Tandem mass spectral metabolic profiling of 54 actinobacterial strains and their 459 mutants.

Natural products encompass a diverse range of compounds with high impact applications in consumer care, agriculture and most notably, therapeutics. However, despite the expansive chemical repertoire indicated in genomic information of microbes, only a small subset can be obtained under laboratory conditions. To increase accessible chemical space and realize Nature's full chemical potential, a multi-pronged genetic- and cultivation-based strategy has been employed to activate and upregulate natural product biosyntheses in native and heterologous strains. This data descriptor documents a characterized collection of 2,138 liquid chromatography-tandem mass spectrometry (LC/MS-MS) spectra of fermentation extracts from 54 native actinobacterial strains collected from soil and marine environments in Singapore, and their 459 activated mutants in 3 to 5 media. A total of 743 unique metabolites have been identified, with the activated mutants demonstrating an approximately 2-fold expansion in accessible chemical space over wild type strains. Interrogating this expanded chemical diversity with cheminformatic tools can provide direction for the discovery of novel natural products with desirable functional activity.

Reversing the directionality of reactions between non-oxidative pentose phosphate pathway and glycolytic pathway boosts mycosporine-like amino acid production in Saccharomyces cerevisiae.

BACKGROUND: Mycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production. RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells. CONCLUSION: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

Application of Cas12j for Streptomyces Editing.

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.

Exploring a general multi-pronged activation strategy for natural product discovery in Actinomycetes.

Natural products possess significant therapeutic potential but remain underutilized despite advances in genomics and bioinformatics. While there are approaches to activate and upregulate natural product biosynthesis in both native and heterologous microbial strains, a comprehensive strategy to elicit production of natural products as well as a generalizable and efficient method to interrogate diverse native strains collection, remains lacking. Here, we explore a flexible and robust integrase-mediated multi-pronged activation approach to reliably perturb and globally trigger antibiotics production in actinobacteria. Across 54 actinobacterial strains, our approach yielded 124 distinct activator-strain combinations which consistently outperform wild type. Our approach expands accessible metabolite space by nearly two-fold and increases selected metabolite yields by up to >200-fold, enabling discovery of Gram-negative bioactivity in tetramic acid analogs. We envision these findings as a gateway towards a more streamlined, accelerated, and scalable strategy to unlock the full potential of Nature's chemical repertoire.

Transcriptomic and metabolomic characterization of antibacterial activity of Melastoma dodecandrum.

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 P.aeruginosa swimming and swarming motility. A comparative analysis of bacterial transcriptomes exposed to M.dodecandrum extracts and four distinct antibiotics indicates that the extracts may trigger similar transcriptomic responses as triclosan, a fatty acid synthesis 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 of antibacterial metabolites in M.dodecandrum.

Natural Products from Singapore Soil-Derived Streptomycetaceae Family and Evaluation of Their Biological Activities.

Natural products have long been used as a source of antimicrobial agents against various microorganisms. Actinobacteria are a group of bacteria best known to produce a wide variety of bioactive secondary metabolites, including many antimicrobial agents. In this study, four actinobacterial strains found in Singapore terrestrial soil were investigated as potential sources of new antimicrobial compounds. Large-scale cultivation, chemical, and biological investigation led to the isolation of a previously undescribed tetronomycin A (1) that demonstrated inhibitory activities against both Gram-positive bacteria Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) (i.e., MIC90 of 2-4 µM and MBC90 of 9-12 µM), and several known antimicrobial compounds, namely nonactin, monactin, dinactin, 4E-deacetylchromomycin A3, chromomycin A2, soyasaponin II, lysolipin I, tetronomycin, and naphthomevalin. Tetronomycin showed a two- to six-fold increase in antibacterial activity (i.e., MIC90 and MBC90 of 1-2 µM) as compared to tetronomycin A (1), indicating the presence of an oxy-methyl group at the C-27 position is important for antibacterial activity.

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces hosts for natural product discovery.

