Uridine diphosphate glucose (UDP-glucose) is not only a precursor for cell wall synthesis, but also an important precursor for many sugar donors. It provides a variety of sugar donors for the synthesis of oligosaccharides, polysaccharides, glycosides and other bioactive macromolecules, and realize the diversification of their structures. Metabolic engineering has the ability to produce specific chemicals from cheap and readily available raw materials. In this study, Escherichia coli BL21(DE3) with clear genetic background was selected as the starting strain, the metabolic pathway of sucrose utilization and UDP-glucose synthesis in E. coli was modified by metabolic engineering technology. E. coli BL21(DE3) used sucrose as the only carbon source in order to achieve one-step synthesis from sucrose to UDP-glucose in vivo, and the hydrolyzed product fructose can be consumed for cell growth at the same time. By introducing sucrose permease (CscB), fructokinase (CscK) from E. coli ATCC 13281 non-phosphoenolpyruvate-dependent phosphotransferase system (non-PEP-PTS system) and sucrose synthase (SuSy) from Nitrosospira multiformis, a new pathway of UDP-glucose synthesis using sucrose as substrate was designed and constructed. On this basis, with the help of systematic metabolic engineering strategies, high copy number plasmid(pMD19-T) and promoter tandem strategy (Pcsc-PT7) were identified as the optimal strategy for UDP-glucose synthesis, the engineering strain ESBK09 which can produce UDP-glucose from sucrose was constructed successfully. In addition, in order to further increase the stability of the strain, the cscB gene was integrated into the genome of E. coli BL21(DE3) to construct the engineered strain EPT03, the recombinant strain EPT03 grew well in sucrose medium, with an OD600 of about 4.8 and a sucrose consumption rate of about 70%. The strain was shown to be able to produce UDP-glucose from sucrose. The utilization of sucrose is achieved through chromosome integration and plasmid construction, so that the E. coli strains unable to use sucrose can use sucrose stably and efficiently. The construction of an industrial strain with the ability to effectively utilize sucrose will promote the use of sucrose as a carbon source, contribute to the transition from petrochemical economy to biological economy, and provide a universal chassis microbial strain for the production of glucose-based biomolecules using UDP-glucose in the future. It also provides a key link for the subsequent construction of metabolic network for UDP-glucose synthesis of related macromolecular substances.
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