大肠杆菌木糖生物合成琥珀酸的代谢工程研究

目的:对大肠杆菌进行代谢网络改造,考察木糖好氧发酵生产琥珀酸的可行性。方法:以有氧条件下大肠杆菌木糖生物合成琥珀酸的代谢途径分析为基础,以大肠杆菌BL21为出发菌株,通过P1噬菌体一步敲除法敲除琥珀酸脱氢酶基因(sdhA)、磷酸转乙酰基酶基因(pta)、丙酮酸脱氢酶基因(poxB)及异柠檬酸裂解酶阻遏物基因(iclR),构建木糖好氧发酵生产琥珀酸的大肠杆菌工程菌JLS400(△poxB△pta△iclR△sdhA)。将携带磷酸烯醇式丙酮酸羧化酶基因的质粒pJW225转化到JLS400中。结果:摇瓶发酵结果表明,构建的工程菌能以木糖为碳源,在好氧发酵条件下琥珀酸产率较高,副产物仅有少量乙酸和丙酮酸。结论:基因工程大肠杆菌JLS400pJW225的构建,为有氧条件下以木糖为原料生产琥珀酸的进一步研究奠定了基础。

Metabolic Engineering of Succinate Biosynthesis from Xylose in Escherichia coli

Objective： The metabolic network of Escherichia coli was modified to evaluate the possibility of producing succinate from xylose under aerobic conditoins.Methods： Genes encoding pyruvate oxidase,aceBAK operon repressor,phosphotransacetylase and succinate dehydrogenase of E.coli BL21 were knocked out respectively by P1 phage transduction based on metabolic pathway analysis of succinate biosynthesis from xylose to create E.coli JLS400（△poxB△pta△iclR△sdhA）.Plasmid pJW225 which expresses phosphoenolpyruvate carboxylase was transformed to JLS400pJW225.Results： Fermentation results in shake flask culture showed that the yield of succinate from xylose was high,and little acetate and pyruvate was detected as byproduct.Conclusion： The construction of E.coli JLS400pJW225 lays the foundation for the production of succinate from xylose under aerobic conditions.