gene, preferably Such 40 mation of an amino acid producer. The cells obtained by Sequences, which re-inforce the gene expression. The regu transformation which are mainly transformed cells from latory gene Sequences comprise a nucleotide Sequence Corynebacterium, contain the gene in reproducible form, which codes for the amino acid sequence SEQ ID No.(B)3 that is, with additional copies on the chromosome wherein and given in table 1 or the allele Variations thereof or the gene copies are integrated at any point of the genome by respectively, a nucleotide Sequence 1421 to 2293 according 45 homologous recombination and/or on a plasmid or to SEQ ID No. (B)1shown in table 2 or a DNA sequence respectively, vector. which is effective essentially in the same manner. A multitude of Sequences is known which code for Allele variations or, respectively, equally effective DNA membrane proteins of unknown function. By providing in Sequences comprise particularly functional derivatives accordance with the invention export genes Such as the which can be obtained by deletion(s) insertion(s) and/or 50 export gene with the nucleotide Sequence of nucleotide 1016 Substitution(S. of nucleotides of corresponding sequences, to 1726 in accordance with SEQ ID No. 1 and table 2 or wherein however the regulator protein activity or function is respectively, the corresponding export proteins for example retained or even increased. In this way, the effectiveness of that with the amino acid sequence according to SEQID No. the interaction of the regulatory protein to the DNA of the 2, it is now possible to identify by Sequence comparison export gene to be regulated can be influenced by mutating membrane proteins, whose function is the transport of amino the regulatory gene Sequence Such that the transcription is 55 acids. The export gene identified in this way can Subse Strengthened and, consequently, the gene expression is quently be used to improve the amino acid production in increased. In addition, also So-called enhancers may be accordance with the process of the invention. assigned to the export gene as regulatory Sequences The membrane proteins known from the state-of-the-art whereby, via an improved correlation between RNA poly generally include 12, Some also only 4 transmembrane merase and DNA, also the export gene expression is 60 helices. However, it has now been found Surprisingly that increased. the membrane proteins responsible or Suitable for the export For the insertion of the export gene into a gene construct, of amino acids include 6 transmembrane helices (see for the gene is preferably isolated from a microorganism Strain example, the amino acid Sequence of an export protein listed of the type Corynebacterium and, with the gene construct in SEQ ID No. 2 and table 3, wherein the 6 transmembrane including the export gene, a microorganism Strain, espe 65 areas have been highlighted by underlining). Consequently, cially Corynebacterium, producing the respective amino there is a new class of membrane proteins present, which has acid is transformed. The isolation and transformation of the not yet been described. US 6,858,406 B1 S 6 BRIEF DESCRIPTION OF DRAWINGS The indicator plates were incubated at 30° C. and exam ined after 15, 24 and 48 hours. In this way, altogether 29 FIG. 1 shows the fragments in pMV6-3 and pMV8-5-24 clones were obtained which showed on the indicator plate obtained by the cloning which cause the lysine Secretion and provided with methionine a growth court by the indicator the subclone pMV2-3 made from pMV6-3, which also strain 49/3. The clones were examined individually and then causes the lysine 30 Secretion and which was Sequenced. again as described above, for reestablishment of the growth BBamHl; Sm, SmaI; Se, Sac1; S1, Sal III, HindII; X, XhoI. court. In this way, the two clones NA8 pMVB-5-24 and NA8 FIG. 2 shows a comparison of the derivated amino acid pMV6-3 were obtained which had again received the capa Sequence of LySE from C. glutamicum (above), with a gene bility to excrete lysine. product of So far unknown function from Escherichi coli From these clones, plasmid preparations were performed (below), which is identified thereby as export carrier. as described in Schwarzer et al. (Bio/Technology (1990)9; FIG.3 shows increased lysine export by pMV2-3 with C. 84-87). By retransformation in NA8, the plasmid-connected glutamicum NA8. On top, the control with low excretion and effect of the excretion of L-lysine was confirmed. Both cellinternal backup of lysine up to about 150 mM. Below, plasmids were Subjected to a restriction analysis. Plasmid the high secretion caused by pMV2-3 with cell internally pMVB-5-24 carries an insert of 8.3 kb, and pMV6-3 one of only small backup of about 30 mM. 15 9.5 kb. The physical charter of the inserts is shown in FIG. FIG. 4 shows the increase of the lysine accumulation in C. 1. glutamicum by lys Elys G(pMV2-3) (middle curve), and the b) Subcloning of an DNA fragment which reconstitutes accumulation caused by lysE(plySE) (upper curve). the lysine export. From the insert of the plasmid pMV6-3 individual Sub EXAMPLES clones were prepared utilizing the restriction Severing point a) Cloning of an export gene and cloning of a regulator of as determined. In this way, the 3.7 kb XhoI-SalI-fragment, Corynebacterium glutamicum. the 2.3 kb BamHI-fragment and the 7.2 kb BamHI fragment Chromosomal DNA from C. glutamicum R127 (FEMS were ligated with the correspondingly Severed and treated Microbiol lett. (1989)65:299-304) was isolated as described vectorp)C1 (Mol Gen. Genet.