Advances in microbial culturing conditions to activate silent biosynthetic gene clusters for novel metabolite production

  • Hailey A. Tomm
  • Lorena Ucciferri
  • Avena C. RossEmail author
Natural Products - Mini Review


Natural products (NPs) produced by bacteria and fungi are often used as therapeutic agents due to their complex structures and wide range of bioactivities. Enzymes that build NPs are encoded by co-localized biosynthetic gene clusters (BGCs), and genome sequencing has recently revealed that many BGCs are “silent” under standard laboratory conditions. There are numerous methods used to activate “silent” BGCs that rely either upon altering culture conditions or genetic modification. In this review, we discuss several recent microbial cultivation methods that have been used to expand the scope of NPs accessible in the laboratory. These approaches are divided into three categories: addition of a physical scaffold, addition of small molecule elicitors, and co-cultivation with another microbe.


Silent biosynthetic gene clusters Natural products Co-culturing Scaffolds Elicitors 



Funding is gratefully acknowledged from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2015-06078) (Discovery Grant to ACR) and Queen’s University (Research Initiation Grant to ACR and Graduate award to LU).


  1. 1.
    Afiyatullov SS, Zhuravleva OI, Antonov AS et al (2018) Prenylated indole alkaloids from co-culture of marine-derived fungi Aspergillus sulphureus and Isaria felina. J Antibiot 71:846–853. CrossRefPubMedGoogle Scholar
  2. 2.
    Akhter N, Liu Y, Auckloo BN et al (2018) Stress-driven discovery of new angucycline-type antibiotics from a marine Streptomyces pratensis NA-ZhouS1. Mar Drugs 16:331. CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Akone SH, Mándi A, Kurtán T et al (2016) Inducing secondary metabolite production by the endophytic fungus Chaetomium sp. through fungal–bacterial co-culture and epigenetic modification. Tetrahedron 72:6340–6347. CrossRefGoogle Scholar
  4. 4.
    Alonso S, Rendueles M, Díaz M (2017) Tunable decoupled overproduction of lactobionic acid in Pseudomonas taetrolens through temperature-control strategies. Process Biochem 58:9–16. CrossRefGoogle Scholar
  5. 5.
    Ancheeva E, Mándi A, Király SB et al (2018) Chaetolines A and B, Pyrano[3,2-f]isoquinoline alkaloids from cultivation of Chaetomium sp. in the presence of autoclaved Pseudomonas aeruginosa. J Nat Prod 81:2392–2398. CrossRefPubMedGoogle Scholar
  6. 6.
    Ancheeva E, Küppers L, Akone SH et al (2017) Expanding the metabolic profile of the fungus Chaetomium sp. through co-culture with autoclaved Pseudomonas aeruginosa. Eur J Org Chem 2017:3256–3264. CrossRefGoogle Scholar
  7. 7.
    Anjum K, Sadiq I, Chen L et al (2018) Novel antifungal janthinopolyenemycins A and B from a co-culture of marine-associated Janthinobacterium spp. ZZ145 and ZZ148. Tetrahedron Lett 59:3490–3494. CrossRefGoogle Scholar
  8. 8.
    Auckloo BN, Pan C, Akhter N et al (2017) Stress-driven discovery of novel cryptic antibiotics from a marine fungus Penicillium sp. BB1122. Front Microbiol 8:1450. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Bai J, Mu R, Dou M et al (2018) Epigenetic modification in histone deacetylase deletion strain of Calcarisporium arbuscula leads to diverse diterpenoids. Acta Pharm Sin B 8:687–697. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ballouz S, Francis AR, Lan R, Tanaka MM (2010) Conditions for the evolution of gene clusters in bacterial genomes. PLoS Comput Biol 6:e1000672. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Baltz RH (2017) Gifted microbes for genome mining and natural product discovery. J Ind Microbiol Biotechnol 44:573–588. CrossRefPubMedGoogle Scholar
  12. 12.
