Genome Mining Strategy Research Articles

Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy.

Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 μg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.

Discovery of Prenyltransferase-Guided Hydroxyphenylacetic Acid Derivatives from Marine Fungus Penicillium sp. W21C371.

Traditional isolation methods often lead to the rediscovery of known natural products. In contrast, genome mining strategies are considered effective for the continual discovery of new natural products. In this study, we discovered a unique prenyltransferase (PT) through genome mining, capable of catalyzing the transfer of a prenyl group to an aromatic nucleus to form C-C or C-O bonds. A pair of new hydroxyphenylacetic acid derivative enantiomers with prenyl units, (±)-peniprenydiol A (1), along with 16 known compounds (2-17), were isolated from a marine fungus, Penicillium sp. W21C371. The separation of 1 using chiral HPLC led to the isolation of the enantiomers 1a and 1b. Their structures were established on the basis of extensive spectroscopic analysis, including 1D, 2D NMR and HRESIMS. The absolute configurations of the new compounds were determined by a modified Mosher method. A plausible biosynthetic pathway for 1 was deduced, facilitated by PT catalysis. In the in vitro assay, 2 and 3 showed promising inhibitory activity against Escherichia coli β-glucuronidase (EcGUS), with IC50 values of 44.60 ± 0.84 μM and 21.60 ± 0.76 μM, respectively, compared to the positive control, D-saccharic acid 1,4-lactone hydrate (DSL). This study demonstrates the advantages of genome mining in the rational acquisition of new natural products.

Genomics- and Transcriptomics-Guided Discovery of Clavatols from Arctic Fungi Penicillium sp. MYA5.

Clavatols exhibit a wide range of biological activities due to their diverse structures. A genome mining strategy identified an A5cla cluster from Penicillium sp. MYA5, derived from the Arctic plant Dryas octopetala, is responsible for clavatol biosynthesis. Seven clavatols, including one new clavatol derivate named penicophenone F (1) and six known clavatols (2-7), were isolated from Penicillium sp. MYA5 using a transcriptome mining strategy. These structures were elucidated by comprehensive spectroscopic analysis. Antibacterial, aldose reductase inhibition, and siderophore-producing ability assays were conducted on compounds 1-7. Compounds 1 and 2 demonstrated inhibitory effects on the ALR2 enzyme with inhibition rates of 75.3% and 71.6% at a concentration of 10 μM, respectively. Compound 6 exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli with MIC values of 4.0 μg/mL and 4.0 μg/mL, respectively. Additionally, compounds 1, 5, and 6 also showed potential iron-binding ability.

Metabolic Blockade-Based Genome Mining of Sea Anemone-Associated Streptomyces sp. S1502 Identifies Atypical Angucyclines WS-5995 A-E: Isolation, Identification, Biosynthetic Investigation, and Bioactivities.

Marine symbiotic and epiphyte microorganisms are sources of bioactive or structurally novel natural products. Metabolic blockade-based genome mining has been proven to be an effective strategy to accelerate the discovery of natural products from both terrestrial and marine microorganisms. Here, the metabolic blockade-based genome mining strategy was applied to the discovery of other metabolites in a sea anemone-associated Streptomyces sp. S1502. We constructed a mutant Streptomyces sp. S1502/Δstp1 that switched to producing the atypical angucyclines WS-5995 A-E, among which WS-5995 E is a new compound. A biosynthetic gene cluster (wsm) of the angucyclines was identified through gene knock-out and heterologous expression studies. The biosynthetic pathways of WS-5995 A-E were proposed, the roles of some tailoring and regulatory genes were investigated, and the biological activities of WS-5995 A-E were evaluated. WS-5995 A has significant anti-Eimeria tenell activity with an IC50 value of 2.21 μM. The production of antibacterial streptopyrroles and anticoccidial WS-5995 A-E may play a protective role in the mutual relationship between Streptomyces sp. S1502 and its host.

A myo-inositol dehydrogenase involved in aminocyclitol biosynthesis of hygromycin A.

Hygromycin A is a broad-spectrum antibiotic that contains a furanose, cinnamic acid, and aminocyclitol moieties. The biosynthesis of the aminocyclitol has been proposed to proceed through six enzymatic steps from glucose 6-phosphate through myo-inositol to the final methylenedioxy-containing aminocyclitol. Although there is some in vivo evidence for this proposed pathway, biochemical support for the individual enzyme activities is lacking. In this study, we verify the activity for one enzyme in this pathway. We show that Hyg17 is a myo-inositol dehydrogenase that has a unique substrate scope when compared to other myo-inositol dehydrogenases. Furthermore, we analyze sequences from the protein family containing Hyg17 and discuss genome mining strategies that target this protein family to identify biosynthetic clusters for natural product discovery.

