Regultory Mechanism Of Oxygen Availability On Riboflavin Biosynthesis By Bacillus Subtilis Based On Integration Of Mixomics

Riboflavin production by Bacillus subtilis became more and more popular instead of synthetic method.However,some shortcomings still exist in riboflavin fermentation,including decreased riboflavin production rate at late phase,accumulation of secreted acidic intermediates and low efficiency of carbon conversion into riboflavin.In previous studies,it was found that the oxygen uptake rate became too high and the riboflavin formation rate subsequently decreased when the dissolved oxygen(DO)tension was below critic level.To get a better understand about riboflavin producing strain,genomics was performed and transcriptomics and metabolomics were integrated to figure out the regulatory mechanism of oxygen availability affect riboflavin production.The results are shown below.1)Comparative genome analysis between B.subtilis A29 and B.subtilis 168.Based on the genomic bioinformation of riboflavin industrial strain B.subtilis A29 and wild type strain B.subtilis 168,two SNPs were found to be involved in riboflavin biosynthesis and were considered as possible key factors to riboflavin synthesis and accumulation.While the whole riboflavin operon from NCBI database was aligned with genome of riboflavin producing strain,at least one more riboflavin operon which contained high ratio SNPs was found.The SNPs located in regulatory region were speculated to cause the malfunction of riboflavin feedback regulation and high expression of riboflavin operon.2)Influence of DO on riboflavin biosynthesis was analyzed based on transcriptomics and metabolomics.Combining the transcriptomics and metabolomics technologies,the influence of oxygen availability on riboflavin fermentation was unveiled.Under low DO condition,a membrane-anchored protein,ResE can phosphorylate ResD,a regulation protein which can activate genes such as narK?narJ?narl?narH?narG and fnr.Gene fnr encodes the fumarate and nitrate reduction regulatory(FNR)protein which can regulate the genes involved in central carbon metabolism.This protein was thought to lead to the upregulation of gene alsS and downregulation of genes ykgB?mdh?sdhA?sucD under low DO tension.It was speculated that the downregulation of gene ykgB was the main cause of carbon fluxes shift,decline of intracellular 6-phosphogluconate concentration and reduction of riboflavin production under low DO tension.The upregulation of gene alsS was the key factor that can balance the ratio of NADH/NAD+ and produce ATP anaerobically.In addition,the accumulation of downstream metabolites in glycolysis pathway especially pyruvate can explain the phenomenon that the flux through TCA cycle was higher under low DO condition.The bioinformatics including genomics,transcriptomics and metabolomics of an industrial strain B.subtilis A29 grown under different DO tensions was analyzed,and elucidated how the oxygen availability affect transcription level of some key genes,leading to changes in enzymes involved in central carbon metabolic pathway and fluxes shift.The results could play a solid ground for new fermentation process development and genetic modification of strain.