(1990)220: 478-480). With the by Scharzer et al. (Bio/Technology (1990) 9:84–87). The 25 ligation products C. glutamicum NA8 was directly DNA was then split with the restriction enzyme Sau3A and transformed, the transformants were tested for having the Separated by Saccharose gradient centrifugation as described lysine excretion properties and the presence of the Subclone in Sambrook et al. (Molecular cloning, A laboratory manual was confirmed by plasmid preparation and restriction analy (1989) Cold Spring Harbour Laboratory Press). The various sis. In this way, the strain with plasmid pMV2-3 (FIG. 1) fractions were analyzed gel electrophoretically with respect was obtained as Smallest Subclone. This fragment resulting to their size and the fraction with a fragment size of about in lysine export contains as insert the 2.3 kb Bam fragment 6-10 kb was used for the ligation with the vector pCl. In from pMV6-3. addition, the vector pCl was linearized with BamHI and c) Sequence of the lysine export gene lys E and its dephosphorylized. Five ng thereof was ligated with 20 ng of regulators lySG. the chromosomal 6-10 kb fragments. With the whole liga The nucleotide sequence of the 2.3 kb BamH1 fragment tion preparation, the export defective mutant NA8 (J. Bacte 35 was performed according to the dideoxy-chain termination rol. (1995)177:4021-4027) was transformed by electropo method of Sanger et al. (Proc. Natl. Acad. Sci USA(1977) ration (FEMS Microbiol Lett(1989)65:299-304). The 74:5463-5467) and the sequencing reaction with the Auto transformants were selected for LBHIS(PEMS Microbiol. Read Sequencing kit from Pharamcia (Uppsala, Sweden). Lett. (1989)65:299-304) with 15 lug kanamycin per ml. The electrophoretic analysis occurs with the automatic laser These transformants were Subjected to extensive plasmid 40 fluorescence DNA sequencing apparatus (A.L.F) from analyses in that 200 of the altogether 4500 clones obtained Pharmacia-LKB(Piscataway, N.J., USA). The nucleotide were individually cultivated and their plasmid content and Sequence obtained was analyzed by the program packet Size was determined. On average, about half of the HUSAR (Release 3.0) of the German Cancer Research kanamycin-resistant clones carried a recombinant plasmid Center (Heidelberg). The nucleotide sequence and the result with an insert of the average size of 8 kb. This provides for 45 of the analysis is presented in SEQ ID No 1 and FIG. 2. The a probability of 0.96 for the presence of any gene of C. analysis results in two fully open reading frames (ORF) on glutamicum in the established gene bank. The 4500 obtained the sequenced DNA piece. ORF1 codes for a protein with a transformants were all individually checked for renewed length of 236 amino acids, OFR2 codes for a protein with a presence of lysinee Secretion. For this purpose, the System length of 290 amino acids. The protein derived from ORF1 described by Vrljic for the induction of the Llysinee excre 50 includes an accumulation of hydrophobic amino acids as tion in Corynebacterium glutamicum was utilized (J. Bac they are characteristic for membrane-embedded proteins. teriol (1995) 177:4021-4027). For this purpose, so-called The detailed analysis of the distribution of the hydrophobic minimal-medium-indicator plates were prepared, which and hydrophilic amino acids by the programs PHD.HTM contained per liter 20 g (NH)2SO4, 5 g uric acid, 1 g (Protein Science(1995)4:521-533) is shown in table 3. It is KHPO, 1 g KHPO, 0.25 g MgSOx7H2O, 42 g mor apparent therefrom that the protein contains Six hydrophobic pholino propane sulfonic acid, 1 ml CaCl (1 g/100 ml), 750 55 helix areas which extend through the membrane. ml dest., 1 ml Cg trace salts, 1 ml biotin (20 ug/1001), pH7, Consequently, this protein is the Searched for exporter of the 4% glucose, 1.8 mg protocatechuic acid, 1 mg FeSO4x7 amino acid L-lysine. The corresponding gene will therefore HO, 1 mg MnSOXHO, 0.1 mg ZnSOx7 HO, 0.02 mg be designated below as lysE. In table 2, it is marked CuSO, 0.002 mg NiClax6 H2O, 20g agar-agar, as well as accordingly. ORF2 is transcribed in a direction opposite to 107 cells/ml of the lysine-auxotrophene C. glutamicum 60 ORF1. The sequence analysis shows that ORF2 has a high mutant 49/3. The original 4500 transformants were all identity with regulator genes which are combined as a Single individually pinned, by toothpicks onto the indicator plates family (Ann Rev Microbiol(1993) 597–626). Genes of this with, in each case, a check of the original non-excretor NAB family regulate the expression processes of the various (J.Bacteriol (1995)177:4021-4027) and the original strain genes involved in catabolic or anabolic processes in a R127. At the same time, always 2 plates were inoculated of 65 positive way. For this reason, ORF2 will below be desig which only one contained additionally SmM L-methionine nated as lysG (Govern=regulating). Because of the coordi in order to induce the lysine excretion in this way. nation and because IySE could be cloned (See a)) and US 6,858,406 B1 7 8 Subcloned (see b)) together with lysG, lysG is regulator of f) Increased accumulation of L-lysine by lysE or LysEG. lySE and consequently also participates in the lysine export. From the subclone pMV2-3 which contains the sequenced The gene lySG and the amino acid Sequence derived there 2374 bp Bam HI-fragment in plCI (see FIG. 1), the lysE from are also shown in SEQ ID No(B)1 and table 2 and, carrying 1173 bpPvulI fragment was ligated in pZ1 (Appl. respectively, SEQ ID No.3. Env. Microbiol (1989)55:684–688) according to the d) Identification of an unknown membrane protein from Sequence information and in this way, the plasmid plySE was Escherichia coli by Sequence comparison. obtained. This plasmid as well as the lySE lySG carrying With the established sequences according to table 3 plasmid pMV2-3 was introduced into C. glutamicum already existing Sequence banks can be searched in order to Strained by electroporation wherein the chromosomal areas assign the proteins derived in this way from Sequenced areas were deleted. The obtained strains C. glutamicum d pMV2 a certain function. Correspondingly, the amino acid 3, C. glutamicum d plySE, C. glutamicum plCl were, as Sequence of the lysine exporters consisting of C. glutamicum described under e) precultivated on a complex medium, then were compared with derivated protein Sequences of all the cultivated in production minimal medium CGX11 together DNA sequences deposited there utilizing the program packet with 4% glucose and 5 mM 1-methionin and samples were HUSAR (Release 3.0) of the German Cancer Research taken to determine the accumulated lysine. AS apparent from Center (Heidelberg). A high homology of 39.3% identical 15 FIG. 4 with lysElysG an increase of the lysine accumulation amino acids and 64.9% similar amino acids was found to a with respect to a control sample is achieved. With plysE an Single Sequence of So far unknown function of E. coli. extraordinarily increased accumulation of from 4.8 to 13.2 The comparison is shown in FIG. 2. The open read frame mM L-lysine is achieved with this method. of E. coli So far not characterized is consequently identified by way of this process as an amino export gene. LEGENDS OF THE TABLES e) Increased export of intracellularly accumulated Table 1: The amino acid Sequence of the lysine exporter L-lysine. regulator from Corynebacterium glutamicum with the helix The strain C. glutamicum NA8 (J. Bacteriol(1995) 177: turn-helix motive typical for DNA-binding proteins. 4021-4027 was transformed with plasmid pMV2-3 and the 25 Table 20three pages): The nucleotide sequence of C. L-lysine excretion of the StrainS was compared. For this glutamicum coding for the lysine exporter and lysine export purpose, NA8 and NA8pMV2-3 in complex medium were regulators. utilized as described in Vrljic et al. (J. Bacteriol (1995) 177:4021-40277) and the fermentation medium CGXII Table 3: the amino acid Sequence of the lysine exporter (Bacteriol (1993)175:5595-5603 were each separately from Corynebacterium glutamicum with the identified trans inoculated. The medium additionally contained 5rnM membrane helices TMH1 to TMH6. L-methionin in order to induce the intracellular L-lysine Sequence Protocol A: biosynthesis. After cultivation for 24 hours at 30° C. on a SEQ ID No. 1: Nucleotide sequence of the coding DNA rotary vibrator at 140 rpm, the cell internal and external Strand and the amino acid Sequence of the Lysine-exporter L-lysine determinations were performed. For the cell LysE derived therefrom. internal determination Silicon oil centri-fugations were per 35 SEQ ID No. 2: Amino acid sequence of the Lysine-exporter formed (Methods Enzymology LV(1979) 547-567); the LysE. determination of the amino acids occurred by high pressure Sequence Protocol B: liquid chromatography (J. Chromat (1983) 266:471-482). SEQ ID No.(B)1: Nucleotide sequence of the anti-sense These de... terminations were performed at different times as Strand and indicated in FIG. 3. In accordance with the process used the 40 retained cell internal L-lysine is excreted also by pMV2-3 to Amino acid Sequences of the Lysine-exporter-regulator a greater degree and is accumulated. Accordingly, also the LysG, derived therefrom and a ORF3. cell internally present L-lysine is greatly reduced. SEQ ID No.(B)2: Amino acid sequence of the open reading Consequently, the utilization of the newly discovered and frame (partial) ORF3. described exporter represents a proceSS for greatly improv SEQ ID No. 3: Amino acid sequence of the Lysine exporter ing the L-lysine production. Regulator LySG.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 5

