(191x) Efficient Production of (Z)-?-Santalol with Multi-Pathway Engineering in Saccharomyces Cerevisiae

Sandalwood oil fragrance is one of the world’s most highly prized fragrances produced by tropical sandalwoods, which is not only broadly applied in the fragrance industry, but also has various biological activities, such as anti-cancer, anti-tumor, anti-viral, anti-diarrhoeal, etc. (Z)-α-santalol and (Z)-β-santalol are the most important contributors to sandalwood oil fragrance. Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds. As a multi-substrate P450, SaCYP736A167 stereo-selectively produces (Z)-a-santalol from α-santalene. In this study, we constructed the (Z)-α-santalol pathway by heterologous expressions of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR), a santalene synthase (SaCYP736A167), and an NADPH-dependent cytochrome P450 reductase (CPR) in Saccharomyces cerevisiae. Further efforts were paid to increase the production of (Z)-α-santalol production with down regulation of the competitive pathways by expressing ERG9 in the ergosterol pathway with the weak HXT1 promoter and deleting LPP1 and DPP1 in the farnesol pathway to divert the carbon flux from sterol synthesis towards a-santalene. Finally, with the fusion of eleven different signal peptides targeting mitochondrion with 8 genes in MVA pathway and santalene synthas to boost acetyl-CoA utilization and the deletion of PAH1 to expand the nuclear/ER membrane to anchor more SaCYP736A167, the production of (Z)-α-santalol was improved greatly. The present work paved one road to produce this attracting fragrance component in industry.