Abstract
Although transcriptional activation of pathwayspecific positive regulatory genes and/or biosynthetic genes is primarily important for enhancing secondary metabolite production, reinforcement of substrate supply, as represented by primary metabolites, is also effective. For example, partial inhibition of fatty acid synthesis with ARC2 (an analog of triclosan) was found to enhance polyketide antibiotic production. Here, we demonstrate that this approach is effective even for industrial high-producing strains, for example enhancing salinomycin production by 40%, reaching 30.4 g/l of salinomycin in an industrial Streptomyces albus strain. We also hypothesized that a similar approach would be applicable to another important antibiotic group, nonribosomal peptide (NRP) antibiotics. We therefore attempted to partially inhibit protein synthesis by using ribosome-targeting drugs at subinhibitory concentrations (1/50∼1/2 of MICs), which may result in the preferential recruitment of intracellular amino acids to the biosynthesis of NRP antibiotics rather than to protein synthesis. Among the ribosome-targeting drugs examined, chloramphenicol at subinhibitory concentrations was most effective at enhancing the production by Streptomyces of NRP antibiotics such as actinomycin, calcium-dependent antibiotic (CDA), and piperidamycin, often resulting in an almost 2-fold increase in antibiotic production. Chloramphenicol activated biosynthetic genes at the transcriptional level and increased amino acid pool sizes 1.5- to 6-fold, enhancing the production of actinomycin and CDA. This “metabolic perturbation” approach using subinhibitory concentrations of ribosome-targeting drugs is a rational method of enhancing NRP antibiotic production, being especially effective in transcriptionally activated (e.g., rpoB mutant) strains. Because this approach does not require prior genetic information, it may be widely applicable for enhancing bacterial production of NRP antibiotics and bioactive peptides.
Similar content being viewed by others
References
Adrio JL, Demain AL (2006) Genetic improvement of processes yielding microbial products. FEMS Microbiol Rev 30:187–214
Ahmed S, Craney A, Pimentel-Elardo SM, Nodwell JR (2013) A synthetic, species-specific activator of secondary metabolism and sporulation in Streptomyces coelicolor. Chembiochem 14:83–91
Baltz RH (2011) Strain improvement in actinomycetes in the postgenomic era. J Ind Microbiol Biotechnol 38:657–666
Bentley SD, Chater KF, Cerdeno-Tarraga AM, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D, Bateman A, Brown S, Chandra G, Chen CW, Collins M, Cronin A, Fraser A, Goble A, Hidalgo J, Hornsby T, Howarth S, Huang CH, Kieser T, Larke L, Murphy L, Oliver K, O’Neil S, Rabbinowitsch E, Rajandream MA, Rutherford K, Rutter S, Seeger K, Saunders D, Sharp S, Squares R, Squares S, Taylor K, Warren T, Wietzorrek A, Woodward J, Barrell BG, Parkhill J, Hopwood DA (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417:141–147
Brakhage AA, Schroeckh V (2011) Fungal secondary metabolites—strategies to activate silent gene clusters. Fungal Genet Biol 48:15–22
Craney A, Ozimok C, Pimentel-Elardo SM, Capretta A, Nodwell JR (2012) Chemical perturbation of secondary metabolism demonstrates important links to primary metabolism. Chem Biol 19:1020–1027
Craney A, Ahmed S, Nodwell J (2013) Towards a new science of secondary metabolism. J Antibiot 66:387–400
Davies J, Spiegelman GB, Yim G (2006) The world of subinhibitory antibiotic concentrations. Curr Opin Microbiol 9:445–453
Doekel S, Marahiel MA (2001) Biosynthesis of natural products on modular peptide synthetases. Metab Eng 3:64–77
Goh EB, Yim G, Tsui W, McClure J, Surette MG, Davies J (2002) Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc Natl Acad Sci U S A 99:17025–17030
Gomez-Escribano JP, Bibb MJ (2011) Engineering Streptomyces coelicolor for eterologous expression of secondary metabolite gene clusters. Microb Biotechnol 4:207–215
