HrdB Site-directed Mutagenesis On Biological Characteristics Of Streptomyces Avermitilis

Avermectin,the macrolide secondary metabolites produced by Streptomyces avermitilis,is a kind of green biological insecticides,can kill nematodes and mites efficiently.It is widely used in agriculture and livestock husbandry.HrdB is the global regulator sigma factor of Streptomyces,responsible for transcriptional regulation of most of the genes for metabolic processes.The pathway-specific regulatory gene of avermectin biosynthesis aveR could be recognized specifically by HrdB.The hrdB 415,488,1066 three A nucleotides were changed to G by site-directed mutagenesis.The 415,488 loci were located at the region 1.1 of the sigma factor.The 1066 locus was located at region 2.4.Region 1.1 had the function of inhibiting the formation of double-stranded DNA and increasing the efficiency of transcription initiation.Region 2.4 had the function of recognizing the-10 region of the promoter.Integrated mutant plasmids were constructed and introduced into the parent strain Streptomyces avermitilis Q and the high-producing strain Streptomyces avermitilis CL respectively.Two series Streptomyces avermitilis mutants were obtained as a result.They were QM1,QM2,QM3,QM4,QM5,QM6,QM7,CM1,CM2,CM3,CM4,CM5,CM6 and CM7.Real-time transcription levels of hrdB,aveR,aveA Ⅰ and aveAlll in fermentation process were studied in Q series mutants and CL series mutants.The results showed that the transcription levels of hrdB,aveR,aveA Ⅰ and aveAⅢ were promoted in hrdB 415A>G 488A>G double points mutation.The transcription levels of hrdB were increased by 35.77%and 32.31%respectively at 48h.The transcription levels of aveR were increased by 73.45%and 84.76%respectively at 120h.The transcription levels of aveA I were increased by 54.27%and 63.39%respectively at 120h.The transcription levels of aveAlll were increased by 35.81%and 41.3 7%respectively at 120h.The fermentation of avermectin was studied in Q series mutants and CL series mutants.The results showed that production of avermectin was promoted in hrdB 415A>G 488A>G double points mutation.The relative potency of avermectin were increased by 62.11%and 52.32%respectively after 192h.The residual sugar of Q series mutants and CL series mutants at the end of fermentation was studied.The utilization ability of substrates was studied by the SF-P2 MicroPlate.The results showed that the utilization ability of substrates of hrdB 415A>G 488A>G double points mutant was higher than that of the other mutants and the parent strain.