Engineering Cofactor Metabolism For More Efficient Conversion Of Xylose To Ethanol In Saccharomyces Cerevisiae

Bioethanol is a kind of clean renewable energy, have widespread attention.Xylose is one of the most abundant pentose in nature which widely exist in thematerials containing hemicellulose from forestry and agriculture plants, the traditionalproduction costs of ethanol will greatly reduced when xylose was used to produceethanol.Saccharomyces cerevisiae has many advantages in ethanol production, but cannot utilize xylose. In the early work, our research group had successfully constructedthe xylose-assimilating S.cerevisiae strains KAM-6X in which the xylose genes XYL1and XYL2of Pichia stipitis were introduced. The conversion rate of xylose to ethanolis low in recombinant S.cerevisiae as XR and XDH have different cofactorspreference of NADPH and NAD+respectively during xylose metabolic pathway,which create cofactor imbalance. NADPH consumed in the first step of xylosepathway is mainly produced in the oxidize part of PPP pathway coupling with CO2formation, which waste carbon source. And the cofactors NADPH and NADH can nottransform to each other as S.cerevisiae do not have transhydrogenases. So in thisstudy, we improved the cofactor regeneration and transformation of xylose-assimilating S.cerevisiae through two strategies as the following:Firstly, the zwf1gene encoding glucose-6-phosphate dehydrogenase (G6PDH) inPPP pathway was deleted in S.cerevisiae to block the formation of NADPH in thisway, then the foreign genes GAPN and GDP1which encoding NADP+dependentglyceraldehyde-3-phosphate dehydrogenase was introduced into these yeastssimultaneously to replace zwf1for NADPH production without CO2. The xylose andmixture sugar fermentation results showed that the strains can not utilize xylose whenzwf1gene was deleted Which indicated zwf1gene has a pivotal role in the PPPpathway. As a result, the gene GAPN and GDP1were transformed into KAM-6X andKAM-6X-NOXE respectively in which the gene zwf1was not deleted, the geneGAPN was overexpressed with the promoter pPGK1, pHXT7, pFBA1and GDP1wasexpressed with the promoters pPGK1, pCCW12, pHXT7respectively. Thefermentation result showed that the production of xylitol and glycerol reduced and theyield of ethanol increased, which illustrated the increased abilities of xylose to ethanol,and more carbon flowed to ethanol. However, the ethanol improvement in the yeastswith GAPN was more evident than the strains with GDP1in xylose fermentation. Secondly, the gene NNT from Aspergillus niger was introduced into recombinantS.cerevisiae. The transhydrogenase NNT was coupled with fluorescent protein andtransferred into S.cerevisiae, the result of fluorescence experiments proved the NNTprotein was localized in chondriosome. Then the gene NNT was expressed with thepromoters pPGK1, pCCW12and pHXT7respectively. The recombinant plasmidswere transferred into S.cerevisiae for xylose fermentation, the results showed that theformation of glycerol and xylitol decreased and the yield of ethanol increased.In conclusion, the exogenous genes GAPN and GDP1was introduced intoS.cerevisiae to make up the NADPH consumed in the first step of xylose fermentation;The transhydrogenase gene NNT was introduced to realize the mutual transformationbetween NADH and NADPH. In general, these strategies above had positive effectson the aspects of improving ethanol production of xylose fermented.