Abstract
Corynebacterium glutamicum R efficiently produces valuable chemicals from glucose under oxygen-deprived conditions. In an effort to reduce acetate as a byproduct, acetate productivity of several mutant-disrupted genes encoding possible key enzymes for acetate formation was determined. Disruption of the aceE gene that encodes the E1 enzyme of the pyruvate dehydrogenase complex resulted in almost complete elimination of acetate formation under oxygen-deprived conditions, implying that acetate synthesis under these conditions was essentially via acetyl-coenzyme A (CoA). Simultaneous disruption of pta, encoding phosphotransacetylase, and ack, encoding acetate kinase, resulted in no measurable change in acetate productivity. A mutant strain with disruptions in pta, ack and as-yet uncharacterized gene (cgR2472) exhibited 65% reduced acetate productivity compared to the parental strain, although a single disruption of cgR2472 exhibited no effect on acetate productivity. The gene cgR2472 was shown to encode a CoA-transferase (CTF) that catalyzes the formation of acetate from acetyl-CoA. These results indicate that PTA-ACK as well as CTF is involved in acetate production in C. glutamicum. This study provided basic information to reduce acetate production under oxygen-deprived conditions.
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Andersch W, Bahl H, Gottschalk G (1983) Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum. Eur J Appl Microbiol Biotechnol 18:327–332
Blombach B, Schreiner ME, Holatko J, Bartek T, Oldiges M, Eikmanns BJ (2007) L-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol 73:2079–2084
Buu LM, Chen YC, Lee FJ (2003) Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae. J Biol Chem 278:17203–17209
Chotani G, Dodge T, Hsu A, Kumar M, LaDuca R, Trimbur D, Weyler W, Sanford K (2000) The commercial production of chemicals using pathway engineering. Biochim Biophys Acta 1543:434–455
Dittrich CR, Bennett GN, San KY (2005a) Characterization of the acetate-producing pathways in Escherichia coli. Biotechnol Prog 21:1062–1067
Dittrich CR, Vadali RV, Bennett GN, San KY (2005b) Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate. Biotechnol Prog 21:627–631
Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H (2004a) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254
Inui M, Murakami S, Okino S, Kawaguchi H, Vertès AA, Yukawa H (2004b) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196
Kinoshita S, Udaka S, Shimono M (1957) Studies on the amino acid fermentation: Part I. Production of L-glutamic acid by various microorganisms. J Gen Appl Microbiol 3:193–205
Liedvogel B, Stumpf PK (1982) Origin of acetate in spinach leaf cell. Plant Physiol 69:897–903
Liu X, Zhu Y, Yang ST (2006) Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants. Enzyme Microb Technol 38:521–528
Mack M, Buckel W (1997) Conversion of glutaconate CoA-transferase from Acidaminococcus fermentans into an acyl-CoA hydrolase by site-directed mutagenesis. FEBS Lett 405:209–212
Nakata K, Inui M, Kos P, Vertès AA, Yukawa H (2003) Vectors for genetic engineering of Corynebacteria. In: Saha B (ed) American Chemical Society Symposium Series 862: fermentation biotechnology. American Chemical Society, Washington, DC, pp 175–191
Nishimura T, Vertès AA, Shinoda Y, Inui M, Yukawa H (2007) Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor. Appl Microbiol Biotechnol 75:889–897
Ohara H (2003) Biorefinery. Appl Microbiol Biotechnol 62:474–477
Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480
Presecan-Siedel E, Galinier A, Longin R, Deutscher J, Danchin A, Glaser P, Martin-Verstraete I (1999) Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis. J Bacteriol 181:6889–6897
Reinscheid DJ, Schnicke S, Rittmann D, Zahnow U, Sahm H, Eikmanns BJ (1999) Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase. Microbiology 145:503–513
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Scherf U, Buckel W (1991) Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum. Appl Environ Microbiol 57:2699–2702
Schreiner ME, Eikmanns BJ (2005) Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization. J Bacteriol 187:862–871
Schreiner ME, Fiur D, Holatko J, Patek M, Eikmanns BJ (2005) E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum: molecular analysis of the gene and phylogenetic aspects. J Bacteriol 187:6005–6018
Schreiner ME, Riedel C, Holatko J, Patek M, Eikmanns BJ (2006) Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects. J Bacteriol 188:1341–1350
Sohling B, Gottschalk G (1996) Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri. J Bacteriol 178:871–880
Terasawa M, Yukawa H (1993) Industrial production of biochemicals by native immobilization. In: Tanaka A, Tosaka O, Kobayashi T (eds) Industrial application of Immobilized biocatalysts. Marcel Dekker, New York, pp 37–52
Wendisch VF, Spies M, Reinscheid DJ, Schnicke S, Sahm H, Eikmanns BJ (1997) Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes. Arch Microbiol 168:262–269
Wolfe AJ (2005) The acetate switch. Microbiol Mol Biol Rev 69:12–50
Yang YT, Aristidou AA, San KY, Bennett GN (1999) Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase. Metab Eng 1:26–34
Yukawa H, Omumasaba CA, Nonaka H, Kos P, Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vertès AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058
Acknowledgments
We thank C. A. Omumasaba (RITE) for helpful comments on the manuscript. We are also grateful to S. Murakami for technical support. This research was financially supported in part by the New Energy and Industrial Technology Development Organization (NEDO), Japan.
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An erratum to this article can be found at https://doi.org/10.1007/s00253-007-1316-y
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Yasuda, K., Jojima, T., Suda, M. et al. Analyses of the acetate-producing pathways in Corynebacterium glutamicum under oxygen-deprived conditions. Appl Microbiol Biotechnol 77, 853–860 (2007). https://doi.org/10.1007/s00253-007-1199-y
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DOI: https://doi.org/10.1007/s00253-007-1199-y