With the advent of rapid automated in silico identification 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 circumvented via intensive 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-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 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 alternative NP hosts. 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.

ß-Cryptoxanthin Production in Escherichia coli by Optimization of the Cytochrome P450 CYP97H1 Activity.

Cytochromes P450, forming a superfamily of monooxygenases containing heme as a cofactor, show great versatility in substrate specificity. Metabolic engineering can take advantage of this feature to unlock novel metabolic pathways. However, the cytochromes P450 often show difficulty being expressed in a heterologous chassis. As a case study in the prokaryotic host Escherichia coli, the heterologous synthesis of ß-cryptoxanthin was addressed. This carotenoid intermediate is difficult to produce, as its synthesis requires a monoterminal hydroxylation of ß-carotene whereas most of the classic carotene hydroxylases are dihydroxylases. This study was focused on the optimization of the in vivo activity of CYP97H1, an original P450 ß-carotene monohydroxylase. Engineering the N-terminal part of CYP97H1, identifying the matching redox partners, defining the optimal cellular background and adjusting the culture and induction conditions improved the production by 400 times compared to that of the initial strain, representing 2.7 mg/L ß-cryptoxanthin and 20% of the total carotenoids produced.

Cytochrome P450 Surface Domains Prevent the ß-Carotene Monohydroxylase CYP97H1 of Euglena gracilis from Acting as a Dihydroxylase.

Molecular biodiversity results from branched metabolic pathways driven by enzymatic regioselectivities. An additional complexity occurs in metabolites with an internal structural symmetry, offering identical extremities to the enzymes. For example, in the terpene family, ß-carotene presents two identical terminal closed-ring structures. Theses cycles can be hydroxylated by cytochrome P450s from the CYP97 family. Two sequential hydroxylations lead first to the formation of monohydroxylated ß-cryptoxanthin and subsequently to that of dihydroxylated zeaxanthin. Among the CYP97 dihydroxylases, CYP97H1 from Euglena gracilis has been described as the only monohydroxylase. This study aims to determine which enzymatic domains are involved in this regioselectivity, conferring unique monohydroxylase activity on a substrate offering two identical sites for hydroxylation. We explored the effect of truncations, substitutions and domain swapping with other CYP97 members and found that CYP97H1 harbours a unique N-terminal globular domain. This CYP97H1 N-terminal domain harbours a hydrophobic patch at the entrance of the substrate channel, which is involved in the monohydroxylase activity of CYP97H1. This domain, at the surface of the enzyme, highlights the role of distal and non-catalytic domains in regulating enzyme specificity.

Antibacterial Spirotetronate Polyketides from an Actinomadura sp. Strain A30804.

Large scale cultivation and chemical investigation of an extract obtained from Actimonadura sp. resulted in the identification of six previously undescribed spirotetronates (pyrrolosporin B and decatromicins C-G; 7-12), along with six known congeners, namely decatromicins A-B (1-2), BE-45722B-D (3-5), and pyrrolosporin A (6). The chemical structures of compounds 1-12 were characterized via comparison with previously reported data and analysis of 1D/2D NMR and MS data. The structures of all new compounds were highly related to the spirotetronate type compounds, decatromicin and pyrrolosporin, with variations in the substituents on the pyrrole and aglycone moieties. All compounds were evaluated for antibacterial activity against the Gram-negative bacteria, Acinetobacter baumannii and Gram-positive bacteria, Staphylococcus aureus and were investigated for their cytotoxicity against the human cancer cell line A549. Of these, decatromicin B (2), BE-45722B (3), and pyrrolosporin B (7) exhibited potent antibacterial activities against both Gram-positive (MIC90 between 1-3 µM) and Gram-negative bacteria (MIC90 values ranging from 12-36 µM) with weak or no cytotoxic activity against A549 cells.

Chemical elicitation as an avenue for discovery of bioactive compounds from fungal endophytes.

