On the role of GAPDH isoenzymes during pentose fermentation in engineeredSaccharomyces cerevisiae

Abstract

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway.

Xylose-induced dynamic effects on metabolism and gene expression in engineered Saccharomyces cerevisiae in anaerobic glucose-xylose cultures

“…at 6.5 h in the glucose cultures), as previously reported (Runquist et al 2009;Matsushika et al 2014). TDH1 encodes a glyceraldehyde 3-phosphate dehydrogenase isoform, which is responsive to NADH stress (Valadi et al 2004), and downregulated in evolved strains as a consequence of improved xylose utilisation (Karhumaa et al 2009;Linck et al 2014). GPM2 encodes a phosphoglycerate mutase which, however, is not the major isoform in glycolysis (Heinisch et al 1998).…”

Aberrant Intracellular pH Regulation Limiting Glyceraldehyde-3-Phosphate Dehydrogenase Activity in the Glucose-Sensitive Yeast tps1 Δ Mutant

“…The deletion of TDH3 in the tps1 Δ strain, on the other hand, completely abolished growth at low glucose concentrations, down to 0.5 mM, with which the tps1 Δ strain still grew to some extent. Since Tdh3 is responsible for most of the GAPDH activity when yeast is growing with glucose ( 39 ), this aggravated glucose sensitivity suggests that the deletion of TDH3 increases the metabolic bottleneck at the level of GAPDH. Unexpectedly, however, the additional deletion of TDH3 in the tps1 Δ strain also completely abolished growth on galactose and allowed only residual growth on glycerol.…”

“…The expression of TDH1 is rapidly enhanced under various stress conditions ( 35 – 38 ). The catalytic activities of the three isoenzymes are different, with Tdh1 ( K m = 0.86 mM; k cat = 29.04 s −1 ) having the highest, Tdh2 intermediate ( K m = 0.42 mM; k cat = 16.22 s −1 ), and Tdh3 the lowest activity ( K m = 0.25 mM; k cat = 9.12 s −1 ), while the affinity ranks in the opposite order ( 39 ). The combined deletion of TDH2 and TDH3 is synthetically lethal, likely due to the low expression of TDH1 , whereas the other double deletion strains are viable ( 34 ).…”

Hxt13, Hxt15, Hxt16 and Hxt17 from Saccharomyces cerevisiae represent a novel type of polyol transporters

“…Conversion of xylitol to xylulose was achieved by plasmid-based overexpression of the xylitol dehydrogenase XYL2 16 . To eliminate any limitations in the downstream pathway 17 18 , the strain overexpresses xylulokinase ( XKS1 ), D-ribulose-5-phosphate epimerase ( RPE1 ), D-ribulose-5-phosphate ketol-isomerase ( RKI1 ), transketolase ( TKL1 ) and transaldolase ( TAL1 ) from a cassette integrated into the PYK2 locus 15 . In this strain background, among all transporters tested, only Hxt11 and Hxt15 could support growth on agar plates containing xylitol ( Supplementary Fig.…”