Enhancing delta-tocotrienol production in Saccharomyces cerevisiae via metabolic engineering strategies in conjunction with the mutagenesis of tocopherol cyclase

Delta (δ)-tocotrienol is a member of the vitamin E family and exhibits bioactivities such as antioxidant, anti-inflammatory, and neuroprotective activities. As a nutrient with protective effects on human health, δ-tocotrienol has broad application prospects in food, cosmetic, and pharmaceutical industries. The construction of efficient microbial cell factories capable of δ-tocotrienol production using synthetic biology approaches is an effective strategy for supplementing or even replacing the vitamin E supply chain in the future. The current study successfully enhanced the biosynthesis of δ-tocotrienol in <i>Saccharomyces cerevisiae</i> by combining metabolic engineering and enzyme engineering strategies. Specifically, the substrate channel constructed by the sequential fusion of the enzymes PaCrtE and SyHPT successfully increased the supply of the key precursor MGGBQ, resulting in a significant increase in the production of δ-tocotrienol. <i>In situ</i> extraction and optimization of the expression of transporter protein PDR1 increased the efflux of δ-tocotrienol, directing the metabolic flux toward the product δ-tocotrienol. To enhance the catalytic activity of the key rate-limiting enzyme tocopherol cyclase from <i>Arabidopsis thaliana</i> (AtTC), semirational protein design was conducted herein. The mutant AtTC^T87S was found to increase the production of δ-tocotrienol by 2.3 times compared to that obtained with the wild-type enzyme. AtTC^T87S can thus be universally used for synthetic biology strategies in future studies to enhance the microbial heterologous production of δ-tocotrienol. The strain T08 was finally obtained herein; the numerous metabolic engineering strategies discussed in this study were integrated into this strain, allowing the production of 4337.3 μg/L of δ-tocotrienol in a shake-flask fermentation, which is 8.9 times that of the yield obtained with the initial strain T03. Scaling up to a 5-L fermentation tank resulted in a δ-tocotrienol yield of 16.9 mg/L.