Unprecedented acetoacetyl-coenzyme A synthesizing enzyme of the thiolase superfamily involved in the mevalonate pathway.
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.
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Eiji
E
Okamura
Takeo
T
Tomita
Ryuichi
R
Sawa
Makoto
M
Nishiyama
Tomohisa
T
Kuzuyama
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006885:Hydroxybutyrates / Q000378:metabolism; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics; D007700:Kinetics; D008798:Mevalonic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D011091:Polyesters / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013729:Terpenes / Q000737:chemistry; D000094122:Polyhydroxybutyrates
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D003066:Coenzyme A Ligases; D006885:Hydroxybutyrates; D006904:Hydroxymethylglutaryl-CoA Synthase; D008798:Mevalonic Acid; D011091:Polyesters; D011994:Recombinant Proteins; D013729:Terpenes; C010667:acetoacetyl CoA; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C550924:acetoacetyl-CoA synthase, Streptomyces