Accumulation of Homolanthionine and Activation of a Novel Pathway for Isoleucine Biosynthesis in Corynebacterium glutamicum McbR Deletion Strains

ABSTRACT In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) in Corynebacterium glutamicum, which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol. 103:51-65, 2003), were studied. C. glutamicum ATCC 13032 ΔmcbR showed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry as l-homolanthionine {S-[(3S)-3-amino-3-carboxypropyl]-l-homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the ΔmcbR strain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-γ-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine (Km = 260 μM) than for homocysteine (Km = 540 μM). The cell is able to cleave homolanthionine at low rates via cystathionine-β-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis of C. glutamicum ΔmcbR revealed that only 24% of the O-acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis; the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion of metB completely prevents homolanthionine accumulation, which is regarded as an important step in the development of C. glutamicum strains for biotechnological methionine production.

[1]  H. Huang.  Accumulation of 1-homolanthionine by an Escherichia coli mutant. , 1963, Biochemistry.

[2]  M. Merten,et al.  Homocysteine Increases Methionine Synthase mRNA Level in Caco-2 Cells , 2004, Cellular Physiology and Biochemistry.

[3]  l-Isoleucine Production with Corynebacterium glutamicum: Further Flux Increase and Limitation of Export , 1996, Applied and environmental microbiology.

[4]  H. Tallan,et al.  Homolanthionine synthesis by human liver cystathionase. , 1971, Biochemical and biophysical research communications.

[5]  J. Büchs,et al.  Effect of Oxygen Limitation and Medium Composition on Escherichia coli Fermentation in Shake‐Flask Cultures , 2004, Biotechnology progress.

[6]  C. Hoischen,et al.  Carrier-mediated glutamate secretion by Corynebacterium glutamicum under biotin limitation. , 1992, Biochimica et biophysica acta.

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

[8]  M. Saier,et al.  Export of l-Isoleucine from Corynebacterium glutamicum: a Two-Gene-Encoded Member of a New Translocator Family , 2002, Journal of bacteriology.

[9]  J. Thompson,et al.  Toxicity of Bordetella avium beta-cystathionase toward MC3T3-E1 osteogenic cells. , 1993, The Journal of biological chemistry.

[10]  Huang Ht.  Accumulation of 1-homolanthionine by an Escherichia coli mutant. , 1963 .

[11]  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.

[12]  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.

[13]  S. Ravanel,et al.  Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli. , 1998, The Biochemical journal.

[14]  H. Sahm,et al.  l-Glutamate and l-lysine: traditional products with impetuous developments , 1999, Applied Microbiology and Biotechnology.

[15]  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.

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

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

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

[19]  Christoph Wittmann,et al.  In vivo analysis of intracellular amino acid labelings by GC/MS. , 2002, Analytical biochemistry.

[20]  H. Kanzaki,et al.  Syntheses of S-substituted L-homocysteine derivatives by cystathionine γ-lyase of Streptomyces phaeochromogenes , 1986 .

[21]  C. Wittmann,et al.  Metabolic Flux Analysis in Corynebacterium glutamicum , 2005 .

[22]  R. Matthews,et al.  Transcriptional regulation of methionine synthase by homocysteine and choline in Aspergillus nidulans. , 2003, The Biochemical journal.

[23]  A. Pühler,et al.  Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans , 1992, Journal of bacteriology.

[24]  C. Dwivedi,et al.  Cloning, purification, and characterization of beta-cystathionase from Escherichia coli. , 1982, Biochemistry.

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

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

[27]  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.

[28]  W. Leuchtenberger.  Amino Acids – Technical Production and Use , 2001 .

[29]  G. Ellman,et al.  A precise method for the determination of whole blood and plasma sulfhydryl groups. , 1979, Analytical biochemistry.

[30]  C. Miller,et al.  Preparation of L-homocysteine from L-homocysteine thiolactone. , 1966, Analytical biochemistry.

[31]  R. C. Greene,et al.  Purification and properties of cystathionine gamma-synthase from overproducing strains of Escherichia coli. , 1990, Biochemistry.

[32]  C. Wittmann,et al.  In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome , 2004, Journal of bacteriology.

[33]  Homolanthionine in fungi: accumulation in the methionine-requiring mutants of Aspergillus nidulans. , 1975, Acta biochimica Polonica.

[34]  S. Ravanel,et al.  Methionine biosynthesis in higher plants. I. Purification and characterization of cystathionine gamma-synthase from spinach chloroplasts. , 1995, Archives of biochemistry and biophysics.