Abstract
The Embleya genus is a new member of the Streptomycetaceae family formed by only two species isolated from soil (Embleya scabrispora and Embleya hyalina). Strain NF3 is an endophytic actinobacterium obtained from the medicinal tree Amphipterygium adstringens. By 16S rRNA gene analysis, NF3 strain was identified as Embleya sp., closely related to E. hyalina. In our interest to deep into the NF3 strain features, a bioinformatic study was performed on the Embleya genus based on their genome information to produce secondary metabolites. A comparative analysis of the biosynthetic gene clusters (BGCs) of NF3 with the two released Embleya genomes revealed that NF3 has 49 BGCs, E. scabrispora DSM41855 has 50 BGCs, and E. hyalina NBRC13850 has 46 BGCs. Although bearing similar cluster numbers, the three strains shared only 25% of the BGCs information. NF3 encoded the nybomycin cluster detected in E. hyalina NBRC13850 and lacked the hitachimycin cluster present in E. scabrispora DSM41855. On the contrary, strain NF3 contained a cluster for the anthracycline steffimycin, neither encoded by E. hyalina NBRC13850 nor by E. scabrispora DSM41855. Our results and previous characterization studies supported strain NF3 as a new member of the genus Embleya. The chemical analysis of the steffimycins produced by strain NF3 showed the production of eight compounds of the steffimycins and steffimycinone families. Four of these molecules have already been described: steffimycin B, steffimycin C, 8-demethoxy-10-deoxysteffimycinone, and 7–deoxiesteffimycinone, and four are new natural products: 8-demethoxysteffimycin B, 8-demethoxy-10-deoxysteffimycin B, 7-deoxy-8-demethoxysteffimycinone, and 7-deoxy-10-deoxysteffimycinone. With this information, we proposed an alternative pathway to produce StefB. Among steffimycins, StefB was the main compound produced by this strain (29.8%) and showed the best cytotoxic activity.
Key points
• The Embleya genus and its biosynthetic potential
• An alternative biosynthetic pathway for steffimycins biosynthesis
• Four new natural products of the steffimycin family
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. https://doi.org/10.1093/nar/25.17.3389
Barka E, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Klenk H-P, Clément C, Ouhdouch Y, van Wezel G (2015) Taxonomy, physiology, and natural products of Actinobacteria. Microbiol Mol Biol Rev 80:1–43. https://doi.org/10.1128/MMBR.00019-15
Bérdy J (2012) Thoughts and facts about antibiotics: where we are now and where we are heading. J Antibiot 65:385–395. https://doi.org/10.1038/ja.2012.27
Bergy ME, Reusser F (1967) A new antibacterial agent (U-20,661) isolated from a streptomycete strain. Experientia 23:254–255. https://doi.org/10.1007/BF02135666
Blin K, Kim HU, Medema MH, Weber T (2019) Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters. Brief Bioinform 20:1103–1113. https://doi.org/10.1093/bib/bbx146
Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP, Medema MH, Weber T (2021) antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res 49:W29–W35. https://doi.org/10.1093/nar/gkab335
Blum M, Chang HY, Chuguransky S, Grego T, Kandasaamy S, Mitchell A, Nuka G, Paysan-Lafosse T, Qureshi M, Raj S, Richardson L, Salazar GA, Williams L, Bork P, Bridge A, Gough J, Haft DH, Letunic I, Marchler-Bauer A, Mi H, Natale DA, Necci M, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A, Finn RD (2021) The InterPro protein families and domains database: 20 years on. Nucleic Acids Res 49:D344–D354. https://doi.org/10.1093/nar/gkaa977
Brodasky T, Mizsak S (1985) Steffimycin C, a new member of the steffimycin anthracyclines isolation and structural characterization. J Antibiot 38(7):849–855. https://doi.org/10.7164/antibiotics.38.849
Caicedo-Montoya C, Gómez-Román MP, Vázquez-Hernández M, Mora-Rincón RA, Rodriguez-Luna SD, Rodríguez-Sanoja R, Sanchez S (2021) Evolutionary genomics and biosynthetic potential of novel environmental Actinobacteria. Appl Microbiol Biotechnol 105(23):8805–8822. https://doi.org/10.1007/s00253-021-11659-3
Ceapă CD, Vázquez-Hernández M, Rodríguez-Luna SD, Cruz-Vázquez AP, Jiménez-Suárez V, Rodríguez-Sanoja R, Alvarez-Buylla ER, Sánchez S (2018) Genome mining of Streptomyces scabrisporus NF3 reveals symbiotic features including genes related to plant interactions. PLoS ONE 13:1–27. https://doi.org/10.1371/journal.pone.0192618
Chandra S (2012) Endophytic fungi: novel sources of anticancer lead molecules. Appl Microbiol Biotechnol 95:47–59. https://doi.org/10.1007/s00253-012-4128-7
Charousová I, Medo J, Halenárová E, Javoreková S (2017) Antimicrobial and enzymatic activity of actinomycetes isolated from soils of coastal islands. J Adv Pharm Technol Res 8:46–51. https://doi.org/10.4103/japtr.JAPTR-161-16
Doroghazi JR, Metcalf WW (2013) Comparative genomics of actinomycetes with a focus on natural product biosynthetic genes. BMC Genomics 14:611–624. https://doi.org/10.1186/1471-2164-14-611
Ganaphathy A, Natesan S (2018) Metabolic potential and biotechnological importance of plan associated endophytic Actinobacteria. In: Pratap B, Kumar V, Kumar A (ed) New and future developments in microbial biotechnology and bioengineering. Actinobacteria: diversity and biotechnological applications. Elsevier, Amsterdam, Chapter 14, pp 207–224. https://doi.org/10.1016/B978-0-444-63994-3.00014-X.