    Barnhart MM, Lynem J, Chapman MR (2006) GlcNAc-6P levels modulate the expression of Curli fibers by Escherichia coli. J Bacteriol 188:5212–5219. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Biggins JB, Ternei MA, Brady SF (2012) Malleilactone, a polyketide synthase-derived virulence factor encoded by the cryptic secondary metabolome of Burkholderia pseudomallei group pathogens. J Am Chem Soc 134:13192–13195. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Boffa LC, Vidali G, Mann RS, Allfrey VG (1978) Suppression of histone deacetylation in vivo and in vitro by sodium butyrate. J Biol Chem 253:3364–3366PubMedGoogle Scholar
  15. 15.
    Boruta T, Bizukojc M (2019) Application of aluminum oxide nanoparticles in Aspergillus terreus cultivations: evaluating the effects on lovastatin production and fungal morphology. Biomed Res Int 2019:1–11. CrossRefGoogle Scholar
  16. 16.
    Bosello M, Zeyadi M, Kraas FI et al (2013) Structural Characterization of the heterobactin siderophores from Rhodococcus erythropolis PR4 and elucidation of their biosynthetic machinery. J Nat Prod 76:2282–2290. CrossRefPubMedGoogle Scholar
  17. 17.
    Brinkmann C, Kearns P, Evans-Illidge E, Kurtbӧke D (2017) Diversity and bioactivity of marine bacteria associated with the sponges Candidaspongia flabellata and Rhopaloeides odorabile from the Great Barrier Reef in Australia. Diversity 9:39. CrossRefGoogle Scholar
  18. 18.
    Craney A, Ozimok C, Pimentel-Elardo SM et al (2012) Chemical perturbation of secondary metabolism demonstrates important links to primary metabolism. Chem Biol 19:1020–1027. CrossRefPubMedGoogle Scholar
  19. 19.
    Dashti Y, Grkovic T, Abdelmohsen UR et al (2017) Actinomycete metabolome induction/suppression with N-acetylglucosamine. J Nat Prod 80:828–836. CrossRefPubMedGoogle Scholar
  20. 20.
    Davie JR (2003) Inhibition of histone deacetylase activity by butyrate. J Nutr 133:2485S–2493S. CrossRefPubMedGoogle Scholar
  21. 21.
    Davies J, Spiegelman GB, Yim G (2006) The world of subinhibitory antibiotic concentrations. Curr Opin Microbiol 9:445–453. CrossRefPubMedGoogle Scholar
  22. 22.
    de Oca-Mejía MM, Castillo-Juárez I, Martínez-Vázquez M et al (2015) Influence of quorum sensing in multiple phenotypes of the bacterial pathogen Chromobacterium violaceum. Pathog Dis 73:1–4. CrossRefPubMedGoogle Scholar
  23. 23.
    Du C, van Wezel GP (2018) Mining for microbial gems: integrating proteomics in the postgenomic natural product discovery pipeline. Proteomics 18:1700332. CrossRefPubMedCentralGoogle Scholar
  24. 24.
    El-Hawary SS, Sayed AM, Mohammed R et al (2018) New Pim-1 kinase inhibitor from the co-culture of two sponge-associated Actinomycetes. Front Chem 6:538. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Everest GJ, Meyers PR (2011) Evaluation of the antibiotic biosynthetic potential of the genus Amycolatopsis and description of Amycolatopsis circi sp. nov., Amycolatopsis equina sp. nov. and Amycolatopsis hippodromi sp. nov. J Appl Microbiol 111:300–311. CrossRefPubMedGoogle Scholar
  26. 26.
    Feng X, He C, Jiao L et al (2019) Analysis of differential expression proteins reveals the key pathway in response to heat stress in Alicyclobacillus acidoterrestris DSM 3922T. Food Microbiol 80:77–84. CrossRefPubMedGoogle Scholar
  27. 27.
