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.

An Evaluation of the Anti-Carcinogenic Response of Major Isothiocyanates in Non-Metastatic and Metastatic Melanoma Cells

“…Specifically, it has been reported that the consumption of cruciferous vegetables is associated with reduced risk of cancer development, a capacity associated with their high content of sulfur-containing phytochemicals known as isothiocyanates (ITCs) [23][24][25][26]. These are secondary metabolites obtained from hydrolysis of their precursor molecules (glucosinolates), by myrosinase, an enzyme activated after plant tissue disruption [27] (Figure 1). The importance of ITCs as nutraceutical agents is reflected upon their capacity to modulate enzymes involved in (i) detoxification, (ii) apoptotic induction, (iii) cell cycle growth arrest and (iv) interactions with various other cellular pathways of tumor growth and invasion known to be deregulated in various cancers including MM [28][29][30][31][32][33][34][35][36][37][38].…”

Gut Glucosinolate Metabolism and Isothiocyanate Production

“…Given that many of the identified metabolizers of the GSLs include groups of beneficial bacteria such as the lactobacilli and bifidobacteria that are often utilized as probiotics, this opens up potential opportunities to exploit such bacteria as dietary supplements with GSL‐containing foods that would provide health benefits particularly to low ITC secreting individuals as part of their personalized nutrition. Recently, a study has looked at the potential for expressing the glucotropaeolin biosynthetic pathway in E. coli together with a myrosinase of B. brassicae with some success as the authors were able to show the in vitro formation of benzylisothiocyanate . If this technology can be developed where ITCs are actually produced during fermentation then it could potentially allow the delivery of sulforaphane in the gut.…”

Gene Engineering of Lamiaceae Family: Basic and Applied Research

“…In addition, the desulphoglucosinolate: PAPS-SULT from Arabidopsis thaliana (Col-0 ecotype) and the myrosinase from Brevicorynebrassicae (aphid) were used. The biosynthesis of benzyl isothiocyanates by the combined expression of in vitro optimized enzymes was confirmed by GC-MS analysis (Liu et al, 2016).…”