Screening High Yield Inosine-producing Strains In Artificial Mutagenesis

The inosine can be available to synthetic food aid fresheners5’-inosinic acid, can be for the treatment of many diseases such as platelet decrease, leukopenia, acute hepatitis,cirrhosis and chronic hepatitis, it is a very important nucleoside drugs. Inosine is currently produced by fermentation what is typical metabolic control, so excellent strain is the key to improve the yield. Selected saved laboratory strain of inosine of Bacillus subtilis HSD1206as starting strain,obtained a new mutant strain and screened high-yielding strains through natural breeding method,ultraviolet mutagenesis,DES mutagenesis and UV-diethyl sulfate compound mutation treatment.(1) Breeded the sulfaguanidine resistant strains through Gradient plate method,after several rounds of screening,obtained sulfadoxine resistance strains,the resistance increased to300mg/L from50mg/L of the original departure strains.Obtained strains whose production glycosides ability was improved through fermentation for primary and secondary screening, the production glycosides level of strain K25reached12.13g/L which is27.95%higher than the starting strain production,its sulfaguanidine resistance and Yield Traits performance stability.(2) The mutant strains were screened in the selection medium by mutagenesis treatment method such as UV, DES, UV+DES and so on, and then high-yielding strains was selected from the variant strains. The results showed that it is more difficult for the mutagenic treatment of UV or DES single factor to obtain new mutants, but it still played a role in the restoration of the typical genetic traits of the original strain and increasing production.For example,after UV or DES single factor treatment, the traits of thiamine defects strains got some recovery,its production increased; new defects-histidine-deficient could be got through UV+DES composite processing, production had also been dramatically increased.(3) Determined the optimal UV mutagenesis:20W UV lamp,30cm, the irradiation time60s. high-yield strains UV23was obtained by UV mutagenesis after primary and secondary screening. After passaged6times, strains production glycosides capacity remained stable, reached19.96g/L which was17.62%higher than the starting strain production.(4) Determined the best DES mutagenesis:1%of the DES treatment for10min high-yielding strains D4was obtained by DES mutagenesis after the primary and secondary screening. After passaged strains, producing glycosides capacity remained stable,reached19.47g/L which was22.53%higher than the starting strain production.(5) Determined the best UV-DES mutagenesis:firstly radiate UV for50s, and then1%of the DES treatment for8min. New auxotrophic strain DU65were obtained by UV-DES, the strain was identified as adenine, thiamine, mutagenesis triple deficient strain.The strain had glycosides production capacity in the non-optimized medium,which improved than the starting strain.(6) After single factor and orthogonal experiments of carbon, nitrogen,and growth factor,the fermentation medium of DU65strains:18%glucose, yeast extract17g/L, corn steep liquor15g/L, peptone6g/L, soybean meal hydrolyzate9g/L, adenine150mg/L thiamine5.4mg/L, histidine75mg/L, potassium dihydrogen phosphate,30g/L, magnesium sulfate0.1g/L, manganese sulfate0.1g/L, antifoaming agent1g/L. Glycosides production of DU65strains reached22.95g/L in the medium of shake flask fermentation, which was31.97%higher than the original strain.(7)3overproducing strains by mutagenesis screening was larger experimental in50L fermentor.Results showed that the level of the reactor still yield performance of three strains of high-yielding strains,including a new auxotrophic strain DU65glycoside production speed, short fermentation time the final product of glycosides37.43g/L, than the starting strain yield17.82%.