Cloning Of Xylose Reductase Gene (xyl1) And Construction Of Yeast Expression Vector

Xylose reductase(XR) is one of key enzymes in yeast xylose metabolism. The first step in xylose metabolism is catalyzed by XR, which reduced xylose to xylitol. The different coenzyme specificity of XR makes the metabolism lean to xylitol or ethanol, based on the metabolic flux in the xylose-fermentation recombinant strains. It is recognized that Saccharomyces cerevisiae itself does not have XR, and can not utilize xylose, but has the ability to metabolize xylulose, So xylose reductase gene can be cloned and expressed in Saccharomyces cerevisiae.In this paper, molecular cloning techniques can be used to construct the integratinon vector pYX-AK-xyl1, which can used as the expression of xylose reductase. The plasmid containing xyl1 gene was transformed into Saccharomyces cerevisiae W5, the activity analysis was carried out. The process of plasmid construction as follows, first of all, using Candida shehatae genomic DNA and plasmid PKT0150 as templates cloned xylose reductase gene xyl1, G418 resistance gene KanR and ADHt terminator sequence, the length of them are 1.1kb, 1.5kb, 370bp. Saccharomyces cerevisiae integration Plasmid p406ADH1 as the basic skeleton, insert ADHt terminator by gene recombination technology, replace cyc1 terminator to form a complete ADH promoter - terminator sequence, the plasmid pYX-ADH were constructed, than insert the G418 resistance gene and xyl1 gene, were ligated into the plasmid pYX-AK and the low copy of integrating plasmid pYX-AK-xyl1 which contains URA3 as homologous recombination sequence. The plasmid containing xyl1 gene was transfomed into Saccharomyces cerevisiae W5 and the G418 resistance gene acted as a dominant selectable marker, the recombinant strains are named YX-1, YX-2, YX-3 and YX-4, induced by different coenzyme, Activities for transformants (NADH, NADPH) were respectively 6.070 U/mg and 6.128U/mg、5.419 U/mg and 5.814 U/mg、6.513 U/mg and 7.080 U/mg、4.707 U/mg and 6.285 U/mg. The results of enzyme activities in YEPD without G418 show that transformants still use two kinds of coenzyme, in which strain YX-3 is the highest activity in using NADH and NADPH, were 6.513 U/mg and 7.080U/mg, than the receptor strain W5 to increase by 1.4 times and 1.3 times, but the activity less than original strain 20335, the activity ratio were 0.919. This shows that the xylose reductase of Candida shehatae can use NADH and NADPH coenzyme two, but mainly dependent on NADPH.The successful construction of recombinant plasmid pYX-AK-xyl1, integrated xylose reductase gene into the chromosome of Saccharomyces cerevisiae genome W5. The research of coenzyme specificity of xylose reductase established the foundation for the follow study, Including construction of using xylose recombinant Saccharomyces cerevisiae strains, xylose-metabolic engineering of microorganisms and the bioconversion of lignocellulosics to ethanol.