<21 Oc SEQ ID NO 1 <211 LENGTH 2374. <212> TYPE DNA <213> ORGANISM: Corynebacterium glutamicum <22O > FEATURE <221 NAME/KEY: gene <222> LOCATION: CDS (1016) . . (1726) <223> OTHER INFORMATION: (LysE) <400 SEQUENCE: 1

ccatttgct g aaggtgttac totgcctggc ccaattic ct g c gogcgaaga agtgaaaaac 60

cctgaac citt ttcagaagta actaaggcc g caatcc.citcg attgctgcat caacgacggc 120 gtotgtgagt citagctagag atctagattic caggcgc.cat cqttgccaat acatcggtgt

US 6,858,406 B1 11 12

-contin ued titc cc.g. citg gtg ggt titC ggC gCa gca goa ttg to a cqc cog citg to c 642 Phe Pro Leu Val Gly Phe Gly Ala Ala Ala Leu Ser Arg Pro Teu Ser 195 200 2O5 agc ccc aag gtg togg C gc tigg atc aac gtc gto gtg gca gtt gtg atg 69 O. Ser Pro Lys Val Trp Arg Trp Ile Asn Wal Wall Wall Ala Wall Wal Met 210 2 15 220 225 acc goa ttg gcc atc a aa citg atg ttg at g g gt tag tittitc.gc ggg 736 Thir Ala Leu Ala Ile Lys Lieu Met Leu Met Gly 230 235 ttittggaatc ggtggcct to gcc caaatgt tgatgcc.ggc gtogtgggaa atcto atcga 796 togcctccaa citcggcgt.ca gaaaactc.ca agttgttgag tgaatcaagg citgttgtc.ca 856 gctgctcaac tgacgaagca ccaatcaatg cactggtoac ggitatcc.gc.g cc.gtacticto 916 cittgctcqcg cago acco at gcaa.gc.gc.ca totg.cgcaag tgactg.ccc.g cgttcctggg 976 cgatgtcatt gag Cttgcgg accatatocaa. tattgttcac gttcaac atg cc citcagaca ggg acttacc Ctggctgg.cg cgg galacc ct citggaatticc atc.gagatat ttgtc.cgtga 2.096 gCaggCCCtg cgcaagtggit gagaaagcaa. tgacgc.caag accattgttg gcagotgact 21.56 gcaacaagtt citcaccgtca togc.ccggitt ccitccaccca acgattaatg atggaatago 221 6 ttggct gatg aatcagaa.gc ggg Cagcc ct cctcc.gc.cat gaacticago c gcc to cqctg 2276 tgagctctgg accgtaggaa gaaataccca cgtaaagagc cittitccagac gcaacaatgt 2336 cacgcaatgc gtacatggitt tottccaaag gagtatct 2374.

