Metabolic Engineering Of Succinate Biosynthesis From Xylose In Escherichia Coli

Succinate which is widely used in medicine, food, chemistry industry, etc. has a potentialmarket demand. But it costs a great deal of nonrenewable resource such as fossil oil if weproduce succinate in traditional chemical method. What’s more, those traditional methods leadto environmental pollution. So a new way not to contaminate the environment should beseeked. In order to reach that goal, production of succinate by microbial fermentation usingrenewable resources is studied. Now we have gotten initial progress in producing succinate byfermentaion. Though producing succinate by glucose has been well studied, there are rarelyreports about producing succinate by other carbon source, especially by xylose in E. coli.In this thesis, succinate synthesized from xylose in E. coli was studied so as not to strivefor carbon source with humanbeing. The purpose of this experiment is to construct somestrains that can produce succcinate by xylose. And we’d like to make a contribution to producesuccinate efficiently.We analysed the biosynthesis pathway of succinate from xylose in E. coli by pathwayanalysis method firstly. On the basis of the results in pathway analysis, some gene-knockoutstrains were constructed by one step knockout way.and checked by PCR using E. coli BL21asthe starting strain. The concentrations of the organic acids of fermentation broth in the shakeflasks were determined by HPLC.We get conclusions as follows:(1) With the understanding of the structure of cell metabolism and genome information,the pathway model in wich E. coli produced succinate using xylose in aerobic condition wasbuilt.(2) The metabolic pathway of succinic acid synthesis from xylose was analysed, and theperfect pathway in producing succinate from xylose was found. The maximum convesion rateof0.83mol/mol xylose was calculated theoretically and the genes need to be knocked out or tobe expressed were determined. (3) According to the results of the pathway analysis, target genes including succinatedehydrogenase(SDH), phosphotransacetylase(Pta), pyruvate oxidase(PoxB) were knocked outfrom E.coli BL21to build strain ZXY542; and target genes including succinatedehydrogenase(SDH), phosphotransacetylase(Pta), pyruvate oxidase(PoxB), aceBAK operonrepressor(IclR)are knocked out from E.coli BL21to get ZXY412; target genes includingsuccinate dehydrogenase(SDH), phosphotransacetylase(Pta), pyruvate oxidase(PoxB), acetatekinase (Ack)are knocked out from E.coli BL21to get ZXY354612.(4) On the basis of pathway analysis, phosphoenolpyruvate carboxylase gene from E.coliMG1655was overexpressed and strains ZXY412-JL225-9, ZXY542-JL225-9andZXY354612-JL225-9were obtained.(5) Results of shake flask fermentation showed that those new strains can producesuccinic acid under aerobic condition from xylose.