Engineering Riboflavin Biosynthesis Midstream And Downstream Pathways In Escherichia Coli For Riboflavin Production

Focusing on the riboflavin biosynthesis midstream and downstream pathways,Escherichia coli K-12MG1655was engineered as the starting strain by metabolicengineering. The ribofavin biosynthetic genes and some important genes of purinepathway were modified to increase the riboflavin biosynthis.Ribofavin biosynthetic genes from Bacillus subtilis and E. coli were engineeredgenetically. Plasmid p20C-Bsrib was constructed by overexpressing rib operon fromB. subtilis and introduced into wild-type E. coli MG1655, creating strain RF01B.Similarly, plasmid p20C-EC10was constructed by overexpressing rib operon from E.coli and introduced into wild-type E. coli MG1655, creating strain RF01S. StrainRF01B produced200.5mg/L riboflavin and RF01S produced225.1mg/L riboflavinin shake-flask fermentations. This showed overexpressing riboflavin biosynthesisgenes from E. coli had more effective on riboflavin accumulation. Thus, RF01S waschosen as the starting strain for further engineering.On the basic, availablity of3,4-dihydroxy-2-butanone4-phosphate (DHBP) andGTP，which are immediate precursors for increasing ribofavin biosynthesis, may bethe limiting factor for robiflavin production. Here improving the precursors supply forriboflavin production was attempted by enhancing ribB expression and engineeringpurine pathway in strain RF01S. Firstly, ribB gene was overexpressed by promotorreplacement to increase the flux from ribulose5-phosphate (Ru-5-P) to DHBP.Secondly, ndk and gmk genes were overexpressed to enhance GTP supply. Thirdly, aR419L mutation was introduced into purA gene to reduce the flux from IMP to AMP.Finally, co-overexpression of mutant purF and prs genes increased the flux fromRu-5-P to IMP and removed feedback regulation of some end-products. The finalstrain RF18S produced387.6mg/L riboflavin with a yield of44.8mg/g glucose inshake-flask fermentations. The titer and yield were72.2%and55.6%higher thanRF01S, respectively. These results demonstrate that simultaneously enhancing DHBPand GTP supply can efficiently boost riboflavin production by rational metabolicengineering in E. coli and offered some valuable information for other studies.