Golinska P, Wypij M, Agarkar G, Rathod D, Dahm H, Rai M (2015) Endophytic Actinobacteria of medicinal plants: diversity and bioactivity. Antonie Van Leeuwenhoek 108:267–289. https://doi.org/10.1007/s10482-015-0502-7
Gullón S, Olano G, Abdelfattah MS, Braña AF, Rohr J, Méndez C, Salas JA (2006) Isolation, characterization, and heterologous expression of the biosynthesis gene cluster of the antitumor anthracycline steffimycin. Appl Environ Microbiol 72:4172–4183. https://doi.org/10.1128/AEM.00734-06
Hiramatsu K, Igarashi M, Morimoto Y, Baba T, Umekita M, Akamatsu Y (2012) Curing bacteria of antibiotic resistance: reverse antibiotics, a novel class of antibiotics in nature. Int J Antimicrob Agents 39:478–485. https://doi.org/10.1016/j.ijantimicag.2012.02.007
Joseph B, Priya R (2011) Bioactive compounds from endophytes and their potential in pharmaceutical effect: a review. Am J Biochem Mol Biol 1:291–309. https://doi.org/10.3923/ajbmb.2011.291.309
Kelly R (1976) Steffimycinone, 7-deoxysteffimycinone and derivatives. United States Patent 3(976):667
Kelly R, Schletter I, Koert J, MacKellar F, Wiley P (1977) Structures of steffimycin and steffimycin B. J Org Chem 42:3591–3596. https://doi.org/10.1021/jo00442a032
Komaki H, Hosoyama A, Kimura A, Ichikawa N, Igarashi Y, Tamura T (2020) Classification of ‘Streptomyces hyalinum’ Hamada and Yokoyama as Embleya hyalina sp. nov., the second species in the genus Embleya, and emendation of the genus Embleya. Int J Syst Evol Microbiol 70:1591–1595. https://doi.org/10.1099/ijsem.0.003941
Kudo F, Kawamura K, Uchino A, Miyanaga A, Numakura M, Takayanagi R, Eguchi T (2015) Genome mining of the hitachimycin biosynthetic gene cluster: Involvement of a phenylalanine-2,3-aminomutase in biosynthesis. ChemBioChem 16:909–914. https://doi.org/10.1002/cbic.201500040
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) Mega X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Kusari S, Hertweck C, Spiteller M (2012) Chemical ecology of endophytic fungi: origins of secondary metabolites. Chem Biol 19:792–798. https://doi.org/10.1016/j.chembiol.2012.06.004
Lee N, Hwang S, Kim J, Cho S, Palsson B, Cho BK (2020) Mini review: genome mining approaches for the identification of secondary metabolite biosynthetic gene clusters in Streptomyces. Comput Struct Biotechnol J 18:1548–1556. https://doi.org/10.1016/j.csbj.2020.06.024
Liu C, Han C, Jiang S, Zhao X, Tian Y, Yan K, Wang X, Xiang W (2018) Streptomyces lassi sp. nov., a novel actinomycete with antifungal activity isolated from the head of an ant (Lasius flavus). Curr Microbiol 75:353–358. https://doi.org/10.1007/s00284-017-1388-6
Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey A, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47:W636–W641. https://doi.org/10.1093/nar/gkz268
