Leveraging a large microbial strain collection for natural product discovery

Abstract

Throughout history, natural products have significantly contributed to the discovery of novel chemistry, drug leads, and tool molecules to probe and address complex challenges in biology and medicine. Recent microbial genome sequencing efforts have uncovered many microbial biosynthetic gene clusters without an associated natural product. This means that the natural products isolated to date do not fully reflect the biosynthetic potential of microbial strains. This observation has rejuvenated the natural product community and inspired a return to microbial strain collections. Mining large microbial strain collections with the most current technologies in genome sequencing, bioinformatics, and high-throughput screening techniques presents new opportunities in natural product discovery. In this review, we report on the newly expanded microbial strain collection at The Scripps Research Institute, which represents one of the largest and most diverse strain collections in the world. Two complementary approaches, i.e. structure-centric and function-centric, are presented here to showcase how to leverage a large microbial strain collection for natural product discovery and to address challenges and harness opportunities for future efforts. Highlighted examples include the discovery of alternative producers of known natural products with superior growth characteristics and high titers, novel analogs of privileged scaffolds, novel natural products, and new activities of known and new natural products. We anticipate that this large microbial strain collection will facilitate the discovery of new natural products for many applications.

Keywords: bioinformatics; biosynthesis; drug screening; function-centric approaches; genome mining; high-throughput screening (HTS); microbial strain collection; natural product; strain prioritization; structure-centric.

Figure 1.

A, total number of strains in the current microbial strain collection at TSRI is as follows: actinobacteria (62,328, 28.7%); fungi (92,225, 42.4%); other bacteria (14,465, 6.7%); and unidentified bacteria or fungi (48,334, 22.2%). B, total number of extracts and fractions in the current NPL at TSRI is as follows: fungal extracts (136,123 made from 12,060 strains, 57.1%); fungal fractions (24,980 made from 2,498 strains, 10.7%); actinobacterial extracts (42,612 made from 8,020 strains, 18.2%); and actinobacterial fractions (32,832 made from 3,025 strains, 14.0%). C, collection dates of strains by decade: 1940s (A, 6.1%; F, 4.6%); 1950s (A, 26.8%; F, 15.0%); 1960s (A, 35.9%; F, 10.4%); 1970s (A, 7.4%; F, 4.2%); 1980s (A, 5.1%; F, 2.4%); 1990s (A, 6.7%; F, 63.2%); 2000s (A, 0.3%; F, 0.2%); and 2010s (A, 11.8%; F, 0.002%), where A indicates actinobacteria; F indicates fungi. These statistics are based on 77% of the actinobacteria, 46% of fungi, 80% of other bacteria, and 12% of unidentified bacteria or fungi from the total collection, for which collection dates could be traced. D, location of collection of strains is as follows: Brazil (F, 2.2%); Canada (A, 0.9%; F, 1.6%); China (A, 49.9%; F, 7.5%); Comoros Island (A, 0.8%); Egypt (F, 1.4%); Fiji (A, 0.9%); French Guiana (F, 2.3%); Indonesia (A, 13.6%; F, 4.4%); Italy (A, 0.8%); Japan (A, 0.9%); Korea (A, 4.2%); Malaysia (F, 2.6%); New Caledonia (F, 2.1%); Singapore (F, 1.6%); South America (A, 7.2%; F, 1.7%); Togo (A, 2.0%); United States (A, 12.2%; F, 56.0%); Venezuela (F, 1.3%); and other countries (A from 58 countries, 6.7%; F from 89 countries, 15.2%). These statistics are based on 20% of the actinobacteria and 41% of fungi from the total collection, for which collection locations could be traced. E, taxonomic information for actinobacteria is as follows: Actinomadura, 4.9%; Actinoplanes, 6.4%; Microbispora, 2.6%; Micromonaspora, 9.7%; Micropolyspora, 1.4%; Nocardia, 6.7%; Rhodococcus, 0.9%; Streptomyces, 55.2%; Streptosporangium, 1.1%; and the 79 other genera, 11.1%. Taxonomic information for fungi is as follows: Acremonium, 1.3%; Candida, 5.5%; Chaetomium, 1.6%; Fusarium, 4.4%; Mortierella, 1.1%; Mucor, 3.1%; Penicillium, 9.9%; Rhisopus, 1.2%; Trichoderma, 1.2%; and the 2,665 other genera; 70.7%. These statistics are based on 20% of the actinobacteria and 66% of fungi from the total collection for which some forms of the taxonomic data could be traced.

