Abstract
Cosmid libraries constructed from environmental metagenome samples are powerful tools for capturing the genomic diversity of complex microbial communities. The large insert size (∼35 kb) of such libraries means they are compatible with downstream expression of large biosynthetic gene clusters (BGCs). This allows the discovery of previously undescribed natural products that would be inaccessible using traditional culture-based discovery pipelines. Here we describe methods for the construction of a cosmid metagenome library from a soil sample, and the process of screening that library for individual cosmid clones containing aromatic polyketide BGCs using degenerate primers that target the ketosynthase alpha (KSα) gene.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Torsvik V, Daae FL, Sandaa RA, Øvreås L (1998) Novel techniques for analysing microbial diversity in natural and perturbed environments. J Biotechnol 64:53–62
Rappé MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394
Katz M, Hover BM, Brady SF (2016) Culture-independent discovery of natural products from soil metagenomes. J Ind Microbiol Biotechnol 43:129–141
Hover BM, Kim SH, Katz M, Charlop-Powers Z, Owen JG, Ternei MA, Maniko J, Estrela AB, Molina H, Park S, Perlin DS, Brady SF (2018) Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant gram-positive pathogens. Nat Microbiol 3:415–422
Stevenson LJ, Owen JG, Ackerley DF (2019) Metagenome driven discovery of nonribosomal peptides. ACS Chem Biol 14:2115–2126
Owen JG, Reddy BVB, Ternei MA, Charlop-Powers Z, Calle PY, Kim JH, Brady SF (2013) Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products. Proc Natl Acad Sci U S A 110:11797–11802
Owen JG, Charlop-Powers Z, Smith AG, Ternei MA, Calle PY, Reddy BVB, Montiel D, Brady SF (2015) Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors. Proc Natl Acad Sci 112:4221–4226
Hertweck C, Luzhetskyy A, Rebets Y, Bechthold A (2007) Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork. Nat Prod Rep 24:162–190
Hertweck C (2009) The biosynthetic logic of polyketide diversity. Angew Chem Int Ed 48:4688–4716
King RW, Bauer JD, Brady SF (2009) An environmental DNA-derived type II polyketide biosynthetic pathway encodes the biosynthesis of the pentacyclic polyketide erdacin. Angew Chem Int Ed 48:6257–6261
Feng Z, Kallifidas D, Brady SF (2011) Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites. Proc Natl Acad Sci U S A 108:12629–12634
Metsä-Ketelä M, Salo V, Halo L, Hautala A, Hakala J, Mäntsälä P, Ylihonko K (1999) An efficient approach for screening minimal PKS genes from Streptomyces. FEMS Microbiol Lett 180:1–6
Kang HS, Brady SF (2014) Mining soil metagenomes to better understand the evolution of natural product structural diversity: Pentangular polyphenols as a case study. J Am Chem Soc 136:18111–18119
Kang HS (2017) Phylogeny-guided (meta)genome mining approach for the targeted discovery of new microbial natural products. J Ind Microbiol Biotechnol 44:285–293
Winn R, Norris M (2010) Analysis of mutations in λ transgenic medaka using the cII mutation assay. In: Techniques in aquatic toxicology, vol 2. CRC Press, Boca Raton, Florida
Nurk S, Bankevich A, Antipov D, Gurevich AA, Korobeynikov A, Lapidus A, Prjibelski AD, Pyshkin A, Sirotkin A, Sirotkin Y, Stepanauskas R, Clingenpeel SR, Woyke T, Mclean JS, Lasken R, Tesler G, Alekseyev MA, Pevzner PA (2013) Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comput Biol 20:714–737
Acknowledgments
This work was supported by the Health Research Council of New Zealand (contract 16/172 to D.F.A. and J.G.O.) and the New Zealand Ministry of Business, Innovation and Employment (Smart Ideas contract RTVU1908 to J.G.O.). J.G.O. was additionally supported by a Rutherford Discovery Fellowship awarded by the Royal Society of New Zealand, and L.J.S. by a Maurice Wilkins Centre Doctoral Scholarship. We thank Drs Magnani, Marabelli, and Paradisi for all their hard work editing this volume and their kind invitation to contribute a chapter.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Stevenson, L.J., Ackerley, D.F., Owen, J.G. (2022). Preparation of Soil Metagenome Libraries and Screening for Gene-Specific Amplicons. In: Magnani, F., Marabelli, C., Paradisi, F. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 2397. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1826-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1826-4_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1825-7
Online ISBN: 978-1-0716-1826-4
eBook Packages: Springer Protocols