基因组发掘策略指导铁载体类化合物的发现
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摘要
目的利用基因组发掘策略指导铁载体类化合物的发现。方法首先,在获得灰产色链霉菌(Streptomyces griseochromogenes)CPCC 200274全基因组序列的基础上,利用antiSMASH对其编码的潜在次级代谢产物生物合成基因簇进行生物信息学分析,通过Blastp比对,判断该菌株中是否存在铁阻遏蛋白(Dmd R1)同源基因,并借助于MEME、FIMO等软件寻找包含Dmd R1蛋白结合位点(iron box)的基因簇,则该基因簇可能为铁载体类化合物的生物合成基因簇;其次,摸索合适的发酵条件,利用实时定量RT-PCR(q RT-PCR)技术检测相关生物合成基因的表达水平;最后分离纯化目标铁载体类化合物,验证此基因组发掘策略的可行性。结果利用antiSMASH从灰产色链霉菌CPCC 200274基因组中发现了5个含有iucA_C基因、标注为铁载体的生物合成基因簇。Blastp比对提示该菌株基因组中存在Dmd R1的同源蛋白SGC5642,可能参与铁载体类化合物生物合成的调控。结合MEME、FIMO软件分析结果,5个含iucA_C的基因簇中只有cluster 29含有iron box序列,推测cluster 29可能编码铁载体类化合物。根据以上信息,利用q RT-PCR方法确定了该基因簇表达的发酵条件,分离纯化获得了3个铁载体类化合物,经结构鉴定分别为去铁敏B,去铁敏E和N1-羟基-N1-琥珀酰基尸胺。结论以基因组发掘策略为指导,从微生物基因组中发现铁载体类化合物生物合成基因簇和铁阻遏蛋白结合位点,并通过qRT-PCR方法在转录水平确定目标基因簇的方法,成功获得3个铁载体类化合物。该策略为解决目前antiSMASH对于铁载体类化合物生物合成基因簇预测的局限性和该类化合物基因簇的快速定位、新生物合成元件的发掘提供了新思路,为新结构铁载体类化合物的寻找及合成生物学改造奠定了基础。
Objective To discover siderophores using genome mining strategy.Methods Firstly, the antiSMASH was used to analyze the whole genome sequence of Streptomyces griseochromogenes CPCC200274 for identifying the biosynthetic gene cluster of potential secondary metabolites. Blastp was used to determine the homologues of iron dependent regulatory protein Dmd R1 from S. coelicolor. Using MEME and FIMO analysis, the biosynthetic gene clusters of siderophore were identified which contain DmdR1 protein binding site(s)(iron box). Secondly, to explore suitable fermentation conditions, real-time RT-PCR(qRT-PCR) was used to detect the expression level of related biosynthetic genes. Finally, the target siderophores were isolated and purified to verify the feasibility of this strategy.Results Five clusters containing iucA_C gene were identified from the genome of S. griseochromogenes CPCC 200274 by antiSMASH. By Blastp analysis, SGC5642 was indicated to be homologous with DmdR1, which is involved in the regulation of siderophore's biosynthesis. Cluster 29 might encode siderophores, as it was the only iron box containing cluster among the five predicted siderophore clusters by MEME and FIMO analysis. Based on the above information, the fermentation conditions for the siderophore production were determined by qRT-PCR. Three compounds were isolated and purified and their structures were identified as deferoxamine B, deferoxamine E and N1-hydroxy-N1-succinyl cadaver amine.Conclusion Under the guidance of our siderophore mining strategy, the biosynthetic gene clusters of siderophores are predicted in the microbial genome and the transcriptional level are determined. Three siderophore compounds are successfully obtained from S.griseochromogenes CPCC 200274. This strategy provides a new idea for the rapid identification of siderophore biosynthetic gene clusters and new biosynthetic elements, and lays a new foundation for the search and synthesis of new structural siderophores through biosynthetic biology.
