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Modification of Xylose Reductase from Pichia Stipitis and Primary Study on Ethanol Fermentation from Xylose Using Recombinant Saccharomyce Cerevisiae

Author: DongLiLi
Tutor: DengXiaoZhao
School: Nanjing Medical University
Course: Biochemistry and Molecular Biology
Keywords: Saccharomyces cerevisiae Xylose reductase Coenzyme Ⅰ (NADPH) Coenzyme Ⅱ (NADH) Ethanol
CLC: TQ223.122
Type: Master's thesis
Year: 2009
Downloads: 49
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Abstract


Natural yeast (Saccharomyces cerevisiae) can make effective use of hexose fermentation to produce ethanol, but can not utilize xylose. Xylose in lignocellulosic hydrolyzate rich content, second only to glucose, therefore, to promote the full use of lignocellulosic materials, reduce the cost of ethanol production one of the key links xylose to ethanol bio-transformation. Import expression Pichia yeast (Pichia stipitis) xylose reductase (xylose reductase, XR) and xylitol dehydrogenase (Xylitol dehydrogenase XDH) gene recombinant Saccharomyces cerevisiae can metabolize xylose, but these recombinant strains ferment xylose weak, one of the main reasons is because of XR activity is mainly dependent on the coenzyme NADPH, XDH activity dependent coenzyme NAD both cofactor the intracellular electron redox imbalance, resulting in xylitol accumulation affect wood glucose metabolism. Therefore, by genetic engineering methods transform XR and / or XDH coenzyme preference XR and XDH coenzyme can be coupled with each other, this is an exploratory study of improved recombinant Saccharomyces cerevisiae to ferment xylose to ethanol production. The first part of the of Pichia pastoris xylose reductase gene cloning, mutation screening sites and prokaryotic expression, purification and enzymatic activity detection based on the Pichia yeast xylose reductase (Pichia stipitis xylose reductase, PsXR) dual coenzyme characteristics (ie on NADH and NADPH were affinity), site-directed mutagenesis of the gene PsXR get NADH high affinity xylose reductase the (recombination xylose reductase rXR), improve the coenzyme different yeast intracellular redox imbalance . Cloned PsXR coding genes XYL1 homology search by BLAST tool, sequence comparison and structure analysis and biological software, to determine the mutation point. Fusion PCR site-directed mutagenesis and fusion expression in E. coli expression system, and HIS-TAG affinity purified expression product, the spectrophotometric detection of enzyme activity to calculate the specific activity. Detection of enzyme activity and specific activity calculations show that, the the three mutant enzyme affinity for both coenzyme has changed to some extent. With unmutated PsXR compared the affinity of the three mutant enzymes of coenzyme NADPH are significantly decreased affinity of of mutant enzymes M3 on the coenzyme NADH is not changed, but significantly increased the affinity of the M1 and M4 of the mutant enzymes of coenzyme NADH which mutations enzyme M1 affinity improved significantly decreased affinity for NADPH of NADH, and its activity is mainly dependent on the coenzyme NADH, suggesting that the key role of the K270R sites XR with coenzyme binding. The second part of the recombinant strain of Saccharomyces cerevisiae xylose fermentation Preliminary using the yeast two-hybrid system, NADH the high affinity rXR mutation gene m1 instead xyl1 with xylitol dehydrogenase gene xyl2 in S. cerevisiae AH109 co-transfection, transfection xyl1 recombinant yeast cells and xyl2 plasmid as control to construct recombinant Saccharomyces cerevisiae to ferment xylose strains. Recombinant yeast in shake flask cultures co-fermentation of xylose and glucose limit oxygen ventilation conditions, the outputs of the HPLC detection fermentation substrate consumption and metabolite. The test results show that, compared with transfection xyl1, and xyl2 gene recombinant strain AH-XR, containing the m1 and xyl2 gene recombinant Saccharomyces cerevisiae AH-M xylose utilization has increased significantly, a 16% increase in ethanol yield The xylitol generation decreased by 41.4%. The results confirmed the key enzyme xylose metabolism by protein engineered Saccharomyces cerevisiae, can be used to ferment xylose to ethanol production, and can improve the Saccharomyces cerevisiae intracellular redox imbalance problem effectively improve xylose utilization and ethanol yield.

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CLC: > Industrial Technology > Chemical Industry > Basic Organic Chemistry Industry > The production of aliphatic compounds ( acyclic compounds) > Aliphatic alcohols (alcohols, hydroxy compounds) and its derivatives > Aliphatic alcohol > Fatty Alcohols > Ethanol ( alcohol )
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