Breeding Of High Daptomycin-Producing Strain By Ribosome Engineering Technology And Analysis Of Its Transcriptome

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus.It is mainly used in medicine to treat drug-resistant bacterial infections,but the yield of daptomycin in wild strains is very low.In this study,a high yield strain of daptomycin was selected by ribosomal engineering technique that introduced step by step by a variety of antibiotics and combined with ARTP-DES compound mutagenesis.After verifying the industrial potential of the high-yielding strain in a 5 L fermentor,the transcriptional differences between the mutant strain and the original strain were analyzed by RNA-Seq,and the key genes related to the high yield of daptomycin were found,which can lay a foundation for further exploring its high yield mechanism.The contents and results of the study are as follows:1.Breeding of high daptomycin-producing strain by ribosomal engineering.Streptomycin,gentamicin and rifamycin were selected by resistant plate experment.The minimum inhibitory concentrations of these three antibiotics to strain ZC-63 were 1.4μg/m L,8.0μg/m L and 9.0μg/m L,respectively.After the gene mutation library was constructed by compound mutagenesis of ARTP-DES,streptomycin was used for the first round of ribosomal engineering.The yield of daptomycin of the mutant strain was 163.8%higher than that of the original strain ZC-63.Gentamicin was used for the second round of ribosomal engineering,and the yield of daptomycin of the mutant strain was 88.3%higher than that of the first round of ribosomal engineering.Rifamycin was used for the third round of ribosomal engineering,a daptomycin high-producing strain RG-904 was obtained,and achieved daptomycin production of 99.58 mg/L in shake-flask fermentation,which was27.8%higher than that of the second round of ribosomal engineering.The results of genetic stability experiment showed that the high yield performance of mutant RG-904 was stably inherited.2.Morphological and genetic analysis of high-yielding strains.The morphological comparison between the mutant RG-904 and the original strain ZC-63 showed that the mutant RG-904 produced pink-white pigment.Compared with the original strain ZC-63,the colony morphology is more regular,the mycelium is more loose,and the ability of spore production is weaker.The comparison of rps L genes of the two strains showed that 440^thbase G of RG-904 was deleted,resulting in a frameshift mutation,which led to the early termination of translation.3.Study on 5 L fermentor of high-yield strain.Temperature,p H,and ventilation remain constant throughout the fermentation process,and the precursor sodium decanoate is gradually added to the tank.In the first 30 hours after the start of fermentation,sodium decanoate solution was not added to the fermentor.Between 30 and 72 hours of fermentation,add 25 ml of sodium decanoate solution to the fermentor every 6 hours.After 72 hours of fermentation,25 ml of sodium decanoate solution was added to the fermentor every 12 hours.When strain RG-904 was fermented for 132 hours,the content of daptomycin in the fermentation broth reached the maximum,which was 473.33 mg/L.4.Comparative Transcriptomics between high yielding strain RG-904 and original strain ZC-63.Analysis of differentially expressed genes,utilizing GO and KEGG annotation,revealed 7 genes potentially associated with a high yield of daptomycin.They are K7395＿RS24500,K7395＿RS27480,K7395＿RS26500,K7395＿RS30220,K7395＿RS27885,K7395＿RS17965 and K7395＿RS03680,respectively.Verification of these seven genes via RT-q PCR revealed that the relative expression levels of these distinctially expressed genes were consistent with the transcriptome results.