Objective To discover siderophores using genome mining strategy.Methods Firstly, the antiSMASH was used to analyze the whole genome sequence of Streptomyces griseochromogenes CPCC200274 for identifying the biosynthetic gene cluster of potential secondary metabolites. Blastp was used to determine the homologues of iron dependent regulatory protein Dmd R1 from S. coelicolor. Using MEME and FIMO analysis, the biosynthetic gene clusters of siderophore were identified which contain DmdR1 protein binding site(s)(iron box). Secondly, to explore suitable fermentation conditions, real-time RT-PCR(qRT-PCR) was used to detect the expression level of related biosynthetic genes. Finally, the target siderophores were isolated and purified to verify the feasibility of this strategy.Results Five clusters containing iucA_C gene were identified from the genome of S. griseochromogenes CPCC 200274 by antiSMASH. By Blastp analysis, SGC5642 was indicated to be homologous with DmdR1, which is involved in the regulation of siderophore's biosynthesis. Cluster 29 might encode siderophores, as it was the only iron box containing cluster among the five predicted siderophore clusters by MEME and FIMO analysis. Based on the above information, the fermentation conditions for the siderophore production were determined by qRT-PCR. Three compounds were isolated and purified and their structures were identified as deferoxamine B, deferoxamine E and N1-hydroxy-N1-succinyl cadaver amine.Conclusion Under the guidance of our siderophore mining strategy, the biosynthetic gene clusters of siderophores are predicted in the microbial genome and the transcriptional level are determined. Three siderophore compounds are successfully obtained from S.griseochromogenes CPCC 200274. This strategy provides a new idea for the rapid identification of siderophore biosynthetic gene clusters and new biosynthetic elements, and lays a new foundation for the search and synthesis of new structural siderophores through biosynthetic biology.
引文
[1]Abbaspour N,Hurrell R,Kelishadi R.Review on iron and its importance for human health.J Res Med Sci,2014,19(2):164-174.
[2]Khan A,Singh P,Srivastava A.Synthesis,nature and utility of universal iron chelator-Siderophore:a review.Microbiol Res,2018,212-213:103-111.
[3]Dias PJ,Sá-Correia I.The drug:H+antiporters of family 2(DHA2),siderophore transporters(ARN)and glutathione:H+antiporters(GEX)have a common evolutionary origin in hemiascomycete yeasts.BMCGenomics,2013,14:901-933.
[4]Dhusia K,Bajpai A,Ramteke PW.Overcoming antibiotic resistance:Is siderophore Trojan horse conjugation an answer to evolving resistance in microbial pathogens?J Control Release,2018,269:63-87.
[5]M?llmann U,Heinisch L,Bauernfeind A,et al.Siderophores as drug delivery agents:application of the“Trojan Horse”strategy.Biometals,2009,22(4):615-624.
[6]Straubinger M,Blenk H,Naber KG,et al.Urinary concentrations and antibacterial activity of BAL30072,a novel siderophore monosulfactam,against uropathogens after intravenous administration in healthy subjects.Antimicrob Agents Chemother,2016,60(6):3309-3315.
[7]Jacobs A.Iron chelation therapy for iron loaded patients.Br JHaematol,1979,43(1):1-5.
[8]Choi SS,Katsuyama Y,Bai L,et al.Genome engineering for microbial natural product discovery.Curr Opin Microbiol,2018,45:53-60.
[9]Chiang YM,Chang SL,Oakley BR,et al.Recent advances in awakening silent biosynthetic gene clusters and linking orphan clusters to natural products in microorganisms.Curr Opin Chem Biol,2011,15(1):137-143.
[10]Huang TT,Lin SJ,Deng ZX.Recent advances in mechanism of siderophore biosynthesis in actinomycetes.Microbiol China,2011,38(5):765-773.(in Chinese)黄婷婷,林双君,邓子新.放线菌中铁载体生物合成机制研究进展.微生物学通报,2011,38(5):765-773.
[11]de Lorenzo V,Neilands JB.Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli.J Bacteriol,1986,167(1):350-355.
[12]Flores FJ,Martín JF.Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.Biochem J,2004,380(Pt 2):497-503.
[13]Escolar L,Pérez-Martín J,de Lorenzo V.Opening the iron box:transcriptional metalloregulation by the Fur protein.J Bacteriol,1999,181(20):6223-6229.
