Department of Chemical and Environmental Engineering, University of California, 900 University Avenue, Bourns Hall, Suite A220, Riverside, California 92521, United States.
ACS Synth Biol. 2023 Apr 21;12(4):1109-1118. doi: 10.1021/acssynbio.2c00599. Epub 2023 Mar 27.
Campesterol is a major phytosterol that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites, such as the phytohormone brassinosteroids. Recently, we established a campesterol-producing yeast strain and extended the bioproduction to 22-hydroxycampesterol and 22-hydroxycampest-4-en-3-one, the precursors to brassinolide. However, there is a trade-off in growth due to the disrupted sterol metabolism. In this study, we enhanced the growth of the campesterol-producing yeast by partially restoring the activity of the sterol acyltransferase and engineering upstream FPP supply. Furthermore, genome sequencing analysis also revealed a pool of genes possibly associated with the altered sterol metabolism. Retro engineering implies an essential role of ASG1, especially the C-terminal asparagine-rich domain of ASG1, in the sterol metabolism of yeast especially under stress. The performance of the campesterol-producing yeast strain was enhanced with the titer of campesterol to 18.4 mg/L, and the stationary OD was improved by ∼33% compared to the unoptimized strain. In addition, we examined the activity of a plant cytochrome P450 in the engineered strain, which exhibits more than 9-fold higher activity than when expressed in the wild-type yeast strain. Therefore, the engineered campesterol-producing yeast strain also serves as a robust host for the functional expression of plant membrane proteins.
菜油甾醇是一种主要的植物甾醇，在调节膜性质方面发挥着重要作用，并作为多种特殊代谢物的前体，如植物激素油菜素甾体。最近，我们建立了一株能够生产菜油甾醇的酵母菌株，并将生物合成扩展到 22-羟基菜油甾醇和 22-羟基菜甾-4-烯-3-酮，这是油菜素内酯的前体。然而，由于固醇代谢的紊乱，生长会出现权衡。在这项研究中，我们通过部分恢复甾醇酰基转移酶的活性和工程上游 FPP 供应来增强生产菜油甾醇的酵母的生长。此外，基因组测序分析还揭示了一组可能与改变的固醇代谢有关的基因。回溯工程表明 ASG1 特别是 ASG1 的 C 端富含天冬酰胺的结构域在酵母固醇代谢中起着至关重要的作用，尤其是在应激条件下。与未经优化的菌株相比，生产菜油甾醇的酵母菌株的性能得到了增强，菜油甾醇的产量达到 18.4mg/L，静止 OD 提高了约 33%。此外，我们还检测了工程菌株中植物细胞色素 P450 的活性，其活性比在野生型酵母菌株中表达时高出 9 倍以上。因此，经过工程改造的生产菜油甾醇的酵母菌株也可以作为植物膜蛋白功能表达的强大宿主。