    Gilmore SA, Naseem S, Konopka JB, Sil A (2013) N-Acetylglucosamine (GlcNAc) triggers a rapid, temperature-responsive morphogenetic program in thermally dimorphic fungi. PLoS Genet. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Gonciarz J, Bizukojc M (2014) Adding talc microparticles to Aspergillus terreus ATCC 20542 preculture decreases fungal pellet size and improves lovastatin production. Eng Life Sci 14:190–200. CrossRefGoogle Scholar
  29. 29.
    Hegemann JD, Zimmermann M, Xie X, Marahiel MA (2015) Lasso peptides: an intriguing class of bacterial natural products. Acc Chem Res 48:1909–1919. CrossRefPubMedGoogle Scholar
  30. 30.
    Jiang J, Sun Y-F, Tang X et al (2018) Alkaline pH shock enhanced production of validamycin A in fermentation of Streptomyces hygroscopicus. Bioresour Technol 249:234–240. CrossRefPubMedGoogle Scholar
  31. 31.
    Ju K-S, Zhang X, Elliot MA (2017) New kid on the block: LmbU expands the repertoire of specialized metabolic regulators in Streptomyces. J Bacteriol. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kawai K, Wang G, Okamoto S, Ochi K (2007) The rare earth, scandium, causes antibiotic overproduction in Streptomyces spp. FEMS Microbiol Lett 274:311–315. CrossRefPubMedGoogle Scholar
  33. 33.
    Keilhofer N, Nachtigall J, Kulik A et al (2018) Streptomyces AcH 505 triggers production of a salicylic acid analogue in the fungal pathogen Heterobasidion abietinum that enhances infection of Norway spruce seedlings. Antonie Van Leeuwenhoek 111:691–704. CrossRefPubMedGoogle Scholar
  34. 34.
    Kharel MK, Pahari P, Shepherd MD et al (2012) Angucyclines: biosynthesis, mode-of-action, new natural products, and synthesis. Nat Prod Rep 29:264–325. CrossRefPubMedGoogle Scholar
  35. 35.
    Kim SD, Park SK, Cho JY et al (2006) Surfactin C inhibits platelet aggregation. J Pharm Pharmacol 58:867–870. CrossRefPubMedGoogle Scholar
  36. 36.
    Lehmann LH, Jebessa ZH, Kreusser MM et al (2018) A proteolytic fragment of histone deacetylase 4 protects the heart from failure by regulating the hexosamine biosynthetic pathway. Nat Med 24:62–72. CrossRefPubMedGoogle Scholar
  37. 37.
    Ligon BL (2004) Penicillin: its discovery and early development. Semin Pediatr Infect Dis 15:52–57. CrossRefPubMedGoogle Scholar
  38. 38.
    Luger K, Richmond TJ (1998) The histone tails of the nucleosome. Curr Opin Genet Dev 8:140–146CrossRefGoogle Scholar
  39. 39.
    Manczinger M, Bocsik A, Kocsis GF et al (2015) The absence of N-acetyl-d-glucosamine causes attenuation of virulence of Candida albicans upon interaction with vaginal epithelial cells in vitro. Biomed Res Int. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Mao D, Okada BK, Wu Y et al (2018) Recent advances in activating silent biosynthetic gene clusters in bacteria. Curr Opin Microbiol 45:156–163. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Matilla MA, Daddaoua A, Chini A et al (2018) An auxin controls bacterial antibiotics production. Nucleic Acids Res 46:11229–11238. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Mo S, Kim J, Oh C-H (2013) Different effects of acidic pH shock on the prodiginine production in Streptomyces coelicolor M511 and SJM1 mutants. J Microbiol Biotechnol 23:1454–1459. CrossRefPubMedGoogle Scholar
  43. 43.
