Acetoacetyl-coenzyme A (CoA) is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. It is also the precursor of poly-β-hydroxybutyrate, a polymer belonging to the polyester class that is procuced 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 the nphT7 gene 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 acecyl-CoA. The site directed mutagenesis experiments suggested that the NphT7-catalyzed reaction proceeds through almost identical reaction mehanisms to that of KAS III and Type III PKS, where the Cys-His-Asn catalytic triad is involved.Based on these properites of NphT7, we propose to name this novel enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Co-expression of the nphT7 gene 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 gene and the HMG-CoA reductase gene were expressed. This result suggests that the nphT7 gene can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leadin to the production of useful terponoids and poly-β-hydroxybutyrate.
