Combined metabolic and enzymatic engineering for de novo biosynthesis of δ-tocotrienol in Yarrowia lipolytica

δ-Tocotrienol, an isomer of vitamin E with anti-inflammatory, neuroprotective and anti-coronary arteriosclerosis properties, is widely used in health care, medicine and other fields. Microbial synthesis of δ-tocotrienol offers significant advantages over plant extraction and chemical synthesis methods, including increased efficiency, cost-effectiveness and environmental sustainability. However, limited precursor availability and low catalytic efficiency of key enzymes remain major bottlenecks in the biosynthesis of δ-tocotrienol. In this study, we assembled the complete δ-tocotrienol biosynthetic pathway in Yarrowia lipolytica and enhanced the precursor supply, resulting in a titre of 102.8 mg/L. The catalytic efficiency of the rate-limiting steps in the pathway was then enhanced through various strategies, including fusion expression of key enzymes homogentisate phytyltransferaseand and tocopherol cyclase, semi-rational design of SyHPT and multi-copy integration of pathway genes. The final a δ-tocotrienol titre in a 5 L bioreactor was 466.8 mg/L following fed-batchfermentation. This study represents the first successful de novo biosynthesis of δ-tocotrienol in Y. lipolytica, providing valuable insights into the synthesis of vitamin E-related compounds.