Complete degradation of PET waste using a thermophilic microbe-enzyme system

Enzymatic degradation has been proposed as a suitable solution for addressing PET pollution, but approximately 10 % of PET is left as nonbiodegradable. Microbes can completely degrade PET at the gram level per year. Based on the complementary benefits of microbes and enzymes, a microbe-enzyme system was created to completely degrade PET. Here, a thermophilic microbe-enzyme (TME) system composed of Bacillus thermoamylovorans JQ3 and leaf-branch compost cutinase variant (ICCG) was used to demonstrate the synergistic degradation of PET, enabling 100 % degradation of PET waste at a high PET loading level (360 g/L). Six endogenous PET hydrolases of strain JQ3 were discovered by employing an ester bond hydrolysis function-first genome mining (EGM) strategy and first successfully expressed in E. coli. These hydrolases could release TPA as the final product from PET and preferentially degraded BHET instead of MHET. Of these, carboxylesterase 39_5 and ICCG could degrade PET in a synergistic manner to generate 50 μM of TPA, which was greater than the sum of the individual treatments. Finally, the degradation pathway of the TME system was speculated to include biofilm formation, PET degradation and utilization. The successful implementation of this study rendered a scale-up degradation feasible of PET at a lower cost.

Bacterial Cytochrome P450 Catalyzed Post‐translational Macrocyclization of Ribosomal Peptides**

AbstractRibosomally synthesized and post‐translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450‐catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule‐based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co‐occurrence and co‐conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450‐catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp‐Trp‐Tyr (one C−C and two C−N bonds), Tyr‐Trp (C−C bond), Trp‐Trp (C−N bond), and His‐His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non‐native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.

Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Here, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence, co-conservation, and interactions between precursors and P450s, successfully facilitating the identification of diverse P450 catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can respectively catalyze Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s in expanding the chemical diversity of strained macrocyclic peptides and enriching biocatalytic tools for peptide macrocyclization.

Targeted genome mining for microbial antitumor agents acting through DNA intercalation

Microbial natural products have been one of the most important sources for drug development. In the current postgenomic era, sequence-driven approaches for natural product discovery are becoming increasingly popular. Here, we develop an effective genome mining strategy for the targeted discovery of microbial metabolites with antitumor activities. Our method employs uvrA-like genes as genetic markers, which have been identified in the biosynthetic gene clusters (BGCs) of several chemotherapeutic drugs of microbial origin and confer self-resistance to the corresponding producers. Through systematic genomic analysis of gifted actinobacteria genera, identification of uvrA-like gene-containing BGCs, and targeted isolation of products from a BGC prioritized for metabolic analysis, we identified a new tetracycline-type DNA intercalator timmycins. Our results thus provide a new genome mining strategy for the efficient discovery of antitumor agents acting through DNA intercalation.

Synthesis of biologically active compounds derived from natural products

Natural products and their structural analogues have historically made a major contribution to pharmacotherapy, especially for cancer and infectious diseases. Nevertheless, natural products also present challenges for drug discovery, such as technical barriers to screening, isolation, characterization and optimization, which contributed to a decline in their pursuit by the pharmaceutical industry from the 1990s onwards. In recent years, several technological and scientific developments including improved analytical tools, genome mining and engineering strategies, and microbial culturing advances are addressing such challenges and opening up new opportunities. Consequently, interest in natural products as drug leads is being revitalized, particularly for tackling antimicrobial resistance. Here, we summarize recent technological developments that are enabling natural product-based drug discovery, highlight selected applications and discuss key opportunities.The first total synthesis and development of a variety of bioactive natural products have been accomplished by using carbohydrates as a chiral source. In addition, practically useful intermediates have been created, analogs of natural products have been prepared, their structure-activity relationships studied, and the large-scale preparations of medicinally useful compounds established.

