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BackgroundRiboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions.ResultsThe basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose.ConclusionsThe engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

BackgroundPoly(3-hydroxybutyrate) (PHB), have been considered to be good candidates for completely biodegradable polymers due to their similar mechanical properties to petroleum-derived polymers and complete biodegradability. Escherichia coli has been used to simulate the distribution of metabolic fluxes in recombinant E. coli producing poly(3-hydroxybutyrate) (PHB). Genome-scale metabolic network analysis can reveal unexpected metabolic engineering strategies to improve the production of biochemicals and biofuels.ResultsIn this study, we reported the discovery of a new pathway called threonine bypass by flux balance analysis of the genome-scale metabolic model of E. coli. This pathway, mainly containing the reactions for threonine synthesis and degradation, can potentially increase the yield of PHB and other acetyl-CoA derived products by reutilizing the CO2 released at the pyruvate dehydrogenase step. To implement the threonine bypass for PHB production in E. coli, we deregulated the threonine and serine degradation pathway and enhanced the threonine synthesis, resulting in 2.23-fold improvement of PHB titer. Then, we overexpressed glyA to enhance the conversion of glycine to serine and activated transhydrogenase to generate NADPH required in the threonine bypass.ConclusionsThe result strain TB17 (pBHR68) produced 6.82 g/L PHB with the yield of 0.36 g/g glucose in the shake flask fermentation and 35.92 g/L PHB with the yield of 0.23 g/g glucose in the fed-batch fermentation, which was almost 3.3-fold higher than the parent strain. The work outlined here shows that genome-scale metabolic network analysis can reveal novel metabolic engineering strategies for developing efficient microbial cell factories.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0369-3) contains supplementary material, which is available to authorized users.