Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering

Abstract

In this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 strain was constructed by the overexpression of zwf harboring the constructed plasmid pAC-Z in the R1 strain. Thus, the level of riboflavin in the R2 strain increased to 319.01 ± 20.65 mg/l (74.66% increase). To further enhance ribB transcript levels and riboflavin production, the FMN riboswitch was deleted from E. coli BL21 with CRISPR/Cas9 to generate the R3 strain. The R4 strain was constructed by cotransforming pET-AE and pAC-Z into the R3 strain. Compared to those of E. coli BL21, the ribB transcript levels of R2 and R4 improved 2.78 and 3.05-fold, respectively. The R4 strain accumulated 437.58 ± 14.36 mg/l riboflavin, increasing by 37.17% compared to the R2 strain. These results suggest that the deletion of the FMN riboswitch can improve the transcript level of ribB and facilitate riboflavin production. A riboflavin titer of 611.22 ± 11.25 mg/l was achieved under the optimal fermentation conditions. Ultimately, 1574.60 ± 109.32 mg/l riboflavin was produced through fed-batch fermentation with 40 g/l glucose. This study contributes to the industrial production of riboflavin by the recombinant E. coli BL21.

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

“…Another example is the bifunctional enzyme RibA, which bears both GTP cyclohydrolase II (GTPCHII) and dihydroxybutanone phosphate synthase (DHBPS) activities and is utilized by plants and the Gram-positive bacterium Bacillus subtilis to catalyze the formation of the two precursors [ 35 , 36 ]. In contrast, Escherichia coli and yeast use two separate monofunctional enzymes (ribA and ribB) [ 37 ]. The fused enzyme has been suggested to have a kinetic advantage to consume the two substrates stoichiometrically [ 38 ].…”