Discovery of new microbial natural products by genome mining

Microbial natural products are an important source of new drug creation. As more and more microbial whole genomes are sequenced, bioinformatics analysis shows that the natural prod-uct resources contained therein are greatly underestimated. Genome mining is a new strategy for natural product discovery guided by gene cluster sequences. At the same time, it can di-rectly associate the structure of natural products with synthesis pathways, which is conven-ient for biosynthesis and combinatorial biosynthesis research. This paper reviews the repre-sentative examples of successful discovery of novel natural products using genome mining strategy in recent years, which fully shows the great advantage of this strategy in exploring the potential of microbial natural metabolites. It is believed that with the continuous devel-opment and improvement of genome mining methods and technologies, it will be possible to realize the in-depth development of microbial natural product resources.This paper describes the application of combinatorial biosynthesis in new drugs, points out the existing problems and puts forward some feasible suggestions.

Somalactams A-D: Anti-inflammatory Macrolide Lactams with Unique Ring Systems from an Arctic Actinomycete Strain.

Four new PKS-NRPS-derived macrolide lactams with three unique ring fusion types were discovered from the Arctic sponge associated actinomycete Streptomyces somaliensis 1107 using a genome mining strategy. Their structures were elucidated by a combination of MS, NMR spectroscopic analysis, and single-crystal X-ray diffraction. Biosynthetically, a novel gene cluster sml consisting of three polyketide synthases and one hybrid polyketide synthase-nonribosomal peptide synthetase together with cytochrome P450s and flavin-containing monooxygenases and oxidoreductases was demonstrated to assemble the unique skeleton. Pharmacological studies revealed that compound 1 displayed a potent anti-inflammatory effect without cytotoxicity. It inhibited IL-6 and TNF-α release in the serum of LPS-stimulated RAW264.7 macrophage cells with IC50 values of 5.76 and 0.18 μM, respectively, and modulated the MAPK pathway. Moreover, compound 1 alleviated LPS-induced systemic inflammation in our transgenic fluorescent zebrafish model.

Somalactams A–D: Anti‐inflammatory Macrolide Lactams with Unique Ring Systems from an Arctic Actinomycete Strain

AbstractFour new PKS‐NRPS‐derived macrolide lactams with three unique ring fusion types were discovered from the Arctic sponge associated actinomycete Streptomyces somaliensis 1107 using a genome mining strategy. Their structures were elucidated by a combination of MS, NMR spectroscopic analysis, and single‐crystal X‐ray diffraction. Biosynthetically, a novel gene cluster sml consisting of three polyketide synthases and one hybrid polyketide synthase‐nonribosomal peptide synthetase together with cytochrome P450s and flavin‐containing monooxygenases and oxidoreductases was demonstrated to assemble the unique skeleton. Pharmacological studies revealed that compound 1 displayed a potent anti‐inflammatory effect without cytotoxicity. It inhibited IL‐6 and TNF‐α release in the serum of LPS‐stimulated RAW264.7 macrophage cells with IC50 values of 5.76 and 0.18 μM, respectively, and modulated the MAPK pathway. Moreover, compound 1 alleviated LPS‐induced systemic inflammation in our transgenic fluorescent zebrafish model.

MALDI Imaging Assisted Discovery of a Di-O-glycosyltransferase from Platycodon grandiflorum Root.

A matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di-O-glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two β-1,6-linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP-glucose is the preferred sugar donor for PgGT1, it could also utilize UDP-xylose and UDP-N-acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.

MALDI Imaging Assisted Discovery of a Di‐O‐glycosyltransferase from Platycodon grandiflorum Root

AbstractA matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di‐O‐glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two β‐1,6‐linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP‐glucose is the preferred sugar donor for PgGT1, it could also utilize UDP‐xylose and UDP‐N‐acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.

Catalytic Site Proximity Profiling for Functional Unification of Sequence-Diverse Radical S-Adenosylmethionine Enzymes.

The radical S-adenosylmethionine (rSAM) superfamily has become a wellspring for discovering new enzyme chemistry, especially regarding ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report a compendium of nearly 15,000 rSAM proteins with high-confidence involvement in RiPP biosynthesis. While recent bioinformatics advances have unveiled the broad sequence space covered by rSAM proteins, the significant challenge of functional annotation remains unsolved. Through a combination of sequence analysis and protein structural predictions, we identified a set of catalytic site proximity residues with functional predictive power, especially among the diverse rSAM proteins that form sulfur-to-α carbon thioether (sactionine) linkages. As a case study, we report that an rSAM protein from Streptomyces sparsogenes (StsB) shares higher full-length similarity with MftC (mycofactocin biosynthesis) than any other characterized enzyme. However, a comparative analysis of StsB to known rSAM proteins using "catalytic site proximity" predicted that StsB would be distinct from MftC and instead form sactionine bonds. The prediction was confirmed by mass spectrometry, targeted mutagenesis, and chemical degradation. We further used "catalytic site proximity" analysis to identify six new sactipeptide groups undetectable by traditional genome-mining strategies. Additional catalytic site proximity profiling of cyclophane-forming rSAM proteins suggests that this approach will be more broadly applicable and enhance, if not outright correct, protein functional predictions based on traditional genomic enzymology principles.

