| The "oil crisis" and environment pollution by rapid economy society development caused concerns about problems of resource depletion and environmental globally deteriorating.Fuel ethanol is found to be one of the renewable and clean energy sources.There are abundant lignocellulosic feedstocks on the earth, however the cheap resource is not utilized effectively.Lignocellulosic feedstocks are composed predominantly of cellulose,hemicellulose and lignin.Xylose is the predominant pentose sugar derived from the hemicellulose and is about 30%in the lignocellulose hydrolysates,which is the second plentiful sugar after glucose in nature. Consequently,the efficient bioconversion of xylose is one of the major keys for efficient utilization of lignocellulosic hydeolysates to produce fuel ethanol.The Gram-negative Zymomonas mobilis is the only microorganism known as the natural ethanologenic bacterium converting glucose to ethanol at high efficiencies by the unique Entner-Doudoroff parthway which is coupled to pyruvate decarboxylase and alcohol dehydrogenase.This organism demonstrates many of the traits sought in an ideal biocatalyst for fuel ethanol production,such as high ethanol yield and tolerance,high fermentation selectivity and specific productivity.However its substrate range is restricted to the fermentation of glucose,fructose and sucrose,and as such,wild-type strains are unable to ferment the pentose sugars derived from lignocellulosic feedstocks because they lack the essential pentose assimilation and metabolism pathways.Consequently,metabolic engineering of xylose fermentation of Z.mobilis is an essential step toward development as a biocatalyst for fuel ethanol production from lignocellulosic feedstocks.Although there has been success in engineering Z.mobilis for xylose metabolism,the strains do not grow and produce ethanol as well on xylose as on glucose for the reasons listed below:the growth inhibited by by-product xylitol,the slow rate of xylose assimilation and metabolism, the loss of carbon and energy and so on.In this study,we developed a xylose-fermenting strain of Z.mobilis and on this basis,clarified the xylitol mechanism and build the platform for genetic evolution of glucose facilitator protein, provided the foundation of improving the properties of xylose-fermenting.The major results of the thesis are as follows:1.Metabolic engineering of Zymomonas mobilis for xyiose utilizationXylose could enter the ED pathway by pentose phosphate.Xylulose-5-P is the important intermediate product and it can be catalyzed by xylulokinase,transaldolase and transketolase further,and then enter ED pathway finally.Z mobilis lacks the essential pentose assimilation and metabolism pathways.Consequently,in this study we introduced genes that encode the xylose isomerase(xylA) and xylulokinase(xylB) precisely under the control of a strong,constitutive glyceraldehydes-3-phosphate (GAP) promoter by PCR-mediated overlap extension,which is in charge of xylose assimilation;and also we introduced genes that encode the transaldolase(talB) and transketolase(tktA) precisely under the control of enolase(ENO) promoter,which is in charge of xylose utilization,then transformed plasmid with the two operons into Z. mobilis.Thus we completed a pentose phosphate pathway in Z mobilis.Enzymatic analyses of recombinant Z.mobilis demonstrated the presence of the four enzymes activities,thereby we developed a xylose-fermenting strain of Z.mobilis CP4 (pBBR-xyl).2.Fermentation performance of recombinant Z.mobilis CP4(pBBR-xyl) with xyloseThe recombinant Z.mobilis CP4(pBBR-xyl) produced 19.5 g/l ethanol from 3.2%of glucose and 3.4%of xylose within 60h,the xylose utilization rate is 32.3%, and the total sugar utilization rate is 65.36%,it achieved 57.6%of theoretical ethanol yield from glucose and xylose.Although the recombinant strain we developed could not convert xylose to ethanol with high-efficient because of the growth inhibited by by-product xylitol,the slow rate of xylose assimilation and metabolism,the loss of carbon and energy and so on.,it has the ability to convert xylose to ethanol,and it provided the foundation of improving the properties of xylose-fermenting through every means such as metabonomics.3.Mechanism of catalyzed formation of xylitol in Z.mobilisXylitol has been shown to be a major by-product of