Characteristics of methionine production by an engineered Corynebacterium glutamicum strain.

A methionine-producing strain was derived from a lysine-producing Corynebacterium glutamicum through a process of genetic manipulation in order to assess its potential to synthesize and accumulate methionine during growth. The strain carries a deregulated hom gene (hom(FBR)) to abolish feedback inhibition of homoserine dehydrogenase by threonine and a deletion of the thrB gene (delta thrB) to abolish threonine synthesis. The constructed C. glutamicum MH20-22B/hom(FBR)/delta thrB strain accumulated 2.9 g/l of methionine by batch fermentation and showed resistance to methionine analogue ethionine at concentrations up to 30 mM. The growth of the strain was apparently impaired as a result of the accumulation of methionine biosynthetic intermediate, homocysteine. Production assays also revealed that the accumulation of methionine in the growth medium was transient and declined as the carbon source was depleted. During the period of methionine disappearance, the methionine biosynthetic genes were completely repressed in the engineered strains but not in the parental strain. After all, we have not only successfully constructed a methionine-producing C. glutamicum strain by genetic manipulation, but also revealed cellular constraints in attaining high yield and productivity.

[1]  A L Demain,et al.  Small bugs, big business: the economic power of the microbe. , 2000, Biotechnology advances.

[2]  A Danchin,et al.  Sulfur metabolism in Escherichia coli and related bacteria: facts and fiction. , 2000, Journal of molecular microbiology and biotechnology.

[3]  J. Kalinowski,et al.  The McbR repressor modulated by the effector substance S‐adenosylhomocysteine controls directly the transcription of a regulon involved in sulphur metabolism of Corynebacterium glutamicum ATCC 13032 , 2005, Molecular microbiology.

[4]  H. Sahm,et al.  Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate , 1989, Applied and environmental microbiology.

[5]  M. Ikeda.  Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering , 2006, Applied Microbiology and Biotechnology.

[6]  T. Hermann.  Industrial production of amino acids by coryneform bacteria. , 2003, Journal of biotechnology.

[7]  A. Sinskey,et al.  Design of a defined medium for growth ofCorynebacteriumglutamicum in which citrate facilitates iron uptake , 2005, Biotechnology Letters.

[8]  Christoph Wittmann,et al.  Accumulation of Homolanthionine and Activation of a Novel Pathway for Isoleucine Biosynthesis in Corynebacterium glutamicum McbR Deletion Strains , 2006, Journal of bacteriology.

[9]  J. Kalinowski,et al.  The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum. , 2003, Journal of biotechnology.

[10]  J. Kalinowski,et al.  Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. , 1994, Gene.

[11]  B. Bathe,et al.  Characterization of Methionine Export in Corynebacterium glutamicum , 2005, Journal of bacteriology.

[12]  S. Ravanel,et al.  Methionine biosynthesis in higher plants. II. Purification and characterization of cystathionine beta-lyase from spinach chloroplasts. , 1995, Archives of biochemistry and biophysics.

[13]  B. Hwang,et al.  Methionine biosynthesis and its regulation in Corynebacterium glutamicum: parallel pathways of transsulfuration and direct sulfhydrylation , 2003, Applied Microbiology and Biotechnology.

[14]  L. Eggeling,et al.  Handbook of Corynebacterium glutamicum , 2005 .

[15]  U. Sauer,et al.  Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum , 2005, Applied Microbiology and Biotechnology.

[16]  Heung-Shick Lee.  Sulfur Metabolism and Its Regulation , 2005 .

[17]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[18]  H. Sahm,et al.  Control of the Lysine Biosynthesis Sequence in Corynebacterium glutamicum as Analyzed by Overexpression of the Individual Corresponding Genes , 1991, Applied and environmental microbiology.

[19]  H Sahm,et al.  Metabolic engineering for L-lysine production by Corynebacterium glutamicum. , 2001, Advances in biochemical engineering/biotechnology.

[20]  W. Leuchtenberger,et al.  Biotechnological production of amino acids and derivatives: current status and prospects , 2005, Applied Microbiology and Biotechnology.

[21]  Y. Tani,et al.  L-Methionine Overproduction by Ethionine-resistant Mutants of Obligate Methylotroph Strain OM 33 , 1982 .

[22]  A J Sinskey,et al.  Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain , 1994, Applied and environmental microbiology.

