Metabolic Engineering Of Pentose Phosphate Pathway For Improving Xylose Fermentation Performance In Saccharomyces Cerevisiae

As an alternative and renewable energy resource, lignocellulosic ethanol hasdrawn increasing attention from both academic and industrial communities. However,Saccharomyces cerevisiae, traditionally used for bio-ethanol production, is unable toutilize xylose, the major component of lignocellulosic hydrogenates, but only itsketol-isomer xylulose. Therefore, improving xylose utilization in S. cerevisiae strainsso as to co-fermentation of both glucose and xylose is the top priority for theindustrialization of lignocellulosic ethanol.In the early work, our research group had successfully constructed xyloseassimilating S. cerevisiae strain AYHNEW2that can efficiently utilize xylose, andtheir fermentation performance was studied on different conditions. However, xyloseconsumption rate of recombinant yeasts was significantly reduced when glucose andxylose were utilized as the carbon source. After the comparative study of thisphenomenon, we pointed out that ethanol produced by glucose fermentation might beresponsible for the slower xylose conversion, and we hypothesized that the effect ofethanol on xylose metabolism might have something to do with the activities of PPPenzymes. As a result, two protocols were adopted in parallel to improve theperformance of strains AYHNEW2in this work, as described below.On the one hand, Glucose phosphate isomerase PGI1, catalyzing the key step ofglucose metabolism was disrupted to construct the exclusive xylose-utilizing yeaststrains AYHNEW2-pgi1, and hence to eliminate the inhibitory effect of glucose onxylose utilization. Fermentation results indicated that glucose metabolism wasseriously deteriorated in PGI1-knockout yeast when the small amount of glucose wasadded, and only6%glucose was utilized after100h cultivation. While growth ofrecombinant strains was completely stopped when the higher concentration of glucosewas used. Where, however, the significant slower growth rate was observed whenxylose was utilized as the sole carbon source.On the other hand, in order to improve the performance of S.cerevisiae strains onxylose condition, the non-oxidase Pentose Phosphate Pathway(PPP) genes wereoverexpressed, using four stronger promoters selected according to their transcriptlevels on xylose condition. Plasmids with the PPP gene RKI1, RPE1, TAL1and TKL1under the control of promoter pFBA1, pHXT7, pCCW12and pGPM1were established, and used to construct the recombinant strains with different combination of theoverexpressed PPP genes. Fermentation performance was comparatively studied,results showed that yeasts with the PPP genes overproduced performed much betteron xylose condition, and their ethanol tolerance was also improved to a large extent.but xylose utilization of the engineered strains was still severely damaged whenglucose and xylose were used. where, however, we found that at the range of ethanolproduced during the glucose fermentation, there was no significant inhibition ofxylose utilization, although their xylose consumption rate was a little bit lowered, allthe recombinants can still use up5%xylose within the48h to72h.Put these all together we can see that the inhibitory effect of ethanol can onlypartially account for the lower xylose utilization in glucose and xylose condition, andthere must be some other factors available in the overall process that influencing thexylose fermentation.