title: Biosynthesis of the High-Value Plant Secondary Product Benzyl Isothiocyanate via Functional Expression of Multiple Heterologous Enzymes in Escherichia coli.
abstract: Plants produce a wide variety of secondary metabolites that are highly nutraceutically and pharmaceutically important. Isothiocyanates, which are found abundantly in cruciferous vegetables, are believed to reduce the risk of several types of cancers and cardiovascular diseases. The challenges arising from the structural diversity and complex chemistry of these compounds have spurred great interest in producing them in large amounts in microbes. In this study, we aimed to synthesize benzyl isothiocyanate in Escherichia coli via gene mining, pathway engineering, and protein modification. Two chimeric cytochrome P450 enzymes were constructed and functionally expressed in E. coli. The E. coli cystathionine β-lyase was used to replace the plant-derived C-S lyase; its active form cannot be expressed in E. coli. Suitable desulfoglucosinolate:PAPS sulfotransferase from Arabidopsis thaliana ecotype Col-0 and myrosinase from Brevicoryne brassicae were successfully mined from the database. Biosynthesis of benzyl isothiocyanate by the combined expression of the optimized enzymes in vitro was confirmed by gas chromatography-mass spectrometry analysis. This study provided a proof of concept for the production of benzyl isothiocyanate by microbially produced enzymes and, importantly, laid the groundwork for further metabolic engineering of microbial cells for the production of isothiocyanates.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Feixia
authors.initials: F
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Han
authors.initials: H
authors.lastname: Yang
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Limin
authors.initials: L
authors.lastname: Wang
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D017879:Isothiocyanates / Q000378:metabolism*; D008190:Lyases / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D064210:Secondary Metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D017879:Isothiocyanates; D011994:Recombinant Proteins; C031403:benzyl isothiocyanate; D003577:Cytochrome P-450 Enzyme System; C408883:CYP79A2 protein, Arabidopsis; C431937:CYP83B1 protein, Arabidopsis; C022321:PAPS sulfotransferase; D015238:Sulfotransferases; D006026:Glycoside Hydrolases; C022739:thioglucosidase; D008190:Lyases; C000604089:AT2G20610 protein, Arabidopsis; D013437:Carbon-Sulfur Lyases; C019965:cystathionine beta-lyase
keywords: benzyl isothiocyanate; microbial synthesis; plant secondary compound; protein engineering