    Montaser R, Luesch H (2011) Marine natural products: a new wave of drugs? Future Med Chem 3:1475–1489. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Moore JM, Bradshaw E, Seipke RF et al (2012) Chapter eighteen—use and discovery of chemical elicitors that stimulate biosynthetic gene clusters in Streptomyces bacteria. In: Hopwood DA (ed) methods in enzymology. Academic Press, Cambridge, pp 367–385Google Scholar
  45. 45.
    Motoyama T, Osada H (2016) Biosynthetic approaches to creating bioactive fungal metabolites: pathway engineering and activation of secondary metabolism. Bioorg Med Chem Lett 26:5843–5850. CrossRefPubMedGoogle Scholar
  46. 46.
    Moussian B (2008) The role of GlcNAc in formation and function of extracellular matrices. Comp Biochem Physiol B Biochem Mol Biol 149:215–226. CrossRefPubMedGoogle Scholar
  47. 47.
    Munshi A, Shafi G, Aliya N, Jyothy A (2009) Histone modifications dictate specific biological readouts. J Genet Genom 36:75–88. CrossRefGoogle Scholar
  48. 48.
    Nah H-J, Pyeon H-R, Kang S-H et al (2017) Cloning and heterologous expression of a large-sized natural product biosynthetic gene cluster in Streptomyces species. Front Microbiol 8:1–10. CrossRefGoogle Scholar
  49. 49.
    Naseem S, Parrino SM, Buenten DM, Konopka JB (2012) Novel roles for GlcNAc in cell signaling. Commun Integr Biol 5:156–159. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Ochi K, Hosaka T (2013) New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters. Appl Microbiol Biotechnol 97:87–98. CrossRefPubMedGoogle Scholar
  51. 51.
    Okada BK, Seyedsayamdost MR (2017) Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules. FEMS Microbiol Rev 41:19–33. CrossRefPubMedGoogle Scholar
  52. 52.
    Özkaya FC, Peker Z, Camas M et al (2017) Marine fungi against aquaculture pathogens and induction of the activity via co-culture: biotechnology. CLEAN Soil Air Water 45:1700238. CrossRefGoogle Scholar
  53. 53.
    Pacwa-Płociniczak M, Płaza GA, Piotrowska-Seget Z, Cameotra SS (2011) Environmental applications of biosurfactants: recent advances. Int J Mol Sci 12:633–654. CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Pál C, Hurst LD (2004) Evidence against the selfish operon theory. Trends Genet 20:232–234. CrossRefPubMedGoogle Scholar
  55. 55.
    Park HB, Kwon HC, Lee C-H, Yang HO (2009) Glionitrin A, an antibiotic–antitumor metabolite derived from competitive interaction between abandoned mine microbes. J Nat Prod 72:248–252. CrossRefPubMedGoogle Scholar
  56. 56.
    Pimentel-Elardo SM, Sørensen D, Ho L et al (2015) Activity-independent discovery of secondary metabolites using chemical elicitation and cheminformatic inference. ACS Chem Biol 10:2616–2623. CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Rateb ME, Hallyburton I, Houssen WE et al (2013) Induction of diverse secondary metabolites in Aspergillus fumigatus by microbial co-culture. RSC Adv 3:14444–14450. CrossRefGoogle Scholar
  58. 58.
    Ren H, Wang B, Zhao H (2017) Breaking the silence: new strategies for discovering novel natural products. Curr Opin Biotechnol 48:21–27. CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Rigali S, Anderssen S, Naômé A, van Wezel GP (2018) Cracking the regulatory code of biosynthetic gene clusters as a strategy for natural product discovery. Biochem Pharmacol 153:24–34. CrossRefPubMedGoogle Scholar
  60. 60.
    Romano S, Jackson S, Patry S, Dobson A (2018) Extending the “One Strain Many Compounds” (OSMAC) principle to marine microorganisms. Mar Drugs 16:244. CrossRefPubMedCentralGoogle Scholar
  61. 61.
