Research On The Biosynthetic Gene Clusters Of Dithiolopyrrolone Antibiotics Thiolutin And Aureothricin

Dithiolopyrrolone(DTP)group antibiotics have an electronically unique dithiolopyrrolone heterobicyclic core.The reduced DTP is capable of chelating metal ions from zinc-dependent enzymes,which affects the process of ubiquitination and RNA transcription in cells,and thus exhibits high inhibitory activity against Gram-positive and Gram-negative bacteria,fungi and tumor cells.The dithiolopyrrolone biosynthesis remains unclear yet.Only the biosynthetic pathway of holomycin in Streptomyces clavuligerus DSM3585 was partially elucidated.Unlike holomycin-type dithiolopyrrolones,the endocyclic pyrrolone nitrogen of thiolutin-type compounds was methylated,which led to much stronger inhibition against fungi and tumor cells.Therefore,elucidation of the synthetic pathways of thiolutin-type dithiolopyrrolones has important research significance.A dithiolopyrrolone biosynthetic gene cluster(aut)was isolated from the aureothricin and thiolutin producer Streptomyces thioluteus DSM 40027 in the previous study.Two pyrrothine derivatives without N4-methylation,holomycin and N-propionylholothine,are the heterologous expression products of this cluster in Streptomyces albus.In order to make the functional study,the putative biosynthetic genes,oxidoreductase genes and transcriptional regulator genes of the aut cluster were in-frame deleted.The absence of the thioesterase gene autC,the nonribosomal peptide synthase(NRPS)gene autE,the decarboxylase gene autF or the thioredoxin reductase gene autI abolished the biosynthesis of two heterologous expression products,indicating that they are essential for dithiolopyrrolone biosynthesis.The analogs of these four essential genes were also found in the previously identified thi and hlm clusters,but the heterologous expression products are different.To investigate functional difference of the homologous genes in these three clusters,gene complementation experiments with its own and the analogs from other clusters were conducted.It demonstrated that the thioesterase gene hlmC and thiC could take the place of autC to release intermediate products from the T domain of NRPS by hydrolysis.The decarboxylase genes hlmF and thiF could substitute for autF to hydrolyze the carboxyl groups of cysteine dipeptides.These homologous genes were verified to play the same functions.Acyltransferase can transfer different kinds of acyl groups to the N7 position of DTP core,resulting in different structures of DTP antibiotics.When the acyltransferase gene autA was knocked out,the yield of holomycin declined and N-propionylholothine vanished.Two heterologous expression products were restored by autA only.The homologous genes thiA1 and thiA2 could not complement the function of autA.The complementation with hlmA only restored the yield of holomycin,but still no N-propionylholothine produced.All results indicated the substrate specificity differences between the four acyltransferases.Bioinformatics analysis revealed that AutA has an additional PLP-dependent lyase domain,suggesting that AutA might have additional functions.NRPS is the key enzyme for dithiolopyrrolone biosynthesis to catalyze the formation of dithiolopyrrolone backbone by condensation of two cysteine.NRPS gene thiE could substitute for autE to recover the dithiolopyrrolone biosynthesis,while the homologous gene hlmE could not.It indicated functional differences between HlmE,ThiE and AutE.The thi and aut cluster were identified from the thiolutin producing strains,while the hlm cluster was isolated from the holomycin producer.Functional difference of the NRPS genes implied that the biosynthetic pathways of thiolutin-type and holomycin-type dithiolopyrrolones might be not exactly the same.In addition,it was found that knockout of the oxidoreductase genes orf8 and orf9 improved the products yield instead of destroy the holomycin and N-propionylholothine biosynthesis.Knockout of the transcriptional regulator genes orf1,orf3,orf4 and orf5 had no effect on the products,indicating that they were not involved in the biosynthesis of the heterologous expression products.