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.
retinolSaccharomyces cerevisiaefusion proteinretinol transport
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