Screening Of High-yield Rimocidin Resistant Mutants Of Streptomyces Rimosus M527 And Detection And Overexpression Of Related Genes

Streptomyces rimosus M527 was shown to produce the polyene macrolide antibiotic rimocidin,which was found to be highly efficient against a broad range of plant pathogenic fungi.However,the rimocidin production is very low in this strain,it was difficult to meet the need of large-scale industrial production.Therefore,in this study ribosome engineering technology was employed to screen the resistant mutants with high rimocidin production level.Mutants were analyzed with respect to rimocidin production,morphological characterization,transcriptional levels of genes involved in rimocidin biosynthesis.The main contents were performed as follows:Firstly,rifampicin?Rif?,gentamicin?Gen?were utilized for screening different kinds of antibiotic resistant mutants of S.rimosus M527.The experimental results showed that 23 of 25?rifampicin-resistance,Rif^r?mutants had an increase of22.3%²7.1%in the rimocidin production comparing with the wild-type?WT?strain M527.All of 23 Rif^r mutants showed the same mutation C1309G in rpoB gene.The alteration of corresponding amino acid was His437Asp.The isolated 93?gentamicin-resistance,Gen^r?mutants exhibited genetic instability or reduced production of rimocidin.Only Gen^r mutant M527-G37 showed good genetic stability,and its rimocidin production was 34.4%higher than that of WT strain M527.Based on the Gen^r mutant M527-G37,the combined antibiotics-resistant mutants with Gen and Rif was isolated.Among obtained 43 Gen^r-Rif^r mutants,Gen^r-Rif^r mutant M527-GR7 with the highest yield of rimocidin 399.7 mg/L,which was a 74.7%increase in rimocidin production comparing with that of control strain M527-G37.Comparison of morphological differentiation in Gen^r-Rif^r mutant M527-GR7,Gen^r mutant M527-G37 and the WT strain M527 revealed that Gen^r mutant M527-G37produced a soluble pigment,whereas the Gen^r-Rif^r mutant M527-GR7 produced deeper soluble pigments.Scanning electron microscopy was employed to observe the morphological differentiation of WT strain M527 and mutants M527-G37 and M527-GR7.Comparing with the WT strain M527,the mycelium and spores of the Gen^r mutant M527-G37 were arranged neatly to form a reticular space structure with gaps.The arrangement of the mycelium and spores of the Gen^r-Rif^r mutant M527-GR7 was irregular and the sporulation capacity was reduced comparing with Gen^r mutant M527-G37.Secondly,based on the sequence of genes rimA^rimK involved in rimocidin biosynthesis in Streptomyces diastaticus var.108 published on the NCBI,the corresponding ten rim genes(rimA_sr-rimK_sr)were cloned from S.rimosus M527 by using degenerated primers.In order to investigate the correlations between the transcriptional level of rim genes and rimocidin biosynthesis,the relative transcriptional levels of rim genes in the Gen^r-Rif^r mutant M527-GR7,Gen^r mutant M527-G37 and WT strain M527 at 48 h were examined.The results indicated that the relative transcriptional levels of rimA_sr^rimK_sr in the mutants M527-GR7 and M527-G37 were significantly higher than those of WT strain M527.Compared with M527-G37,the transcriptional levels of rimB_sr,rimD_sr,rimE_srr and rimG_sr genes of M527-GR7 were much high.Finally,conjugal transfer system for S.rimosus M527 was established and optimized:donor strain ET12567?pUZ8002,pGUS-ermE^*?/receptor spores 10⁸/10⁷;receptor spores were heat shocked at 50°C for 10 min and incubated at 37°C for 2-3h;using 2CMC medium as the conjugation transfer plate and covering a final concentration of 300?g/mL apramycin and 50?g/mL nalidixic acid and after cultivating 28°C for 16¹8 h.Under optimal conditions,a maximal conjugation frequency of 1.67×10^-5 per recipient was obtained.The expression of reporter gene GUS verified not only the applicability of conjugation transformation for S.rimosus M527,but also the efficiency of promoter permE^*for heterologous gene expression in S.rimosus M527.Subsequently,rim genes rimB_sr(rimD_sr,rimE_sr,rimG_sr)were respectively placed under the control of the promoter permE^*to construct the recombinant plasmids pIB139-rimB_sr(rimD_sr,rimE_sr,rimG_sr)was transferred into S.rimosus M527 by using conjugational transfer system to obtain the recombinants M527-RIMB,M527-RIMD,M527-RIME and M527-RIMG,respectively.The results showed that rimocidin production of recombinant strains M527-RIMB,M527-RIME,and M527-RIMG was 95.3%,1.3 times,and 85.3%higher than that of WT strain M527,respectively,There was no remarkable difference between recombinant M527-RIMD and WT strain M527 in term of rimocidin production.The results indicated that the transcriptional levels of rimB_sr,rimG_sr and rimE_srr genes were positively correlated with the rimocidin biosynthesis.