Effects Of Related Metabolic Pathway Modification On Uridine Fermentation By Bacillus Subtilis

Uridine is an intermediate in the synthesis of antineoplastic and antiviral drugs.It is also directly used as a drug and has a demand for application in the field of medicine.Chemical synthesis of uridine still has insurmountable technical defects.Relatively speaking,microbial fermentation has potential technological advantages of low cost,less pollution and good quality.On the basis of previous studies,metabolic engineering of uridine-producing Bacillus subtilis was studied in this paper.In Bacillus subtilis producing uridine strains,both the loss of glutamate dehydrogenase and the blocking of citrulline to arginine reaction led to a decrease in uridine production.On this basis,N-acetyl amino acid transferase（arg J）and N-acetylglutamate gamma-semialdehyde dehydrogenase（arg C）were further knocked out,and the uridine yield of TD300 was increased to 11.11 g/L.The results showed that under certain conditions,the metabolic flux of arginine synthesis increased,resulting in an increase in glutamate consumption,and a decrease in intracellular glutamate level,resulting in a decrease in uridine production.When TD300 strain was knocked out of the aspartase（ans B）,the growth of the derived strain TD301 was damaged,the cell was prone to autolysis,and the yield of uridine decreased significantly.Supplementary glutamate could alleviate this phenomenon.The results showed that the physiological function of aspartase was to catalyze the amination of fumaric acid to aspartate.Loss of ans B may result in inadequate intracellular aspartate level,affect the synthesis of peptidoglycan,and lead to cell autolysis.The deletion of Cod Y regulatory element of IMP dehydrogenase gene gua B resulted in a 0.04-fold decrease in gua B transcription level,a 19.8-fold increase in pur operon transcription level,a 49.7%decrease in uridine production,and the disappearance of inhibition of branched-chain amino acids on uridine synthesis.The results showed that branched-chain amino acids activate gua B expression through Cod Y mediation,thereby increasing the metabolic flux of de novo IMP synthesis,resulting in a decrease in uridine production.By weakening or blocking pur operon expression,the metabolic flux of de novo IMP synthesis was reduced,and the yield and yield of uridine were significantly increased.The pur operon decreased by 0.81times,and the production of uridine of its derivative strain TD312 increased to 11.81g/L,and the yield increased from 0.19 to 0.27 g/g of glucose.The results showed that there was a competitive relationship between the de novo IMP synthesis pathway and the de novo UMP synthesis pathway for the common precursor PRPP.In constitutive expression of puc R gene by P_{ccp A}resulted in a 194.01-fold increase in the transcription level,and a 349.71-fold increase in the transcription level of ure operon,resulting in a 13.06 g/L uridine production of the derived strain TD314.The results showed that Puc R protein could activate the transcription of ure operon effectively.Using urea as a supplementary nitrogen source to increase the supply of ammonia could significantly increase the yield of urea.P_{ccp A2}was used to overexpress the 6-phosphogluconate lactone gene ykg B,which increased the transcription level by 27.86 times and the uridine yield of TD316 by18.0%,reaching 15.42 g/L.The results showed that ykg B expression level affected the metabolic flux of PP pathway and the intracellular level of PRPP,and then affected the synthesis of uridine.