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Antibacterial resistance poses a significant global threat, necessitating the discovery of new therapeutic agents. Plants are a valuable source of secondary metabolites with demonstrated anticancer and antibacterial properties. In this study, we reveal that Melastoma dodecandrum exhibits both bacteriostatic and bactericidal effects against Pseudomonas aeruginosa and Staphylococcus aureus. Treatment with plant extracts results in membrane damage and a reduction in Pseudomonas swimming and swarming motility. A comparative analysis of bacterial transcriptomes exposed to Melastoma extracts and four distinct antibiotics indicates that the extracts trigger similar transcriptomic responses as triclosan, a fatty acid inhibitor. Activity-guided fractionation suggests that the antibacterial activity is not attributable to hydrolyzable tannins, but to unidentified minor compounds. Additionally, we identified 104 specialized metabolic pathways and demonstrated a high level of transcriptional coordination between these biosynthetic pathways and phytohormones, highlighting potential regulatory mechanisms in plant metabolism.

Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 Ascomycetes fungi isolated from St. John's Island, Singapore were investigated for their general biosynthetic potential and their ability to produce antimicrobial secondary metabolites (SMs). All the 30 fungal isolates belong to the Phylum Ascomycota and are distributed into 6 orders and 18 genera with Order Hypocreales having the highest number of representative (37%). Screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes using degenerate PCR led to the identification of 23 polyketide synthases (PKSs) and 5 nonribosomal peptide synthetases (NRPSs) grouped into nine distinct clades based on their reduction capabilities. Some of the identified PKSs genes share high similarities between species and known reference genes, suggesting the possibility of conserved biosynthesis of closely related compounds from different fungi. Fungal extracts were tested for their antimicrobial activity against S. aureus, Methicillin-resistant S. aureus (MRSA), and Candida albicans. Bioassay-guided fractionation of the active constituents from two promising isolates resulted in the isolation of seven compounds: Penilumamides A, D, and E from strain F4335 and xanthomegnin, viomellein, pretrichodermamide C and vioxanthin from strain F7180. Vioxanthin exhibited the best antibacterial activity with IC50 values of 3.0 μM and 1.6 μM against S. aureus and MRSA respectively. Viomellein revealed weak antiproliferative activity against A549 cells with an IC50 of 42 μM. The results from this study give valuable insights into the diversity and biosynthetic potential of fungi from this unique habitat and forms a background for an in-depth analysis of the biosynthetic capability of selected strains of interest with the aim of discovering novel fungal natural products. Agency for Science, Technology and Research (A*STAR) Published version This research was funded by the Singapore Institute of Food and Biotechnology Innovation, Agency for Science, Technology and Research (A*STAR), Singapore.

Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound. Ministry of Education (MOE) Nanyang Technological University Published version I.J. is supported by Nanyang Biologics, M.M. is supported by a NTU Start‐Up Grant and Singaporean Ministry of Education grant MOE2018‐T2‐2‐053. J.M.D (NTU‐PPF‐2019). Y.K. is supported by Natural Product Research Laboratory Biomedical Research Council of A*STAR (Agency for Science, Technology and Research) Transition Fund (H16/99/b0/004), the Singapore Institute of Food and Biotechnology Innovation core fund.

With the advent of rapid automatedin silicoidentification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers,Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumventedviaintensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-richStreptomycesgenomes whose lengths range from 7.1 to 12.1 Mb at an average N50 of 5.9 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as NP chassis. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.Graphic abstractSchematic of hybrid long- and short read assembly workflow for genome sequencing of GC-richStreptomyces. Boxes shaded blue and grey correspond to experimental andin silicoworkflows, respectively.HighlightsA cost-effective genome sequencing approach for GC-richStreptomycesis presentedHybrid assembly improves BGC annotation and identificationA new species,Streptomyces sydneybrenneri, identified by taxonomic analysisGenomes of 8Streptomycesspecies are reported and analysed in this study

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112’s type V-A Cas (FnCpf1).

Background Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. Results Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. Conclusions We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene. Electronic supplementary material The online version of this article (10.1186/s12864-019-5762-6) contains supplementary material, which is available to authorized users.