    Ross AC, Gulland LES, Dorrestein PC, Moore BS (2015) Targeted capture and heterologous expression of the Pseudoalteromonas alterochromide gene cluster in Escherichia coli represents a promising natural product exploratory platform. ACS Synth Biol 4:414–420. CrossRefPubMedGoogle Scholar
  62. 62.
    Rutledge PJ, Challis GL (2015) Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat Rev Microbiol 13:509–523. CrossRefPubMedGoogle Scholar
  63. 63.
    Seyedsayamdost MR (2014) High-throughput platform for the discovery of elicitors of silent bacterial gene clusters. Proc Natl Acad Sci USA 111:7266–7271. CrossRefPubMedGoogle Scholar
  64. 64.
    Sharma R, Jamwal V, Singh VP et al (2017) Revelation and cloning of valinomycin synthetase genes in Streptomyces lavendulae ACR-DA1 and their expression analysis under different fermentation and elicitation conditions. J Biotechnol 253:40–47. CrossRefPubMedGoogle Scholar
  65. 65.
    Shi Y, Pan C, Auckloo BN et al (2017) Stress-driven discovery of a cryptic antibiotic produced by Streptomyces sp. WU20 from Kueishantao hydrothermal vent with an integrated metabolomics strategy. Appl Microbiol Biotechnol 101:1395–1408. CrossRefPubMedGoogle Scholar
  66. 66.
    Shu C-H, Tseng K, Jaiswal R (2018) Effects of light intensity and wavelength on the production of lactobionic acid from whey by Pseudomonas taetrolens in batch cultures: effects of light intensity and wavelength on the production of lactobionic acid. J Chem Technol Biotechnol 93:1595–1600. CrossRefGoogle Scholar
  67. 67.
    Skellam E (2018) Strategies for engineering natural product biosynthesis in fungi. Trends in Biotechnol. CrossRefGoogle Scholar
  68. 68.
    Slawson C, Housley MP, Hart GW (2006) O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks. J Cell Biochem 97:71–83. CrossRefPubMedGoogle Scholar
  69. 69.
    Smetanina OF, Yurchenko AN, Afiyatullov SS et al (2012) Oxirapentyns B–D produced by a marine sediment-derived fungus Isaria felina (DC.) Fr. Phytochem Lett 5:165–169. CrossRefGoogle Scholar
  70. 70.
    Stierle AA, Stierle DB, Decato D et al (2017) The berkeleylactones, antibiotic macrolides from fungal coculture. J Nat Prod 80:1150–1160. CrossRefPubMedPubMedCentralGoogle Scholar
  71. 71.
    Sung A, Gromek S, Balunas M (2017) Upregulation and identification of antibiotic activity of a marine-derived Streptomyces sp. via co-cultures with human pathogens. Mar Drugs 15:250. CrossRefPubMedCentralGoogle Scholar
  72. 72.
    Tanaka Y, Hosaka T, Ochi K (2010) Rare earth elements activate the secondary metabolite–biosynthetic gene clusters in Streptomyces coelicolor A3(2). J Antibiot 63:477–481. CrossRefPubMedGoogle Scholar
  73. 73.
    Timmermans M, Paudel Y, Ross A (2017) Investigating the biosynthesis of natural products from marine Proteobacteria: a survey of molecules and strategies. Mar Drugs 15:235. CrossRefPubMedCentralGoogle Scholar
  74. 74.
    Timmermans ML, Picott KJ, Ucciferri L, Ross AC (2018) Culturing marine bacteria from the genus Pseudoalteromonas on a cotton scaffold alters secondary metabolite production. Microbiol Open. CrossRefGoogle Scholar
  75. 75.