The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of fungal endophytes obtained from the A*STAR Natural Organism Library (NOL) and previously isolated from Sungei Buloh Wetland Reserve, Singapore. Phylogenetic analysis based on ITS2 gene suggests that these isolates belong to 46 morphotypes and are affiliated to 23 different taxa in 17 genera of the Ascomycota phylum. Colletotrichum was the most dominant fungal genus accounting for 37% of all the isolates, followed by Diaporthe (13%), Phyllosticta (10.9%) and Diplodia (8.7%). Chemical elicitation using 5-azacytidine, a DNA methyltransferase inhibitor and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor resulted in an increase in the number of active strains. Bioassay-guided isolation and structural elucidation yielded pestahivin and two new analogues from Bartalinia sp. F9447. Pestahivin and its related analogues did not exhibit antibacterial activity against Staphylococcus aureus but displayed strong antifungal activities against Candida albicans and Aspergillus brasiliensis, with IC50 values ranging from 0.46 ± 0.06 to 144 ± 18 µM. Pestahivin and its two analogues furthermore exhibited cytotoxic activity against A549 and MIA PACA-2 cancer cell lines with IC50 values in the range of 0.65 ± 0.12 to 42 ± 5.2 µM. The finding from this study reinforces that chemical epigenetic induction is a promising approach for the discovery of bioactive fungal secondary metabolites encoded by cryptic gene clusters.

Diethyl phthalate (DEP) perturbs nitrogen metabolism in Saccharomyces cerevisiae.

Phthalates are ubiquitously used as plasticizers in various consumer care products. Diethyl phthalate (DEP), one of the main phthalates, elicits developmental and reproductive toxicities but the underlying mechanisms are not fully understood. Chemogenomic profiling of DEP in S. cerevisiae revealed that two transcription factors Stp1 and Dal81 involved in the Ssy1-Ptr5-Ssy5 (SPS) amino acid-sensing pathway provide resistance to DEP. Growth inhibition of yeast cells by DEP was stronger in poor nitrogen medium in comparison to nitrogen-rich medium. Addition of amino acids to nitrogen-poor medium suppressed DEP toxicity. Catabolism of amino acids via the Ehrlich pathway is required for suppressing DEP toxicity. Targeted metabolite analyses showed that DEP treatment alters the amino acid profile of yeast cells. We propose that DEP inhibits the growth of yeast cells by affecting nitrogen metabolism and discuss the implications of our findings on DEP-mediated toxic effects in humans.

Antibacterial Thiopeptide GE2270-Congeners from Nonomuraea jiangxiensis.

Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopeptides (1−9) were obtained from a Nonomuraea jiangxiensis isolated from a terrestrial soil sample collected in Singapore. Even though some of these compounds were previously synthesized or isolated from engineered strains, herein we report the unprecedented isolation of these thiopeptides from a native Nonomuraea jiangxiensis. A comparison with the literature and a detailed analysis of the NMR and HRMS of compounds 1−9 was conducted to assign their chemical structures. The structures of all new compounds were highly related to the thiopeptide antibiotics GE2270, with variations in the substituents on the thiazole and amino acid moieties. Thiopeptides 1−9 exhibited a potent antimicrobial activity against the Gram-positive bacteria, Staphylococcus aureus with MIC90 values ranging from 2 µM to 11 µM. In addition, all compounds were investigated for their cytotoxicity against the human cancer cell line A549, none of the compounds were cytotoxic.

Lipid accumulation facilitates mitotic slippage-induced adaptation to anti-mitotic drug treatment.

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.

Role of the serotonergic pathway in uterotonic activity of Ananas comosus (L.) Merr. - An in vitro and in vivo study.

