Abstract
BackgroundSaccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate.ResultsOverexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1).ConclusionsWe demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.
Highlights
Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels
Among three pyruvate dehydrogenase (PDH) complexes that have been expressed in the cytosol of yeast, PDH from Enterococcus faecalis was demonstrated to enable complete replacement of the native pathway for cytosolic acetyl-CoA synthesis, and was less sensitive to high NADH/NAD+ ratios compared with other PDH complexes [16,17,18]
The goal of this study is to investigate the effect of expressing the cytosolic PDH complex on Free fatty acid (FFA) production in yeast strains with deregulated fatty acid metabolism and optimized cytosolic redox balance (Fig. 1)
Summary
Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Endogenous synthesis of cytosolic acetyl-CoA, the main C2 metabolite, is often limited and several heterologous enzymes have been expressed to increase acetyl-CoA supply for FFA synthesis, including N AD+-dependent alcohol dehydrogenase (AdhE), NAD+-dependent acylating acetaldehyde dehydrogenase (EutE) [10], ATP citrate lyases (ACLs) [6, 7, 11,12,13], xylulose-5-phosphate phosphoketolase (XpkA) and phosphotransacetylase (Pta) [14] Compared to these strategies, expression of cytosolic pyruvate dehydrogenase (PDH) is attractive because of its lower energy cost [15]. One concern with PDH expression for FFA production is the redox imbalance between redox couples of NAD+/NADH and NADP+/NADPH
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