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Discovery of microbial natural products by activation of silent biosynthetic gene clusters

Key Points

  • Microorganisms produce an array of specialized metabolites with a wide range of biological activities and potential applications, such as antibiotics, anticancer agents and agrochemicals.

  • Next-generation sequencing (NGS) technologies have led to an exponential increase in microbial genome data; mining of genome databases with bioinformatics tools enables biosynthetic gene clusters (BGCs) encoding specialized metabolites to be identified.

  • Genomic analysis reveals that many microorganisms have far greater potential to produce specialized metabolites than is suggested by classic bioactivity screens. Many BGCs in the genome are silent and are therefore not expressed under standard laboratory growth conditions.

  • A range of strategies have been developed to activate these silent BGCs and thereby gain access to their metabolic products.

  • Pleiotropic strategies induce organism-wide changes to trigger BGC expression; such strategies include variation in growth conditions, the introduction of competing species, the upregulation of global transcriptional regulators and epigenetic perturbation. These approaches can be high throughput, but are often empirical in nature and generally offer less control or predictability.

  • Pathway-specific strategies enable a more targeted approach, but are generally lower throughput. Methods include inducing the expression of pathway-specific activator genes, deleting genes encoding pathway-specific repressors, refactoring a BGC of interest to replace the natural promoters, or BGC expression in a heterologous host.

  • High-pressure liquid chromatography (HPLC), mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy are the key techniques used to isolate and characterize the metabolic products when a silent BGC has been successfully activated.

Abstract

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities and a wide variety of applications in medicine and agriculture, such as the treatment of infectious diseases and cancer, and the prevention of crop damage. Genomics has revealed that many microorganisms have far greater potential to produce specialized metabolites than was thought from classic bioactivity screens; however, realizing this potential has been hampered by the fact that many specialized metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures. In this Review, we discuss the strategies that have been developed in bacteria and fungi to identify and induce the expression of such silent BGCs, and we briefly summarize methods for the isolation and structural characterization of their metabolic products.

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Figure 1: Drugs developed from microbial specialized metabolites since the turn of the century.
Figure 2: The genome mining approach for natural product discovery in microorganisms.
Figure 3: Pleiotropic strategies that have been used to activate silent biosynthetic gene clusters encoding specialized metabolites.
Figure 4: Pathway-specific strategies that have been used to induce the expression of silent biosynthetic gene clusters encoding specialized metabolites.

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Acknowledgements

P.J.R. thanks the Royal Society of Chemistry Journals Grants for International Authors for supporting a sabbatical visit to the University of Warwick, Coventry, UK. G.L.C. gratefully acknowledges the UK Biotechnology and Biological Sciences Research Council (BBSRC; grants BB/K002341/1, BB/L010852/1 and BB/L023342/1), the UK Medical Research Council (MRC; grant MR/N501839/1), the Royal Society (grant WM130033) and Syngenta for their financial support of ongoing research concerning genomics-driven natural product discovery.

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Glossary

Agrochemicals

Chemical products that are used in agriculture, such as pesticides, growth enhancers and fertilizers.

Nonribosomal peptide synthetases

(NRPSs). A family of modular multi-enzymes that catalyse the biosynthesis of many specialized peptide metabolites via a ribosome- and mRNA-independent mechanism.

Polyketide synthases

(PKSs). Multi-enzyme complexes that are responsible for the biosynthesis of polyketides via a similar mechanism to fatty acid biosynthesis. In this mechanism, polyketides (which are specialized metabolites) are assembled via the decarboxylative condensation of (alkyl)malonyl-CoA-derived extender units with an acyl-CoA-derived starter unit.

Terpene synthases

Enzymes that catalyse the formation of terpene natural products from an oligo-isoprenyl diphosphate precursor (for example, geranyl diphosphate or farnesyl diphosphate).

NRPS-independent siderophore synthetases

A family of ATP-dependent enzymes that catalyse the biosynthesis of siderophores without the involvement of nonribosomal peptide synthetases (NRPSs).

antiSMASH

(Antibiotics and secondary metabolite analysis shell). A web-based program that identifies biosynthetic gene clusters in unannotated DNA sequence assemblies.

Lanthipeptide

A type of peptide natural product that is ribosomally biosynthesised and post-translationally modified; lanthipeptides characteristically contain lanthionine (a non-proteinogenic amino acid of formula HO2CCH(NH2)CH2SCH2CH(NH2)CO2H; the monosulfide analogue of cystine) or methyl-lanthionine residues in the processed peptide. Lanthipeptides that display antibacterial activity are called lantibiotics.

Bacteriocins

Proteinaceous toxins that are produced by bacteria to inhibit competing strains.

Whole-genome shotgun sequencing

A sequencing technique in which the DNA of interest is sheared into random fragments, size selected and ligated to adaptor molecules or cloned into an appropriate vector; DNA fragments are sequenced, and the original DNA sequence is reconstructed using sequence assembly software.

Mate pair

The two sequences from opposite ends of an insert within a single clone of a genomic library. These sequences are used in conjunction with whole-genome shotgun sequencing to aid sequence assembly.

