Metabolic Engineering Of Saccharomyces Cerevisiae For Efficient Retinol Synthesis

Retinol,the major active form of vitamin A,is crucial for maintaining vision,immune function,growth,and development,inhibiting tumor growth,and improving anemia.Furthermore,it is widely used in various fields such as food,drugs,and cosmetics.Currently,the synthesis of retinol mainly includes natural extraction and chemical synthesis.However,retinol extraction from animal tissues is scattered,the steps are cumbersome,and the cost is high.Chemical synthesis has a high technical threshold and high energy consumption.Microbial synthesis has many advantages,including short cycles,low cost,mild fermentation conditions,high safety.Therefore,constructing microbial cell factories has become an effective way to synthesize many high-value chemicals.In this paper,a heterologous de novo synthesis pathway of retinol was constructed using Saccharomyces cerevisiae CEN.PK2-1C as a chassis strain.Strategies such as multimodule engineering,transporter engineering,and enzyme engineering were employed to enhance retinol synthesis in S.cerevisiae by enhancing precursor supply,increasing the expression of key enzymes,increasing retinol turnover,and stability.The main findings are as follows:（1）Five heterologous essential genes were integrated into the genome of S.cerevisiae CEN.PK2-1C to realize the synthesis of retinol in S.cerevisiae,and the titer of retinol reached0.9 mg·L^-1.The metabolic fluxes of endogenous mevalonate synthesis module,central carbon metabolism module,squalene synthesis module,exogenousβ-carotene synthesis module,and retinol synthesis module in retinol metabolism pathway was regulated by multimodule engineering.The metabolic network of retinol was systematically optimized,and the titer of retinol reached 153.6 mg·L^-1.（2）Transporter engineering was used to modulate the efflux of the precursor retinal,thereby increasing retinol production.The transporter species involved in the efflux of the precursor were verified as ABC transporters by adding transporter inhibitors and knocking out the transporter Pdr5p,which increased the intracellular precursor retinal production by 170.1%and the total retinol production by 7.2%,to 164.6 mg·L^-1.（3）The key retinol dehydrogenase ENV9 was semi-rationally modified by enzyme engineering after screening it out through protein sequence comparison mining and fermentation verification.Thr99 of ENV9 was mutated into alanine,and the titer of retinol reached 254.2 mg·L^-1.Overexpressing the mutated ENV9 and the retinol dehydrogenase Ybbo derived from Escherichia coli improved the conversion rate of the retinal to retinol,further improving the titer to 425.7 mg·L^-1.（4）The fermentation extraction conditions were optimized according to the product characteristics.Retinol has relatively active chemical properties and is easily oxidized.The effect of adding dodecane,olive oil and antioxidants on retinol titer was compared.Olive oil was used as an extractant in two-phase fermentation to retard retinol oxidation and improve retinol stability.After 120 h of fermentation,the shake flask level retinol production reached1.2 g·L^-1.