[23]  G. Stephanopoulos,et al.  Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799 , 1995, Applied and environmental microbiology.

[24]  R. Heinrikson,et al.  Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. , 1984, Analytical biochemistry.

[25]  G. Stephanopoulos,et al.  Strain improvement by metabolic engineering: lysine production as a case study for systems biology. , 2005, Current opinion in biotechnology.

[26]  H. Sahm,et al.  Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. , 2001, Journal of molecular microbiology and biotechnology.

[27]  Analysis of Corynebacterium glutamicum methionine biosynthetic pathway: isolation and analysis of metB encoding cystathionine gamma-synthase. , 1999, Molecules and cells.

[28]  M. S. Lee,et al.  Properties of the Corynebacterium glutamicum metC gene encoding cystathionine beta-lyase. , 2001, Molecules and cells.

[29]  Nina L Tuite,et al.  Homocysteine Toxicity in Escherichia coli Is Caused by a Perturbation of Branched-Chain Amino Acid Biosynthesis , 2005, Journal of bacteriology.

[30]  R. Walker,et al.  S-adenosylhomocysteine metabolism in various species. , 1975, Canadian journal of biochemistry.

[31]  H. Sahm,et al.  Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase , 1991, Journal of bacteriology.

[32]  A. Pühler,et al.  The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha, beta-elimination activity that degrades aminoethylcysteine , 1992, Journal of bacteriology.

[33]  M. Inui,et al.  Manipulating Corynebacteria, from Individual Genes to Chromosomes , 2005, Applied and Environmental Microbiology.

[34]  J. Uren.  Cystathionine β-lyase from Escherichia coli , 1987 .

[35]  H. Sahm,et al.  Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids , 1994, Applied and environmental microbiology.

[36]  F. Neidhardt,et al.  Physiology of the bacterial cell : a molecular approach , 1990 .

[37]  A. Pühler,et al.  Genome-wide analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation. , 2003, Journal of biotechnology.

[38]  B. Hwang,et al.  Corynebacterium glutamicum Utilizes both Transsulfuration and Direct Sulfhydrylation Pathways for Methionine Biosynthesis , 2002, Journal of bacteriology.

[39]  Kartik Subramanian,et al.  Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium. , 2005, Bioresource technology.

[40]  A. Rodal,et al.  Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum , 2001, Applied Microbiology and Biotechnology.

[41]  M. Bally,et al.  A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa. , 1995, Microbiology.

[42]  Y. Kim,et al.  Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in corynebacterium glutamicum. , 1998, Molecules and cells.

[43]  A. Ghosh,et al.  Threonine analogue resistant mutants of Escherichia coli K-12 , 1995, Biotechnology Letters.

[44]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[45]  P. Brigidi,et al.  Use of protoplast fusion to introduce methionine overproduction into Saccharomyces cerevisiae , 1988, Applied Microbiology and Biotechnology.

[46]  J. Ohnishi,et al.  A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant , 2001, Applied Microbiology and Biotechnology.

[47]  Albert A. de Graaf,et al.  Pathway Analysis and Metabolic Engineering in Corynebacterium glutamicum , 2000, Biological chemistry.

[48]  Gregory Stephanopoulos,et al.  Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum , 2002, Applied and Environmental Microbiology.

[49]  H. Takagi,et al.  Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes , 1999, Applied Microbiology and Biotechnology.

[50]  A. Pühler,et al.  Manipuiation of Corynebacterium glutamicum by Gene Disruption and Replacement , 1991, Bio/Technology.

[51]  H. Kase,et al.  l-Methionine Production by Methionine Analog-resistant Mutants Of Corynebacterium glutamicum , 1975 .

[52]  H. Sahm,et al.  Isolation and prominent characteristics of an L-lysine hyperproducing strain of Corynebacterium glutamicum , 1992, Applied Microbiology and Biotechnology.

[53]  H. Kase,et al.  Fermentation production of L-methionine and regulation of L-methionine biosynthesis on Corynebacterium glutamicum. I. Production of O-acetyl-L-homoserine by methionine analogresisttant mutants and regulation of homoserine-O-transacetylase in Corynebacterium glutamicum. , 1974 .

[54]  M. Follettie,et al.  Gene structure and expression of the Corynebacterium flavum N13 ask-asd operon , 1993, Journal of bacteriology.