Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes

For the biotechnological production of l-lysine, mainly strains of Corynebacterium glutamicum are used, which have been obtained by classical mutagenesis and screening or selection or by metabolic engineering. Gene targets for the amplification and deregulation of the lysine biosynthesis pathway, for the improvement of carbon precursor supply and of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) regeneration, are known. To identify novel target genes to improve lysine production, the transcriptomes of the classically obtained lysine producing strain MH20-22B and several other C. glutamicum strains were compared. As lysine production by the classically obtained strain, which possesses feedback-resistant aspartokinase and is leucine auxotrophic, exceeds that of a genetically defined leucine auxotrophic wild-type derivative possessing feedback-resistant aspartokinase, additional traits beneficial for lysine production are present. NCgl0855, putatively encoding a methyltransferase, and the amtA-ocd-soxA operon, encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized enzyme, were among the genes showing increased expression in the classically obtained strain irrespective of the presence of feedback-resistant aspartokinase. Lysine production could be improved by about 40% through overexpression of NCgl0855 or the amtA-ocd-soxA operon. Thus, novel target genes for the improvement of lysine production could be identified in a discovery-driven approach based on global gene expression analysis.

[1]  A. Gornall,et al.  Determination of serum proteins by means of the biuret reaction. , 1949, The Journal of biological chemistry.

[2]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

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

[4]  Valentino Bontempo,et al.  Branched-chain amino acids. , 2015, Methods in enzymology.

[5]  G. Kohlhaw.  Alpha-isopropylmalate synthase from yeast. , 1988, Methods in enzymology.

[6]  M. Ashburner.  A Laboratory manual , 1989 .

[7]  C. Tricot,et al.  Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways. , 1991, Journal of general microbiology.

[8]  H Sahm,et al.  A functionally split pathway for lysine synthesis in Corynebacterium glutamicium , 1991, Journal of bacteriology.

[9]  A. Sinskey,et al.  Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum , 1993 .

[10]  Lothar Eggeling,et al.  Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems , 1993, Applied and environmental microbiology.

[11]  M. Pátek,et al.  Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis , 1994, Applied and environmental microbiology.

[12]  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.

[13]  H. Sahm,et al.  Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. , 1994, Microbiology.

[14]  H. Sahm,et al.  Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum , 1995, Journal of bacteriology.

[15]  B. Eikmanns,et al.  Functional and Genetic Characterization of the (Methyl)ammonium Uptake Carrier of Corynebacterium glutamicum(*) , 1996, The Journal of Biological Chemistry.

[16]  H Sahm,et al.  Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing , 1996, Biotechnology and bioengineering.

[17]  A. D. de Graaf,et al.  C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum , 1996, Archives of Microbiology.

[18]  A. D. de Graaf,et al.  Response of the central metabolism of Corynebacterium glutamicum to different flux burdens. , 1997, Biotechnology and bioengineering.

[19]  Volker F. Wendisch,et al.  Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum. , 1997, Microbiology.

[20]  H Sahm,et al.  the Czech Republic, , 2022 .

[21]  J. Kalinowski,et al.  Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product , 1998, Archives of Microbiology.

[22]  H Sahm,et al.  Response of the central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase. , 1999, Metabolic engineering.

[23]  Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins. , 1999, FEMS microbiology letters.

[24]  F. S. Mathews,et al.  Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme. , 1999, Structure.

[25]  M. Baucher,et al.  Cloning of the Malic Enzyme Gene fromCorynebacterium glutamicum and Role of the Enzyme in Lactate Metabolism , 2000, Applied and Environmental Microbiology.

[26]  Albert A. de Graaf,et al.  Metabolic Flux Analysis of Corynebacterium glutamicum , 2000 .

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

[28]  R. Krämer,et al.  AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum , 2000, Molecular microbiology.

[29]  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.

[30]  H Sahm,et al.  Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production. , 2001, Journal of molecular microbiology and biotechnology.

[31]  A. Khodursky,et al.  Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays. , 2001, Analytical biochemistry.

[32]  落合 恵子,et al.  The novel glucose-6-phosphate dehydrogenase , 2001 .

[33]  H. Sahm,et al.  Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum , 2002, Applied and Environmental Microbiology.