Figure 2.

Schematic representation of how to leverage a microbial strain collection for natural product discovery by two complementary approaches. A, structure-centric approach utilizes genomic information from the strain collection, along with bioinformatics, to prioritize privileged strains based on a unique pharmacophore or scaffold of the target natural products. B, function-centric approach utilizes biological activity, via HTS against targeted biology, to prioritize privileged strains based on unique targets or mechanism of action. Upon identification of these privileged strains, correlation of the targeted natural products or biology to specific BGCs and exploitation of these BGCs via enabling technology, such as cluster activation or heterologous expression, allow for the characterization of novel natural products, alternative producers of known natural products, and novel enzymes for combinatorial biosynthesis and biocatalysis.

Figure 3.

Examples of structure-centric approaches to leverage the microbial strain collection at TSRI for natural product discovery. A, PTM and PTN contain unique diterpenoid scaffolds, the biosynthesis of which is encoded by terpene cyclases, T1, T2, T3, and T4. An RT-PCR screen of a fraction of the actinobacteria strains in the collection prioritized strains containing all four cyclase genes, resulting in the identification of alternative PTM and PTN producers with high titers and superior growth characteristics. B, enediyne natural products contain a unique pharmacophore, consisting of two triple bonds in conjugation with a double bond or incipient double bond, the biosynthesis of which is encoded by the enediyne PKS gene cassette, E, E10, E3, E4, and E5. An RT-PCR screen of a fraction of the actinobacterial strains in the collection prioritized strains containing the enediyne PKS gene cassette, resulting in the identification of alternative C-1027 producers with high titers and new anthraquinone-fused enediynes TNMs. The TNM producer, with its high TNM titers and superior growth characteristics, has enabled the development of a platform strain for engineered biosynthesis and production of the anthraquinone-fused family of enediyne natural products. C, LNM contains a unique sulfur-containing heterocycle, where the sulfur incorporation chemistry is encoded by a DUF-SH didomain. An RT-PCR screen of a fraction of the actinobacterial strains in the collection prioritized strains containing the DUF-SH didomain, resulting in the identification of a family of modular biosynthetic pathways that exemplify how Nature does combinatorial biosynthesis for the LNM family of natural products.

Figure 4.

Examples of function-centric approaches to leverage the microbial strain collection at TSRI for natural product discovery. Selected strains from the collection are fermented in diverse media to make the crude extracts, and upon HPLC analysis, the most chemically diverse conditions are selected, scaled up, and subjected to chromatography to afford the partially purified fractions. The current NPL consists of crude extracts, partially purified fractions, and pure natural products (also see Fig. 1B) and has been subjected to HTS, against emerging biology, for natural product discovery. A, NPL was screened for cytotoxicity and prolactin-initiated phosphorylation of ERK1/2. Bioassay-guided dereplication of the active hits led to the isolation of two new bafilomycin congeners, along with nine known ones. B, NPL was screened for parasitic AsnRS inhibition. Bioassay-guided dereplication of the hits identified TAMs as potent and selective AsnRS inhibitors, three of which were new congeners, and two were previously characterized. C, NPL was screened for inhibition of protein translation initiation via a high-throughput bicistronic mRNA translation assay. Bioassay-guided dereplication of the hits led to the identification of actiphenol and cycloheximide as potent inhibitors, importantly, from the same Streptomyces strain, shedding new insights into their biosynthesis. D, pure natural product collection of the NPL was subjected to a high-content image-based screening to discover inhibitors of Wolbachia via a Drosophila infection model, resulting in the identification of kirromycin as a potent and specific inhibitor of Wolbachia. E, partially-purified fraction collection of the NPL was subjected to a high-content image-based screen to search for inhibitors of Cryptosporidium via a JW18 infection model, leading to the discovery of the herbicidins as a promising scaffold for anti-Cryptosporidium drug development.