<210> SEQ ID NO 2 &2 11s LENGTH 236 &212> TYPE PRT <213> ORGANISM: Corynebacterium glutamicum &220s FEATURE <223> OTHER INFORMATION: (LysE) <400 SEQUENCE: 2 Met Wall Ile Met Glu I le Phe Ile Thr Gly Leu Leu Lleu Gly Ala Ser 1 5 10 15

Teu Telu Telu Ser Ile G ly Pro Glin Asn. Wall Leu Val Ile Lys Glin Gly 2O 25 3O

Ile Lys Arg Glu Gly L. eu. Ile Ala Wall Leu Lieu Val Cys Lieu Ile Ser 35 40 45

Asp Val Phe Leu Phe I le Ala Gly Thr Leu Gly Val Asp Lieu Teu Ser 5 O 55 60

Asn Ala Ala Pro Ile W all Lieu. Asp Ile Met Arg Trp Gly Gly Ile Ala 65 70 75 8O

Tyr Lieu Lieu Trp Phe Ala Val Met Ala Ala Lys Asp Ala Met Thr Asn 85 90 95

Lys Val Glu Ala Pro G lin Ile Ile Glu Glu Thr Glu Pro Thr Wall Pro 100 105 110

Asp Asp Thr Pro Leu G ly Gly Ser Ala Wall Ala Thr Asp Thr Arg Asn 115 120 125

Arg Val Arg Wall Glu W al Ser Wall Asp Lys Glin Arg Val Trp Wall Lys 130 135 1 4 0

Pro Met Leu Met Ala I le Wall Leu Thr Trp Leu Asn. Pro Asn Ala Tyr 145 1 50 155 160 Leu Asp Ala Phe Val Phe Ile Gly Gly Val Gly Ala Glin Tyr Gly Asp 1.65 170 175

Thr Gly Arg Trp Ile Phe Ala Ala Gly Ala Phe Ala Ala Ser Lieu. Ile 185 19 O US 6,858,406 B1 13

-continued

Trp Phe Pro Leu Val Gly Phe Gly Ala Ala Ala Leu Ser Arg Pro Leu 195 200 2O5 Ser Ser Pro Llys Val Trp Arg Trp Ile Asin Val Val Val Ala Val Val 210 215 220 Met Thr Ala Lieu Ala Ile Lys Lieu Met Leu Met Gly 225 230 235

