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

Dithiolopyrrolone group antibiotics are a class of compounds possessing a special structure,which have been reported to display broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria,fungi and parasites by interfering with RNA polymerase.Moreover,some kinds of dithiolopyrrolone compounds exhibit antitumor activity.However,the exact mechanism of action,biosynthesis,regulation and resistance of this class of compounds have yet to be elucidated.The biosynthetic gene cluster of dithiolopyrrolone antibiotic holomycin was first isolated from Streptomyces clavuligerus in 2010.As the genome sequencing technology develops,more gene clusters of dithiolopyrrolone antibiotics were identified.Elucidation of the dithiolopyrrolone biosynthetic pathways will give an insight on combinatorial biosynthesis of new dithiolopyrrolones with different structures and biological activities by genetic engineering.On the basis of substituent species at the endo-cyclic pyrrolone N4,all dithiolopyrrolone compounds could be classified into two types:holomycin-type and thiolutin-type.Since thiolutin type dithiolopyrrolones display better biological activities,we constructed two genomic libraries for two thiolutin-producing strains,Streptomyces thioluteus DSM 40027 and Saccharothrix algeriensis DSM 44581,and selected out the aut cluster and the thi cluster,respectively.By comparison with the holomycin biosynthetic gene cluster(hlm)in S.clavuligerus,we found that all of these three clusters contains one nonribosomal peptide synthetase gene,one thioesterase gene and four flavin-dependent oxidoreductases genes,which might be responsible for assembly of the dithiolopyrrolone core.Heterologous expression and in-frame deletion of the key genes confirmed that these two gene clusters are involved in dithiolopyrrolone antibiotic biosynthetic pathways.Two standalone thioesterase genes,hlmC and hlmK,were identified in the hlm cluster,but only HlmC was proposed to be involved in holomycin biosynthesis.The hlmK gene was considered invalid in the previous study based on a key amino acid residue mutation at the conserved catalytic active sites of thioesterases.Intriguingly,gene deletion experiments indicted that HlmC was essential for dithiolopyrrolone biosynthesis,while HlmK might serves as an active type ? thioesterase which was responsible for high yield production of dithiolopyrrolones in S.albus.In addition,heterologous expression of hlmK in the heterologous expression strain of the aut cluster significantly improved yield of the dithiolopyrrolone products.Contrary to the previous studies,the available evidences of our studies suggested that HlmK is an active enzyme that plays a corrective role by removal the misloaded substrates or aberrant intermediates from carrier protein and renders the assembly line active again to increase product yield in dithiolopyrrolone biosynthesis.Meanwhile,HlmC acted as TEI to release synthesized products from the NRPSs by hydrolytic reaction.Acyltransferase is responsible for transferring distinct acyl groups to exo-cyclic pyrrolone N7 and contributes to produce different kinds of dithiolopyrrolone compounds.Deletion of the acyltransferase gene hlmA abolished the biosynthesis of holomycin,and holomycin production was restored by complementation of hlmA into the deletion mutant strain.The aut cluster is derived from S.thioluteus DSM 40027 which produces two dithiolopyrrolone compounds,aureothricin and thiolutinin,The heterologous expression of this cluster in S.albus also produce two dithiolopyrrolone compounds,holomycin and N-propionylholotine.There is only one acyltransferase gene autA found in the aut cluster,deletion of this gene abolished biosynthesis of N-propionylholotine and significantly decreased the production yield of holomycin,which indicated that both acetyl CoA and propionyl CoA are native substrates of the acyltransferase AutA.However,holomycin rather than N-propionylholotine was produced by complementation of autA into the hlmA-deletion mutant,which suggested that AutA was not able to utilize propionyl CoA as the substrate in this system.Sa.algeriensis DSM 44581 was reported to be able to produce at least five pyrrothine derivatives.Two acyltransferase genes thiA1 and thiA2 obtained from this strain was respectively expressed into the hlmA-deletion mutant and only holomycin was produced as well.All these data implied that the acylation modification of exo-cyclic pyrrolone N7 is not only catalyzed by the acyltransferase,but also affected by other genes in this cluster.It has been proposed that dithiolopyrrolone compounds are prodrugs which require enzymatic or chemical reduction of the disulfide bond into the active dithiol form to produce biological effects in the cell.The thioredoxin reductase plays an important role in self-protection of the producing strains by oxidizing the reduced dithiol form of dithiolopyrrolones to the disulfide form.Methylation of the thiol group may be an alternative self-protection mechanism when the thioredoxin reductase is absent.The fact that heterologous expression of the thi cluster in S.albus produced two new thiol methylation compounds further verified the self-protection mechanism by methylation of the thiol groups.Although the thi cluster harbors a thioredoxin oxidoreductase gene thiI,the disulfide bond of heterologous expression products is not performed.RT-PCR experiments established that all oxidoreductase genes including the thioredoxin reductase gene were transcribed as normal.Furthermore,heterogentic complementation of the thioredoxin reductase genes hlmI and autI in the heterologous expression strain failed to catalyze the formation of disulfide bond.Additionally,a unique flavin-dependent oxidoreductase gene orf7t was found in the thi cluster,deletion of orf7t drastically reduced the production of dithiolopyrrolones,indicating that orf7t was involved in dithiolopyrrolone biosynthesis.Since that there are multiple oxidation reactions during the dithiolopyrrolone biosynthesis pathways,we cannot determine which step Orf7t was involved in only based on the bioinformatics analyses.Additional in vivo and in vitro experiments will not only unveil the exact function of this gene but also help us for intensive understanding of the detailed synthesis,regulation and resistance mechanisms of thiolutin type dithiolopyrrolone compounds.