Screening Of Bacillus Subtilis For Riboflavin Production By ARTP And Optimization Of Riboflavin Fermentation

Bacillus subtilis is an important part in the industrial production of riboflavin.However,the export and application of riboflavin have been restricted due to the large number of antibiotic labels on traditional industrial production.In this study,high riboflavin-producing B.subtilis was bred by Atmospheric and Room Temperature Plasma(ARTP),while explored the genetic basis of riboflavin-producing by genomic sequencing,combined with metabolic engineering and optimized fermentation conditions to increase riboflavin production.In this work,BSG1 was obtained which treated the parent strain BS120 and screened with 40 mg/L 8-azaguanine with increase of production(83.59%)and yield(78.76%)than BS120,respectively.BSG3 which generated by treating BSG1 and screening with 300 mg/L oligomycin were increased of production(83.59%)and yield(78.76%)compared with BS120,respectively.BSG5 which obtained by transformed riboflavin operon expression plasmid p MX45 into BSG3,of which riboflavin production was 4467.08 ± 99.47 mg/L,and the yield was 42.56 ± 1.25 mg/g glucose,which was 140.94% and 120.52% higher than that of BS120.And these three mutant strains exhibited excellent riboflavin fermentation performance and genetic stability.There were no SNV between BS120 and BSG1,and dha S(G376R)was found between BSG1 and BSG3 by the second generation sequencing.Results showed that dhaS(G376R)increased the riboflavin production of BSG6(7.63%)and BSG7(5.37%)obtained by introducing dhaS(G376R)into BS120 and BSG1 respectively,but the production of BSG7 was 5.81% lower than BSG3.The third generation sequencing will be used to in-depth analyze of genomic sequence and structural differences obtaining the complete genomic information of the mutant strains.The production of fermentation conditions optimized by statistical strategy was increased to 6014.12 ± 45.88 mg / L,and the yield was 44.44 ± 0.08 mg / g glucose after 144 h.According to the optimal medium formula,the culture was expanded in a 5L fermenter and fed-batch fermentation resulting to its production was 11842.68 mg/L,yield was 51.99 mg/g glucose,increased by 96.91% and 16.75%,respectively.