<210> SEQ ID NO 3 &2 11s LENGTH 2374 &212> TYPE DNA <213> ORGANISM: Corynebacterium glutamicum &220s FEATURE <221 NAME/KEY: unsure <222> LOCATION: CDS (2) . . (652) &223> OTHER INFORMATION or f3 (complement to SEQ ID No. 1) &220s FEATURE <221s NAME/KEY: gene <222> LOCATION: CDS (1421) . . (2293) <223> OTHER INFORMATION: LysG <400 SEQUENCE: 3 a gat act cot ttg gaa gaa acc atg tac goa ttg cqt gac att gtt gcg 49 Asp Thr Pro Leu Glu Glu Thr Met Tyr Ala Leu Arg Asp Ile Val Ala 1 5 10 15 tot gga aag got citt tac gtg ggt att tot toc tac ggit coa gag citc 97 Ser Gly Lys Ala Leu Tyr Val Gly Ile Ser Ser Tyr Gly Pro Glu Lieu 2O 25 3O aca gC g gag gC g gct gag titc at g gcg gag gag ggc tigc cc g citt citg 145 Thr Ala Glu Ala Ala Glu Phe Met Ala Glu Glu Gly Cys Pro Leu Lieu 35 40 45 att cat cag coa agc tat toc atc att aat cqt togg gtg gag gaa cog 193 Ile His Gln Pro Ser Tyr Ser Ile Ile Asin Arg Trp Val Glu Glu Pro 5 O 55 60 ggc gat gac ggit gag aac ttg ttg cag to a got gcc aac aat ggit citt 241 Gly Asp Asp Gly Glu Asn Lieu Lieu Glin Ser Ala Ala Asn. Asn Gly Lieu 65 70 75 8O ggc gtc att got titc. tca cca citt gcg cag ggc citg citc acg gac aaa 289 Gly Val Ile Ala Phe Ser Pro Leu Ala Glin Gly Lieu Lieu. Thir Asp Lys 85 90 95 tat citc gat gga att coa gag ggit toc cqc goc agc cag ggit aag to c 337 Tyr Lieu. Asp Gly Ile Pro Glu Gly Ser Arg Ala Ser Glin Gly Lys Ser 100 105 110 citg tot gag ggc atg ttgaac gitg aac aat att gat at g g to cqc aag 385 Leu Ser Glu Gly Met Lieu. Asn Val Asn. Asn. Ile Asp Met Val Arg Lys 115 120 125 citc aat gac atc gcc cag gaa cqc ggg cag to a citt gcg cag at g gog 433 Lieu. Asn Asp Ile Ala Glin Glu Arg Gly Glin Ser Leu Ala Glin Met Ala 130 135 1 4 0 Ctt gca togg gtg Ctg C gC gag caa gga gag tac ggC gCg gat acc gtg 481 Leu Ala Trp Val Lieu Arg Glu Glin Gly Glu Tyr Gly Ala Asp Thr Val 145 15 O 155 160 acc agit gca ttg att got gct tcg to a gtt gag cag citg gac aac agc 529 Thir Ser Ala Lieu. Ile Gly Ala Ser Ser Val Glu Glin Lieu. Asp Asn. Ser 1.65 170 175 citt gat to a citc aac aac ttg gag titt tot gac goc gag ttg gag gog 577 Leu Asp Ser Lieu. Asn. Asn Lieu Glu Phe Ser Asp Ala Glu Lieu Glu Ala 18O 185 19 O atc gat gag att toc cac gac goc ggc atc aac att togg gc g aag goc 625 Ile Asp Glu Ile Ser His Asp Ala Gly Ile Asn. Ile Trp Ala Lys Ala 195 200 2O5

US 6,858,406 B1 17

-continued gac ggg cqc gtc gat ggit cott gt g g g g c gc agg cqc gta toc att gtc 2O68 Asp Gly Arg Val Asp Gly Pro Val Gly Arg Arg Arg Val Ser Ile Val 420 425 430 cc.g. tcg gC g gala ggt titt got gag goa att cqc cqa ggc citt ggit togg 2116 Pro Ser Ala Glu Gly Phe Gly Glu Ala Ile Arg Arg Gly Lieu Gly Trp 435 4 40 4 45 gga citt citt coc gaa acc caa got gct coc atg cita aaa goa gga gaa 21 64 Gly Lieu Lieu Pro Glu Thr Glin Ala Ala Pro Met Leu Lys Ala Gly Glu 450 455 460 465 gtg atc ctic ctic gat gag ata coc att gac aca ccg atg tat togg caa 2212 Val Ile Leu Leu Asp Glu Ile Pro Ile Asp Thr Pro Met Tyr Trp Gln 470 475 480 cga togg cqc ct g gaa tot aga tot cita got aga citc aca gac goc gtc 2260 Arg Trp Arg Lieu Glu Ser Arg Ser Lieu Ala Arg Lieu. Thir Asp Ala Val 485 490 495 gtt gat gca goa atc gag gga ttg cqg cct tag titactitctga aaaggttcag 2313 Val Asp Ala Ala Ile Glu Gly Lieu Arg Pro 5 OO 505 ggitttittcac ttctitcgc.cc gcaggaattg ggc.cagg cag agtaacacct tcago: aaatg 2373 g 2374.

