Study On The Key Factors Affecting The Biosynthesis Of Riboflavin In B.subtilis

Riboflavin(vitamin B2)is an important vitamin not only for pharmaceuticals but also for animal feed or food additives.The riboflavin produced by microbiological fermentation process,in which recombinant B.subtilis is the main strain.Although the study of riboflavin fermentation by B.subtilis over 20 years,the performance of recombinant bacteria could get better.Some metabolic and molecular genetic mechanisms affecting the synthesis of riboflavin have not been fully revealed.Based on the previous work,this article studies the relationship of nitrogen metabolism,xanthine decomposition,riboflavin conversion,gluconeogenesis and glycolytic metabolism to excessive synthesis of riboflavin.The ure ABC operon was overexpressed in B.subtilis LXZ7,get LR1.Overexpress the tnr A gene in LXZ7,the global nitrogen regulatory protein constitutive expression strains LR2 and LR3 were constructed.The results show that these two change disadvantage to synthesis of riboflavin.The puc ABCDE operon was knocked out in B.subtilis LXZ7,get LR01.The rib R was knocked out in LR01,construct strain LR11.The results showed that blocked the decomposition of xanthine and the conversion of riboflavin,which partly release the inhibitory effect of organic nitrogen sources on the synthesis of riboflavin.Knockout ccp N and pck A in B.subtilis LR11,construct strains LR22.Overexpress gap B by P_cdd and P_{lyt R} respectively,construct LR23 and LR24.The riboflavin yield of strain LR22 increased to 3.33 g/L.The riboflavin yield of strain LR23 is 3.54 g/L,and the yield of LR24 was 2.18 g/L,which decreased by 31.5%.Knock out the sr1 of the above strains and construct LR32,LR33 and LR34 strains.The yield of riboflavin in strain LR33 was the highest,reaching 3.78 g/L.Overexpress fbp in LR11,construct different expression level of fbp strains:LR25,LR26,LR27 and LR28.Riboflavin of LR28 increase to 2.82 g/L.Modify gap A of B.subtilis LR11,construct LR35,LR36,LR37,LR38,LR39 and LR40 with different gap A expression levels.The results showed that too low expression of gap A affected cell growth and decreased riboflavin production.Mild reduction of gap A gene expression slightly affect cell growth and riboflavin biosynthesis.Overexpress gap A gene in B.subtilis LR34,construct different gap A expression level:LR41 and LR42.LR42 gap A expression level lower than LR41,whose riboflavin yield was slightly higher than LR34,reaching 3.19 g/L.Therefore,maybe the 3-phospho glyceraldehyde dehydrogenase catalyzed by the gluconeogenesis consumed an additional NADPH,lead to the high metabolic flux of pentose phosphate pathway and the improvement of the supply of riboflavin precursors and the high production of riboflavin.