Efficient production of (S)-limonene and geraniol by peroxisomal compartmentalization of the monoterpene biosynthetic pathway in Saccharomyces cerevisiae

Abstract

Many monoterpenoids have valuable applications in the cosmetics, food, fuel, and pharmaceutical industries (e.g. geraniol, (S)-limonene and (S)-perillyl alcohol). Due to their versatility, the market demands for monoterpenoids have been growing over the past decades, highlighting the need for an environmentally friendly, stable, and cost-effective synthesis of these molecules. With the rapid development of metabolic engineering tools, microbial hosts have emerged as a promising alternative to produce valuable molecules. The bakers yeast S. cerevisiae possesses an efficient endogenous mevalonate (MVA) pathway, produces naturally high amounts of sterols, and is resistant to toxic chemicals and stressful industrial fermentation conditions, making it suitable for large-scale production of monoterpenoids. In this study, metabolic engineering of the yeast S. cerevisiae was carried out to build robust platform strains for geraniol and (S)-limonene synthesis. Monoterpenoids are produced from geranyl pyrophosphate (GPP) through the MVA pathway. Erg20 is a farnesyl pyrophosphate synthetase catalyzing two sequential condensations of isopentenyl pyrophosphate (IPP); first, with dimethylallyl pyrophosphate (DMAPP) to produce GPP, and second, with GPP to produce farnesyl pyrophosphate (FPP). As the GPP node is critical in monoterpenoid production, the carbon flux was redirected to the product formation by fusing Erg20WWG, a novel mutant with reduced FPP synthesis activity, to a truncated (S)-limonene or geraniol synthase lacking their plastid-targeting sequence. Then, peroxisomal compartmentalization of the whole MVA pathway and the Erg20WWG-fused monoterpenoid synthases increased the product formation through better precursor utilization. In addition, wild-type ERG20 was downregulated using the glucose-sensing HXT1 promoter to redirect the carbon flux of the GPP node towards product formation more efficiently. After further optimizations and multicopy integration of key genes, the final (S)-limonene and geraniol platform strains produced 1062.96 mg/L of (S)-limonene, the best-achieved titer in a yeast host, and 1233.54 mg/L of geraniol after a 6-day fed-batch cultivation through glucose and ethanol feeding. These strains reached a gram-scale monoterpenoid titer, making them suitable to produce diverse valuable derivatives of geraniol and (S)-limonene.