<210> SEQ ID NO 4 <211& LENGTH: 216 &212> TYPE PRT <213> ORGANISM: Corynebacterium glutamicum &220s FEATURE <223> OTHER INFORMATION: (orf3) <400 SEQUENCE: 4 Asp Thr Pro Leu Glu Glu Thr Met Tyr Ala Lieu Arg Asp Ile Val Ala 1 5 10 15 Ser Gly Lys Ala Leu Tyr Val Gly Ile Ser Ser Tyr Gly Pro Glu Lieu 2O 25 3O Thr Ala Glu Ala Ala Glu Phe Met Ala Glu Glu Gly Cys Pro Leu Lieu 35 40 45 Ile His Gln Pro Ser Tyr Ser Ile Ile Asin Arg Trp Val Glu Glu Pro 5 O 55 60 Gly Asp Asp Gly Glu Asn Lieu Lieu Glin Ser Ala Ala Asn. Asn Gly Lieu 65 70 75 8O Gly Val Ile Ala Phe Ser Pro Leu Ala Glin Gly Lieu Lieu. Thir Asp Lys 85 90 95 Tyr Lieu. Asp Gly Ile Pro Glu Gly Ser Arg Ala Ser Glin Gly Lys Ser 100 105 110 Leu Ser Glu Gly Met Lieu. Asn Val Asn. Asn. Ile Asp Met Val Arg Lys 115 120 125 Lieu. Asn Asp Ile Ala Glin Glu Arg Gly Glin Ser Leu Ala Glin Met Ala 130 135 1 4 0 Leu Ala Trp Val Lieu Arg Glu Glin Gly Glu Tyr Gly Ala Asp Thr Val 145 15 O 155 160 Thir Ser Ala Lieu. Ile Gly Ala Ser Ser Val Glu Glin Lieu. Asp Asn. Ser 1.65 170 175 Leu Asp Ser Lieu. Asn. Asn Lieu Glu Phe Ser Asp Ala Glu Lieu Glu Ala 18O 185 19 O Ile Asp Glu Ile Ser His Asp Ala Gly Ile Asn. Ile Trp Ala Lys Ala 195 200 2O5 Thr Asp Ser Lys Thr Arg Glu Asn US 6,858,406 B1 19 20

-continued

210 215

SEQ ID NO 5 LENGTH 290 TYPE PRT ORGANISM: Corynebacterium glutamicum FEATURE: OTHER INFORMATION: (LysG)

<400 SEQUENCE: 5

Met Asin Pro Ile Glin Teu Asp Thr Telu Telu Ser Ile Ile Glu Gly 1 5 10 15

Ser Phe Glu Gly Ala Ser Teu Ala Telu Ser Ile Ser Pro Ser Ala Wall 25 3O

Ser Glin Arg Wall Lys Ala Teu Glu His His Wall Gly Arg Wall Telu Wall 35 40 45

Ser Arg Thr Glin Pro Ala Lys Ala Thr Glu Ala Gly Glu Wall Telu Wall 5 O 55 60

Glin Ala Ala Arg Lys Met Wall Telu Telu Glin Ala Glu Thr Ala Glin 65 70 75 8O

Teu Ser Gly Arg Teu Ala Glu Ile Pro Telu Thr Ile Ala Ile Asn Ala 85 90 95

Asp Ser Telu Ser Thr Trp Phe Pro Pro Wall Phe Asn Glu Wall Ala Ser 100 105 110

Trp Gly Gly Ala Thr Teu Thr Telu Telu Glu Asp Glu Ala His Thr 115 120 125

Teu Ser Telu Telu Gly Asp Wall Telu Gly Ala Wall Thr Arg Glu 130 135 1 4 0

Ala Asn Pro Wall Ala Gly Cys Glu Wall Wall Glu Teu Gly Thr Met Arg 145 15 O 155 160