Sortase A (SrtA) is a membrane-associated enzyme that anchors surface-exposed proteins to the cell wall envelope of Gram-positive bacteria such as Staphylococcus aureus. As SrtA is essential for Gram-positive bacterial pathogenesis but dispensable for microbial growth or viability, SrtA is considered a favorable target for the enhancement of novel anti-infective drugs that aim to interfere with key bacterial virulence mechanisms, such as biofilm formation, without developing drug resistance. Here, we used virtual screening to search an in-house natural compound library and identified two natural compounds, N1287 (Skyrin) and N2576 ((4,5-dichloro-1H-pyrrol-2-yl)-[2,4-dihydroxy-3-(4-methyl-pentyl)-phenyl]-methanone) that inhibited the enzymatic activity of SrtA. These compounds also significantly reduced the growth of S. aureus but possessed moderate mammalian toxicity. Furthermore, S. aureus strains treated with these compounds exhibited reduction in adherence to host fibrinogen, as well as biofilm formation. Hence, these compounds may represent an anti-infective therapy without the side effects of antibiotics.

Sortase A (SrtA) is a membrane-associated enzyme that anchors surface-exposed proteins to the cell wall envelope of Gram-positive bacteria such as Staphylococcus aureus. As SrtA is essential for Gram-positive bacterial pathogenesis but dispensable for microbial growth or viability, SrtA is considered a favorable target for the enhancement of novel anti-infective drugs that aim to interfere with key bacterial virulence mechanisms, such as biofilm formation, without developing drug resistance. Here, we used virtual screening to search an in-house natural compound library and identified two natural compounds, N1287 (Skyrin) and N2576 ((4,5-dichloro-1H-pyrrol-2-yl)-[2,4-dihydroxy-3-(4-methyl-pentyl)-phenyl]-methanone) that inhibited the enzymatic activity of SrtA. These compounds also significantly reduced the growth of S. aureus but possessed moderate mammalian toxicity. Furthermore, S. aureus strains treated with these compounds exhibited reduction in adherence to host fibrinogen, as well as biofilm formation. Hence, these compounds may represent an anti-infective therapy without the side effects of antibiotics.

Notonesomycin A is a 32-membered bioactive glycosylated macrolactone known to be produced by Streptomyces aminophilus subsp. notonesogenes 647-AV1 and S. aminophilus DSM 40186. In a high throughput antifungal screening campaign, we identified an alternative notonesomycin A producing strain, Streptomyces sp. A793, and its biosynthetic gene cluster. From this strain, we further characterized a new more potent antifungal non-sulfated analogue, named notonesomycin B. Through CRISPR-Cas9 engineering of the biosynthetic gene cluster, we were able to increase the production yield of notonesomycin B by up to 18-fold as well as generate a strain that exclusively produces this analogue. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This work was supported by the Agency for Science, Technology and Research (A*STAR), Singapore. WLY, EH, MMZ, FTW are supported by National Research Foundation, Singapore [NRF2013-THE001-094].

Modern medicine is unthinkable without antibiotics; yet, growing issues with microbial drug resistance require intensified search for new active compounds. Natural products generated by Actinobacteria have been a rich source of candidate antibiotics, for example anthracimycin that, so far, is only known to be produced by Streptomyces species. Based on sequence similarity with the respective biosynthetic cluster, we sifted through available microbial genome data with the goal to find alternative anthracimycin-producing organisms. In this work, we report about the prediction and experimental verification of the production of anthracimycin derivatives by Nocardiopsis kunsanensis, a non-Streptomyces actinobacterial microorganism. We discovered N. kunsanensis to predominantly produce a new anthracimycin derivative with methyl group at C-8 and none at C-2, labeled anthracimycin BII-2619, besides a minor amount of anthracimycin. It displays activity against Gram-positive bacteria with similar low level of mammalian cytotoxicity as that of anthracimycin.

Streptomyces are a genus of Actinobacteria capable of producing structurally diverse natural products. Here we report the isolation and characterization of a biosynthetically talented Streptomyces (Streptomyces sp. SD85) from tropical mangrove sediments. Whole-genome sequencing revealed that Streptomyces sp. SD85 harbors at least 52 biosynthetic gene clusters (BGCs), which constitute 21.2% of the 8.6-Mb genome. When cultivated under lab conditions, Streptomyces sp. SD85 produces sceliphrolactam, a 26-membered polyene macrolactam with unknown biosynthetic origin. Genome mining yielded a putative sceliphrolactam BGC (sce) that encodes a type I modular polyketide synthase (PKS) system, several β-amino acid starter biosynthetic enzymes, transporters, and transcriptional regulators. Using the CRISPR/Cas9–based gene knockout method, we demonstrated that the sce BGC is essential for sceliphrolactam biosynthesis. Unexpectedly, the PKS system encoded by sce is short of one module required for assembling the 26-membered macrolactam skeleton according to the collinearity rule. With experimental data disfavoring the involvement of a trans-PKS module, the biosynthesis of sceliphrolactam seems to be best rationalized by invoking a mechanism whereby the PKS system employs an iterative module to catalyze two successive chain extensions with different outcomes. The potential violation of the collinearity rule makes the mechanism distinct from those of other polyene macrolactams.

