Construction Of Riboflavin Overproduced Bacillus Subtilis

The key factor of Bacillus subtilis oversynthesized riboflavin is the riboflavinoperon (rib operon) expression. rib operon expression is strictly regulated in wild-type B. Subtilis, so it can not excessive accumulated riboflavin. To make the strainoverproduce riboflavin, the rib operon regulation should be released. Through doublestrain homologous recombination, the rib operon promoter-35zone first base Tchange to base C, this change can increase the strength of rib promoter; strong RBSreplace the regulatory rib operon leader region, to lift the control and at the same timeit can increasing the stability of rib operon mRNA, riboflavin operon transcript levelsare increased1602fold.ypaA genes encoding riboflavin transport proteins, knocked-out the ypaA gene inorder to reduce absorption of riboflavin. ywlF encoding ribose-5-phosphate isomerase,catalytic ribulose-5-phosphate isomers into ribose-5-phosphate. The purpose of theover-expression of this enzyme, is to increase production of ribose-5-phosphate. Afterreplacing the gene ypaA with ywlF riboflavin production from158.1mg/L down to61.4mg/L, a decrease of61.2%. Gene ypaA encoding riboflavin bidirectionaltransporter proteins, not only involved in the absorption of riboflavin, but alsoinvolved in riboflavin excretion, may explain this phenomenon.Bacillus subtilis has two electron transfer chain, one through a oxidase aa3,another through bd oxidase, but oxidase aa3has a higher efficiency of energyproduction. After the knockout bd oxidase gene cyd, electron transfer have to passthrough aa3oxidase. It can improve efficiency of energy production in the cell.Riboflavin production reached to194.3mg/L.prs gene encoding ribose phosphate pyrophosphate (PRPP) kinase, is a keyenzyme of riboflavin biosynthesis pathway. prs into the xylR locus increased prs genecopy number, thereby increasing expression of the ribose phosphate pyrophosphatekinase, can increasing the concentration of PRPP. High PRPP concentration canreduce the inhibition of AMP on purF, also can lift purR repression of purine de novosynthesis pathway. After the modification riboflavin production is212.5mg/L, improved by9.3%compared to cyd knock-out strain.rbsK genes encoding ribosomal kinase in the phosphate pentose pathwaycatalyzes production of D-ribose. ndk and gmk encode GDP and GTP synthase. Afterreplacement of rbsK genes with gmk, ndk gene, consumption of ribose-5-phosphatewas reducing in branch pathway, while GMP to GTP generate flux increased,riboflavin production increased by15.6%, up to246.2mg/L.