Development of Marker Recycling Systems for Sequential Genetic Manipulation in Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7.

Marine-derived fungi are emerging as prolific workhorses of structurally novel natural products (NPs) with diverse bioactivities. However, the limitation of available selection markers hampers the exploration of cryptic NPs. Recyclable markers are therefore valuable assets in genetic engineering programs for awaking silent SM clusters. Here, both pyrG and amdS-based recyclable marker cassettes were established and successfully applied in marine-derived fungi Aspergillus sp. SCSIO SX7S7 and Spiromastix sp. SCSIO F190, respectively. Using pyrG recyclable marker, a markerless 7S7-∆depH strain with a simplified HPLC background was built by inactivating a polyketide synthase (PKS) gene depH and looping out the pyrG recyclable marker after depH deletion. Meanwhile, an amdS recyclable marker system was also developed to help strains that are difficult to use pyrG marker. By employing the amdS marker, a backbone gene spm11 responsible for one major product of Spiromastix sp. SCSIO F190 was inactivated, and the amdS marker was excised after using, generating a relatively clean F190-∆spm11 strain for further activation of novel NPs. The collection of two different recycle markers will guarantee flexible application in marine-derived fungi with different genetic backgrounds, enabling the exploitation of novel structures in various fungi species with different genome mining strategies.

Mammalian Commensal Streptococci Utilize a Rare Family of Class VI Lanthipeptide Synthetases to Synthesize Miniature Lanthipeptide-type Ribosomal Peptide Natural Products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products with remarkable chemical and functional diversities. These peptides are often synthesized as signals or antibiotics and frequently associated with quorum sensing (QS) systems. With the increasing number of available genomes, many hitherto unseen RiPP biosynthetic pathways have been mined, providing new resources for novel bioactive compounds. Herein, we investigated the underexplored biosynthetic potential of Streptococci, prevalent bacteria in mammal-microbiomes that include pathogenic, mutualistic, and commensal members. Using the transcription factor-centric genome mining strategy, we discovered a new family of lanthipeptide biosynthetic loci under the control of potential QS. By in vitro studies, we investigated the reaction of one of these lanthipeptide synthetases and found that it installs only one lanthionine moiety onto its short precursor peptide by connecting a conserved TxxC region. Bioinformatics and in vitro studies revealed that these lanthipeptide synthetases (class VI) are novel lanthipeptide synthetases with a truncated lyase, a kinase, and a truncated cyclase domain. Our data provide important insights into the processing and evolution of lanthipeptide synthetase to tailor smaller substrates. The data are important for obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of lanthipeptides.

Biosynthesis of selenium-containing small molecules in diverse microorganisms

Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids,via selenocysteine and 2-selenouridine, respectively1. However, despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.

A Silent Biosynthetic Gene Cluster from a Methanotrophic Bacterium Potentiates Discovery of a Substrate Promiscuous Proteusin Cyclodehydratase.

Natural product-encoding biosynthetic gene clusters (BGCs) within microbial genomes far outnumber the known natural products; chemical products from such BGCs remain cryptic. These silent BGCs hold promise not only for the elaboration of new natural products but also for the discovery of useful biosynthetic enzymes. Here, we describe a genome mining strategy targeted toward the discovery of substrate promiscuous natural product biosynthetic enzymes. In the genome of the methanotrophic bacterium Methylovulum psychrotolerans Sph1T, we discover a transcriptionally silent natural product BGC that encoded numerous ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These cryptic RiPP natural products were accessed using heterologous expression of the substrate peptide and biosynthetic enzyme-encoded genes. In line with our genome mining strategy, the RiPP biosynthetic enzymes in this BGC were found to be substrate promiscuous, which allowed us to use them in a combinatorial fashion with a similarly substrate-tolerant cyanobactin biosynthetic enzyme to introduce head-to-tail macrocyclization in the proteusin family of RiPP natural products.