Revealling The Biosynthetic Mechanism Of Antibiotic Anisomycin

Anisomycin,a pyrrolidine antibiotic,was first isolated from Streptomyces roseochromogenes and Streptomyces.griseolus in 1954.Owing to its reversible 60S ribosomal subunit binding,anisomycin can block peptide bond formation and shows potent selective activity against pathogenic protozoa and fungi.It has been successfully used in the clinics for the treatment of amoebic dysentery and trichomoniasis and it is also one of the important effective components in Agricultural Antibiotic 120,which has been widely used to treat the crops decay in China.These diverse biological activities and structural features of anisomycin have attracted the attention of many chemists and biologists.But its biosynthetic studies had been limited to isotope feeding experiments.This study aims to clarify the unique mechanism for anisomycin biosynthesis at molecular level and provide an opportunity to generate libraries of derivative compounds for structure-activity relationship studies.We employed a bioactivity-guided high-throughput library screening approach to identify the anisomycin biosynthetic gene cluster?BGC?.In order to identify the genes responsible for its biosynthesis,we constructed a series of gene deleted mutant cosmids and transferred them to Streptomyces lividans TK24.Metabolite profile analysis of the S.lividans TK24 strains harboring the mutant cosmids suggested that eight of the genes?aniF,aniI,aniK,aniL,aniN,aniO,aniP,and aniQ?are essential for anisomycin biosynthesis as strains lacking these individual genes lost the ability to produce it.We also identified six intermediates structure using HPLC,Q-TOF and NMR.Further bioinformatics analysis predicted that aniF,aniI,aniK and aniL are respectively involved in transcriptional regulation,tailoring modifications and transport,while aniN,aniO,aniP and aniQ may be responsible for biosynthesis of its core scaffold.Besides,we also confirmed aniG was another necessary gene through in vitro assay,so the BGC of anisomycin contains nine essential genes.We identified six biosynthetic enzymes?AniN,O,P,Q,G and K?through biochemical characterization and isotope feedings experiment.AniN is a bifunctional dehydrogenase that mediates pyrrolidine formation and we proposed the mechanism of AniN reaction.The aminotransferase AniQ catalyzed deamination and transamination reactions and the two substrates had significant difference in chemical sturcture and polarity.The glycosyltransferase AniO catalyzed a cryptic glycosylation step.To the best of our knowledge,glucose,which is the main carbon source,is used to mediate the biosynthesis of natural products has not been previously reported in microbial natural product pathways.Besides,the glucosyl unit plays an important role as AniN and AniQ conveted the glycosylated substrates much faster than the non-glycosylated equivalents.We also performed three tailoring reactions,deglycosylation by AniG,O-acetylation by AniI,and O-methylation by AniK.Besides,we fed1,3-¹³C₂-labeled glycerol into S.hygrospinosus var.beijingensis culture and confirmed that AniP may catalyzed a transketolase-type condensation reaction using 4-hydroxyphenylpyruvic acid and G3P as substrates.In summary,using a combination of bioinformatic analysis,reverse genetics and biochemical assays,we identified the BGC of anisomycin and proposed an overview of its biosynthetic pathway.The anisomycin producer strain is very‘clean'?no accumulation of pathway intermediates in the WT strain?as the flexible enzymes could redirect shunt intermediates back into the pathway to anisomycin.Overall,AniQ,AniP,AniO and AniN formed a new set of enzyme ensemble capable of forming pyrrolidine ring systems.The findings provided new opportunities for genome mining of novel pyrrolidine natural products and set the foundation for engineering the biosynthesis of anosimycin to advance drug development in the future.