Modulating Flux-Competing Enzyme Squalene Synthase By Protein Degradation Mechanism to Improve Sesquiterpene Production in Saccharomyces Cerevisiae

Sesquiterpenes have functions as fragrances, flavours, and semiochemicals, and as active molecules in pharmaceuticals and traditional herbs. Because of their high energy content, sesquiterpenes can also be applied as future alternative aviation fuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Methods to avoid competition between production of endogenous isoprenoids and the introduced sesquiterpene are required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used as the platform organism to produce an acyclic sequeterpene alcohol, trans-nerolidol. Nerolidol production was firstly improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP produced was instead directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1 % biomass. It was shown that Erg9p is located on endoplasmic reticulum (ER) membrane through a C-terminal ER-targeted transmembrane peptide. An endoplasmic reticulum-associated protein degradation mechanism was developed to decrease cellular levels of Erg9p. This improved nerolidol titre by 86 % to ~100 mg L^-1. To further improve titres, the strong inducible CUP1 promoter was applied for overexpression of the mevalonate pathway and nerolidol synthase. This resulted in a nerolidol titre of 248 mg L^-1 in the pathway-enhanced, Erg9p-destabilized strain. In this strain, squalene levels were similar to the wild-type control strain, and there was no negative effect on cell growth. These data demonstrate that the protein degradation mechanism can be a useful strategy to balance the flux competition between the endogenous sterol pathway and introduced bio-product pathways at the FPP node for metabolic engineering applications.