Research On The Biosynthetic Mechanism Of Chuangxinmycin

The emerging of bacterial drug resistances in bacteria has severe threaten the human health,and the development of new antibiotics is urgent.The rediscovery separation of natural products in recent years has prompted scientists to focus their attention on previously studied antibiotics.The development and recognition of new targets has gradually become a new research hotspot.Aminoacyl-tRNA synthetase(aaRS)catalyzes the attachment of specific amino acids to the corresponding tRNA molecules,which are commonly distributed in living cells.There is a large evolutionary difference between the different species but the same species is relatively conservative,so tRNA synthetase(aaRS)is an ideal target for antibiotic development.Mupirocin produced by Pseudomonas fluorescens has been marketed as a commercial antibiotic(Bactroban or Centany),which selectively inhibits bacterial isoleucyl-tRNA synthetase(IleRS).Now it is widely used for topical treatment of methicillin-resistant Staphylococcus aureus(MRSA),which incentives scientists to screen for aminoacyl-tRNA synthetase inhibitors from microbial natural products for drug development.Chuangxinmycin is an indole alkaloid antibiotic firstly isolated from actinomycetes Actinoplanes tsinanensis 40 years ago.Due to its structural resemblance to tryptophan,chuangxinmycin is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase(TrpRS),which is responsible for its antibacterial activity.It showed antibacterial activity against several Gram-positive and Gram-negative bacteria in vitro and effective inhibition of Escherichia and infections in mice.Chuangxinmycin was also effective against E.coli septicemia,urinary and biliary infections in a preliminary clinical trial.Furthermore,it showed low toxicity in mice.To understand how chuangxinmycin is formed and to set the basis for further efforts to improve yield and generate non-natural derivatives,we identified the biosynthetic gene cluster of chuangxinmycin using heterologous expression of a candidate cluster coupled with systematic in vivo gene inactivation.The complete genome of A.tsinanensis was sequenced by PacBio technology.Generally,biosynthesis,regulation and resistance genes for a specialized secondary metabolites are clustered in the genome of bacteria.Therefore,to isolate the gene cluster for chuangxinmycin biosynthesis,we used genome scanning to target the TrpRS gene that confers resistance to chuangxinmycin or other TrpRS inhibitors such as indolmycin.Through bioinformatics analysis of the resulting complete genomic sequence,we identified two putative TrpRS genes within the genome.Among them,cxm0(YDGM004646)attracted our attention.This gene is located in a putative ten-gene cluster(YDGM004646-YDGM004655)that spans an 11 kb DNA region.The GC content(64.9%)of this putative gene cluster is significantly lower than the average GC content(70.3%)of the genome,suggesting that it might have been acquired from a foreign source by horizontal gene cluster.Sequence analysis of this gene cluster,named as cxm cluster,predicts that cxml encodes a LysR family transcriptional regulator for regulation of chuangxinmycin production,cxm2 encodes a MFS(Major Facilitator Superfamily)transporter for efflux of chuangxinmycin,cxm4 encodes a sulfur carrier protein(SCP),cxm5 encodes a cytochrome P450,cxm6 encodes a ketopantoate reductase,cxm7 encodes a pyridoxal 5'-phosphate(PLP)-dependent aminotransferase,cxm8 encodes a B12-dependent radical S-adenosylmethionine(SAM)protein,and cxm3 and cxm9 encode a hypothetical protein,respectively.To connect this gene cluster with chuangxinmycin biosynthesis,we directly cloned this DNA region(cxm0-cxm9)into an E.coli plasmid using linear plus linear homologous recombination.and transferred it into heterologous hosts S.coelicolor A3(2),S.albus J1074 and S.lividans K4-114 for functional expression.Metabolic profiling showed that chuangxinmycin was produced in all three strains containing cxm0-cxm9,which shows that cxm0-cxm9 are sufficient to direct the biosynthesis of chuangxinmycin in these bacteria.Subsequently,nine mutants with in-frame deletion of cxm1-9,respectively,were generated in E.coli by Red/ET recombineering and transferred into S.coelicolor for comparative metabolite analysis.All mutants except ?cxm2,?cxm6 and ?cxm7,had completely abolished chuangxingmycin production.The cxm2 and cxm7 genes encode a transporter and aminotransferase,respectively,whose function was be complemented by by their homologs in the heterologous host S.coelicolor A3(2),thus their deletion mutants did not affected the production of chuangxinmycin.Mutant?cxm6 produced trace levels of chuangxinmycin(-3%yield of intact cxm gene cluster),potentially due to its function not being efficiently complemented by homologs in S.coelicolor.Inactivation of cxm1 resulted in elimination of chuangxinmycin demonstrates that cxml encodes a positive transcriptional