Construction Of Recombinant Saccharomyces Cerevisiae Strains For Glycerol Utilization And Efficient Xylose Fermentation By Genetic Engineering And Genome Shuffling

The climate change caused by CO2emition coupled with imminent depletion offossil fuel reserves have made human spent more and more energy in looking foralternative energy sources. As a pollution-free and renewable resource, bioethanol isone of the most promising alternatives to conventional fuels. However, theconventional method of ethanol fermentation by food would cause food shortage.Meanwhile, converting glycerol from biodiesel coproduct and lignocellulosic biomassfrom agricultural and agro-industrial residues represents abundant xylose tobioethanol is one of the current research hot spot. It is of great significance to find outhow to efficiently convert these materials to ethanol for solving the environmental andenergy problems.In the present study，Saccharomyces cerevisiae was used as model organism tostudy glycerol and xylose metabolism. First of all, we intergrate noxE, encodingwater-forming NADH oxidase, in the chromosome of a strain LGE73carryingdeletion in glycerol production pathway to modify the cell redox balance, overexpressDAK1、DAK2encoding dihydroxyacetone kinase and GCY1encoding glyceroldehydrogenase. Then we cultured the strain on YPG plate, results demonstrate thatthe intergration of noxE increase the strain growth on YPG plate. The determinationof glycerol utilizaition gene transcrition by RT-PCR demonstrates that the genetranscriptional level of DAK1，DAK2，GCY1enhanced significantly but still in a lowexpression level. Glycerol fermentation research demonstrates that the engineeredstrain needs a long term fermentation of144h to utilize16g/l glycerol and produce0.6g/l ethanol.We deleted PHO13gene encoding para-nitrophenyl phosphatase in anrecombinant xylose-utilizing strain, KAM-6X, obtatined a strain named KAM-6X(△PHO13). The results demonstrate that deletion of PHO13has a positive effect onxylose utilization. The maximum specific growth rate of KAM-6X(△PHO13) was0.261h1，it can consume50g/l xylose in48h and ethanol yield reached to0.313g/lwith less glycerol production and high acetate level. Then we applied a new genomeshuffling method meiotic mediated by recombination developed in our laboratory on axylose-utilizing recombinant yeast strain KAM-6X(K270R). The strain was first transformed to be a diploid ste2/ste2strain (the STE2gene encodesα-factor receptor),then subjected to EMS mutagenesis followed by meiotic recombination-mediatedgenome shuffling and evolutionary engineering. We obtained a strain with enhancedxylose-fermenting ablity named WCY1which can consume100g/l xylose in48hwith an ethanol yield of0.395g/l, and the maximum specific growth rate was0.294h-1.These figures exceed by a significant margin any other performance metrics onxylose utilization by S. cerevisiae reported to-date.. The fermentation in medium withmixed sugar of WCY1showed that xylose-utilization was inhibited. Measurement ofthe xylose-utilizing enzymes activities showed no significant change suggesting a newgenetic trait in the strain had been generated in favor of xylose metabolism. Finally,we tested the mating type of evolved strains. Result meets the expectation of thedesigned experiment which verified that genome shuffling is an efficient strategy toimprove xylose fermenting property of Saccharomyces cerevisiae strain.