His Telu Ala Ile Ala Thr Pro Ser Telu Arg Asp Ala Met Wall Asp 1.65 170 175

Gly Lys Telu Asp Trp Ala Ala Met Pro Wall Teu Arg Phe Gly Pro 18O 185 19 O

Asp Wall Telu Glin Asp Telu Asp Gly Arg Wall Asp Gly Pro Wall 195 200

Gly Arg Arg Wall Ser Ile Wall Pro Ser Ala Glu Gly Phe Gly Glu 210 215 220

Ala Ile Gly Teu Gly Trp Gly Telu Teu Pro Glu Thr Glin Ala 225 230 235 240

Ala Pro Met Telu Lys Ala Gly Glu Wall Ile Teu Teu Asp Glu Ile Pro 245 250 255

Ile Asp Thr Pro Met Tyr Trp Glin Arg Trp Arg Teu Glu Ser Arg Ser 260 265 27 O

Teu Ala Arg Telu Thr Ala Wall Wall Asp Ala Ala Ile Glu Gly Telu 275 280 285

Arg Pro 29 O

What is claimed is: 60 amino acid sequence SEO ID NO: 2, and the export 1. A process for the microbacterial production of amino carrier activity is endogenous to Said microbial acids, comprising the Steps of 1) providing a microbial organism having amino acid organism, and export carrier activity and having export gene 3) increasing the export gene expression of Said export expression, 65 carrier, wherein Said gene comprising nucleotides 1016 2) increasing one of the export carrier activities of said to 1726 of SEQ ID NO: 1 by means of one of the steps microbial organism, wherein Said export carrier has the Selected from the group of US 6,858,406 B1 21 22 i) increasing the number of gene copies of the export enzymes which participate in the Synthesis of the corre carrier gene, sponding amino acids are deregulated. ii) modifying regulatory signals assigned to the export 10. The process according to claim 7, wherein, for the carrier gene, and transformation, a microorganism is utilized, which contains 5 an increased amount of the metabolites of the central iii) amplifying regulatory signals assigned to the export metabolism. carrier gene, whereby amino acids are produced by Said 11. The proceSS according to claim 2, wherein the export microbial organism with increased efficiency, and gene is isolated from a microorganism Strain of the type 4) recovering the amino acids from the culture. Corynebacterium. 2. The proceSS according to claim 1, wherein the export 12. The proceSS according to claim 1, wherein the export carrier gene expression of the export carrier is increased by gene expression is increased by amplifying the transcription increasing the number of gene copies, whereby the export Signals. carrier gene is expressed from the additional gene copies. 13. The process according to claim 1 for the manufacture 3. The process according to claim 2, wherein, in order to of L-lysine. increase the number of export carrier gene copies, the export 14. A process for the increased microbial production of gene is inserted into a gene construct. 15 amino acids using an export carrier gene, comprising the 4. The proceSS according to claim 3, wherein the export Steps of carrier gene is inserted into a vector with a low number i) constructing a gene construct including an export copies. carrier gene, 5. The proceSS according to claim 3, wherein the export ii) inserting said construct into a Suitable vector, carrier gene is inserted into a gene construct, which includes regulatory gene Sequences operably linked to the export iii) transforring a Suitable host cell with said vector, carrier gene. iv) cultivating said transformed host cell in a culture 6. The process according to claim 5, wherein the regula medium, tory gene Sequence includes a nucleotide Sequence V) recovering the amino acid(s) from the culture, and 1421–2293, coding for the amino acid Sequence as given in 25 Vi) determining the desired amino acid(s) amount. SEO ID NO: 3. 15. The proceSS according to claim 14, wherein the gene 7. The process according to claim 3, wherein the micro construct additionally carries regulatory gene Sequences. organism producing the respective amino acid is trans 16. The process according to claim 14, wherein an export formed with the gene construct. carrier gene from Corynebacteriumn is utilized. 8. The process according to claim 7, wherein the micro 17. The process according to claim 14, wherein Coryne organism is the type Corynebacterium. bacterium is used as amino acid producing microorganism. 9. The process according to claim 7, wherein, for the transformation, a microorganism is utilized in which the k k k k k