The economic viability of bioprocesses is constrained by the limited range of operating conditions that can be tolerated by the cell factory. Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular components that contribute to maintenance of appropriate membrane function, such as: flotillin-like proteins, membrane structural proteins, and membrane lipids. Specifically, we exploited the promiscuity of squalene hopene cyclase (SHC) to produce polycyclic terpenoids with properties analogous to cholesterol. Strains producing these cholesterol-like molecules were visualized by AFM and height features were observed. Production of these cholesterol-like molecules was associated with increased tolerance towards a diversity of chemicals, particularly alcohols, and membrane trafficking processes such as lipid droplet accumulation and production of extracellular vesicles. This engineering approach improved the production titers for wax-esters and ethanol by 80- and 10-fold, respectively. Expression of SHC resulted in the production of steroids. Strains engineered to also express truncated squalene synthase (tERG9) produced diplopterol and generally did not perform as well. Increased expression of several membrane-associated proteins, such as YqiK, was observed to impact vesicle trafficking and further improve tolerance relative to SHC alone, but did not improve bio-production. Deletion of YbbJ increased lipid droplet accumulation as well as production of intracellular wax esters. This work serves as a proof of concept for engineering strategies targeting membrane physiology and trafficking to expand the production capacity of microbial cell factories.

生物工艺的经济可行性受到细胞工厂可以容忍的有限操作条件范围的限制。微生物细胞膜的工程化是一种可以提高稳健性并因此改变这一范围的策略。在这项工作中，我们针对有助于维持适当膜功能的细胞成分，例如：flotillin 样蛋白、膜结构蛋白和膜脂。具体来说，我们利用角鲨烯环化酶 (SHC) 的混杂性来生产具有类似于胆固醇的特性的多环萜类化合物。产生这些胆固醇样分子的菌株通过 AFM 可视化并观察到高度特征。这些类胆固醇分子的产生与对多种化学物质的耐受性增加有关，特别是酒精，和膜运输过程，例如脂滴积累和细胞外囊泡的产生。这种工程方法将蜡酯和乙醇的生产效价分别提高了 80 倍和 10 倍。SHC 的表达导致类固醇的产生。经工程改造以表达截短角鲨烯合酶 (tERG9) 的菌株产生双氯特罗，但通常表现不佳。观察到几种膜相关蛋白（如 YqiK）的表达增加会影响囊泡运输并进一步提高相对于单独的 SHC 的耐受性，但并未改善生物生产。YbbJ 的缺失增加了脂滴的积累以及细胞内蜡酯的产生。