Background & Aims Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. Methods We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. Results Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non–small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation–related metabolites to generate ATP. Conclusions In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.

SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as “crown-like” structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.

Transcription factors are important drivers of cancer but the development of therapeutics against these factors has had limited success. We developed a stringent high-throughput chemical genetic screening platform to identify compounds that target oncogenic transcription factor SALL4 dependency in liver cancer. The platform comprises SALL4 low- and high-expressing endogenous and engineered isogenic liver cancer cell lines. Unexpectedly, from screening 21,575 natural product extracts, the top hits were four oxidative phosphorylation inhibitors that selectively reduced SALL4-dependent cell viability. The ATP synthase inhibitor oligomycin suppressed SALL4-expressing cancer in lung and liver cancer cell culture models, and in patient-derived xenograft models of liver cancer. Oligomycin also synergized with sorafenib, the standard-of-care targeted therapy in liver cancer, to effectively suppress SALL4-driven tumorigenesis in vivo. When aberrantly expressed in cancer, SALL4 binds ~50% of mitochondrial genes, including many oxidative phosphorylation genes, to predominantly upregulate their expression. SALL4 upregulation also alters the levels of oxidative phosphorylation-related metabolites and functionally increases oxidative phosphorylation activity. Application of our endogenous/isogenic transcription factor-screening platform revealed a therapeutically actionable oxidative phosphorylation vulnerability in SALL4-expressing cancers. Citation Format: Justin L. Tan, Feng Li, Joanna Z. Yeo, Kol Jia Yong, Mahmoud A. Bassal, Guo Hao Ng, May Yin Lee, Chung Yan Leong, Hong Kee Tan, Chan-Shuo Wu, Bee Hui Liu, Hon Man Chan, Zi Hui Tan, Yun Shen Chan, Siyu Wang, Zhi Han Lim, Tan Boon Toh, Lissa Hooi, Kia Ngee Low, Siming Ma, Nikki R. Kong, Alicia J. Stein, Yue Wu, Matan T. Thangavelu, Atsushi Suzuki, Giridharan Periyasamy, John M. Asara, Yock Young Dan, Glenn K. Bonney, Edward K. Chow, Guo-Dong Lu, Huck Hui Ng, Yoganathan Kanagasundaram, Siew Bee Ng, Wai Leong Tam, Li Chai, Daniel G. Tenen. A high-throughput chemical genetic screen reveals SALL4-induced metabolic vulnerabilities in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1788.

Aberrant lipid accumulation is a hallmark of cancer known to contribute to its aggressiveness and malignancy. Emerging studies have demonstrated context-dependent changes in lipid metabolism during chemotherapy. However, there is little known regarding the mechanisms linking lipid metabolism to chemotherapy-induced cell fates. Here, we describe lipid accumulation in cells following antimitotic drug treatment. Cells arrested in mitosis, as well as cells that escaped mitotic arrest and underwent mitotic slippage, showed elevated cytoplasmic lipid droplets. Interestingly, we found that TOFA, a lipid biosynthesis inhibitor that targets acetyl-CoA carboxylase (ACC) and blocks lipid accumulation, promoted early slippage, reduced cellular stress and enhanced survival of antimitotic-treated cells. Our work previously revealed that cells that survive after mitotic slippage can become senescent and confer pro-tumourigenic effects through paracrine signalling. Modulating lipid biosynthesis in cells post slippage by TOFA amplified their inflammatory secretion profiles and accelerated the development of tumourigenic behaviour, particularly cell migration and invasion, in a paracrine-dependent manner. In contrast to TOFA, inhibition of lipid accumulation by C75, a drug targeting fatty acid synthase (FASN), significantly reduced the production of pro-tumourigenic factors and associated phenotypic effects. This suggests that discrete lipid biosynthesis pathways could contribute differentially to the regulation of pro-tumourigenic inflammation. The divergent effects of TOFA and C75 may be attributed to the opposing regulation of Malonyl-CoA, an intermediate in fatty acid synthesis that serves as a mediator of fatty acid oxidation. Taken together, our data reveal a previously unappreciated role for lipid accumulation in the cellular adaptation to antimitotic drug treatment. Targeting lipid biosynthesis in cells post slippage may reprogramme its secretory profile such that it not only negates tumour-promoting effects, but may also promote anti-tumour inflammation for clearance of post-slippage senescent cells.