Metabolic Engineering of Saccharomyces cerevisiae for the Production of Isopentenol, a Valuable Biochemical and Potential Biofuel

Abstract

The rising concern about limited fossil fuel resources and the environmental factors associated with it has motivated the development of alternative fuels. Ethanol is currently the primary substitute to gasoline, however the intrinsic properties of ethanol have limited its ability to fully replace petroleum based gasoline. Microbial production of higher chain alcohols is currently being explored to circumvent the problems associated with ethanol. Isoprenol is a 5-carbon alcohol that has a higher energy density and a lower water affinity when compared to ethanol. These characteristics allow isoprenol to fit well with existing fuel infrastructure. However, reported yields of isoprenol remain low and the process thus requires further optimization to increase production. Microbial production of isoprenol has been limited to synthetic pathways introduced into Escherichia coli. The approach in this study is to utilize the endogenous, Saccharomyces cerevisiae’s isoprenoid pathway and apply metabolic engineering approaches to manipulate gene expression and protein stability to increase flux through the pathway, maximizing isopentenyl pyrophosphate production. Isopentenyl pyrophosphate is an intermediate in the isoprenoid pathway that can be dephosphorylated to produce isoprenol. NudF, an exogenous phosphatase, with potential activity towards isopentenyl pyrophosphate, was expressed in Saccharomyces cerevisiae to produce isoprenol. Optimization of the isoprenoid pathway in Saccharomyces cerevisiae and expression of NudF did not lead to isoprenol production but did yield production of isopentyl alcohol. This work emphasizes the existence of inherent limitations when introducing novel processes into the cell.