Efficient production of retinol in Yarrowia lipolytica via synergistic enzyme and redox engineering

Retinol (Vitamin A) is a micronutrient that is essential for human health. This study has engineered the oleaginous yeast Yarrowia lipolytica for high-level de novo retinol biosynthesis. The precursor flux based on a previously established high-lycopene strain was further strengthened by overexpressing GGPP synthase and the β-carotene synthesis pathway, which achieved 822.7 mg·L −1 β-carotene. The retinol pathway was subsequently established by introducing β-carotene dioxygenase (Blh) and retinol dehydrogenase (RDH12). The catalytic activity of Blh was significantly improved using directed evolution in order to address the excessively low catalytic efficiency of the key rate-limiting enzymes. Furthermore, retinol production was improved using co-factor engineering to increase the availability of NADPH and knocking out the oxidase gene to maintain the cellular redox balance. Finally, the retinol titer reached 8.92 g·L −1 using butylated hydroxytoluene (BHT) as an antioxidant in a 5-L fed-batch fermentation process, which represents the highest reported microbial titer, establishing Y. lipolytica as a potent platform for sustainable vitamin A production.