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Methods for the preparation of lysine by fermentation of corynebacterium glutamicum

Methods for the preparation of lysine by fermentation of corynebacterium glutamicum description/claims

The present application is divisional of U.S. application Ser. No. 10/579,690, filed May 18, 2006, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/IB2004/004429, filed Dec. 17, 2004, which claims priority to International Application No. PCT/IB2003/006456, filed Dec. 18, 2003. The entire contents of each of these applications are hereby incorporated by reference herein.

This application incorporates herein by reference the sequence listing filed concurrently herewith, i.e., the file “SEQLIST.txt” (45.0 KB) created on Dec. 29, 2007).

The industrial production of the amino acid lysine has became an economically important industrial process. Lysine is used commercially as an animal feed supplement, because of its ability to improve the quality of feed by increasing the absorption of other amino acids, in human medicine, particularly as ingredients of infusion solutions, and in the pharmaceutical industry.

Commercial production of this lysine is principally done utilizing the gram positive Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum (Kleemann, A., et. al., “Amino Acids,” in ULLMANN'S ENCYCLOPEDIA OF INDUSTRIAL CHEMISTRY, vol. A2, pp. 57-97, Weinham: VCH-Verlagsgesellschaft (1985)). These organisms presently account for the approximately 250,000 tons of lysine produced annually. A significant amount of research has gone into isolating mutant bacterial strains which produce larger amounts of lysine. Microorganisms employed in microbial process for amino acid production are divided into 4 classes: wild-type strain, auxotrophic mutant, regulatory mutant and auxotrophic regulatory mutant (K. Nakayama et al., in Nutritional Improvement of Food and Feed Proteins, M. Friedman, ed., (1978), pp. 649-661). Mutants of Corynebacterium and related organisms enable inexpensive production of amino acids from cheap carbon sources, e.g., molasses, acetic acid and ethanol, by direct fermentation. In addition, the stereospecificity of the amino acids produced by fermentation (the L isomer) makes the process advantageous compared with synthetic processes.

Another method in improving the efficiency of the commercial production of lysine is by investigating the correlation between lysine production and metabolic flux through the pentose phosphate pathway. Given the economic importance of lysine production by the fermentative process, the biochemical pathway for lysine synthesis has been intensively investigated, ostensibly for the purpose of increasing the total amount of lysine produced and decreasing production costs (reviewed by Sahm et al., (1996) Ann. N.Y. Acad. Sci. 782:25-39). There has been some success in using metabolic engineering to direct the flux of glucose derived carbons toward aromatic amino acid formation (Flores, N. et al., (1996) Nature Biotechnol. 14:620-623). Upon cellular absorption, glucose is phosphorylated with consumption of phosphoenolpyruvate (phosphotransferase system) (Malin & Bourd, (1991) Journal of Applied Bacteriology 71, 517-523) and is then available to the cell as glucose-6-phosphate. Sucrose is converted into fructose and glucose-6-phosphate by a phosphotransferase system (Shio et al., (1990) Agricultural and Biological Chemistry 54, 1513-1519) and invertase reaction (Yamamoto et al., (1986) Journal of Fermentation Technology 64, 285-291).

During glucose catabolism, the enzymes glucose-6-phosphate dehydrogenase (EC 1.1.14.9) and glucose-6-phosphate isomerase (EC 5.3.1.9) compete for the substrate glucose-6-phosphate. The enzyme glucose-6-phosphate isomerase catalyses the first reaction step of the Embden-Meyerhof-Parnas pathway, or glycolysis, namely conversion into fructose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase catalyses the first reaction step of the oxidative portion of the pentose phosphate cycle, namely conversion into 6-phosphogluconolactone.

In the oxidative portion of the pentose phosphate cycle, glucose-6-phosphate is converted into ribulose-5-phosphate, so producing reduction equivalents in the form of NADPH. As the pentose phosphate cycle proceeds further, pentose phosphates, hexose phosphates and triose phosphates are interconverted. Pentose phosphates, such as for example 5-phosphoribosyl-1-pyrophosphate are required, for example, in nucleotide biosynthesis. 5-Phosphoribosyl-1-pyrophosphate is moreover a precursor for aromatic amino acids and the amino acid L-histidine. NADPH acts as a reduction equivalent in numerous anabolic biosyntheses. Four molecules of NADPH are thus consumed for the biosynthesis of one molecule of lysine from oxalacetic acid. Thus, carbon flux towards oxaloacetate (OAA) remains constant regardless of system perturbations (J. Vallino et al., (1993) Biotechnol. Bioeng., 41, 633-646).

