灵芝酸3-羟基-8,24-羊毛甾二烯-26-酸（HLDOA）是蘑菇真菌灵芝特有的三萜类抗肿瘤化合物。鉴于蘑菇真菌遗传操作困难，实现HLDOA的异源生物合成将为其高效生产奠定基础。我们前期发现过表达灵芝的细胞色素P450酶CYP5150L8首次在酿酒酵母中实现HLDOA的异源生产，并利用双可调式系统表达CYP5150L8和P450还原酶大幅提升了单位细胞的产量，但菌株生长受到抑制。构建适配的细胞生理代谢及调控机制，使细胞生长与产物合成平衡进行，是高效生产HLDOA的关键。本项目拟采用可追踪式基因组扰动的方法对上述工程菌进行定向进化，筛选生长不受抑制且高产HLDOA的突变体，并发现协同菌株生理代谢与HLDOA生物合成的关键靶点。再基于多组学和代谢流量分析等技术手段，解析筛选所获关键突变体生理代谢与HLDOA生物合成的适配机制并指导菌株改造，为其他重要灵芝酸以及三萜类化合物的高效生产提供借鉴。

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26 oic acid (HLDOA), a unique secondary metabolite of mushroom Ganoderma lucidum, is an anti-tumor triterpenoid. Owing to the difficulties in genetic manipulation of mushroom, biosynthesis of HLDOA in a heterologous host lays the foundation for its efficient bioproduction. In our previous work, we firstly found overexpression of a cytochrome P450 (CYP5150L8) from Ganoderma lucidum enabled heterologous production of HLDOA by Saccharomyces cerevisiae. On this basis, HLDOA titer produced by per cell was significantly enhanced by using a dual tunable system to express CYP5150L8 and a P450 reductase, but cell growth of the engineered strains were suppressed. Construction of an adaptive cellular physiological metabolism and regulation, with balanced cell growth and efficient product biosynthesis, is crucial for hyperproduction of HLDOA. In this proposal, a trackable genome-wide perturbation strategy will be used for directed evolution of the above mentioned engineered strains to screen for mutants with improved cell growth and higher HLDOA production, and to discover the determinants of coordinating cell metabolism and HLDOA biosynthesis. Based on the multi-level omics analyses and metabolic flux analyses, the adaptation mechanism between cell physiological metabolism and HLDOA biosynthesis in key mutants will be resolved to guide strain engineering. This study will serve as a reference and provide insights to efficient bioproduction of other important ganoderic acids and triterpenoids.