Genome Shuffling Was Used To Screen Saccharomyces Cerevisiae That Uses Xylose Glucose To Produce High Ethanol

Conversion of renewable lignocellulosic biomass into bio-ethanol has become a research hotspot recently,and the xylose fermentation is the foundation and the restraining factor for this technology.But the traditional ethanol fermentation strain Saccharomyces cerevisiaeis can only use the glucose of lignocellulose hydrolysate to produce ethanol,and not be able to utilize xylose,therefore,the xylose metabolic related genes needed to be introduced and expressed in the S.cerevisiae in order to make this strain to effectively consume xylose and convert it to ethanol.This paper was focus on a novel methodology of genome shuffling which could intergrate three key genes of xylose metabolism(xylose reductase gene xyl1,xylitol dehydrogenase gene xyl2 and xylulose kinase gene xks1)into the chromosome of S.cerevisiae to develop engineering S.cerevisiae strains that availably use xylose and glucose to generate ethanol.And the optimized conditions of protoplasts preparation and regeneration were studied based on the original strain S.cerevisiae W5.According to the principle of protoplast damage complementarity,the best inactivated conditions of three parent strains S cerevisiae YX-3(containing xyll),LX-4(containing xyl2)and CXS-5(containing xks1)were researched.And later,the orthogonal test was used to optimize the conditions of protoplast fusion.The production of ethanol was confirmed by the early TTC screening and second fermentation screening.A hereditarily strain S.cerevisiae GS3-10 with high ethanol production by xylose mixed with glucose fermentation was obtained through three rounds of genome shuffling.After fermentation for 120h in the initial amount of sugars 60g/L(glucose 40.75g/L,xylose 20.34g/L),the S.cerevisiae GS3-10 strain(which utilized 70.12%and 100%of the xylose and glucose,respectively)produced 18.98g/L ethanol.34.13%higher than ethanol production by S.cerevisiae W5(14.15g/L).The ethanol yield was 0.350g/g(ethanol/consumed glucose and xylose),increased by 12.54%than the amount generated by S.cerevisiae W5(0.311 g/g).The results of genomic PCR identification showed the xyll and xyl2 were integrated in the recombinant’s chromosome.And the enzyme activities of xylose reductase,xylitol dehydrogenase and xylulose kinase in the recombinant were increased by more than 28.33%,94.08%and 51.68%compared to W5,indicating that the three kinds of genes in recombinant had been effectively expressed,and also the technique of genome shuffling in S.cerevisiae had been successful.The flask fermentation conditions of recombinant including the proportion of mixed-sugars,shaking speed and liquid volume were studied.And some preliminary researches of recombinant’s fermentability in fermenter were.also discussed.These results showed that rigorous and precise oxygen level had a significant impact on the output of ethanol produced by recombinant.In conclusion,we report for the first time the use of genome shuffling as means of improving the xylose metabolic pathways of S.cerevisiae,which is of significant importance in the context of the bio-ethanol conversion of lignocellulose materials into ethanol.