Manzoor A, Wani A, Qazi PH, Rehman S, Mushtaq S, Abid S, Hussain A, Shah A, Qazi AK, Makhdoomi US, Hamid A, Kumar A (2016) Isolation and characterization of alborixina from Streptomyces scabrisporus: a potent cytotoxic agent against human colon (HCT-116) cancer cells. Chem Biol Interact 256:198–208. https://doi.org/10.1016/j.cbi.2016.06.032
Martinez-Klimova E, Rodríguez-Peña K, Sánchez S (2017) Endophytes as source of antibiotics. Biochem Pharmacol 134:1–17. https://doi.org/10.1016/j.bcp.2016.10.010
Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56:185–229. https://doi.org/10.1124/pr.56.2.6
Nagai A, Khan ST, Tamura T, Takagi M, Shin-Ya K (2011) Streptomyces aomiensis sp. nov., isolated from a soil sample using the membrane-filter method. Int J Sys Evol Microbiol 61:947–950. https://doi.org/10.1099/ijs.0.020719-0
Naganawa H, Wakashiro T, Yagi A, Kondo S, Takita T, Hamada M, Maeda K, Umezawa H (1970) Deoxynybomycin from a Streptomyces. J Antibiot 23:365–368. https://doi.org/10.7164/antibiotics.23.365
Nouioui I, Carro L, García-López M, Meier-Kolthoff J, Woyke T, Kyrpides N, Pukall R, Klenk H, Goodfellow M, Göker M (2018) Genome-based taxonomic classification of the phylum Actinobacteria. Front Microbiol 9:2007. https://doi.org/10.3389/fmicb.2018.02007
Olano C, Abdelfattah MS, Gullón S, Braña AF, Rohr J, Méndez C, Salas JA (2008) Glycosylated derivatives of steffimycin: insights into the role of the sugar moieties for the biological activity. ChemBioChem 9:624–633. https://doi.org/10.1002/cbic.200700610
Ping X, Takahashi Y, Seino A, Iwai Y, Omura S (2004) Streptomyces scabrisporus sp. nov. Int J Syst Evol Microbiol 54:577–581. https://doi.org/10.1099/ijs.0.02692-0
Reusser F (1975) Steffimycin B, a DNA binding agent. Biochim Biophys Acta 383:266–273. https://doi.org/10.1016/0005-2787(75)90055-6
Rodríguez M, Myronovskyi M, Gummerlich N, Nadmid S, Luzhetskyy A (2018) Heterologous expression of the nybomycin gene cluster from the marine strain S. albus subsp. chlorinus NRRL B-24108. Mar Drugs 16:1–10. https://doi.org/10.3390/md16110435
Rodríguez-Peña K, Macías-Rubalcava ML, Rocha-Zavaleta L, Trenado-Uribe M, Rodríguez-Sanoja R, Sánchez S (2018) Streptomyces scabrisporus, an endophyte isolated from Amphipterygium adstringens as producer of an anthracycline active against different cancer cell lines. Glob Drugs Therap 3(5):1–10. https://doi.org/10.15761/gdt.1000158
Shirling E, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Sys Bacteriol 16:313–340. https://doi.org/10.1099/00207713-16-3-313
Singh TA, Passari AK, Jajoo A, Bhasin S, Kumar V, Hashem A, Alqarawi AA, Abd_Allah EF, (2021) Tapping into Actinobacterial genomes for natural product discovery. Front Microbiol 12:655620. https://doi.org/10.3389/fmicb.2021.655620
Sriram M, Liaw Y-C, Gao Y-G, Wang H-J (1991) Molecular structure of antitumor drug steffimycin and modeling of its binding to DNA. J Biomol Struct Dyn 9:251–269. https://doi.org/10.1080/07391102.1991.10507911
Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of pacific yew. Science 260:214–216. http://www.jstor.org/stable/2881310.
