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

In this study,the engineering strain B.subtilis 1-1 for high-yield riboflavin production was obtained from B.subtilis X42 and B.subtilis 120 using genome shuffling technology.And the production was furthered improved by medium optimization in shake-flask fermentation.What's more,the fermentation was conducted in 5Lbioreactor and the production of riboflavin was furthered improved by the optimization of fed-batch fermentation process and medium components.The production of B.subtilis 1-1 finally reached 7443.66 ± 19.5 mg/L riboflavin and the yield reached 0.119 mg/g glucose within 120 h.At last,B.subtilis 120 spc was engineered as the starting strain and modified to over-express heterologous genes coding endo-1,4-betaxylanase and ?-xylosidase as well as the gene for xylose utilization.The resulting strain can metabolize xylan as carbon resource to produce riboflavin.The screened stain that restored the production level of riboflavin was used as parent strain.Overexpress spc gene in B.subtilis 120 and obtained the engineering stain B.subtilis 120 spc,which used as the other parent strain.The two parent strains were fused by genome shuffling and one fusant named B.subtilis 1-1 for the highest riboflavin production was screened out.The production of B.subtilis 1-1 can reach 7443.66 ± 102.54 mg/L riboflavin and the yield can reach 93.87± 1.00 mg/g glucose,which is 11.04% and 16.53% higher than B.subtilis X42,respectively.The medium components in shake-flask fermentation were further optimized.with addition of 2 g/L urea and 1 g/L yeast extract powder,the riboflavin production of B.subtilis 1-1 can reach 8557.11 ± 93.45 mg/L and the yield can reach 122.74 mg/g glucose,which is 15.00% and 30.75% higher compared to the values using unoptimized fermentation medium,respectively.Next,we conducted fermentation of the engineering stain B.subtilis 1-1 in 5Lbioreactor.The fed-batch fermentation process and medium components were optimized and the riboflavin production finally reachd 18.5 g/L and the riboflavin yield reached 0.119 g/ glucose within 120 h.In the end,the engineering stain B.subtilis 120 spc was modified to express xyn10 B gene from Clostridium stercorarium and xsa gene from Selenomonas ru minantium GA192,and endogenous gene araE expressing xylose transporter,as well as xylA and xylB from Escherichia coli.The resulting strain named B.subtilis E2X6 PAB can metabolize 49 g/L xylan and 1 g/L xylose to produce 2484.303 ± 25.9 mg/L riboflavin and the yield of riboflavin is 49.67± 0.5 mg/g glucose,which verified the feasibility to produce riboflavin from xylan.