丁酸乙酯是一种具有特殊果香的短链脂肪酸乙酯,是浓香型白酒等酒品中不可或缺的香气成分,在食品饮料等行业中广泛应用。该研究基于前期构建的产丁酸乙酯酿酒酵母E01,采用多层次代谢工程策略,进一步优化其丁酸乙酯合成效率。首先,敲除ADH1减少碳流竞争,但导致氧化还原失衡,丁酸乙酯产量降至原菌株的8.97%。随后,引入NADH调节基因(cPOS5、mPOS5、udhA和noxE),菌株Eu(过表达udhA)丁酸乙酯产量达(69.94±5.04) mg/L,较E01提高42.88%。进一步在引入NADH调节基因的基础上敲除ADH1,菌株EmD丁酸乙酯产量恢复至(54.66±6.73) mg/L,较E01提升11.66%。最后,引入突变型乙酰辅酶A合成酶基因SeACSL641P,菌株EmDS丁酸乙酯产量达(61.82±1.05) mg/L,较E01提升19.56%,但仍低于Eu菌株,表明NADH调节基因能优化乙醇转化和酯类合成,但无法完全弥补ADH1敲除引发的代谢缺陷。该研究通过协同调控碳流、氧化还原平衡和乙酸代谢,显著提升了丁酸乙酯合成效率,也为酿酒酵母乙醇生成阻断和氧化还原平衡调节相关代谢工程改造提供了有益参考。
Ethyl butyrate is a short-chain fatty acid ethyl ester with a distinctive fruity aroma, serving as an essential aroma compound in Nongxiangxing Baijiu and other alcoholic beverages.It is extensively applied in food and beverage industries.This study, based on the previously constructed ethyl butyrate-producing Saccharomyces cerevisiae E01, employed a multi-level metabolic engineering strategy to further optimize its ethyl butyrate biosynthesis efficiency.Firstly, the knockout of ADH1 reduced carbon flow competition but led to redox imbalance, decreasing ethyl butyrate production to 8.97% of the parental strain.Subsequently, NADH-regulating genes (cPOS5, mPOS5, udhA, and noxE) were introduced.Strain Eu (overexpressing udhA) achieved ethyl butyrate production of (69.94±5.04) mg/L, representing a 42.88% increase over E01.Furthermore, ADH1 knockout was implemented in the NADH-regulated background, enabling strain EmD to restore ethyl butyrate production to (54.66±6.73) mg/L, corresponding to an 11.66% improvement compared to E01.Finally, the mutant acetyl-CoA synthase gene SeACSL641P was introduced, enabling strain EmDS to produce (61.82±1.05) mg/L ethyl butyrate, a 19.56% increase over E01 but still lower than strain Eu.These results indicate that NADH-regulated genes can optimize ethanol conversion and ester synthesis but cannot fully compensate for the metabolic disruptions caused by ADH1 knockout.This study achieved significant enhancement of ethyl butyrate biosynthesis through synergistic modulation of carbon flux, redox homeostasis, and acetate metabolism, providing valuable references for metabolic engineering strategies targeting ethanol pathway disruption and redox balance regulation in Saccharomyces cerevisiae.
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