ARTP Mutagenesis And Gene Orientation Transformation Breeding Adenosine High And Stable Yield Strains

Adenosine is an endogenous nucleoside that is spread throughout the human body.Adenosine on the cardiovascular system and the body system and many other organizations have physiological effects.Adenosine is used for the synthesis of intermediates of adenosine triphosphate(ATP),adenine,adenosine,adenosine and many anti viral drug.At present in industry mainly using sugar as raw materials for fermentation production.But the domestic adenosine production efficiency is still a large space to improve,through the method of mutation breeding and genetic engineering to transform strains,optimization of fermentation conditions to improve and improve yield and reduce cost;at the same time in the production process there exists the problem of genetic traits and yield production instability.The laboratory preserved an adenosine accumulation of strain Bacillus subtilis DI4-24,through continuous passaging,,strain degeneration phenomenon is serious,its genetic traits histidine defect is easily lost,is not conducive to the production of adenosine for a long time.So this topic,in which the Degradation Strain B.subtilis DI4-24 R purified as the starting strain,regained increased histidine deficient strains,genetic stability and high yield breeding traits of adenosine by ARTP mutation.and the first attempt by gene knockout technology oriented the transformation strain,to keep the stability of the production strain performance enhancements.The main conclusions include the following aspects:1.Reason analysis of strain degeneration : Through to the high yield adenosine strain of b.subtilis DI4-24 purification and genetic phenotype analysis,we get B.subtilis DI4-24 R of degradation strains that its colony morphology and genetic markers were changed,and B.subtilis DI4-24 R of glycosides performance degradation is due to histidine defect type high frequency back mutation,degradation strains yield greatly reduce to 4.34 g/L.2.They are first screened strains that his-genetic traits restoration through the ARTP mutagenic treatment based on DI4-24 R strain.Experiments show that : ARTP mutagenic treatment of the processing time of 10 s are more likely to get positive mutant strain.The mutant strain B.subtilis AR-27.by s rescreening,its producing adenosine remains high through 6 generations,it shake flask fermentation was reached 15.84 g/L compared with the original strain yield increased by nearly 3 times,its rate of his-backmutation down to 9.3×10-8,Its glycosides production performance and genetic stability are improved significantly.3.Shaking flask fermentation of mutant strain B.subtilis AR-27 through the growth factor,glucose,and single factor experiments of nitrogen sources and multi factors orthogonal test,determine the optimum medium formula: glucose 11 g/L,soybean meal hydrolysate 80 mL/L,corn pulp 60 mL/L,yeast extract 12 g/L,ammonium chloride 15 g/L,potassium dihydrogen phosphate 2 g/L,magnesium Sulfate0.3 g/L,ferrous sulfate 0.02 g/L,manganese sulfate 0.02 g/L,calcium chloride 5 g/L,calcium carbonate 20g/L,isoleucine 0.02 g/L,valine 0.02 g/L,histidine 50 mg/L.With optimize the culture medium formula to produce glycosides can reach 16.12 g/L.It is increased by 10 % than before optimization.4.To retrieve the hisC genes of Bacillus subtilis on NCBI,Respectively designed upstream and downstream primers,PCR amplification genome,get the upstream(TF)and downstream homologous sequences(TB),connect the homologous sequence upstream and downstream integration,the fragment size of about 1000 bp,The purpose fragment was connected with T vector,to construct a recombinant plasmid T-H.5.Extract shuttle plasmid vector pMAD and recombinant plasmid,respectively enzyme digestion using restriction enzymes BamH,Bg l ?,in-fusion which the new technology was transformed into DH5 cells,The homologous recombinant plasmid pMAD-H which hisC gene deletion were screened.The homologous recombinant plasmid p MAD-H was transferred into the recipient cell DI4-24 R,and the clones were screened with the resistant plate.Acquisition of B.subtilis DI4-24R(pMAD-H)of subsequent transformation strain acquired the homologous recombination of engineering bacteria screening laid a solid foundation.