[14]Barona-Gómez F,Lautru S,Francou FX,et al.Multiple biosynthetic and uptake systems mediate siderophore-dependent iron acquisition in Streptomyces coelicolor A3(2)and Streptomyces ambofaciens ATCC23877.Microbiology,2006,152(Pt 11):3355-3366.
[15]Bunet R,Brock A,Rexer HU,et al.Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2).FEMSMicrobiol Lett,2006,262(1):57-64.
[16]Barona-Gómez F,Wong U,Giannakopulos AE,et al.Identification of a cluster of genes that directs desferrioxamine biosynthesis in Streptomyces coelicolor M145.J Am Chem Soc,2004,126(50):16282-16283.
[17]Bergeron RJ,Pegram JJ.An efficient total synthesis of desferrioxamine B.J Org Chem,1988,53(14):3131-3134.
[18]Fujita MJ,Sakai R.Heterologous production of desferrioxamines with a fusion biosynthetic gene cluster.Biosci Biotechnol Biochem,2013,77(12):2467-2472.
[19]Cone MC,Yin X,Grochowski LL,et al.The blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes:sequence analysis,organization,and initial characterization.Chembiochem,2003,4(9):821-828.
[20]Badfar G,Mansouri A,Shohani M,et al.Hearing loss in Iranian thalassemia major patients treated with deferoxamine:a systematic review and meta-analysis.Caspian J Intern Med,2017,8(4):239-249.
[21]Wencewicz TA,Long TE,M?llmann U,et al.Trihydroxamate siderophore-fluoroquinolone conjugates are selective sideromycin antibiotics that target Staphylococcus aureus.Bioconjug Chem,2013,24(3):473-486.
[22]Lin YM,Miller MJ.Practical synthesis of hydroxamate-derived siderophore components by an indirect oxidation method and syntheses of a DIG-siderophore conjugate and a biotin-siderophore conjugate.J Org Chem,1999,64(20):7451-7458.
[23]Wei HJ,Lin ZJ,Li DH,et al.OSMAC(one strain many compounds)approach in the research of microbial metabolites-a review.Acta Microbiol Sinica,2010,50(6):701-709.(in Chinese)韦洪娟,林贞建,李德海,等.单菌多次级代谢产物方法及其在微生物代谢产物研究中的应用.微生物学报,2010,50(6):701-709.
[24]Challis GL.Genome mining for novel natural product discovery.J Med Chem,2008,51(9):2618-2628.
[25]Gaballa A,Helmann JD.Identification of a zinc-specific metalloregulatory protein,Zur,controlling zinc transport operons in Bacillus subtilis.J Bacteriol,1998,180(22):5815-5821.
[26]Patzer SI,Hantke K.The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.Mol Microbiol,1998,28(6):1199-1210.
[2]Khan A,Singh P,Srivastava A.Synthesis,nature and utility of universal iron chelator-Siderophore:a review.Microbiol Res,2018,212-213:103-111.
[3]Dias PJ,Sá-Correia I.The drug:H+antiporters of family 2(DHA2),siderophore transporters(ARN)and glutathione:H+antiporters(GEX)have a common evolutionary origin in hemiascomycete yeasts.BMCGenomics,2013,14:901-933.
[4]Dhusia K,Bajpai A,Ramteke PW.Overcoming antibiotic resistance:Is siderophore Trojan horse conjugation an answer to evolving resistance in microbial pathogens?J Control Release,2018,269:63-87.
[5]M?llmann U,Heinisch L,Bauernfeind A,et al.Siderophores as drug delivery agents:application of the“Trojan Horse”strategy.Biometals,2009,22(4):615-624.
[6]Straubinger M,Blenk H,Naber KG,et al.Urinary concentrations and antibacterial activity of BAL30072,a novel siderophore monosulfactam,against uropathogens after intravenous administration in healthy subjects.Antimicrob Agents Chemother,2016,60(6):3309-3315.
[7]Jacobs A.Iron chelation therapy for iron loaded patients.Br JHaematol,1979,43(1):1-5.
