Study On Production Of Ascomycin By Strain Breeding And Process Optimization

Ascomycin,also called FK520,is a macrolide compound with immunosuppressive activity and produced by Streptomyces hygroscopicus var ascomyceticus.Pimecrolimus,an important derivative of FK520,has become the first-class drug for the treatment of atopic dermatitis and owned broad market prospects.At present,FK520 is mainly produced by microbial fermentation,however,the domestic production process of FK520 is more complex than the foreign one and its yield is much lower.And the study on the microbial stain and its fermentation is seriously insufficient.The main purpose of our research is to improve the production of FK520 through breeding strain by traditional mutation and genetic engineering modification,optimization of culture medium and production process.Firstly,an original strain SIPI-FK520-1 with a stable characteristics was obtained by natural screening.Then the strain was treated by NTG mutation,UV radiation and selected by the streptomycin resistance screening model.Finally,a high-yield mutant strain SIPI-FK520-33 was got,the production of FK520 in which was 2434.2 mg/l in shake flask culture,an increase of about 85% compared with the original strain.To further promote the yield in SIPI-FK520-33,we tried to add 1% macroporous adsorption resin XAD-16 N into the fermentation medium to reduce the feedback inhibition of metabolites,and finally the yield of FK520 rose up to 3272.6 mg/l.In order to solve the problem of limited dissolved oxygen(DO)during the fermentation process in a fermentor,the Vitreoscilla hemoglobin gene(vgb)which encodes Vitreoscilla hemoglobin(VHb)was integrated into SIPI-FK520-1.VHb is a prokaryotic hemoglobin,which can improve the utilization efficiency of oxygen to significantly increase DO in the fermentation process of engineering strain SIPI-FK520-vgb.The yield of FK520 was 1273.1 mg/l in a 5L bio-reactor,an increase of 38% compared with SIPI-FK520-1.Additionally,we tried to overexpress the crotonyl-Co A reductase gene(ccr)inSIPI-FK520-33 to inhibit the generation of FK523,a main impurity of FK520.The impurity produced by engineering strain SIPI-FK520-ccr in the culture medium added with 0.1% crotonic acid,was decreased by 27.4% compared with the control.