Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme’s substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.

中链脂肪醇（MCFOH，C6至C12）是化石燃料（例如柴油和喷气燃料）的潜在替代品，在各种制造过程中具有广泛的应用。如今，MCFOH主要来自石化产品或植物油，而微生物生物合成是可扩展，可靠且可持续的替代品。在这里，我们的目标是建立能够选择性产生MCFOHs的酿酒酵母平台。这可以通过定制来自海洋分枝杆菌的细菌羧酸还原酶的特性来实现。（MmCAR）。实施了广泛的蛋白质工程，包括定向进化，结构指导的半理性设计和合理设计。依赖于酶底物中链脂肪酸（MCFA）的解毒作用，使用生长偶联高通量筛选测定法鉴定了具有增强活性的MmCAR变体。详细的表征表明，MmCAR的特异性和催化活性都得到了成功的改善，具有最佳MmCAR变体的酵母菌株产生的MCFOH比表达未修饰酶的菌株多2.8倍。通过删除天然的MCFA出口基因TPO1，进一步提高了MCFOH的产量，最终菌株的效价为252 mg / L，这表明酿酒酵母在基本培养基中的MCFOH产量有了显着提高。