Metabolic engineering of Escherichia coli for the production of riboflavin

Abstract

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.

Production of riboflavin and related cofactors by biotechnological processes

“…In C. famata, overexpression of the genes RIB1 and RIB7, respectively encoding GTP cyclohydrolase II and riboflavin synthase, led to increased production of RF [30]. In E. coli, overexpression of an artificial RF operon called EC10 (Ptrc-ribABDEC) was used to successfully engineer RF overproduction [54].…”

Optoregulated Drug Release from an Engineered Living Hydrogel

“…Being the workhorse for genetic engineering, E. coli is the most extensively used bacterium for which the highest variety of genetic modules are available. E. coli has been engineered for metabolic production of drugs such as riboflavin, 29 artemisinin, 30 salidroside, 31 cinnamaldehyde, 32 carbapenem, 33 violacein, 34,35 deoxyviolacein (dVio), 34,35 etc. at industrial scales.…”

Genetic-metabolic coupling for targeted metabolic engineering

“…When these enzymes are over-expressed, the supply of energy, cofactors, and carbon building blocks quickly become limiting and can lead to a strong metabolic burden on the cell. Indeed, metabolic burden can lead to low cell density and reduced titers during the production of recombinant vitamins, such as cobalamin (vitamin B 12 ) (Biedendieck et al, 2010) or riboflavin (vitamin B 2 ) (Lin et al, 2014). Furthermore, central cell metabolism, which provides building blocks for vitamin biosynthesis, is subject to complex regulation that can affect pathway optimization (Lin et al, 2014).…”