Overproduction of fatty acids in engineered Saccharomyces cerevisiae

Abstract

The long hydrocarbon fatty acyl chain is energy rich, making it an ideal precursor for liquid transportation fuels and high-value oleo chemicals. As Saccharomyces cerevisiae has many advantages for industrial production compared to Escherichia coli. Here, we attempted to engineer Saccharomyces cerevisiae for overproduction of fatty acids. First, disruption of the beta-oxidation pathway, elimination of the acyl-CoA synthetases, overexpression of different thioesterases and acetyl-CoA carboxylase ACC1, and engineering the supply of precursor acetyl-CoA. The engineered strain XL122 produced more than 120 mg/L of fatty acids. In parallel, we inactivated ADH1, the dominant gene for ethanol production, to redirect the metabolic flux to fatty acids synthesis. The engineered strain DG005 produced about 140 mg/L fatty acids. Additionally, Acetyl-CoA carboxylase was identified as a critical bottleneck of fatty acids synthesis in S. cerevisiae with a cell-free system. However, overexpression of ACC1 has little effect on fatty acids biosynthesis. As it has been reported that phosphorylation of ACC1 may influent its activity, so phosphorylation sites of ACC1 were further identified. Although the regulatory mechanisms remain unclear, our results provide rationale for future studies to target this critical step. All these efforts, particularly the discovery of the limiting step are critical for developing a "cell factory" for the overproduction of fatty acids by using type I fatty acids synthase in yeast or other fungi.

Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites

“…Specifically, less than 2-fold improvement was achieved in most cases simply by ACC1 overexpression [21,53,56,61,75,76]. In some studies, ACC1 overexpression did not improve the production at all [42]. Such results indicated the complicated regulation of ACC1 enzyme, making it necessary to engineer the regulatory machinery.…”

“…By the deletion of major ADHs in the cytosol (∆adh1-∆adh4), acetyl-CoA level was increased around 2-fold and the production of n-butanol, an important biofuel molecule derived from acetyl-CoA, was increased more than 4-fold [43]. In another study, 1.9-fold improvement in fatty acids production was achieved by knocking out ADH1 in a fatty acids producing host [42].…”

“…Such results indicated the complicated regulation of ACC1 enzyme, making it necessary to engineer the regulatory machinery. The activity of ACC1 was post-translationally regulated by SNF1 [42] (Fig. 2).…”

“…The activation of acetyl-CoA to malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), encoded by ACC1 in S. cerevisiae, which was determined to be critical and ratelimiting for FAB [42]. In addition, the ACC activity in S. cerevisiae is highly regulated, at both transcriptional level and post-translational level.…”

“…In addition, the use of A-ALD was only demonstrated by growth complementation and its energetic benefits for the synthesis of acetyl-CoA derived products still needs further evaluation. Recently, EcEutE was introduced into a fatty acids producing host but only marginal improvement was observed in fatty acids production [42].…”