Engineering the Cytochrome P450 Oxidation System To Enhance Benzyl Glucosinolate Production in <i>Saccharomyces cerevisiae</i>

Benzyl isothiocyanate (BITC) belongs to the family of isothiocyanates, a group of natural compounds known for their anticancer, antibacterial, and anti-inflammatory properties. Microbial synthesis offers a promising alternative method to traditional plant extraction. In BITC biosynthesis, the cytochrome P450 enzymes CYP79A2 and CYP83B1 catalyze the rate-limiting steps. This study focused on systematically engineering the P450 oxidation system to enhance the production of benzyl glucosinolate (BGLS)─the direct and stable precursor of BITC─in <i>Saccharomyces cerevisiae</i>. First, a four-copy strain was constructed by integrating the full biosynthetic pathway into the δ sites of the yeast genome, achieving a BGLS production of 28.00 mg/L. Subsequently, the efficiency of the oxidation system was significantly improved by optimizing the P450 reductase (CPR) compatibility, enhancing heme biosynthesis to boost cofactor supply, expanding the endoplasmic reticulum membrane to accommodate P450 enzymes, and elevating intracellular NADPH levels to support redox reactions. With these efforts, the final engineered strain produced 62.95 mg/L of BGLS in shake-flask cultures, representing the highest reported titer to date.