Directive Breeding Of Avermectin-high-producing Strains

Avermectins,produced by Streptomyces avermitilis,are an important type of lactone antibiotic with broad-spectrum bacteriostasis,low toxicity and easy degradation.They are widely used in agriculture,animal husbandry and medicine.It is difficult to improve the production by metabolic engineering due to avermectins are secondary metabolites with complex regulation mechanism.In this study,we established a spore mutant library for Streptomyces avermitilis through physical and chemical mutagenesis.The high-throughput screening?HTS?methods were established,one was integrated with Qpix420 microbial screening system,automated pipette workstation and enzyme labelling instrument detection was established,another method was based on the flow cytometry for efficient single cell sorting.High-producing avermectins mutant was easily obtained with the two methods.Genomic comparative analysis was carried out to investigate the regulation mechanism of high-titer mutant.Main results were presented as follows:?1?It was found that avermectins had a specific absorption peak at 245 nm and the medium had no interference through full wavelength scanning.A high-throughput screening method was set up,combining Qpix420 microbial screening system and related equipments.Streptomyces ATCC 31267 was used as the original strain,treated by atmospheric and room temperature plasma?ARTP?,ethyl methanesulfonate?EMS?and nitroguanidine?NTG?.A large mutant library was constructed and the lethality rate of each mutagenesis method was obtained.The relationship between the colony morphology and the titer of the mutant was studied.Strain G8 was selected with the high-throughput screening method and the fermentation titer of G8 in flasks increased 13.0%,compared with the wild strain.?2?Propidium iodide?PI?and fluorescein diacetate?FDA?were employed to discriminate between dead and live spores using the FACS system.Spore suspension,the concentration of fluorescent dye and staining time was studied.Spores stained with 7?g·m L^-1 PI and 15?g·m L^-1 FDA at 4°C in the dark for 30 min resulted in optimum sorting.The accuracy of the staining method on FACS was verified.Single live spore was sorted and solid-liquid combinatorial microculture was established for high-throughput avermectin culture.With the high-producing strain G8 as the starting strain,mutant G9 was screened in ARTP mutant library,which titer was increased 20.6%in 15-L fermentor compared with the wild strain.?3?Genomic sequencing and comparative genome analysis were carried out for the mutant strain G9,G8,A1 and control strain.The mutant genes were analyzed by statistical analysis of SNV,Indel,SV and CNV.A relative quantitative PCR was used to analyze the changes in the expression level of the mutant genes.Results showed that the expression level of genes aveA1and aveA2 were upregulated 0.54-,2.37-fold in the high-producing strain G9,0.22-,0.36-fold in the strain G8,respectively.The gene SAVERM_RS33605 involved in the intracellular ATP associated with the growth was upregulated 9.6-,4.41-fold in the strain G9 and G8,respectively.Gene SAVERM_RS19670 related to cell tolerance in the low yield strain A1 for lipid transferring was downregulated 0.86-fold.