Construction And Fermentation Optimization Of Pyrimidine Nucleosides Producing Strains

Pyrimidine nucleosides, consisting of uridine and cytidine, are components of RNA and play an important role in vital activity and metabolic processes in all organisms. In recent years, they exhibit an excellent pharmaceutical value by being used as intermediates in the production of antiviral and antitumor drugs, such as IDUR, BUDR, FUDR, CTP, Cyclo C, CDP-Choline, and so on. Producing of pyrimidine nucleosides by microbial fermentation has many advantages, but there are no high yield strains appling to this technology for industrial production. This paper studied the breeding of pyrimidine nucleosides producing strains based on the theory of metabolic engineering, and by using of the statistical method the fermentation medium were optimized. Ultimately, the pyrimidine nucleosides accumulation of the strain improved greatly. This work provides reference and guidance for the industrial production of pyrimidine nucleosides.With the wild type strain, Bacillus subtilis 168, as the starting strain, the upstream and downstream fragments of cytidine deaminase coding gene cdd were amplified by PCR. They were spliced by ovelap-extension PCR, and subsequently joined together with the plasmid vector pKS1, forming the recombinant plasmid pKS1△cdd. The recombinant plasmid was transferred into strain 168. Based on the temperature sensitive characteristic of the plasmid and homologous recombination, the gene cdd of strain 168 was knocked out successfully, creating the strain PYR1. This strain had the possibility to accumulate two pyrimidine nucleosides, and laid the foundation for the next step of breeding.Through the cooperation with metabolic engineering laboratory, Chemical Engineering Institute of Tianjin University, a series of gene engineering strains were constructed. Shake flask fermentation experiement was used to test these strains of pyrimidine nucleosides, and study the influence of each key gene modification on pyrimidine nucleosides yields. By the knockout of gene hom and pyrR, the strain possessed the ability to accumulate pyrimidine nucleosides, but the by-products of uracil accumulation was huge. Overexpression of wild type and mutant prs gene both had positive impacts on the production of pyrimidine nucleosides. Although cytidine accumulation increased slightly, but the accumulation of uridine were increased by 31.93% and 74.79%, and the latter is better than the former, in addition, there were rarely by-products of uracil accumulation. Mutations of pyrAB gene increased the pyrimidine synthesis pathways, uridine production increased by 76.44%, and cytidine production increased by 16.22%. This work also threw light on the assignment and direction for the next work. Through the comparison of the fermentation parameters, strain PYR5 was screened out with a good performance, and by using it as starting strain, we continued to do the next work.Response surface methodology was applied to optimize the pyrimidine nucleosides fermentation medium of PYR5 with the uridine yield as the response. First, yeast extract, monosodium glutamate and soybean meal hydrolysate were found to be significant factors influencing the production of uridine, which was obtained by a Plackett-Burman screening design. The steepest ascent experiment aiming at approaching the optimum region of the response was carried out subsequently. Finally, the effects of the three significant factors on uridine production were investigated by a central composite design and the optimal conditions were determined as follows:25.6 g/L yeast extract,25.2 g/L monosodium glutamate,40.9 mL/L soybean meal hydrolysate. With optimal conditions, a predicted maximum uridine production of 13.76 g/L would be reached. The verification experiment found that, an actual value of 13.52 g/L of uridine yield was obtained in which was in agreement with the prediction and increased by 164.1% compared with that using the original culture medium. The cytidine yield was 0.78 g/L, which was increased by 73.33%. In addition, the biomass, glucose consumption, inversion rate of glucose and pyrimidine nucleosides accumulation per cell were all greatly improved, indicating that the optimized condition is more suitable for cell growth and production of pyrimidine nucleosides.