(553b) Optimizing Campesterol-Producing Yeast Strains for Enhanced Functional Reconstitution of Plant Membrane Enzyme

Campesterol is an important phytosterol regulating membrane properties, which is also the precursor of many phytosteroids of pharmaceutical values. In our previous work, we have established a set of engineered yeast strains accumulating free campesterol. However, the growth of the engineered campesterol-producing strain is deteriorated by the inactivation of the endogenous sterol acyltransferases, which impedes further application of the engineered strain to synthesize downstream phytosteroids of higher economic values. In this study, the growth of campesterol-producing yeast strain was optimized by restoring and tuning sterol esterification and engineering upstream FPP supply. The CRISPR-Cas9 library for inactivation and upregulation of genes of interests at genome-scale was establish and applied in the strain. Strains with enhanced growth was selected. Combination of optimization from different strategies resulted in improved growth and higher production of campesterol, which serves as the foundation to reconstitute the diverse phytosteroid structures. In addition, the campesterol-accumulating yeast strain is a preferred host for the heterologous reconstitution of plant cytochrome P450s, which exhibits higher enzymatic efficiency than expressed in wildtype yeast.