Cloning Of Key Enzyme Genes And Construction Of Engineering Yeasts For Xylose Metabolism

Lignocellulose is a kind of widely spreaded, inexpensive renewable resource on the earth.As the raw material for producing fuel ethanol, lignocellulose can be hydrolyzed into sugarmixture mainly including glucose and xylose, the effective utilization of xylose is one of thecritical barriers in the processes of ethanol transformation. In consideration of the absence ofhigh performance xylose fermenting strains in nature, genetic engineering technologies havebeen applied to construct various xylose metabolizing strains in recent years. But manytechnologies are needed to be improved and there is great research potential in this field.Among xylose-assimilating microorganisms, only a few critical genes have beencharacterized, cloned, and expressed. Based on the homologous analysis of nucleotidesequences encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from variousspecies, degenerate primers have been designed and applied on total RNAof Candidalusitaniae, Pachysolen tannophilus, and Phaffia rhodozyma to amplify their xylose metabolicgenes, and parts of them have been obtained specifically. The highly characterized sequenceswere chosen as the targets for cloning XR and XDH cDNAs from C. lusitaniae. cDNAs with1073bp and1322bp were obtained, encoding957bp and1086bp open reading frames(ORFs). These ORFs are highly similar to that of the corresponding genes from C. shehatae,with similarity75%and72%, respectively. Through phylogenetic analysis, the XR and XDHgenes were confirmed as new ones on the level of RNA.The newly obtained XR and XDH ORFs were cloned and expression vectors withpEASY-E1under the control of T7promoter were constructed. The vectors were thentransformed into Escherichia coli BL21(DE3) and used for expression. Axylose isomerasegene encoding XI-PB8from Thermoanaerobacterium aotearoense was cloned and expressedas well. The expressed XR, XDH, and XI showed molecular weight of35.94KD,38.65KD,and50.22KD. By enzyme activity assay, XR was proved to prefer NADPH30times morethan NADH, while XDH was exclusively depended on NAD+.Atwo-step gene replacement method was applied to insert PGK1promoter in front of thenative XKS1gene in the genome of Saccharomyces cerevisiae, resulting in a strain overexpressing xylulokinase (XK). The expression vectors of XR, XDH, and XI wereconstructed based on yeast episomal plasmid pYES2. These vectors were then transformedinto S. cerevisiae and genetically engineered strains with the XR-XDH pathway and XIpathway were constructed. Enzyme activity assay showed that the activities of XR, XDH,XK, and XI-Pir (original from Piromyces) were0.18U/mg,0.37U/mg,1.91U/mg, and0.11U/mg, respectively. In contrast, the XI-PB8failed to be expressed in S. cerevisiae.Fermentations under aerobic, oxygen-limited, and anaerobic conditions were carried out totest the xylose-utilizing ability of the constructed strains. The expression level of the genesplayed an important role in xylitol accumulation, while oxygen supply showed significanteffect on xylose consumption. Engineered strains Saccharomyces cerevisiae004-PGK(pPGK-XKS1, pTPI1-XYL1, pHXT7-XYL2),004-1917(pTPI1-XYL1, pHXT7-XYL2) and002-PGK (pPGK-XKS1, pTPI1-XYLA-Pir) were capable of utilizing xylose in spite of low rate.The maximum specific growth rate of0.016/h and0.022/h for strains004-PGK and004-1917were achieved under aerobic condition.