Metabolic Engineering Of Bacillus Subtilis For Riboflavin Production

In this study, focusing on overexpressing riboflavin biosynthetic genes, increasing the supply of GTP which is the precursor of riboflavin and modifying respiratory chain, B. subtilis BS110(lab stock) was engineered as the starting strain and modified to increase the riboflavin biosynthis by metabolic engineering. Finally, the riboflavin production of engineering strain reached 4.2 g/L and riboflavin yield reached 40 mg/g glucose.Purine pathway of BS110 was engineered genetically with the purpose of increasing the supply of GTP. Knockout guaC gene, blocking the pathway of GMP to IMP; Overexpress prs and ywl F genes, improve concentration of PRPP and ribulose-5-phosphate(Ru-5-P) increasing the flux to purine pathway; At last, guaB gene was overexpressed by promotor replacement enhance the flux from IMP to GMP, creating engineering strain BS118; Strain BS118 produced 1194.95 ± 45.58 mg/L riboflavin and riboflavin yield reached 11.61 ± 0.47 mg/g glucose in shake-flask fermentations. This showed metabolic engineering of purine pathway had effective on riboflavin accumulation.Metabolic engineering of the respiratory chain based on BS118 can improve the energy efficiency of the engineering strain. Knockout cyd gene coding bd oxidase, creating engineering strain BS119; The riboflavin production of BS119 was 1296 ± 34.03 mg/L and riboflavin yield was 11.72 ± 0.05 mg/g glucose in shake-flask fermentations. After improving the respiratory chain, strain BS119 did not increase the riboflavin production significantly. The expression of riboflavin biosynthetic genes may be the limiting factor for riboflavin production.Finally, to improve the ability of riboflavin biosynthesis, we overexpressed rib operon. Knockout sigE gene to cut off pathway of sporulation based on BS120, creating strain BS121. Riboflavin yield increaseed by 13% compared to BS120. Rib operon was integraed into starch hydrolytic enzyme loci, creating strain BS120, The riboflavin production of BS120 reached 1854 ± 31.87 mg/L. Converted pMX45 plasmid and increased rib operon copy, creating strain BS125; The riboflavin production of BS125 reached 4.2 g/L, riboflavin yield reached 40 mg/g. Riboflavin production of strain BS125 increased by 408% compared to BS110 and riboflavin yield increased by 373%.