S180: Development of whole-cell biocatalyst for styrene monooxygenase reaction

Styrene monooxygenase (SMO) can produce chiral (S)-styrene oxide, an important chemical intermediate for several drugs and functional food ingredients, by epoxidation of the vinyl group of styrene. SMO consists of two separate enzymes, an NADH-FAD oxidoreductase (StyB) and an FAD-dependent hydroxylase (StyA). The reaction requires the continuous regeneration of NADH by carbon metabolism, and the whole cells expressing SMO rather than purified SMO enzymes, are being employed for catalysis. This study focuses on the development of various whole-cell biocatalysts containing SMO genes (styAB) of Pseudomonas putida SN1 and comparison of their performance. With Escherichia coli BL21 as a host, three types of recombinants were developed: pET system with strong promoter T7, pBAD system with arabinose promoter, and pET-chaperon system where various chaperons were co-expressed in pET system. With P. putida SN1, two biocatalysts were developed, a styAB self-cloned recombinant and a styC knockout mutant of SN1 that was devoid of the degradation activity of styrene oxide to phenyl acetaldehyde. In addition, various E. coli strains which have genetic modifications in carbon metabolic pathways and electron transport chain were constructed and evaluated. By choosing a proper expression system for styAB and through a series of metabolic engineering of the host strain, it was possible to obtain a highly active whole-cell SMO biocatalyst with the specific activity > 500 U/ g cell which is more than 5 fold higher than the highest one reported so far.