[34]  H. Sahm,et al.  The Phosphate Starvation Stimulon of Corynebacterium glutamicum Determined by DNA Microarray Analyses , 2003, Journal of bacteriology.

[35]  M. Bott,et al.  Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1. , 2003, The Journal of biological chemistry.

[36]  A. Goesmann,et al.  The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. , 2003, Journal of biotechnology.

[37]  Stephan Hans,et al.  Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. , 2003, Journal of biotechnology.

[38]  H. Sahm,et al.  Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine , 2003, Applied and Environmental Microbiology.

[39]  J. Ohnishi,et al.  Efficient 40 degrees C fermentation of L-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding. , 2003, Applied microbiology and biotechnology.

[40]  H. Sahm,et al.  Fructose-1,6-bisphosphatase from Corynebacterium glutamicum: expression and deletion of the fbp gene and biochemical characterization of the enzyme , 2003, Archives of Microbiology.

[41]  H. Sahm,et al.  DNA Microarray Analyses of the Long-Term Adaptive Response of Escherichia coli to Acetate and Propionate , 2003, Applied and Environmental Microbiology.

[42]  V. Wendisch.  Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. , 2003, Journal of biotechnology.

[43]  Brigitte Bathe,et al.  Biotechnological manufacture of lysine. , 2003, Advances in biochemical engineering/biotechnology.

[44]  J. Ohnishi,et al.  Efficient 40°C fermentation of l-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding , 2003, Applied Microbiology and Biotechnology.

[45]  V. Wendisch,et al.  Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays , 2004, Applied biochemistry and biotechnology.

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

[47]  J. Kalinowski,et al.  Cloning of a DNA fragment fromCorynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase , 2004, Applied Microbiology and Biotechnology.

[48]  M. Follettie,et al.  The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and expression , 1989, Molecular and General Genetics MGG.

[49]  H. Sahm,et al.  Cometabolism of a Nongrowth Substrate: l-Serine Utilization by Corynebacterium glutamicum , 2004, Applied and Environmental Microbiology.

[50]  Birgit Kamm,et al.  Biorefineries – Industrial Processes and Products , 2005 .

[51]  H. Sahm,et al.  Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum , 2005, Journal of bacteriology.

[52]  V. Wendisch,et al.  Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. , 2005, Metabolic engineering.

[53]  Haruo Suzuki,et al.  Corynebacterium sp. U-96 Contains a Cluster of Genes of Enzymes for the Catabolism of Sarcosine to Pyruvate , 2005, Bioscience, biotechnology, and biochemistry.

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

[55]  A. Niebisch,et al.  Respiratory Energy Metabolism , 2005 .

[56]  Uwe Sauer,et al.  The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. , 2005, FEMS microbiology reviews.

[57]  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.

[58]  B. Bathe,et al.  l -Lysine Production , 2005 .

[59]  L. Eggeling,et al.  Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production , 2005, Applied and Environmental Microbiology.

[60]  Masato Ikeda,et al.  A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum. , 2005, FEMS microbiology letters.

[61]  V. Wendisch.  Genetic regulation of Corynebacterium glutamicum metabolism , 2006 .

[62]  V. Wendisch,et al.  Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. , 2006, Current opinion in microbiology.

[63]  Michael Kamm,et al.  Biorefineries - industrial processes and products : status quo and future directions , 2006 .

[64]  N. Glansdorff,et al.  Microbial Arginine Biosynthesis: Pathway, Regulation and Industrial Production , 2006 .

[65]  M. Bott,et al.  Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production , 2006, Applied and Environmental Microbiology.

[66]  J. Ohnishi,et al.  Transcriptome Analysis Reveals Global Expression Changes in an Industrial L-Lysine Producer of Corynebacterium glutamicum , 2006, Bioscience, biotechnology, and biochemistry.

[67]  Takashi Hirasawa,et al.  Production of Glutamate and Glutamate-Related Amino Acids: Molecular Mechanism Analysis and Metabolic Engineering , 2006 .

[68]  W. Wiechert,et al.  Emerging Corynebacterium glutamicum systems biology. , 2006, Journal of biotechnology.

[69]  V. Wendisch.  Amino acid biosynthesis : pathways, regulation and metabolic engineering , 2007 .

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

[71]  V. Wendisch,et al.  Towards Integration of Biorefinery and Microbial Amino Acid Production , 2008 .