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Review
. 2021 Jun 20:90:763-788.
doi: 10.1146/annurev-biochem-081420-102432. Epub 2021 Apr 13.

A Natural Product Chemist's Guide to Unlocking Silent Biosynthetic Gene Clusters

Affiliations
Review

A Natural Product Chemist's Guide to Unlocking Silent Biosynthetic Gene Clusters

Brett C Covington et al. Annu Rev Biochem. .

Abstract

Microbial natural products have provided an important source of therapeutic leads and motivated research and innovation in diverse scientific disciplines. In recent years, it has become evident that bacteria harbor a large, hidden reservoir of potential natural products in the form of silent or cryptic biosynthetic gene clusters (BGCs). These can be readily identified in microbial genome sequences but do not give rise to detectable levels of a natural product. Herein, we provide a useful organizational framework for the various methods that have been implemented for interrogating silent BGCs. We divide all available approaches into four categories. The first three are endogenous strategies that utilize the native host in conjunction with classical genetics, chemical genetics, or different culture modalities. The last category comprises expression of the entire BGC in a heterologous host. For each category, we describe the rationale, recent applications, and associated advantages and limitations.

Keywords: bacteria; biosynthesis; cryptic metabolite; natural product; silent gene cluster.

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Figures

Figure 1
Figure 1
Categories of approaches to activate silent BGCs. In classical genetics (top), forward or reverse genetic manipulations are used to enhance expression of silent BGCs. These can include deletion of transcriptional repressors, as depicted. In chemical genetics (right), libraries of small molecules provide candidate elicitors of silent BGCs, as shown with the example of trimethoprim. Alternatively, the activity of relevant pathways or proteins can be modulated with specific small molecules in reverse chemical-genetic applications. Different culture modalities (bottom), in either mono- or mixed-culture fermentation, can also lead to induction of sparingly expressed BGCs. In heterologous expression (left), the entire BGC is transplanted into a desired host with regulatory elements designed to gain access to the products of a silent BGC. Abbreviation: BGC, biosynthetic gene cluster.
Figure 2
Figure 2
Overview of classical genetic activation approaches. (a) Forward genetics involves untargeted gene mutagenesis, which either directly or indirectly affects the expression of a BGC of interest. (b) Reverse genetic strategies activate cryptic BGCs through targeted gene manipulation, such as promoter insertions (left) or repressor deletions (right). The promoter, RNA polymerase, and repressor are indicated in yellow, blue, and red, respectively. Abbreviation: BGC, biosynthetic gene cluster.
Figure 3
Figure 3
Phases of activation of silent BGCs. Regardless of the method used, the process consists of two steps. (a) A variety of methods can be used for expression of silent BGCs in an endogenous or exogenous host. (b) Upon induction, production of cryptic metabolites can be monitored with several approaches, including visual inspection; genetic reporters; various bioactivity assays including antimicrobial activity; and diverse instrumental methods, such as HPLC-MS, IMS, and UV-visible or NMR spectroscopy. Abbreviations: BGC, biosynthetic gene cluster; HPLC, high-performance liquid chromatography; IMS, imaging mass spectrometry; MS, mass spectrometry; NMR, nuclear magnetic resonance.
Figure 4
Figure 4
Selected cryptic metabolites discussed in this review. Gaudimycin D and thailandene A (blue) were discovered via RGMS. Pseudomonol, venemycin, and malleicyprol (pink) were identified via applications of reverse genetics. Forward chemical genetic strategies gave rise to keratinicyclin B and taylorflavin A (orange). Reverse chemical genetics led to the discovery of piperidamycin D, mutaxanthene A, and the ciromicins (green). Chojalactone A, niizalactam A, amycomicin, and heronapyrroles (purple) emerged from mixed culture fermentation. Arixanthomycin B (light brown) was found via heterologous expression. Note that ciromicin B was also detected with coculture methods. Abbreviations: BGC, biosynthetic gene cluster; RGMS, reporter-guided mutant selection.
Figure 5
Figure 5
Overview of induction of silent BGCs by chemical genetics. (a) Forward chemical-genetic approaches screen libraries of small molecules to identify elicitors of secondary metabolism, which can be monitored by a variety of methods. (b) Reverse chemical-genetic strategies activate cryptic BGCs by using small molecules to selectively inhibit or alter a chosen target, such as the ribosome by the antibiotic streptomycin. Abbreviation: BGC, biosynthetic gene cluster.
Figure 6
Figure 6
Overview of culture modality approaches. (a) Alterations in microbial culture conditions can significantly impact BGC expression. Monoculture variations screen organisms under several different culture conditions to increase secondary metabolite production. Closthioamide was discovered using this approach. (b) Mixed cultures can activate cryptic BGCs through a multitude of complex interactions between competing and/or cooperating organisms. In this example, production of the siderophores myxochelin by Myxococcus xanthus (shown in gold) and desferrioxamine E by Streptomyces coelicolor (shown in blue) is greatly increased when the two strains are cultured together. Abbreviation: BGC, biosynthetic gene cluster.
Figure 7
Figure 7
Overview of exogenous activation. Heterologous approaches use a surrogate organism for the production of secondary metabolites from a BGC of interest, usually targeted based on the predicted novelty or utility of the putative natural product. The general steps involved are as follows: ❶ the BGC is identified and assembled into a mobile, transferable vector; ❷ the regulatory elements in the BGC are refactored to suit the surrogate producer; ❸ the mobile, refactored BGC is cloned and expressed in the surrogate host; and ❹ the resultant natural products are identified and characterized. Abbreviation: BGC, biosynthetic gene cluster.

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