On September 12, 2025, Yanyan Wang et al. from Beijing University of Chemical Technology published a research paper titled “Engineering the Cytochrome P450 Oxidation System To Enhance Benzyl Glucosinolate Production in Saccharomyces cerevisiae” in the Journal of Agricultural and Food Chemistry.
Abstract
Benzyl isothiocyanate (BITC) belongs to the isothiocyanate compound family, a class of natural products exhibiting anticancer, antibacterial, and anti-inflammatory activities. Microbial synthesis offers a highly promising alternative to traditional plant extraction methods. In the BITC biosynthetic pathway, cytochrome P450 enzymes CYP79A2 and CYP83B1 catalyze the rate-limiting steps. This study focuses on the systematic modification of the P450 oxidation system to enhance the yield of benzyl glucosinolate (BGLS) in Saccharomyces cerevisiae—a direct and stable precursor of BITC. The study first integrated the complete biosynthetic pathway into the δ site of the yeast genome, creating a quadruple-copy strain that achieved a BGLS yield of 28.00 mg/L. Subsequently, a series of optimization strategies significantly enhanced the efficiency of the oxidation system: Compatibility of P450 reductase (CPR) was optimized, heme biosynthesis was enhanced to increase cofactor supply, endoplasmic reticulum membrane area was expanded to accommodate more P450 enzymes, and intracellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were elevated to support redox reactions. Following these modifications, the engineered strain achieved a BGLS yield of 62.95 mg/L under shake flask culture conditions, setting the highest reported yield to date.
Introduction
Isothiocyanate compounds (ITCs) are a class of natural products primarily found in cruciferous plants. Based on their precursor amino acids, ITCs can be classified into three categories: aliphatic, aromatic, and indole derivatives. These compounds exhibit diverse biological activities, including anti-inflammatory, anti-cancer, and antioxidant effects. Representative examples include benzyl isothiocyanate (BITC) and sulforaphane (SFN).
Currently, ITCs are primarily obtained through plant extraction methods, which face several critical limitations. First, the cultivation cycle of cruciferous crops (e.g., broccoli) is lengthy (3–6 months), and the yield of target compounds is low, severely restricting large-scale production. Second, the abundance of isothiocyanates in natural plants is extremely low. For instance, broccoli contains only 0.1–1.2 mg/g dry weight of sulforaphane precursors, while phenethyl isothiocyanate constitutes merely 0.05%–0.3% of mustard seeds. Product yields vary across plant species and growth stages. Combined with environmental pressures from large-scale cultivation and pollution from solvent extraction methods, these factors further increase production costs and pose challenges to sustainable production. Furthermore, the active −N=C=S group in isothiocyanate molecules makes them prone to degradation reactions, including hydrolysis (forming amines and hydrogen sulfide), oxidation (forming disulfides), and polymerization. Humidity, high temperatures, and oxygen accelerate these degradation processes, necessitating strict storage conditions to maintain stability. The chemical synthesis process is operationally complex and generates multiple undesirable byproducts (e.g., sulfides, cyanides, and unreacted halogenated alkanes), making it difficult to apply in industrial-scale production. In contrast, microbial heterologous biosynthesis has emerged as a highly promising sustainable production solution. Microbial synthesis not only ensures the sustainability of industrial production but also reduces manufacturing costs. Compared to prokaryotic expression systems, the yeast expression platform offers distinct advantages: First, it enables post-translational modification of eukaryotic cytochrome P450 enzymes. Second, it facilitates stable integration of foreign genes through multiple copy sites on the chromosome. In contrast, the Escherichia coli expression system is often constrained by plasmid instability and protein aggregation issues. Recent advances in yeast genetic engineering have demonstrated this platform's capability to successfully synthesize diverse natural products, highlighting its immense potential to overcome limitations of traditional extraction methods.
The biosynthetic pathways of several isothiocyanates (ITCs) have been elucidated. Taking the biosynthesis of benzyl glucosinolate (BGLS) as an example, its pathway originates from the shikimic acid pathway, using phenylalanine as the substrate. Two consecutive cytochrome P450-mediated oxidation reactions—catalyzed by CYP79A2 and CYP83B1, respectively—convert phenylalanine into an oxynitrile. Subsequently, these oxynitriles react with glutathione in the presence of glutathione S-transferase GSTF9 to form S-alkyl thiolactone adducts. Subsequently, the glucosinolate-γ-glutamyl hydrolase GGP1 and the carbonyl sulfide hydrolase SUR1 cleave this conjugate into thiol hydroxy acid and pyruvate. Uridine diphosphate glucosyltransferase UGT74B1 then catalyzes the attachment of uridine diphosphate glucose (UDP-glucose) to the sulfhydryl group, yielding desulfated benzyl glucosinolate (dsBGLS). Finally, under the action of sulfotransferase SOT16, desulfobenzyl-benzyl-mustard-like glycoside loses its sulfonate group, converting to benzyl-benzyl-mustard-like glycoside (BGLS). Recent studies demonstrate that de novo synthesis of BGLS at 8.30 mg/L was achieved by expressing this biosynthetic pathway in Escherichia coli via plasmid-based expression and optimizing the supply of the cofactor 3'-adenosine 5'-phosphosulfate (PAPS). In Saccharomyces cerevisiae, chromosomal integration technology combined with overexpression of sulfotransferase SOT16 and adenosine 5'-phosphosulfate kinase yielded 5.20 mg/L of BGLS.
Despite these advances, BGLS production remains constrained by multiple factors, including low catalytic efficiency of cytochrome P450 enzymes, insufficient precursor supply, and metabolic flux imbalance, with P450 enzyme activity being the core limiting factor. Multiple strategies have been employed to enhance P450 enzyme catalytic efficiency: In prokaryotic expression systems, methods such as codon optimization, N-terminal modification, fusion tag addition, and co-expression of molecular chaperones can improve the soluble expression levels of P450 enzymes in E. coli. Covalent assembly of P450 with P450 reductase (CPR) using SpyCatcher/SpyTag technology enhances electron transfer efficiency between the two enzymes. In eukaryotic expression systems, Saccharomyces cerevisiae provides an ideal platform for in-depth exploration of P450 enzyme functions due to its native organelle architecture. Optimization strategies focus on modifying NADPH supply pathways, expanding endoplasmic reticulum membrane area, and combinatorial screening of CYP-CPR pairing combinations to construct efficient oxidative metabolic networks. Furthermore, rational design and directed evolution-based protein engineering approaches can further modify the substrate-binding pockets of P450 enzymes, thereby enhancing their catalytic activity and substrate specificity.
This study targets the P450 oxidation system and its initiator enzyme CYP79A2 for optimization. The initial chassis strain was constructed by integrating the benzyl glucosinolate (BGLS) synthesis pathway into the δ multicopy site of Saccharomyces cerevisiae. Subsequent systematic modifications included: screening for CYP79A2-compatible P450 reductase (CPR) isoforms, enhancing heme biosynthesis, expanding the endoplasmic reticulum membrane area, and increasing intracellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels. Through this series of modifications, the engineered strain Y141 was ultimately obtained, achieving a BGLS yield of 62.95 mg/L under shake flask culture conditions. These results demonstrate that targeted optimization of the cytochrome P450 system can significantly enhance the synthetic yield of sinigrin in metabolically engineered yeast.
