Construction Of Cytidine Production Strains Of Bacillus Subtilis

Cytidine, which is the intermediate in the process of chemically synthetizing antiviral and anti-cancer drugs, has important application value.This article has preliminary research on the construction of cytidine production strains of Bacillus subtilis by genetic engineering breeding.In this study, firstly, the neomycin counter-select cassette which is regulated and controlled by AraR protein was amplified by PCR and transformed into original strain Bacillus subtilis 168. The neomycin resisitance transformants in which the chromosomal araR gene was disrupted by the inserted fragment via homologous recombination was selected and named ZY10 N and regarded as the original strain of marker-free modification and produce cytidine.Secondly, we deleted the cdd gene, which code cytidine deaminase, of the strain ZY10 N and named the modified strain ZY11 C.Thirdly, we modified the transcription initiation region of pyrG gene of ZY41 PRS in which the cdd, hom and pyrR genes were deleted and prs gene was highly expressed and got the strains ZY42G3, ZY42G9 and ZY42G12, of which the transcription initiation site, leader region and coding sequence of pyrG had different point mutations. In the same time, we modified the transcription initiation region of pyrG gene of ZY51 PRS in which the cdd, hom, pyrR and pdp-nupC genes were deleted and prs gene was highly expressed and got the strains ZY52G3 and ZY52G9, of which the transcription initiation site, leader region and coding sequence of pyrG had different point mutations as well. Shake flask fermentation results showed that the maximum OD600 of the original strain was 10.08 and ZY42 Gs were 8.72, 7.2 and 10.84, respectively, which indicates that the change of the pyrG gene's state will lead to the decrease of the strains' growing abilities. While, the ZY42 Gs could accumulate cytidine about 42.6?g/mL, 56.6?g/m L, 33.4?g/m L, respectively, improving 63.8%, 117.7%, 28.5% than ZY41 PRS. They produced uracil at the same time, the volume of uracil were 533.4?g/m L, 439.1?g/m L, 314.4?g/mL, respectively. Meanwhile, between 20 to 30 hours, the accumulation of uridine reached the peak, which were 25.9?g/m L, 26.3?g/m L, 23.5?g/m L, respectively. It turned out that the yield of cytidine is relative to the pyrG gene's state and the point mutation of pyrG gene's transcription initiation region has effect on pyrG gene's state. The results of ZY52Gs' shake flask fermentation showed that their growing abilities had no obvious decrease and there were no uracil was detected in the fermentation broth. What's more, the volume of cytidine were both about 27?g/mL, decreasing 44% than the original strain and the volume of uridine were both about 600?g/m L, decreasing 33% than the original strain. The result shows that, against the background of pdp-nupC gene's deficiency, changing the pyrG gene's state has disadvantage to the excessive synthesis of pyrimidine nucleoside.Finally, in order to construct the strain of which pyrG gene is over expressed, we used gsiB element to modify pyrG gene's m RNA leader region and constructed homologous recombination fragment and then transformed ZY51 PRS. As the consequence of this, we got the intermediate strain into which the exogenetic fragment had been transformed, however, we could not get the transformant which occurred intrachromosomal recombination. The result indicates that, against the background of pdp-nupC gene's deficiency, expressed pyrG gene will lead to the cells' death.