High-yield Mechanism Of Actinomycin D Based On Comparative Genomics Analysis

Actinomycin D(Actinomycin D)is an important class of cyclic peptide antibiotics with anti-tumor,anti-viral,anti-bacterial and other biological activities.In this study,Streptomyces luteus TRM45540 isolated from the saline-alkali soil in Lop Nur,Xinjiang,was the subject of study.Its secondary metabolites were mainly actinomycin D.Whole genome sequencing and comparative genomics analysis were carried out.Homologous cluster analysis,screening and analysis of differential functional genes,etc.,conduct more in-depth research to verify the functional genes that may affect the production of actinomycin.This research has mainly made the following research progress:(1)Through whole-genome sequencing and bioinformatics analysis of Streptomyces luteus TRM45540,a circular chromosome with a size of 7189594 bp,GC % content of71.75 %,6547 gene ORF,and 18 r RNA were obtained,including 6 5S r RNA,6 16 S r RNA,623 S r RNA,67 t RNA and 121 nc RNA;the translated protein sequence has 5651,2404,4562 protein sequences in the COG,KEGG,and GO databases,respectively,which have been functionally annotated;Using anti SMASH 5.0 software,24 secondary metabolic gene clusters were predicted,indicating that Streptomyces luteus TRM45540 has the potential to produce useful antibiotics.(2)By screening the phenotypic,molecular and chemical characteristics of the strain obtained by UV mutagenesis of the wild strain TRM45540,a stable genetic high yield actinomycin D strain TRM45540-3515 and a low yield strain TRM45540-0332 were obtained.The high-yielding strain TRM45540-3515 has a strong spore-producing ability,the mycelium is radial,the color is dark yellow after fermentation,and the HPLC analysis content is higher.The yield is 1540.37 mg/L,which is 136.12 % higher than the yield of the wild strain TRM45540;The spore-producing ability of TRM45540-0332 is weak,the mycelium is radial,the color is light yellow after fermentation,the HPLC analysis content is low,the yield is only52.54 mg/L,which is 91.94 % lower than the yield of the wild strain TRM45540.At the same time,the high-yielding strain TRM45540-3515 and the low-yielding strain TRM45540-0332 of actinomycin D were sequenced and analyzed by comparative genomics to find out the different functional genes between the strains.It was found that there are 16 single nucleotide polymorphism(SNP),2 inserts(Ins)and 4 deletion fragments(Del)in the high-yielding strain TRM45540-3515;there are 3 single nucleotide polymorphism(SNP),3 inserts(Ins)and 2deletion fragments(Del)in the low-yielding strain TRM45540-0332.In the high-yielding and low-yielding strains,the occurrence of 5 single nucleotide polymorphism(SNP)and 1deletion(Del)may be related to regulatory genes.It is speculated that these functional genes may be important genes leading to changes in actinomycin D production.(3)After comparative genomics analysis,a key gene that may affect the changes in the production of actinomycin D was found-the H-type transcription repressor nag R deletion gene in the low-yielding strain TRM45540-0332.First,construct the complementing plasmid p IB139-nag R,transform it into ET12567(p UZ8002)competent,obtain transformant ET12567(p UZ8002,p IB139-nag R),and transfer it as a donor strain to the low-yielding strain TRM45540-0332 to obtain recombinant strain nag R::TRM45540-0332.The recombinant strain was fermented and tested by HPLC,and it was found that its actinomycin D production was increased by 4.79 % compared with the wild strain TRM45540,and 12 times higher than that of the low yield strain TRM45540-0332.The results show that the nag R gene is indeed the key gene that causes changes in the production of actinomycin D.In this study,through comparative genomics analysis,we found the key genes that affect the increase of actinomycin D production,which provides a theoretical basis for the further high-yield transformation of actinomycin D producing bacteria in the future.