The Research Of Gre3 Gene Disruption In Saccharomyces Cerevisiae

The un-specific aldose reductase encoded by gre3,is on chromosome III in Saccharomyces cerevisiae,which could reduce xylose to xylitol.Althout xylitol has become an important food additive, and widely added to diffent kinds of foods. If we want to improve the ethanol yield in recombinant Saccharomyces cerevisiae, xylitol is the main of by-products,which reduced the ethanol yield.The gene of gre3 was deteled in the haploid strain AS7 and AS21,which were used by LHF-PCR(Long homology flanking-PCR) and the Piromyces sp xylA, Pichia stipites xks1 were introduced at the gre3 locus.And then the xylose utilization capacity of the recombinant Saccharomyces cerevisiae srains were studied.And we quantitated ethanol yield and xylitol yield and so on in each recombinant stain.The commercial vector of pGAPZ?A was digested by Bgl II and Ase I,and the gene of GAP promoter, ?- signal peptide gene and AOX terminator, TEF1 promoter were removed. The gene of gre3 promoter and terminator from saccharomyces cerevisiae were cloned by PCR and then ligated into the vector of pGAPZ?A. Therefore the gene of Bleomycin could be transcripted by the gre3 promoter,and the fragment of gre3 promoter and terminator were served as homologous arms in knockout vector of pGREZ.We try to constructed the replacement vector, which capable of introducing at the gre3 locus with gene of xylose isomerase, xylulokinase. The xylose isomerase gene(xylA) of Piromyces sp E2 and the xylulokinase gene(xks1) of Pichia stipites were cloned into the replacement vector of pGREZ. Finallly,we constructed the expression vector of pGREZ-xks1, co-expression vector of pGREZ-xyl A-xks1. And the co-expression vector of pGREZ-xylA-xks1 was introduced to enable the haploid strains AS7(MAT?) and AS21(MATa) to convert xylose to D-Xylulose-5-phosphate. The diploid of co-expresing the gene of xylA and xks1 was construted by hybrid fusion.The xylose utilization capacity of the recombinant strains was studied. The recombinant yeasts could grow on xylose.And the parent strain AS2.489 could not grow on YPX basically. Xylose utilization rate of AS7-KA and AS21-KA were 21% and 22% respectively,comparing with the parent strain of 19%. And the xylose- xylitol conversion rate were 83.8% and 87.3% respectively, xylose-xylitol conversion rate decreased by 11.9% and 8.4% respectively,comparing with the original strain AS2.489 of 95.7%. Xylose utilization rate of the biploid recombinant yeast of AS7-KA/AS21-KA was about 22.6% and xylose-xylitol conversion rate was 78.4%, xylose-xylitol conversion rate decreased by 17.3%,comparing with the original strain AS2.489 of 95.7%. In the tenth day of fermentation period, the recombinant yeast producted 0.221-0.253 g / L ethanol.Subsequently EMS mutagenesis and selection in D-xylose-enriched medium(YPX), followed by two rounds of evolutionary engineering in YPX medium with D-xylose in aerobic conditions and limiting oxygen conditions, gradually established more efficient D-xylose fermentation. Thereford the evolved haploid recombinant strains of AS7-KAEAE and AS21-KAEAE,the biploid recombinant strain of AS7-KA/AS21-KA-EAE were gotted obtianed( ‘E' means of EMS mutagenesis treatment; ‘AE' means of adaptive evolution). The evolved strain AS7-KA/AS21-KAEAE displayed cxylose utilization of 28.0% and xylose-xylitol conversion rate decreasing by 72.8%,along with higher ethanol concentration of 0.499 g/L, about 17.9% of the theoretical conversion rate. The evolved strain AS7-KAEAE showed xylose utilization of 30.8% and xylose-xylitol conversion rate decreasing by 78.4%,ethanol concentration of 0.384 g/L, about 11.8% of the theoretical conversion rate. The evolved strain AS21-KAEAE displayed higher ethanol conversion rate, 24.1% of the theoretical conversion rate.