Retinol is one of the main active forms of vitamin A, crucial for the organism's growth, development, and maintenance of eye and skin functions. It is widely used in cosmetics, pharmaceuticals, and feed additives. Although animals lack a complete pathway for synthesizing vitamin A internally, they can obtain vitamin A directly through diet or convert β-carotene acquired from the diet. To boost the research on the biosynthesis of retinol, three different sources of alcohol dehydrogenase were firstly screened based on the β-carotene synthesis platform CAR*1. It was determined that ybbO from Escherichia coli exhibited the highest catalytic activity,with a conversion rate of 95. 6%. To further enhance the reaction rate and yield of retinol, protein fusion technology was employed to merge two adjacent enzymes, blh and ybbO, within the retinol synthesis module. The evaluation was conducted using the high-yield engineered strain CAR*3 of β-carotene. The optimal combination, blh-GGGS-ybbO, was obtained, with a 44. 9% increase in yield after fusion, reaching(111. 1± 3. 5) mg·L~(-1). Furthermore, through the introduction of human-derived retinol-binding protein(RBP4) and transthyretin(TTR), the process of hepatic cell secreting retinol was simulated in Saccharomyces cerevisiae, leading to an increased retinol yield of(158. 0±13. 1)mg·L~(-1). Finally, optimization strategies including overexpressing INO2 to enhance the reaction area for β-carotene synthesis, enhancing hemoglobin VHb expression to improve oxygen supply, and strengthening PDR3m expression to facilitate retinol transport were implemented. A two-stage fermentation process resulted in the successful elevation of retinol production to(2 320. 0±26. 0)mg·L~(-1) in the fermentation tank of 5 L, which provided a significant foundation for the industrial development of retinol.
Keywords: Saccharomyces cerevisiae; fusion protein; retinol; retinol transport.