xylose metabolism causing significant growth inhibition of recombinant strains by Kim et al.They found that only the recombinant strain was growth inhibited with addition of xylose,and this resulted from the accumulation of xylitol phosphate due to a side reaction effected by a cloned xylulokinase.Furthermore,Kim et al.found above concentrations of 5 g of xylitol per liter,recombinant strains could not grow at all.Production of xylitol and the resulting growth inhibition by xylitol phosphate have been considered to be one of the important factors affecting the rates and yields from xylose metabolism by the recombinant Zymomonas mobilis,but the mechanism of xylitol formation is largely unknown.In this study we found that glucose-fructose oxidoreductase(GFOR),a periplasmic enzyme responsible for sorbitol production,catalyzed the reduction of xylose to xylitol in vitro operating via a ping-pong mechanism similar to that in the formation of sorbitol.However,the velocity of xylitol formation was much lower than that of sorbitol(1.27 mmol 1-1 vs 57.45 mmol 1-1 h-1);xylitol formation at different concentrations of xylose demonstrated a typical sigmoidal curve with a Hill coefficient of about 2,implying that a substrate-induced configuration change occurred with GFOR during its catalyzed reduction of xylose,ping-pong kinetics was observed for GFOR in catalyzing the formation of xylitol with parallel sigmoidal curves when the initial concentrations of xylose varied against different concentrations of glucose,suggesting a similar binding competition exist between xylose and glucose;While change of the potential acid-base catalyst Tyr269 to Phe almost completely abolished the activity toward xylose as well as fructose,mutant S116D,which has been shown to lose tight cofactor binding,displayed an even slower catalytic process against xylose.Overall,the results suggested that similar catalytic mechanisms were adopted by GFOR during the reduction of fructose and xylose.While when we prepared to study in vivo further after our paper published,we found a patent applicantion published by NREL on 2008 Nov.at the same time,which reported xylitol production in xylose metabolizing Z.mobilis is predominantly mediated by the enzyme glucose-fructose oxidoreductase(GFOR).And the GFOR knockout mutant consumed more xylose and produced significantly higher concentrations of ethanol.So they applied for patent protection for the technology and contents in light of potential industrialization.So with our study in vitro and NREL's study in vivo we confirm that it is GFOR that catalyze reduction of xylose to xylitol in Z.mobilis.4.Platform for analysis of glucose facilitator protein mediated xylose transporting and genetic evolutionThe glucose facilitator protein(GLF) of Z.mobilis was found to play its part in glucose uptake and also with xylose uptake in a non-energy dependant manner. Kinetic studies on sugar uptake by GLF showed that the affinity for glucose(Km=4.1 mM) is higher than xylose(Km = 40 mM),therefore GLF may prefer to transport glucose to xylose when they are coexist.Competition between sugars for transport via GLF may play a critical role in preferential catabolism of a particular sugar from a mixed sugar fermentation.We knocked out xylFGH gene related with xylose transporting in E.coli ZSC112ΔptsHI-crr(the strain lacks all phosphoenolpyruvate-dependent sugar phosphotransferase system PTS uptake systems and glucokinase,so it could neither transport nor phosphorylated glucose) by homologous recombination,and developed ZSC112ΔptsHI-crr-xylfgh strain which could neither transport glucose nor xylose. Further we transformed GLF-GLK(glucose facilitator protein- glucokinase) derived from Z.mobilis into ZSC112ΔptsHI-crr-xylfgh strain and found that the new recombinant strain can grow again on glucose and xylose.It illustrated GLF-GLK derived from Z mobilis could make up defects on PTS in E.coli functionally,and also GLF could make up defects on xylose transporting(xylFGH gene was deleted) in E.coli.So we acquired glucose-xylose-negative mutant strain ZSC112ΔptsHI-crr-xylfgh,and got a type strain used to study on characterization of various kinds of sugars.It makes foundation to get high glucose-xylose uptake mutant GLF-GLK by chemical mutagenesis or error-prone PCR.
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