Metabolic Engineering, Cofactor Engineering And Enzymatic Engineering For Construction Of Recombinant Xylose-utilizing Saccharomyces Cerevisiae Strains To Produce Ethanol

The imminent depletion of natural fossil fuel reserves coupled with the ever-increasing demand for energy has necessitated the development of sustainable renewable energy sources. Bioethanol is currently one of the most promising alternatives to conventional transport fuels because of its desirable characteristics. However, the conventional method of ethanol fermentation with corns rich in hexose would face the foodstuff competition problem with human beings. Meanwhile, lignocellulosic biomass from agricultural and agro-industrial residues represents abundant pentose (e.g. xylose). It is of great significance to find out how to efficiently convert these petose to ethanol for solving the environmental and energy problems.In the present study, Saccharomyces cerevisiae was used as model organism to study xylose metabolism. First of all, the transcriptional levels of several constitutive promoters in the Saccharomyces cerevisiae was measured through RT-PCR when strains are cultured in the medium containing xylose, and then four strongest promoters named pHXT7, pTPI1, pFBA1, pCCW12 was chosen to overexpress the xylose reductase(XR) gene XYL1, xylitol dehydrogenase(XDH) gene XYL2 (from P.stipitis) and xylulose kinase gene XKS1 (endogenous gene of S.cerevisiae) in varying degrees and eventually two high-througput xylose-fermenting recombinant strains named KAM-3X(XYL1 , XYL2 , XKS1 expression with single promoter) and KAM-6X(XYL1,XYL2,XKS1 expression with double promoters) with XR-XDH system were constructed. Both of the two strains were able to consume about 50g/l xylose in 72h, while the ethanol yield were 0.280g/g and 0.310g/g, respectively. Moreover, strain KAM-6X was capable of fermenting 100g/l xylose in 96h with the ethanol yield been 0.34g/g, while strain KAM-3X could only consume 75g/l xylose with 0.32g/g ethanol yield under the same condition. These results indicated that the xylose consumption rate as well as the ethanol yield of the recombinant strains would increase and the xylitol yield would decrease with the enhancement of XYL1/XYL2/XKS1 transcriptional levels. Whereas, only simply heighten the transcriptional levels could not solve the problem of xylitol accumulation fundamentally, which necessitated the solution of the cofactor imbalance problem existing in the XR-XDH systems to reduce the byproduct xylitol accumulation so as to increase the ethanol yield.For solving the cofactor imbalance problem, our research focused on changing the cofactor preference of XR at first. The XR mutants 2-2C12 and K270R prefers NADH but not NADPH to be its cofactor as reported previously by several paper in which the fermentation performance were not shown. Hence, we introduced the two mutants into strain KAM-6X and then the fermentation performance were tested. The results showed that the XR mutant 2-2C12 was incapable of converting xylose on account of its seriously affected enzyme activity. Meanwhile, the K270R mutation of XR partially affected its enzyme activity as well as its xylose converting rate, but the alteration of the XR cofactor preference caused significant reduction of xylitol yield from 0.105g/g in control strain KAM-6X to 0.026g/g in strain KAM-6X(K270R), while the ethanol yield was increased to 0.312g/g from 0.297g/g, all of which certified the feasibility of altering cofactor preference for reducing the byproduct xylitol accumulation and improving the ethanol yield. In addition, our research directly aimed at modifying the cofactor system. POS5(-17), an endogenous gene of S.cerevisiae encoding NADH kinase without its leading peptide and noxE, an exogenous gene from Lactococcus lactis encoding NADH oxidase were introduced into recombinant xylose-fermenting S.cerevisiae strain KAM-3X, respectively, after which the fermentation performance of KAM-3X carrying the two genes are tested. The data showed that the xylitol yield was increased from 0.177g/g to 0.298g/g, while the ethanol yield was reduced from 0.271g/g to 0.213g/g in comparison with the control strain owing to the introduction of POS5(-17), which was contrary to our expected results. However, the introduction of noxE led to 69.93% decreasing of xylitol yield from 0.191g/g to 0.058g/g, while the ethanol yield was increased by 39.33% from 0.211g/g to 0.294g/g, which was consistent with our expected results.The fermentation performance of the recombinant xylose-fermenting strain KAM-6X was measured in medium containing mixed sugar. The results showed that most of the xylose began to be consumed only after glucose was exhausting and the xylose consuming rate of KAM-6X would become more and more slower with the increase of glucose concentration in the mixed sugar, all of which was proved to be the effect of the ethanol produced by glucose fermentation through our following experimental verification.In the present study, we obtained two mutants of xylose isomerase gene derived form Piromyces sp named XImut5 and XImut8. The xylose-utilizing rate of strains carrying the two mutants was significantly improved in comparison with the reference strain KAM-2XKS(XI) which could only grow with extremely slow rate in medium containing xylose with no ethanol production, while KAM-2XKS(XImut5) and KAM-2XKS(XImut8) was capable of using up 20g/l xylose in 120h with the ethanol production of 4-5g/l. The enhancement of XI activity was proved to be the consequence of its mRNA stability promotion.