System Metabolic Engineering Of Bacillus Subtilis For Improved Riboflavin Production

Based on system biology and metabolic engineering, here we reported the characterization of the riboflavin biosynthesis ability in three riboflavin overproducers. The metabolism and regulation contributed to the riboflavin overproducing was studied. Consequently, a series of genetic modified B. subtilis strains were constructed and the major findings are:In this study, the purF gene was introduced into the amyE gene locus by a double crossover mechanism and the purF locus by a single like mechanism. However, as demonstrated in this work, a significant different effect on the purine pathway and riboflavin production was found according to the used integration mechanism. It was reasoned that the double crossover mechanism would singly up-regulate the expression of purF, in contrast, the single crossover mechanism would simultaneously up-regulate purF and its downstream genes purM, purN, purH, purD, which efficiently increased the supply of the purine precursors into purine pathway.A comparative transcriptome profiling between riboflavin producing strains and the wild type strain was performed, and it found the genotype that contributed to the riboflavin overproducing trait: The strongly up-regulated transcription of rib operon would enforce riboflavin biosynthesis pathway; Down-regulation of gene citZ and the byproducts formation genes would facilitate reducing overflow metabolism; Adoption of the glutamate dehydrogenase system could save one molecule of ATP for the assimilation of one molecule of ammonium; The up-regulation of gdh and gntK modulated carbon flow through the gluconate bypass to provide more precursor ribulose-5-P; Redirection electron flow to high coupling efficiency of terminal oxidase could enhance energy generation.In RH33 and RH44, we selected and co-overexpressed prs and ywlF genes simultaneously, which are involved in the biosynthetic pathway of PRPP from ribulose-5-phosphate. This co-amplification led to an elevated PRPP pool and thus the increased transcript abundances of PurR-regulated genes participated in riboflavin precursor biosynthesis, including purine nucleotides, glycine, glutamine etc. The riboflavin biosynthetic ability was enhanced by 20% and 3.4%, respectively.Since two DHBP molecules and one DARPP molecule were needed for the formation of one riboflavin molecule, DHBP was consumed at a higher rate than DARPP, which resulted in an imbalance supply of riboflavin synthesis precursors. However, enhancing purine biosynthesis pathway or PurR-regulated genes only facilitated the formation DARPP, which is provided by the long and normally tight-regulated metabolic pathway. Therefore, lacking of DHBP, the imbalance of precursors supply would limit the riboflavin overproducing in RH44. To solve this imbalance problem, ribB gene from E. coli that only coding the DHBP synthase was selected and overexpressed. As a result, a 17% increase in the riboflavin production and a 19% increase in the yield were obtained, which suggested that the expression of ribB gene from E. coli would produce more DHBP to recover the balance of the precursors supply.