Stothard P (2000) The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102–1104. https://doi.org/10.2144/00286ir01
Sullivan MJ, Petty NK, Beatson SA (2011) Easyfig: a genome comparison visualizer. Bioinformatics 27:1009–1010. https://doi.org/10.1093/bioinformatics/btr039
Trenado-Uribe M, Silva-Miranda M, Rivero-Cruz JF, Rodríguez-Peña K, Espitia-Pinzón C, Rodríguez-Sanoja R, Sánchez S (2018) Antimycobacterial activity of an anthracycline produced by an endophyte isolated from Amphipterygium adstringens. Mol Biol Rep 45:2563–2570. https://doi.org/10.1007/s11033-018-4424-0
Umezawa I, Takeshima H, Komiyama K, Hoh Y, Yamamoto H, Kawaguchi M (1981) A new antitumor antibiotic, stubomycin. J Antibiot 34:259–265. https://doi.org/10.7164/antibiotics.34.259
Vazquez-Hernandez M, Ceapă CD, Rodríguez-Luna SD, Rodríguez-Sanoja R, Sánchez S (2017) Draft genome sequence of Streptomyces scabrisporus NF3, an endophyte isolated from Amphipterygium adstringens. Genome Announc 5:e00267-e317. https://doi.org/10.1128/genomeA.00267-17
Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, van Sinderen D (2007) Genomics of Actinobacteria: tracing the evolutionary history of an ancient phylum. Microbiol Mol Biol Rev 71(3):495–548. https://doi.org/10.1128/MMBR.00005-07
Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, Gillespie JJ, Gough R, Hix D, Kenyon R, Machi D, Mao C, Nordberg EK, Olson R, Overbeek R, Pusch GD, Shukla M, Schulman J, Stevens RL, Sullivan DE, Vonstein V, Warren A, Will R, Wilson MJ, Yoo HS, Zhang C, Zhang Y, Sobral BW (2014) PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res 42:D581–D591. https://doi.org/10.1093/nar/gkt1099
Zhang C, Ondeyka JG, Zink DL, Basilio A, Vicente F, Salazar O, Genilloud O, Dorso K, Motyl M, Byrne K, Singh SB (2009) Discovery of okilactomycin and congeners from Streptomyces scabrisporus by antisense differential sensitivity assay targeting ribosomal protein S4. J Antibiot 62:55–61. https://doi.org/10.1038/ja.2008.8
Zhao J, Sun W, Shan T, Mou Y, Lou J, Li Y, Wang M, Zhou L (2012) Antimicrobial metabolites from the endophytic fungus Gliomastix murorum Ppf8 associated with the medicinal plant Paris polyphylla var. yunnanensis. J Med Plant Res 6:2100–2104. https://doi.org/10.5897/JMPR11.893
Acknowledgements
We are grateful to Cecilia Aguilar and B. Ruiz-Villafán for their valuable technical support in this work, Abel Blancas and Jesús Villegas Cruz for the Biorreactor facilities, M.A. Ortiz-Jiménez for the actinomycete preservation. The authors recognize the valuable support of Nuria Estrau Escofet, Beatríz Quiroz García, María de la Paz Orta, Lucero Ríos and Carmen Márquez from Instituto de Química, UNAM for recording NMR, IR, UV, and MS analysis and Silvia Ivonne Mora Herrera for the UPLC-TOF-MS tests. We are indebted to Roxana Olguín and Andrea Bedoya from LabNalCit by flow cytometry facilities.
Funding
This work was supported by DGAPA, UNAM, PAPIIT grants IN-202216 and IN-205922. We acknowledge the funding made by the NUATEI program from Instituto de Investigaciones Biomédicas, UNAM. As a doctoral student of the Programa de doctorado en Ciencias Biomédicas, UNAM, KR-P was recipient of the fellowship 161183 from CONACyT, México. This project used the UNAM´s NMR lab: LURMN facilities from IQ-UNAM, co-funded by CONACyT Mexico (Project: 0224747), and was also supported by the projects IN-210920, A1-S-20469, and A1-S-9143.
Author information
Authors and Affiliations
Contributions
KR-P conducted the experimental wok as part of her PhD thesis. Besides, she analyzed and organized all data and wrote the first manuscript draft. MPG-R performed the bioinformatic analysis and contributed to the first draft. MLM-R guided the compounds isolation and chemical characterization. LR-Z assessed cytotoxic activity assays and provided the human cell-lines and facilities. RR-S gave advice, supported in all the essential project fundamentals, and critically read the manuscript. SS directed, supported and led the research group. All authors read, gave feedback, and approved the final version of the manuscript.
Corresponding author
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by the authors.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Rodríguez-Peña, K., Gómez-Román, M.P., Macías-Rubalcava, M.L. et al. Bioinformatic comparison of three Embleya species and description of steffimycins production by Embleya sp. NF3. Appl Microbiol Biotechnol 106, 3173–3190 (2022). https://doi.org/10.1007/s00253-022-11915-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-022-11915-0