Genome Mining in <i>Streptomyces </i><i>c</i><i>oelicolor</i>: Molecular Cloning and Characterization of a New Sesquiterpene Synthase

Abstract

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).