Abstract
Riboflavin (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. The 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. The 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.
Highlights
Riboflavin, 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
Riboflavin is the universal precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), both of which act as hydrogen carriers and are essential for the activity of a wide variety of metabolic enzymes in higher eukaryotes [1,2]
In B. subtilis, introduction of multiple copies of rib operon into the chromosome resulted in improving the riboflavin production [7]
Summary
Riboflavin (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. Riboflavin (vitamin B2) is the universal precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), both of which act as hydrogen carriers and are essential for the activity of a wide variety of metabolic enzymes in higher eukaryotes [1,2]. Riboflavin is synthesized by all plants, fungi and most bacteria, but not by higher animals including humans. Riboflavin was synthesized and produced by chemical. Riboflavin biosynthesis has been studied in both grampositive and gram-negative bacteria, extensively in Bacillus subtilis and E. coli. The genetic background of these strains was usually complicated and the mechanism of riboflavin overproduction was not fully elucidated
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