Production of benzylglucosinolate by engineering and optimizing the biosynthetic pathway in Saccharomyces cerevisiae

Glucosinolates are amino acid-derived defense compounds characteristic of the Brassicales order. Benzylglucosinolate (BGLS) derived from phenylalanine is associated with health-promoting effects, which has primed a desire to produce BGLS in microorganisms for a stable and rich source. In this study, we engineered the BGLS production in Saccharomyces cerevisiae by either stably integrating the biosynthetic genes into the genome or introducing them from plasmids. A comparison of the two approaches exhibited a significantly higher level of BGLS production (9.3-fold) by expression of the genes from genome than from plasmids. Towards optimization of BGLS production from genes stably integrated into the genome, we enhanced expression of the entry point enzymes CYP79A2 and CYP83B1 resulting in a 2-fold increase in BGLS production, but also a 4.8-fold increase in the biosynthesis of the last intermediate desulfo-benzylglucosinolate (dsBGLS). To alleviate the metabolic bottleneck in the last step converting dsBGLS to BGLS by 3-phosphoadenosine-5-phosphosulfate (PAPS)-dependent sulfotransferase, SOT16, we first obtained an increased BGLS production by 1.7-fold when overexpressing SOT16. Next, we introduced APS kinase APK1 of Arabidopsis thaliana for efficient PAPS regeneration, which improved the level of BGLS production by 1.7-fold. Our work shows an optimized production of BGLS in S. cerevisiae and the effect of different approaches for engineering the biosynthetic pathway (plasmid expression and genome integration) on the production level of BGLS.