Effect Of Genetic Modification And Xylose Fermentation On Riboflavin Production In Bacillus Subtilis

Riboflavin is an important B vitamin.The most important industrial production method of riboflavin is using recombinant Bacillus subtilis fermentation.In this paper,in order to further improve riboflavin production of the recombinant strain,the restrictive factors of riboflavin biosynthesis pathway were researched in Bacillus subtilis;The effect of genetic modification of the genes that involved in xylose metabolismand fermentation with xylose as carbon sourceon riboflavin biosynthesis were investigated.Firstly,we use B.subtilis LX34 as the starting strain,introduced G596 A point mutation in the coding sequenceat of flavin kinase / FAD synthase gene(ribC),achieved the genetic effects that the 199 th amino acid coding sequence occurred "Gly? Asp " change constructed the recombinant strain B.subtilis LXZ-1.The results showed that the G596 A point mutation can significantly reduced the flavin kinase activity then induce the accumulation of riboflavin in the bacteria.Use B.subtilis LXZ-1 as the starting strain,we constructed the recombinant strain B.subtilis LXZ-2 by integrated the ribA gene on the site of sacB promoted with the strong promoter Pcdd.Resulted in an increase of riboflavin yield by 80% and a reduce of maximum biomass by 30% because of autolysis.The results showed that the bifunctional enzyme ecoded by ribA gene is rate-limiting enzyme of riboflavin biosynthesis,but its overexpression can lead to some cell autolysis becouse of the accumulation of intermediate metabolite.Use B.subtilis LXZ-1 as the starting strain,we constructed the recombinant strain B.subtilis LXZ-2 by integrated the ribA and ribH genes on the site of sacB promoted with the strong promoter Pcdd.the yield of riboflavin increased by 280%along with the biomass restored and autolysis disappeared.The results showed that in the riboflavin biosynthesis pathway,after the overexpression of gene ribA,the Dioxo-tetrahydro-pteridine synthetase that encoded by ribH become another rate-limiting enzyme,Co-overexpression of ribA and ribH genes avoided the cytotoxicity resulted from the accumulation of intermediate metabolite effectively and further increased riboflavin production.With B.subtilis LXZ-3/pMX45 as the starting strain,we use four recoment carbon as the carbon source fermentation to produce riboflavin.When use xylose as the carbon source,there has a highest production of riboflavin,but a lower biomass.When we use sucrose/xylose as the carbon source,the biomass become normal.The results showed that sucrose/xylose co-metabolism improved the supply of ribulose-5-phosphate,which consequently contributed to an improvement of riboflavin production.Use B.subtilis LXZ-3 as the starting strain,we constructed the recombinant strain B.subtilis LXZ-4 by overexpress araE gene and the recombinant strain B.subtilis LXZ-5 by overexpress rpe gene and the recombinant strain B.subtilis LXZ-6 by constitutive expression of the xylose operon in situ.Compared to the starting strain,riboflavin production of these recombinant bacteria were significantly reduced.The results showed that on the base of the lack of regulatory proteins AraR,absorption and metabolism of xylose is no longer limiting factors for riboflavin production in B.Subtilis.With B.subtilis LXZ-3/pMX45 as the starting strain,use 5 L fermenter riboflavin fermentation.When use 8% sucrose as the carbon source,the highest yield of riboflavin can reach 2 g/L.When use 6.5% xylose fermentation and 1.5% sucrose as the carbon source,the highest riboflavin production reached 3.6 g/L,after 70 h fermentation.The results showed that the main xylose xylose / sucrose co-metabolism,riboflavin to improve fermentation yield had a significant effect.