Towards methionine overproduction in Corynebacterium glutamicum--methanethiol and dimethyldisulfide as reduced sulfur sources.

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

[1]  Heung-Shick Lee,et al.  Characteristics of methionine production by an engineered Corynebacterium glutamicum strain. , 2007, Metabolic engineering.

[2]  C. Wittmann,et al.  Physiological response of Corynebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR. , 2008, Microbiology.

[3]  Christoph Wittmann,et al.  Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum--over expression and modification of G6P dehydrogenase. , 2007, Journal of biotechnology.

[4]  M. Moran,et al.  Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton , 1999, Applied and Environmental Microbiology.

[5]  M. Ikeda.  Amino acid production processes. , 2003, Advances in biochemical engineering/biotechnology.

[6]  Christoph Wittmann,et al.  Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose , 2004, Applied and Environmental Microbiology.

[7]  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.

[8]  C. Wittmann,et al.  The l -Lysine Story: From Metabolic Pathways to Industrial Production , 2007 .

[9]  Christoph Wittmann,et al.  Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source , 2004, Applied and Environmental Microbiology.

[10]  K. Shimizu,et al.  Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS. , 2007, Journal of biotechnology.

[11]  M. Flavin,et al.  Enzymatic synthesis of homocysteine or methionine directly from O-succinyl-homoserine. , 1967, Biochimica et biophysica acta.

[12]  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.

[13]  C. Wittmann,et al.  Sampling for metabolome analysis of microorganisms. , 2007, Analytical chemistry.

[14]  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.

[15]  H. Kanzaki,et al.  Purification and characterization of cystathionine γ‐synthase type II from Bacillus sphaericus , 1987 .

[16]  Christoph Wittmann,et al.  Fluxome analysis using GC-MS , 2007, Microbial cell factories.

[17]  Jeroen S. Dickschat,et al.  Volatiles Released by a Streptomyces Species Isolated from the North Sea , 2005, Chemistry & biodiversity.

[18]  Christoph Wittmann,et al.  Metabolic pathway analysis for rational design of L-methionine production by Escherichia coli and Corynebacterium glutamicum. , 2006, Metabolic engineering.

[19]  S. Mondal,et al.  Methionine production by microorganisms , 2008, Folia Microbiologica.

[20]  C. Wittmann,et al.  Impact of the cold shock phenomenon on quantification of intracellular metabolites in bacteria. , 2004, Analytical biochemistry.

[21]  Christoph Wittmann,et al.  Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources , 2005, Applied and Environmental Microbiology.

[22]  S. Yamagata.  Homocysteine synthesis in yeast. Partial purification and properties of O-acetylhomoserine sulfhydrylase. , 1971, Journal of biochemistry.

[23]  Jeroen S. Dickschat,et al.  Biosynthesis and Identification of Volatiles Released by the Myxobacterium Stigmatella aurantiaca , 2005, Chembiochem : a European journal of chemical biology.

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

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

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

[27]  P. Bonnarme,et al.  Diversity of l-Methionine Catabolism Pathways in Cheese-Ripening Bacteria , 2000, Applied and Environmental Microbiology.

[28]  Christoph Wittmann,et al.  Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum , 2009, Applied and Environmental Microbiology.

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

[30]  C. Wittmann,et al.  In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. , 2005, Analytical biochemistry.