[8]Choi SS,Katsuyama Y,Bai L,et al.Genome engineering for microbial natural product discovery.Curr Opin Microbiol,2018,45:53-60.
[9]Chiang YM,Chang SL,Oakley BR,et al.Recent advances in awakening silent biosynthetic gene clusters and linking orphan clusters to natural products in microorganisms.Curr Opin Chem Biol,2011,15(1):137-143.
[10]Huang TT,Lin SJ,Deng ZX.Recent advances in mechanism of siderophore biosynthesis in actinomycetes.Microbiol China,2011,38(5):765-773.(in Chinese)黄婷婷,林双君,邓子新.放线菌中铁载体生物合成机制研究进展.微生物学通报,2011,38(5):765-773.
[11]de Lorenzo V,Neilands JB.Characterization of iucA and iucC genes of the aerobactin system of plasmid ColV-K30 in Escherichia coli.J Bacteriol,1986,167(1):350-355.
[12]Flores FJ,Martín JF.Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.Biochem J,2004,380(Pt 2):497-503.
[13]Escolar L,Pérez-Martín J,de Lorenzo V.Opening the iron box:transcriptional metalloregulation by the Fur protein.J Bacteriol,1999,181(20):6223-6229.
[14]Barona-Gómez F,Lautru S,Francou FX,et al.Multiple biosynthetic and uptake systems mediate siderophore-dependent iron acquisition in Streptomyces coelicolor A3(2)and Streptomyces ambofaciens ATCC23877.Microbiology,2006,152(Pt 11):3355-3366.
[15]Bunet R,Brock A,Rexer HU,et al.Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2).FEMSMicrobiol Lett,2006,262(1):57-64.
[16]Barona-Gómez F,Wong U,Giannakopulos AE,et al.Identification of a cluster of genes that directs desferrioxamine biosynthesis in Streptomyces coelicolor M145.J Am Chem Soc,2004,126(50):16282-16283.
[17]Bergeron RJ,Pegram JJ.An efficient total synthesis of desferrioxamine B.J Org Chem,1988,53(14):3131-3134.
[18]Fujita MJ,Sakai R.Heterologous production of desferrioxamines with a fusion biosynthetic gene cluster.Biosci Biotechnol Biochem,2013,77(12):2467-2472.
[19]Cone MC,Yin X,Grochowski LL,et al.The blasticidin S biosynthesis gene cluster from Streptomyces griseochromogenes:sequence analysis,organization,and initial characterization.Chembiochem,2003,4(9):821-828.
[20]Badfar G,Mansouri A,Shohani M,et al.Hearing loss in Iranian thalassemia major patients treated with deferoxamine:a systematic review and meta-analysis.Caspian J Intern Med,2017,8(4):239-249.
[21]Wencewicz TA,Long TE,M?llmann U,et al.Trihydroxamate siderophore-fluoroquinolone conjugates are selective sideromycin antibiotics that target Staphylococcus aureus.Bioconjug Chem,2013,24(3):473-486.
[22]Lin YM,Miller MJ.Practical synthesis of hydroxamate-derived siderophore components by an indirect oxidation method and syntheses of a DIG-siderophore conjugate and a biotin-siderophore conjugate.J Org Chem,1999,64(20):7451-7458.
[23]Wei HJ,Lin ZJ,Li DH,et al.OSMAC(one strain many compounds)approach in the research of microbial metabolites-a review.Acta Microbiol Sinica,2010,50(6):701-709.(in Chinese)韦洪娟,林贞建,李德海,等.单菌多次级代谢产物方法及其在微生物代谢产物研究中的应用.微生物学报,2010,50(6):701-709.
[24]Challis GL.Genome mining for novel natural product discovery.J Med Chem,2008,51(9):2618-2628.
[25]Gaballa A,Helmann JD.Identification of a zinc-specific metalloregulatory protein,Zur,controlling zinc transport operons in Bacillus subtilis.J Bacteriol,1998,180(22):5815-5821.
[26]Patzer SI,Hantke K.The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.Mol Microbiol,1998,28(6):1199-1210.