Comparative Genomic and Transcriptomic Analysis Reveal Genetic Characteristics Related to Industrially Riboflavin Production in Bacillus Subtilis

Bacillus subtilis has been successfully engineered as cell factory for riboflavin production, whose construction was primarily carried out by random mutagenesis and high-throughput screening in collaboration with metabolic engineering. However, random mutagenesis is laborious, time-intensive, and suffers from the limitation that most random mutations are neutral or detrimental toward the desired phenotype. Due to the unclear background and unstable performance of mutant strains, it is not easy to further improve their performance by rational metabolic engineering. And herein we attempted to understand the genetic basis of riboflavin accumulation with B. subtilis through a comparative genomic and transcriptomic approach. Two riboflavin-producing strains B. subtilis WS1 and WS2 were conducted whole-genome sequencing to parse out the identities of their mutations. By analyzing dozens of mutations in a combinational way, at least seven specific gene mutations were approved to be directly related to the riboflavin accumulation, including ribC, ribO, purA, ccpN, ywaA, yhcF, yvrH. An unexpected point-mutation in two-component response regulator gene yvrH was firstly reported to greatly prompt riboflavin production. Comparative transcriptomic analysis was employed to assist in elucidating the mechanism involved in riboflavin synthesis. It was highly recommended that an extended application of these beneficial mutations for the production of similar products in microorganisms. Based on the identified beneficial mutations, a strain with minimal mutations was constructed for subsequent improvement in riboflavin production by systems metabolic engineering.