Genome Mining in Streptomyces coelicolor. Molecular Cloning and Characterization of a New Sesquiterpene Synthase

摘要

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene ofStreptomyces coelicolor was cloned by PCR and expressed inEscherichia 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 of¹H,¹³C, COSY, HMQC, HMBC, and NOESY NMR. The steady state kinetic parameters werekcat 0.049 ± 0.001 s⁻¹ and aKm (FPP) of 147 ± 14 nM. Individual incubations of recombinant epiisozizaene synthase with {1,1-²H2}FPP (>1a), (1R)-{1-²H}-FPP (>1b), and (1 S)-{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).