The Genetic Modification Of Purine Nucleotide Biosynthesis And RibC Gene In Bacillus Subtilis

Riboflavin(vitamin B2, VB2) is widely used not only for pharmaceuticals but also foranimal feed additives and food industry. The Gram-positive bacterium Bacillus subtilis,which does not naturally over-produce riboflavin, has been adopted in the currentcommercial riboflavin production processes. The riboflavin producer B. subtilis isdeveloped by the “classical” strain improvement approach that relies on iterative cycles ofgenetic diversity creation through mutagenesis. Although some mutants isolated in thisway exhibited higher riboflavin-producing ability, such conventional methods depend onchance to encounter desired mutants among resulting colonies which inevitably accumulatenumerous unidentifiable and unwanted mutations, and could not reveal the mechanisms onthe basis of molecular biology. In this study, we used genetic engineering breedingmethods to modify the genes that associated with purine nucleotide synthetic pathway andriboflavin conversion reactions in Bacillus subtilis, and then inspect the effects onriboflavin excessive synthesis.Firstly, the gene for GMP reductase (guaC) of B. subtilis LX20in which rib oerponconstitutively expressed was inactivated by marker-free deletions to establish therecombinant strain LX21. On the basis, the LX22strain was obtained in which the gene foradenylosuccinate synthetase (purA) was deleted.Secondly, the LX34strain was constructed by deleting the gene for pur operonrepressor (purR-yabJ) of strain LX22. At the same time, hypoxanthine-deficient strainLX23was obtained after pur operon mRNA leader region was replaced with thechloramphenicol-resistant gene in LX21. Subsequently, the function of pur operon inLX33was restored by the replacement chloramphenicol-resistant gene with cryIIIA mRNAstabilizer in LX23. The strain LX33was engineered to be LX35which purA andpurR-yabJ gene were inactivated by the marker-free deletions. The transcriptional level ofpur operon was detected by RT-PCR. It shows that the intracellular pur operon mRNAexpression level of LX34and LX35were about10.18-fold and53.33-fold higher than thatof LX22respectively. It suggested that knockout of purR-yabJ and replacement of puroperon mRNA leader region with cryIIIA mRNA stabilizer could effectively improve thetranscriptional level of pur operon.Finally, the stains LX24, LX36and LX37were obtained when the first nucleotide of-35sequences of ribC promoter in LX22, LX34and LX35was mutated from “T” to “C”.The transcriptional level of ribC gene was detected by RT-PCR. It indicates that the intracellular ribC gene mRNA expression level dropped to3%of wild type. Shake flasksfermentation results showed that the genetic modification of purA, guaC, purR-yabJ andribC have no significant impact on the growing ability of the strain. However, the specificgrowth rate of strain LX37in which pur operon constitutively expressed has a dramaticdecline. The point mutation of ribC gene promoter could only increase riboflavinproduction by3times, up to160mg/L. It suggested that the point mutation of ribCpromoter could not achieve the effect of point mutation of ribC coding sequences.Meanwhile, it could not fully reflect the effect of constitutive expression of pur operon onriboflavin synthesis on the basis of point mutation of ribC promoter.