regulator.Both mutants ?cxm8 and ?cxm9 had increased compound 3(RT 27.3min,m/z 218.0286[M-H]-)production which was purified and characterized as 3-desmethyl-chuangxinmycin by using HRMS and NMR.This suggests that both are involved in the 3-methyaltion during chuangxinmycin biosynthesis.To verify the concerted action of cxm8 and cxm9,cxm8 and cxm9 were cloned both separately and together under the strong artificial promoter Virolle-al-14 in S.coelicolor A3(2).then fed 3.Only when cxm8 was expressed together with cxm9,was 3 completely converted into CXM.Along with compound 3,a new compound 4(RT 25.7 min,m/z 381.0597[M-H]-)was found in the heterologous hosts containing the cxm gene cluster and was also present in the ?cxm5 mutant.Compound 4 production was abolished in the cxm3 or cxm4 knock-out mutants.while placing cxm3 and cxm4 under a strong artificial promoter in S.coelicolor A3(2)also resulted in the biosynthesis of 4(Figure 2C).4 was purified from the ?cxm5 mutant as a colorless oil and the molecular formula was determined as C16H18N2O5S2(calcd for 381.0584[M-H]-)on the basis of HRESIMS analysis.The 1D NMR data were similar to those of 3,indicating that they share a similar indole-3-propanoic acid skeleton,which was further supported by the COSY and HMBC correlations.The featured thiopyrano[4,3,2-cd]indole scaffold in compound 3 comprises two unique carbon-sulfur bonds,but it biosynthetic enzymes are yet to be discovered.On the basis of gene inactivation,cxmO-2,cxm7(converting Trp 2 to indolepyruvate 5),and cxm89 do not participate in the biosynthesis of 3.The remaining cxm3456 genes are likely responsible for the biosynthesis of this tricyclic scaffold because their mutants had eliminated or substantially decreased 3 production.In addition cxm3456 genes under the control of the strong artificial promoter in S.coelicolor A3(2)resulted in the robust production of 3-desmethyl-chuagnxinmycin 3.Thus we determined the minimal enzymes cxm3-6 are required for production of the unique thiopyrano[4,3,2-cd]indole scaffold in vivo.The exact conversion mechanism from indolepyruvate 5 to 3-desmethyl-chuagnxinmycin 3 is still unclear.Cxm5 is a cytochrome P450 enzyme that would catalyze intermediate thiotryptophan(6)to form 7(2,3-dehydro-3-desmethyl-chuangxinmycin).Whereas the introduction of the sulfur(first C-S bond formation)involves in a mechanism seems to be similar to that found in thiamine,molybdopterin,cysteine,thioquinolobactin and BE-7585A biosynthetic pathway.Cxm4 is a sulfur carrier protein(SCP)but the conserved C-terminal diglycine trail(-GG-COOH)is followed by ten additional amino acids.This variation of a sulfur carrier protein is also found in thioquinolobactin biosynthesis or methionine biosynthesis in Wolinella succinogenes,the metal-dependent hydrolase QbsD or HcyD cleaves the final two or one amino acids from SCP generating the diglycyl C-terminus,respectively.Cxm6 possibly converts intermediate 7 to 3 to give the final 3,5-dihydro-2H-thiopyrano[4,3,2-cd]indole scaffold.Based on the above experimental results and bioinformatic analysis,a biosynthetic model for chuangxinmucin was proposed:Cxm7,or other aminotransferase converts Tip(2)to indolepyruvate(5),Cxm3 and Cxm4 catalyzes sulfur incorporation,Cxm5 catalyzes the S-heterocyclization,Cxm6 catalyzes the reduction of double bond,and cxm8 coupled with Cxm9 finally catalyzes C3-methylation.Furthermore,to elucidate the biosynthetic mechanism of chuangxinmycin by in vitro reconstruction,the single core chuangxinmucin biosynthetic gene cxm3-cxm 7 was cloned into a recombinant expression vector for protein recombinant expression,respectively.The resulting expression constructs were transformed into E.coli rosetta or Rhodococcus sp.RHA1 for heterologous reproduction,and the recombinatnt protein was purified to homogeneity,respectively.The aminotransferase Cxm7 catalyzes the formation of indole-3-pyruvic acid from tryptophan through in vitro transformation experiments,which is the first step in the biosynthetic pathway of chuangxinmycin.The CO-bound reduced difference spectra of Cxm5 protein showed the characteristic peak at 450 nm,confirming the expression of functional P450 enzymes.Unfortunately,the cycliczation assay was not performed due to the inaccessibility and instability of possible substrate with a-SH.In this study,genome seuqnecing,bioinformatics analysis combined with heterologous expression allowed to identify the biosynthetic gene cluster of the chuangxinmycin.systematic in vivo gene inactivation,structure elucidaiton of biosynthetic intermediates.We were able to attribute the function of each enzyme in the chuagnxinmycin biosynthesis,and the timing of each biochemical reaction requires further work.This study provided guidance for the further analysis of the biosynthetic mechanism of the unique thiopyran tricyclic structure of chuangxinmycin,and lays the foundation for the synthesis of biomimetic structural analogs of chuangxinmycin.