Disruption of PHO13 Improves Ethanol Production Via the Xylose Isomerase Pathway

Xylose is the second most abundant sugar in lignocellulosic materials and can be converted to ethanol by recombinant Saccharomyces cerevisiae yeast strains expressing heterologous genes involved in xylose assimilation pathways. The oxidoreductase pathway involves the conversion of xylose to xylitol by xylose reductase (XR) and the conversion of xylitol to xylulose by xylitol dehydrogenase (XDH). In this pathway, the yield of ethanol is poor for the accumulation of xylitol. Another xylose assimilation pathway is catalyzed by xylose isomerase (XI), which directly converts xylose to xylulose. Therefore, XI-expressing strains do not produce excess xylitol, and suitable for high-yield ethanol production from xylose.

Recent research demonstrated that disruption of the alkaline phosphatase gene, PHO13, enhances ethanol production from xylose by a strain expressing the XR and XDH genes; however, the yield of ethanol is poor yet. In this study, PHO13 was disrupted in a recombinant strain harboring multiple copies of the XI gene derived from Orpinomyces sp., coupled with overexpression of the endogenous xylulokinase (XK) gene and disruption of GRE3, which encodes aldose reductase. The resulting strain YΔGP/XK/XI demonstrated a xylose consumption rate of 2.08 g/L/h, 0.88 g/L/h volumetric ethanol productivity, and 86.8% theoretical ethanol yield (0.45 g-ethanol/g-xylose), whereas the xylose consumption rate, volumetric ethanol productivity, and ethanol yield of the control strain YΔG/XK/XI were 1.76 g/L/h, 0.57 g/L/h,and 80.3%, respectively, and only YΔGP/XK/XI demonstrated increase in cell concentration.

In the present study, the effect of PHO13 disruption on intracellular metabolism of the XI-expressing strain was investigated by transcriptome and metabolome analysis using DNA microarray and LC-QqQ-MS methods, respectively. We revealed that the expression of pentose phosphate pathway (PPP) genes was upregulated. And, 125 genes involved in the cell cycle were dramatically altered by PHO13 disruption. Metabolome analysis demonstrated a decrease in the level of S7P, an increase in the level of 6PG, and accumulation of ATP by PHO13 deletion during xylose fermentation.

In previous reports, overexpression of PPP genes has improved ethanol yield in recombinant xylose-fermenting yeast strains. Especially, utilization of S7P is thought to be a rate-limiting step in the non-oxidative PPP.

In the present study, the non-oxidative PPP genes was upregulated by the deletion of PHO13, which should be the reason of the reduction in S7P pool size. And, the oxidative PPP genes (SOL3 and GND1) was also upregulated by PHO13 deletion, which might cause depletion of NADP⁺ and reduction of the oxidative PPP flux. Since the oxidative PPP generates CO₂, reduced flux of the oxidative PPP might increase ethanol yield from xylose consumed. Previously, PHO13 disruptants indicate high growth ability on xylose medium. In the present study, YΔGP/XK/XI demonstated higher cell concentration than the control strain on xylose fermentation.

We found that PHO13 disruption upregulates PPP-gene expression in XI-harboling strain and that transcriptional level of 125 cell cycle genes was altered. NADPH supplied by PPP is required for biosynthetic processes and biomass yield. Thus, PHO13 should be one of the factors affecting yeast cell growth.