Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae

Abstract

Background Saccharomyces 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. Results Overexpression 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). Conclusions We 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.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β‐alanine metabolism

“…To enhance the cytosolic route to acetyl‐CoA, expression of PDH complex in cytosol was attempted by several groups (Nielsen, 2014). However, only the E. faecalis PDH complex was expressed functionally in yeast cytosol, which activity required the presence of lipoic acid in the medium (Kozak et al ., 2014; Zhang et al ., 2020). Herein, pyruvate kinase encoded by CDC19 involving the formation of pyruvate from phosphoenolpyruvate and pyruvate decarboxylase encoded by PDC1 were coexpressed as a fusion protein in strain SQ3‐2 to generate strain SQ3‐3, which produced 35.47 ± 2.25 mg g −1 DCW of squalene (Fig.…”

Metabolic engineering of Saccharomyces cerevisiae for production of medium-chain fatty acids and their derivatives

“…In fed batch fermentations these combined strategies have shown the highest reported titers for LCFA production in S. cerevisiae so far (Yu et al, 2018). In a different strategy, a bacterial PDH complex from Enterococcus faecalis was expressed directly in the cytosol of S. cerevisiae for AcCoA supply and increased free FA production of mainly unsaturated C16:1 and C18:1 FA by 83 % when combined with other engineering strategies (Zhang et al, 2020).…”