    Tong Y, Charusanti P, Zhang L et al (2015) CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth Biol 4:1020–1029. CrossRefPubMedGoogle Scholar
  76. 76.
    van der Meij A, Willemse J, Schneijderberg MA et al (2018) Inter- and intracellular colonization of Arabidopsis roots by endophytic actinobacteria and the impact of plant hormones on their antimicrobial activity. Antonie Van Leeuwenhoek 111:679–690. CrossRefPubMedPubMedCentralGoogle Scholar
  77. 77.
    Wang C, Huang D, Liang S (2018) Identification and metabolomic analysis of chemical elicitors for tacrolimus accumulation in Streptomyces tsukubaensis. Appl Microbiol Biotechnol 102:7541–7553. CrossRefPubMedGoogle Scholar
  78. 78.
    Watrous JD, Dorrestein PC (2011) Imaging mass spectrometry in microbiology. Nat Rev Microbiol 9:683–694. CrossRefPubMedPubMedCentralGoogle Scholar
  79. 79.
    Wright GD (2017) Opportunities for natural products in 21st century antibiotic discovery. Nat Prod Rep 34:694–701. CrossRefPubMedGoogle Scholar
  80. 80.
    Xu D, Han L, Li C et al (2018) Bioprospecting deep-sea Actinobacteria for novel anti-infective natural products. Front Microbiol. CrossRefPubMedPubMedCentralGoogle Scholar
  81. 81.
    Xu D, Nepal KK, Chen J et al (2018) Nocardiopsistins A–C: new angucyclines with anti-MRSA activity isolated from a marine sponge-derived Nocardiopsis sp. HB-J378. Synth Syst Biotechnol 3:246–251. CrossRefPubMedPubMedCentralGoogle Scholar
  82. 82.
    Xu F, Nazari B, Moon K et al (2017) Discovery of a cryptic antifungal compound from Streptomyces albus J1074 using high-throughput elicitor screens. J Am Chem Soc 139:9203–9212. CrossRefPubMedPubMedCentralGoogle Scholar
  83. 83.
    Xu F, Wu Y, Zhang C et al (2019) A genetics-free method for high-throughput discovery of cryptic microbial metabolites. Nat Chem Biol 15:161. CrossRefPubMedPubMedCentralGoogle Scholar
  84. 84.
    Zeng Z, Cai X, Wang P et al (2017) Biofilm formation and heat stress induce pyomelanin production in deep-sea Pseudoalteromonas sp. SM9913. Front Microbiol 8:1822. CrossRefPubMedPubMedCentralGoogle Scholar
  85. 85.
    Zhang MM, Wong FT, Wang Y et al (2017) CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nat Chem Biol 13:607–609. CrossRefGoogle Scholar
  86. 86.
    Zhang Z, He X, Zhang G et al (2017) Inducing secondary metabolite production by combined culture of Talaromyces aculeatus and Penicillium variabile. J Nat Prod 80:3167–3171. CrossRefPubMedGoogle Scholar
  87. 87.
    Zhu H, Sandiford SK, van Wezel GP (2014) Triggers and cues that activate antibiotic production by actinomycetes. J Ind Microbiol Biotechnol 41:371–386. CrossRefPubMedGoogle Scholar
  88. 88.
    Zhuravleva OI, Afiyatullov SS, Vishchuk OS et al (2012) Decumbenone C, a new cytotoxic decaline derivative from the marine fungus Aspergillus sulphureus KMM 4640. Arch Pharmacal Res 35:1757–1762. CrossRefGoogle Scholar
  89. 89.
    Zou A, Liu J, Garamus VM et al (2010) Interaction between the natural lipopeptide [Glu1, Asp5] surfactin-C15 and hemoglobin in aqueous solution. Biomacromolecules 11:593–599. CrossRefPubMedGoogle Scholar
  90. 90.
    Zuck KM, Shipley S, Newman DJ (2011) Induced production of N-formyl alkaloids from Aspergillus fumigatus by co-culture with Streptomyces peucetius. J Nat Prod 74:1653–1657. CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Department of ChemistryQueen’s UniversityKingstonCanada

Personalised recommendations