PacBio SMRT sequencing

A next-generation sequencing technology, developed by Pacific Biosciences, that uses DNA polymerase and fluorescently tagged nucleotides to follow DNA replication nucleotide by nucleotide and thus determine a DNA sequence.

In silico dereplication

The predictive, computer-based analysis of biosynthetic gene clusters to eliminate those that are likely to direct the production of known metabolites or those with closely related structures.

Alkaloids

Specialized metabolites that contain nitrogen, such as morphine, quinine, nicotine and cocaine.

Cathepsin K

A lysosomal cysteine protease involved in bone remodelling and resorption.

Polyphenols

Class of organic compounds that contain multiple phenol (hydroxybenzene) units.

Matrix-assisted laser desorption/ionization–imaging mass spectrometry

(MALDI–IMS). An analytical method that uses a laser to directly desorb and ionize molecules from a specific location within a biological sample (for example, a tissue sample or microbial colony) and detect the resulting ions by mass spectrometry.

Iturin family

A family of fungicidal cyclic lipopeptides that were first identified as products of Bacillus subtilis.

Phenazine

A tricyclic aromatic compound with the structure (C6H4)N2(C6H4); also known as dibenzo-p-diazine or dibenzopyrazine.

Siderophore

A small, high-affinity iron chelator secreted by a microorganism to sequester environmental iron.

Stringent response

A stress response in bacteria to conditions such as fatty acid limitation, amino acid starvation, iron limitation or heat shock.

Velvet complex

A group of regulatory proteins that control specialized metabolite biosynthesis in fungi and include a characteristic 150 amino acid sequence known as the velvet domain. Velvet proteins may combine with themselves (homodimers), with each other (heterodimers) or with other proteins to regulate transcription.

DNA microarrays

Collections of microscopic DNA spots (probes) adhered to a solid surface, forming the basis of fluorescence-labelling techniques to detect and quantify the expression of large numbers of genes at the same time. Also known as DNA chips or biochips.

Chromatin

The complex of DNA, histone proteins and RNA that functions to package DNA into a smaller volume and control gene expression.

Anthraquinone

A type of aromatic compound based on 9,10-dioxoanthracene. Although several isomers are possible, the term anthraquinone usually refers to the 9,10-dioxo isomer in which the keto groups are located on the central ring of the tricyclic system.

Congeners

Chemical substances that are related to each other by origin, structure or function.

Azaphilone

A type of polyketide natural product that contains a highly oxygenated bicyclic pyranoquinone core (usually referred to as isochromene) and a quaternary carbon centre.

Mycaminosylated polyketide

A polyketide with β-D-mycaminose (3,6-dideoxy-3- (dimethylamino)-D-glucopyranose) attached to one or more hydroxyl groups.

LAL family

(Large ATP-binding repressors of the LuxR family). A family of transcriptional regulators that control the expression of biosynthetic gene clusters that direct the production of specialized metabolites. LAL proteins were first identified in proteobacteria and have since been discovered in several actinobacteria.

IC50

(Half-maximal inhibitory concentration).The concentration of a compound that results in inhibition of the activity of its biological target by 50%.

TetR-like repressor proteins

Transcriptional regulators that incorporate a 47 amino acid helix–turn–helix (HTH) binding motif and a ligand-binding domain that shows sequence similarity to the tetracycline-responsive repressor of the tetracycline resistance (tetR) gene.

Quorum sensing

A mechanism by which bacteria alter their gene expression in response to the density of their local population.

E. coli–Streptomyces artificial chromosomes

(ESACs). Bacterial artificial chromosomes that can be shuttled between Escherichia coli (in which they replicate autonomously) and a Streptomyces sp. host (in which they integrate into a specific chromosomal site), thus enabling the cloning and heterologous expression of large segments (up to 140 kb) of DNA in Streptomyces spp. hosts.

Cosmid

A hybrid plasmid containing the cos (cohesive end site) sequence from phage λ; commonly used in genetic engineering as a cloning vector and to build genomic libraries.

Fosmid

A low-copy-number vector based on the Escherichia coli F factor replicon; it can hold DNA inserts of up to 40 kb, and the low copy number affords higher stability than vectors with higher copy numbers (such as cosmids).

High-pressure liquid chromatography

(HPLC). An analytical technique used to separate the components of a mixture by pumping a solution through a column of solid adsorbent material.

m/z value

The mass-to-charge ratio. This is the measured output from a mass spectrometer, which enables the identification and characterization of compounds based on their molecular mass.

NMR spectroscopy

An analytical technique that exploits the magnetic properties of certain nuclei (including those of 1H, 13C and 15N) to determine the molecular structure of compounds that contain these elements.

X-ray crystallography

An analytical technique for elucidating molecular structure, exploiting the fact that atoms in a crystalline sample cause a beam of X-rays to diffract; from the angles and intensities of the diffracted beams, a 3D image of the molecule can be derived.

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Rutledge, P., Challis, G. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat Rev Microbiol 13, 509–523 (2015). https://doi.org/10.1038/nrmicro3496

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