Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis

Abstract

Riboflavin is an essential vitamin widely used in the food, pharmaceutical, and feed industries. However, the insufficient supply of precursors caused by the imbalance of intracellular metabolic flow limits the riboflavin synthesis by industrial strains. Here, we increase riboflavin production by tuning multiple gene expression to balance intracellular metabolic flow. First, we tuned the expression of mCherry and egfp genes within operons by generating libraries of tunable intergenic regions (TIGRs) and confirmed the relative expression of the two reporter genes. The TIGR library can coordinate the expression ratio of reporter genes more than 180 times in Escherichia coli and more than 70 times in Bacillus subtilis. Next, we used this strategy to tune the expression of zwf, ribBA, and ywlf genes within operons through the TIGR library to increase the intracellular precursor pool for riboflavin biosynthesis. Based on the fluorescence characteristics of riboflavin, 96-well plates were used to screen the optimal combination mutants quickly. The best-engineered strain was selected from the library, which produced 2.7 g/L riboflavin, increasing by 64.35% in the shake flask. Finally, the riboflavin titer increased by 59.27% to 11.77 g/L in fed-batch fermentation. The strategy described here will contribute to the industrial production of riboflavin and related products by B. subtilis.

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens

“…The fluorescence values of eGFP of different strains were measured to further identify strong constitutive promoters in S. marcescens as described previously ( You et al, 2022 ). In brief, strain JNB5-1/pUCP18 and recombinant strains containing plasmids pUCP18-promoter-egfp were inoculated in sterile 96 black-well plates (corning 3603) containing 200 μl LB medium and incubated at 37°C for 10 h. Then eGFP fluorescence was determined at the end of the incubation with a microplate Multi-Mode Reader (BIOTEK, Cytation 3) at the absorption wavelengths of 490 nm and 530 nm.…”

Utilizing 5′ UTR Engineering Enables Fine-Tuning of Multiple Genes within Operons to Balance Metabolic Flux in Bacillus subtilis

“…Fluorescence intensities of EGFP and mCherry were determined as previously reported [36]. Single colonies were selected and inoculated into 96-well black plates (Corning 3603), each containing 200 µL of LB medium.…”

Metabolic Engineering of Bacillus subtilis for Riboflavin Production: A Review

“…Zhang et al overexpressed gntP , which encodes gluconate permease, to increase riboflavin production [ 54 ]. By constructing the tunable intergenic regions library, the gene expression of zwf , ribBA and ywlF were adjusted to increase the intracellular Ru5P and produced 2.7 g/L riboflavin in shaking flask, which increased the yield by 64.35% [ 55 ].…”