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(12) United States Patent (10) Patent No.: US 6,858,406 B1 Vrlijc Et Al

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(12) United States Patent (10) Patent No.: US 6,858,406 B1 Vrlijc Et Al 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 48 x 88 a 4th 88 48 488. A 8. pMV6-3 as as as as as a spp. pMV8-5-24 - pMV7-2 al to to a prox pMV3-7 pMV2-5 2000 4000 6000 8000 10000 ----|--|--|--|--|--| | | | | U.S. Patent Feb. 22, 2005 Sheet 1 of 4 US 6,858,406 B1 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 150 O o E 00 s te s 2 2 s e 50 a O 150 C O s 100 F.e s s É 50 > O 5 17.5 20 22.5 TIME (h) O NA8KpMV2-3X FIG 3B U.S. Patent Feb. 22, 2005 Sheet 4 of 4 US 6,858,406 B1 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).
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