Modification Of Xylose Reductase From Pichia Stipitis And Primary Study On Ethanol Fermentation From Xylose Using Recombinant Saccharomyce Cerevisiae

The wild-type strains of Saccharomyces cerevisiae efficiently ferment hexose sugars to ethanol, but they are unable to utilize xylose. Xylose is the second most abundant sugar in lignocellulosic materials. The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals. Recombinant strains containing genes coding for xylose reductase and xylitol dehydrogenase from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One of the main reasons is a intracellular redox imbalance caused by the different coenzyme specificity between PsXR(with NADPH) and PsXDH(with NAD+). Hence, modifying the coenzyme specificity of PsXR and/or PsXDH by protein engineering is an attractive challenge for efficient ethanol fermentation from xylose using recombinant S.cerevisiae.Section I: Expression, Purification and Enzyme Assays of Xylose Reductase from Pichia stipitis and Various Mutants of Xylose Metabolizing EnzymesXylose reductase of Pichia stipitis (PsXR) could accept either NADH or NADPH as a cofactor, therefore, we introduced site-directed mutations designed to obtain the NADH-preferring XR and correct the intracellular redox imbalance caused by coenzyme differences in Saccharomyces cerevisiae. The mutation sites in cloned PsXR gene were identified by BLAST homogenous matching and molecular structure analysis. The mutations were introduced by nested PCR and the mutant proteins were expressed in E.coli then purified with a HIS-TAG column. The specific enzyme activity of the mutant proteins was assayed by spectrophotometry. The results showed that three of the mutants (M1, M3 and M4) differed in the enzyme activity, specific activity and the coenzyme affinity to NADPH or NADH. Comparing to wild-type PsXR, coenzyme affinity to NADPH was remarkably decreased in all three mutants. The coenzyme affinity to NADH in M1 and M4 was increased significantly, but changed mildly in M3. M1 showed the most remarkably increase of NADH-preferring activity and decrease of NADPH-preferring activity, demonstrating a maximum dependency to NADH. This implies that the mutation site K270R in M1 plays an important role in determinin the coenzyme preference of PsXR.Sectionâ…¡: Primary Study on Xylose Fermentation Recombinant Strain of Saccharomyces cerevisiaeThe genes m1 and xyl2 respectively expressing NADH-preferring xylose reductase (NADH-XR) and xylitol dehydrogenase (XDH) were introduced into Saccharomyces cerevisiae AH109 to obtain the cells AH-M, the strains AH-XR expressing xyl1 and xyl2 genes as the control group. In this study, we used the vectors provided in two-hybrid system to screen recombinant strains. The recombinant cells were cultured in shake-flask under O2-limited conditions in a yeast minimal medium with glucose plus xylose, and thanol fermentation was measured by HPLC. Comparing to the strains AH-XR, xylose utilization of the strains AH-M increased significantly, ethanol yield increased by 16%, and xylitol excretion decreased by 41.4%. This study suggested key enzymes constructed using protein engineering in xylose metabolism can increase xylose utilization and ethanol production in recombinant strain of Saccharomyces cerevisiae.