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The purpose of this paper is increasing the precursor of riboflavin in Bacillus subtilis by knocking out rbsK gene, and then examining its yield. At the same time, the knockout of tkt gene causing Transketolase defecting that is constructed, so the knockout of rbsK cannot lead to flux increasing in other branches, which affecting the fermentation results.rbsK gene expresses ribose kinase in pentose phosphate pathway, and further catalyzed the formation of D-ribose. Since the direct formation of riboflavin needs two precursors, that is 2DHPB + DARPP = Riboflavin, riboflavin produces by a long pathway and a short one, and D-ribose-5P is necessary for the two pathway precursors. Also, to avoid the flux increasing in other unwanted by-product pathway, we design to knock tkt gene out. Take the knockout of rbsK gene as example: rbsK gene fragments amplified by PCR obtain by digestion with the same end nuclease-off plasmid pUC18 in E. coli. Then we get the first recombinant plasmid pUC18- rbsK; in pUC18-rbsK the rbsK fragment of the selection which has suitable restriction sites were digested, and then linked with the same enzyme digested resistance gene (Spc, Kan resistance, etc.), there we get the second plasmid pUC18-rbsK-Spc or pUC18-rbsK-Kan, so rbsK gene can not express completely inactivation; Successfully built integrating plasmid pUC18-rbsK-Spc or pUC18-rbsK-Kan in Bacillus subtilis double exchange integration by inserting resistance gene express in Bacillus subtilis finally. Similarly, after a series of enzyme cutting, linking, transformation, screening operation, tkt gene can be knocked in Bacillus subtilis.This paper mailly introduces the knockout of rbsK gene and tkt gene in riboflavin producing strains RH33 and RH44, then we make some testing on the strains. First, some related metabolites in the strains constrcted before and after are examined, and we find D-ribose disappearing and Sedoheptulose-7P decreasing after the knockout of two genes. Then the strains ferment in flasks, and we find the effection of knocking out two strains is greater in RH44, which we get a strain with 4.98 g of riboflavin productin, 14.5% higher than before.
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