The present invention is based, at least in part, on the discovery of key enzyme-encoding genes, e.g., fructose-1,6-bisphosphatase, of the pentose phosphate pathway in Corynebacterium glutamicum, and the discovery that deregulation, e.g., increasing expression or activity of fructose-1,6-bisphosphatase results in increased lysine production. Furthermore, it has been found that increasing the carbon yield during production of lysine by deregulating, e.g., increasing, fructose-1,6 bisphosphatase expression or activity leads to increased lysine production. In one embodiment, the carbon source is fructose or sucrose. Accordingly, the present invention provides methods for increasing production of lysine by microorganisms, e.g., C. glutamicum, where fructose or sucrose is the substrate.

Accordingly, in one aspect, the invention provides methods for increasing metabolic flux through the pentose phosphate pathway in a microorganism comprising culturing a microorganism comprising a gene which is deregulated under conditions such that metabolic flux through the pentose phosphate pathway is increased. In one embodiment, the microorganism is fermented to produce a fine chemical, e.g., lysine. In another embodiment, fructose or sucrose is used as a carbon source. In still another embodiment, the gene is fructose-1,6-bisphosphatase. In a related embodiment, the fructose-1,6-bisphosphatase gene is derived from Corynebacterium, e.g., Corynebacterium glutamicum. In another embodiment, fructose-1,6 bisphosphatase gene is overexpressed. In a further embodiment, the protein encoded by the fructose-1,6-bisphosphatase gene has increased activity.

In another embodiment, the microorganism further comprises one or more additional deregulated genes. The one or more additional deregulated gene can include, but is not limited to, an ask gene, a dapA gene, an asd gene, a dapB gene, a ddh gene, a lysA gene, a lysE gene, a pycA gene, a zwf gene, a pepCL gene, a gap gene, a zwal gene, a tkt gene, a tad gene, a mqo gene, a tpi gene, a pgk gene, and a sigC gene. In a particular embodiment, the gene may be overexpressed or underexpressed. Moreover, the deregulated gene can encode a protein selected from the group consisting of a feed-back resistant aspartokinase, a dihydrodipicolinate synthase, an aspartate semialdehyde dehydrogenase, a dihydrodipicolinate reductase, a diaminopimelate dehydrogenase, a diaminopimelate epimerase, a lysine exporter, a pyruvate carboxylase, a glucose-6-phosphate dehydrogenase, a phosphoenolpyruvate carboxylase, a glyceraldedyde-3-phosphate dehydrogenase, an RPF protein precursor, a transketolase, a transaldolase, a menaquinine oxidoreductase, a triosephosphate isomerase, a 3-phosphoglycerate kinase, and an RNA-polymerase sigma factor sigC. In a particular embodiment, the protein may have an increased or a decreased activity.

In accordance with the methods of the present invention, the one or more additional deregulated genes can also include, but is not limited to, a pepCK gene, a mal E gene, a glgA gene, a pgi gene, a dead gene, a menE gene, a citE gene, a mikE17 gene, a poxB gene, a zwa2 gene, and a sucC gene. In a particular embodiment the expression of the at least one gene is upregulated, attenuated, decreased, downregulated or repressed. Moreover, the deregulated gene can encode a protein selected from the group consisting of a phosphoenolpyruvate carboxykinase, a malic enzyme, a glycogen synthase, a glucose-6-phosphate isomerase, an ATP dependent RNA helicase, an o-succinylbenzoic acid-CoA ligase, a citrate lyase beta chain, a transcriptional regulator, a pyruvate dehydrogenase, an RPF protein precursor, and a Succinyl-CoA-Synthetase. In a particular embodiment, the protein has a decreased or an increased activity.

In one embodiment, the microorganisms used in the methods of the invention belong to the genus Corynebacterium, e.g., Corynebacterium glutamicum.

In another aspect, the invention provides methods for producing a fine chemical comprising fermenting a microorganism in which fructose-1,6-bisphosphatase is deregulated and accumulating the fine chemical, e.g., lysine, in the medium or in the cells of the microorganisms, thereby producing a fine chemical. In one embodiment, the methods include recovering the fine chemical. In another embodiment, the fructose-1,6-bisphosphatase gene is overexpressed. In yet another embodiment, fructose or sucrose is used as a carbon source.

In one aspect, fructose-1,6-bisphosphatase is derived from Corynebacterium glutamicum and comprises the nucleotide sequence of SEQ ID NO:1 and the amino acid sequence of SEQ ID NO:2.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

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