Herbert S, Barry P, Novick PR (2001) Suninhibitory clindamysin differentially inhibits transcription of exoprotein genes in Staphylococcus aureus. Infect Immun 69:2996–3003
Hojati Z, Milne C, Harvey B, Gordon L, Borg M, Flett F, Wilkinson B, Sidebottom PJ, Rudd BAM, Hayes MA, Smith CP, Micklefield J (2002) Structure, biosynthetic origin, and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor. Chem Biol 9:1175–1187
Hopwood DA (2008) Small things considered: the tip of the iceberg. ― http://schaechter.asmblog.org/schaechter/2008/06/the-tip-of-the.html ―
Hosaka T, Ohnishi-Kameyama M, Muramatsu H, Murakami K, Tsurumi Y, Kodani S, Yoshida M, Fujie A, Ochi K (2009) Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12. Nat Biotechnol 27:462–464
Ikeda H, Ishikawa J, Hanamoto A, Shinose M, Kikuchi H, Shiba T, Sakaki Y, Hattori M, Omura S (2003) Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21:526–531
Imai Y, Sato S, Tanaka Y, Ochi K, Hosaka T (2015) Lincomycin at subinhibitory concentrations potentiates secondary metabolite production by Streptomyces spp. Appl Environ Microbiol 81:3869–3879
Katz E (1967) Actinomycin. In: Gottlieb D, Shaw PD (eds) Antibiotics, vol II. Springer, New York, pp 276–341
Keller U, Kleinkauf H, Zocher R (1984) 4-Methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme from actinomycin-producing Streptomyces chrysomallus. Biochemistry 23:1479–1484
Keller U, Lang M, Crnovcic I, Pfenning F, Schauwecker F (2010) The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus: a genetic hall of mirrors for synthesis of a molecule with mirror symmetry. J Bacteriol 192:2583–2595
Kelly WL, Pan L, Li C (2009) Thiostrepton biosynthesis: prototype for a new family of bacteriocins. J Am Chem Soc 131:4327–4334
Komatsu M, Uchiyama T, Omura S, Cane DE, Ikeda H (2010) Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proc Natl Acad Sci U S A 107:2646–2651
Laureti L, Song L, Huang S, Corre C, Leblond P, Challis GL, Aigle B (2011) Identification of a bioactive 51-membered macrolide complex by activation of a silent polyketide synthase in Streptomyces ambofacience. Proc Natl Acad Sci U S A 108:6258–6263
Levner MH, Urbano C, Rubin BA (1980) Lincomycin increases synthetic rate and periplasmic pool size for cholera toxin. J Bacteriol 143:441–447
Liao R, Duan L, Lei C, Pan H, Ding Y, Zhang Q, Chen D, Shen B, Yu Y, Liu W (2009) Thiopeptide biosynthesis featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. Chem Biol 16:141–147
Lin JT, Connelly MB, Amolo C, Otani S, Yaver DS (2005) Global transcriptional response of Bacillus subtilis to treatment with subinhibitory concentrations of antibiotics that inhibit protein synthesis. Antimicrob Agents Chemother 49:1915–1926
Mahlert C, Kopp F, Thirlway J, Micklefield J, Marahiel MA (2007) Stereospecific enzymatic transformation of α-ketoglutarate to (2S, 3R)-3-metyl glutamate during acidic lipopeptide biosynthesis. J Am Chem Soc 129:12011–12018
Marahiel MA, Stachelhaus T, Mootz HD (1997) Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem Rev 97:2651–2673
Martin JF, Liras P (2010) Engineering of regulatory cascades and networks controlling antibiotic biosynthesis in Streptomyces. Curr Opin Microbiol 13:263–273
McKenzie NL, Thaker M, Koteva K, Hughes DW, Wright GD, Nodwell JR (2010) Induction of antimicrobial activities in heterologous streptomycetes using alleles of the Streptomyces coelicolor gene absA1. J Antibiot 63:177–182
Neumann CS, Jiang W, Heemstra JR Jr, Gontang EA, Kolter R, Walsh CT (2012) Biosynthesis of piperazic acid via N5-hydroxy-ornithine in Kutzneria spp. 744. Chembiochem 13:972–976
Nielsen J (1998) The role of metabolic engineering in the production of secondary metabolites. Curr Opin Microbiol 1:330–336
Nierhaus D, Nierhaus KH (1973) Identification of the chloramphenicol-binding protein in Escherichia coli ribosomes by partial reconstitution. Proc Natl Acad Sci U S A 70:2224–2228
Ochi K (1987) Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to a factor. J Bacteriol 169:3608–3616
Ochi K (2007) From microbial differentiation to ribosome engineering. Biosci Biotechnol Biochem 71:1373–1386
