Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l⁻¹ extracellular MCFAs—a more than 250-fold improvement over the original strain.

中链脂肪酸（MCFA； C6-C12）是用于生物燃料和油脂化学生产的有价值的分子。然而，由于MCFA的细胞毒性，使用微生物生物催化剂有效地合成这些脂肪酸具有挑战性。在这项研究中，内源性脂肪酸合酶（FAS）和正交细菌I型FAS都被设计用于酿酒酵母中的MCFA生产。。为了提高细胞对毒性MCFA的耐受性，我们进行了膜转运蛋白Tpo1的定向进化和应变适应性实验室进化，这分别使MCFA产量提高了1.3±0.3-倍和1.7±0.2-倍。因此，我们进一步设计了高抗性菌株，以增加对MCFA的代谢通量。在单一蛋白质/酶水平，途径水平和细胞水平上对酵母进行的多维工程设计，再加上优化的培养过程，导致产生了> 1 g l ^-1的细胞外MCFA，与传统方法相比提高了250倍以上原始的应变。