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Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway

Proceedings of the National Academy of Sciences of the United States of America, 22 June 2010, Vol.107(25), pp.11265-70 [Peer Reviewed Journal]

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  • Title:
    Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway
  • Author: Okamura, Eiji ; Tomita, Takeo ; Sawa, Ryuichi ; Nishiyama, Makoto ; Kuzuyama, Tomohisa
  • Found In: Proceedings of the National Academy of Sciences of the United States of America, 22 June 2010, Vol.107(25), pp.11265-70 [Peer Reviewed Journal]
  • Subjects: Gene Expression Regulation, Bacterial ; Acyl Coenzyme A -- Metabolism ; Bacterial Proteins -- Genetics ; Coenzyme A Ligases -- Genetics ; Escherichia Coli -- Metabolism ; Mevalonic Acid -- Metabolism ; Streptomyces -- Enzymology
  • Language: English
  • Description: Acetoacetyl-CoA is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. Acetoacetyl-CoA is also the precursor of poly-beta-hydroxybutyrate, a polymer belonging to the polyester class produced by microorganisms. The de novo synthesis of acetoacetyl-CoA is usually catalyzed by acetoacetyl-CoA thiolase via a thioester-dependent Claisen condensation reaction between two molecules of acetyl-CoA. Here, we report that nphT7, found in the mevalonate pathway gene cluster from a soil-isolated Streptomyces sp. strain, encodes an unusual acetoacetyl-CoA synthesizing enzyme. The recombinant enzyme overexpressed in Escherichia coli catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-(acyl carrier protein) resulted in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity was detected through the condensation of two molecules of acetyl-CoA. Based on these properties of NphT7, we propose to name this unusual enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Coexpression of nphT7 with the HMG-CoA synthase gene and the HMG-CoA reductase gene in a heterologous host allowed 3.5-fold higher production of mevalonate than when only the HMG-CoA synthase and HMG-CoA reductase genes were expressed. This result suggests that nphT7 can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leading to the production of useful terpenoids and poly-beta-hydroxybutyrate.
  • Identifier: E-ISSN: 1091-6490 ; PMID: 20534558 Version:1 ; DOI: 10.1073/pnas.1000532107

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