Ochi K (2017) Insights into microbial cryptic gene activation and strain improvement: principle, application and technical aspects. J Antibiot 70:25–40
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
Ochi K, Okamoto S (2012) A magic bullet for antibiotic discovery. Chem Biol 19:932–934
Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda H, Yamashita A, Hattori M, Horinouchi S (2008) Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 190:4050–4060
Olano C, Lombo F, Mendez C, Salas JA (2008) Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab Eng 10:281–292
Oliynyk M, Samborskyy M, Lester JB, Mironenko T, Scott N, Dickens S, Haydock SF, Leadlay PF (2007) Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338. Nat Biotechnol 25:447–453
Pootoolal J, Thomas MG, Marshall CG, Neu JM, Hubbard BK, Walsh CT, Wright GD (2002) Assembling the glycopeptide antibiotic scaffold: the biosynthesis of A47934 from Streptomyces toyocaensis NRRL 15009. Proc Natl Acad Sci U S A 99:8962–8967
Sader HS, Streit JM, Fritsche TR, Jones RN (2004) Antimicrobial activity of daptomycin against multidrug-resistant Gram-positive strains collected worldwide. Diagn Microbiol Infect Dis 50:201–204
Scolnick E, Tompkins R, Caskey CT, Nirenberg M (1968) Release factors differing in specificity for terminator codons. Proc Natl Acad Sci U S A 61:768–774
Shibl MA (1993) Effect of antibiotics on production of enzymes and toxins by microorganisms. Rev Infect Dis 5:865–875
Tamehiro N, Hosaka T, Xu J, Hu H, Otake N, Ochi K (2003) Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus. Appl Environ Microbiol 69:6412–6417
Tanaka Y, Komatsu M, Okamoto S, Tokuyama S, Kaji A, Ikeda H, Ochi K (2009) Antibiotic overproduction by rpsL and rsmG mutants of various actinomycetes. Appl Environ Microbiol 75:4919–4922
Tanaka Y, Hosaka T, Ochi K (2010) Rare earth elements activate the secondary metabolite-biosynthetic gene clusters in Streptomyces coelicolor A3(2). J Antibiot (Tokyo) 63:477–481
Tanaka Y, Kasahara K, Hirose Y, Murakami K, Kugimiya R, Ochi K (2013) Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpoB) mutations in actinomycetes. J Bacteriol 195:2959–2970
Tojo S, Kim J-Y, Tanaka Y, Inaoka T, Hiraga Y, Ochi K (2014) The mthA mutation conferring low-level resistance to streptomycin enhances antibiotic production in Bacillus subtilis by increasing the S-adenosylmethionine pool size. J Bacteriol 196:1514–1524
Wang G, Izawa M, Yang X, Xu D, Tanaka Y, Ochi K (2017) Identification of a novel lincomycin resistance mutation associated with activation of antibiotic production in Streptomyces coelicolor A3(2). doi:10.1128/AAC.02247-16.
Yamada S, Suenaga H, Doi K, Yoshino S, Ogata S (2003) Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921. Biosci Biotechnol Biochem 67:797–802
Yim G, Wang HH, Davies J (2006) The truth about antibiotics. Int J Med Microbiol 296:163–170
Zhu H, Sandiford SK, van Wezel GP (2014) Triggers and cues that activate antibiotic production by actinomycetes. J Ind Microbiol Biotechnol 41:371–386
Acknowledgements
This work was supported by grants to K. O. from the National Agriculture and Food Research Organization (Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry) and MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2014 to 2016 (grant S1413002). Y. T. and Y. M. performed experiments for antibiotic production and antibiotic assays, Y. H. analyzed amino acid pool size with a LC-MS system, and M. I. and T. W. performed gene expression analysis by real-time qPCR. K.O. designed the research work and wrote the article. The authors thank Izumi Yamada for providing experimental assistance.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Electronic supplementary material
ESM 1
(PDF 514 kb)
Rights and permissions
About this article
Cite this article
Tanaka, Y., Izawa, M., Hiraga, Y. et al. Metabolic perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs. Appl Microbiol Biotechnol 101, 4417–4431 (2017). https://doi.org/10.1007/s00253-017-8216-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-017-8216-6