BACKGROUND: Ananas comosus (L.) Merr. has been used as a traditional medicine in inducing abortion in many countries. Our previous in vitro experiments showed that the aqueous fraction (F4) of A. comosus extract stimulated the rat and human uterine contractions. PURPOSE: The aim of this study was to identify the bioactive compound and further investigate the molecular mechanism of F4 induced contraction and the in vivo uterotonic effect of F4. MATERIALS AND METHODS: Organ bath studies were employed to compare the stimulatory effect of F4 in non and late pregnant uterine tissue followed by isolation of protein from late pregnant uterine tissue for the western blot analysis. The PhysioTel transmitter was implanted in pregnant SD rats to measure the changes in intrauterine pressure (IUP). Analyses of the crude extract and active principle in F4 was performed using LC-HRMS. RESULTS: Ripe F4 in a similar manner as serotonin produced a greater stimulatory response in late pregnant than non-pregnant uterine tissue without significant change in potency; ripe F4 also increased ERK phosphorylation which eventually led to a significant increase of the final product, MMP-13. In pregnant rats (E18), oral ripe F4 (1.5 g.100 g-1 body weight) and ergometrine (1 mg) did not stimulate the uterine contraction probably due to the low level of estradiol and as a consequence low 5-HT receptors at the time of administration. In contrast, in postpartum rats, oral administration of F4 and ergometrine produced a significant increase in maximal IUP to 4.3 and 4.9 folds of basal IUP respectively. Contrary to the folklore use, unripe F4 did not stimulate the uterine activity during pregnancy and postpartum. Bioassay guided fractionation identified serotonin as a major bioactive compound in ripe F4. CONCLUSIONS: Our data clearly indicate that the uterotonic effect of ripe F4 is mediated via the serotonergic pathway and suggest that serotonin rich diet may increase the peripheral serotonin and implicate in diverse physiological functions, including uterine motility.

Potent tocolytic activity of ethyl acetate fraction of Ananas comosus on rat and human uteri.

The aim of this study was to investigate the tocolytic properties of Ananas comosus extract in rat and human uterine tissue in vitro and in the rat in vivo. Organ bath technique was employed to perform functional studies in vitro. The PhysioTel transmitter was implanted in SD rats to measure the changes in intrauterine pressure (IUP) in vivo. Analyses of F2 was performed using LC-HRMS. F2 produced a non-selective inhibitory response on oxytocin, prostaglandin F2α, acetylcholine and KCl. The inhibitory activity of F2 on oxytocin-induced contraction was not attenuated by propranolol, TEA, glibenclamide and indomethacin. Nω-Nitro-L-arginine, a nitric oxide synthase inhibitor, suppressed the maximal tocolytic activity of F2 by 25%. DIDS, an inhibitor of chloride channels, appeared to suppress the relaxant effect of F2. F2 suppressed the oxytocin-induced contraction in Ca2+ free solution. The in vivo tocolytic activity of F2 and ritodrine were observed in non-pregnant rats during the estrous stage by suppressing the frequency and amplitude of IUP peaks following intrauterine administration. Chemical analysis confirmed the involvement of citric acid in the tocolytic activity of F2. However, another less polar fraction is essential to accompany citric acid to produce such potent inhibitory response of F2. It is likely that F2 exerted tocolytic activity by multiple mechanisms, including antagonizing L-type Ca2+ channels, interfering with the intracellular Ca2+ release mechanism and releasing nitric oxide. F2 would be a promising candidate to develop as a tocolytic agent.

Isolation and Identification of an Anthracimycin Analogue from Nocardiopsis kunsanensis, a Halophile from a Saltern, by Genomic Mining Strategy.

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.

Pyrrole carboxamidine tryptase inhibitors from Leptonychia pubescens.

The root and stem bark extracts of a Nigerian sample of Leptonychia pubescens Keay (Sterculiaceae) were found to inhibit the serine protease tryptase, a potential therapeutic target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Bioassay-guided isolation led to the identification of 1-beta-ribofuranosylbrunfelsamidine as the active component with a tryptase IC (50) of 3 microM. Brunfelsamidine was also isolated, but was only weakly active.

Identification of chelerythrine as an inhibitor of BclXL function.

The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins.