High Throughput Screening, Combined Mutation Breeding And Genetic Engineering Of Streptomyces Lomondensis For High Production Of Lomofungin

Streptomyces lomondensis S015 produces the phenazine compound lomofungin.Lomofungin is a broad-spectrum antibiotic,and can be used in many areas due to its antibacterial,antifungal and antitumor activities.Because of the low production of lomofungin in S015 strain,it is difficult to meet the requirements of research and production.Hence,this thesis aims to establish a high throughput screening method and then enhance the lomofungin production in S015by combined mutation breeding and genetic engineering.In this thesis,according to the phenomenon that there is a characteristic UV absorption peak at 375 nm in 2-butanone solution of lomofungin,it is determined that 375 nm is a characteristic wavelength which can be utilized to detect lomofungin in acidic 2-butanone solution.Then,it is proved that there is a good positive correlation between the concentrations of lomofungin in the solution and the values of A₃₇₅.Thus,the A₃₇₅ value of the upper organic phase of Streptomyces lomondensis fermentation broth can be used as a criterion for rapid determination of its lomofungin content.Combining the advantages of 24-deep-well culture and microplate reader detection,a high throughput screening method for lomofungin high-yield strains was successfully established.Utilizng this high throughput screening method,six rounds of ARTP and UV combined mutation breeding were carried out based on the starting strain S015.Finally,a high-yield and genetically stable strain M6 was screened from 4,320 mutants.Its yield of lomofungin is61.33 mg/L,which is 7.35 times of the yield of starting strain S015.Comparing M6 strain with S015 strain,although there is no significant difference in colony morphology,M6 strain grows much more vigorously.The maximum dry cell weight（DCW）of M6 strain is 12.49g/L,which is 2.21 times the DCW of S015 strain.After the high-yield mutant M6 was screened,the trpE1 and trpE2gene in the lomofungin-precursor-consumption pathway were double knocked out,so that the yield increased to 81.89 mg/L.Based on M6ΔtrpE1ΔtrpE2 mutant,the global regulatory gene afsR was overexpressed,and the yield of lomofungin increased to 109.53 mg/L,which is 33.75%higher than that of M6ΔtrpE1ΔtrpE2 mutant,and13.13 times the yield of S015 strain.After that,eight chorismate-synthesis pathway related genes were overexpressed,respectively,but there was no obvious effect for the yield of lomofungin.Finally,when M6ΔtrpE1ΔtrpE2::afsR mutant was cultured,the concentration of malt extract in medium was optimized to 14 g/L,soybean oil to 1.5%and Fe³⁺ion to 0.08 mmol/L,and the final yield of lomofungin was improved to 212.68 mg/L,which is 25.50 times the yield of S015strain.In this thesis,high throughput screening method for lomofungin high-yield strains,combined mutation breeding and genetic engineering were conducted to isolate and construct the lomofungin high-yield strains.This research provides a good reference for screening other antibiotics or secondary metabolites producing strains.