Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)­catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C­(sp2)–C­(sp3) cross-link between the (hetero)­aromatic ring to Cβ. In addition to 3-CyFEs, we functionally validated an Fe­(II)/α-ketoglutarate-dependent hydroxylase, resulting in β-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.

The rise of antibiotic resistance (ABR) and the drying up of the pipeline for the development of new antibiotics demands an urgent search for new antibiotic leads. While the majority of clinically available antibiotics were discovered from terrestrial Streptomyces, related species from marine sediments as a source of antibiotics remain underexplored. Here, we utilized culture-dependent isolation of thirty-five marine sediment-derived actinobacterial isolates followed by a screening of their antibacterial activity against multidrug-resistant S. aureus ATCC BAA-44. Our results revealed that the crude extract of Streptomyces griseorubens strain DSD069 isolated from marine sediments collected in Romblon, Philippines displays the highest antibacterial activity, with 96.4% growth inhibition. The S. aureus ATCC BAA-44 cells treated with crude extract of Streptomyces griseorubens strain DSD069 showed cell membrane damage as demonstrated by (a) leakage and loss of vital cell constituents, including DNA and proteins, (b) irregular shrinkage of cells, and (c) increase membrane permeability. The antibiotic compounds were identified as Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone with MIC value of 6.25 µg/mL and 50.00 µg/mL, respectively. Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone are shunt metabolites in the biosynthesis of anticancer anthracycline derivatives namely doxorubicin, daunorubicin, and cinerubins. It is rare, however, that shunt metabolites are accumulated during fermentation of marine sediment-derived Streptomyces strain without genetic modification. Thus, our study provides evidence that natural bacterial strain can produce Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone as antibiotic leads to combat ABR.

The Philippine archipelago is geographically positioned in the tropics with rich areas of marine biodiversity. Its marine sediments harbor actinomycetes that exhibit antibacterial activity. Screening of actinomycetes isolated from marine sediments collected near the coast of Islas de Gigantes, Iloilo showed one isolate that exhibited high activity against the multidrug-resistant Staphylococcus aureus (MRSA) strain carrying the Staphylococcal Cassette Chromosome mec (SCCmec) type 1 gene, a biomarker for drug resistance. The isolate was identified as Streptomyces sp. strain DSD011 based on its 16s rRNA and protein-coding genes (atpD, recA, rpoB, and trpB) sequences, and was found to be a new species of salt-tolerant marine Streptomyces. Further, the strain harbors both non-ribosomal peptide synthetase (NRPS) and type II polyketide synthase (PKS) in its genome. The targeted chromatographic isolation and chemical investigations by Liquid Chromatography Mass Spectrometry-Time of Flight (LCMS-TOF), tandem mass spectrometry (MS/MS), and Global Natural Product Social molecular networking (GNPS) of the antibiotics produced by the strain, afforded the two polycyclic aromatic polyketide angucycline glycosides, fridamycin A (1) and fridamycin D (2), which are products of type II PKS biosynthesis. Compounds 1 and 2 displayed antibacterial activity against MRSA with minimum inhibitory concentration(MIC) of 500 μg/mL and 62.5 μg/mL, respectively. These results suggest that the underexplored marine sediments near the coast of Islas de Gigantes, Iloilo offer access to undiscovered Streptomyces species that are invaluable sources of antibiotic leads.

Honey exhibits antibacterial and antioxidant activities that are ascribed to its diverse secondary metabolites. In the Philippines, the antibacterial and antioxidant activities, as well as the bioactive metabolite contents of the honey, have not been thoroughly described. In this report, we investigated the in vitro antibacterial and antioxidant activities of honey from Apis mellifera and Tetragonula biroi, identified the compound responsible for the antibacterial activity, and compared the observed bioactivities and metabolite profiles to that of Manuka honey, which is recognized for its antibacterial and antioxidant properties. The secondary metabolite contents of honey were extracted using a nonionic polymeric resin followed by antibacterial and antioxidant assays, and then spectroscopic analyses of the phenolic and flavonoid contents. Results showed that honey extracts produced by T. biroi exhibits antibiotic activity against Staphylococcal pathogens as well as high antioxidant activity, which are correlated to its high flavonoid and phenolic content as compared to honey produced by A. mellifera. The bioassay-guided fractionation paired with Liquid Chromatography Mass Spectrometry (LCMS) and tandem MS analyses found the presence of the flavonoid isorhamnetin (3-methylquercetin) in T. biroi honey extract, which was demonstrated as one of the compounds with inhibitory activity against multidrug-resistant Staphylococcus aureus ATCC BAA-44. Our findings suggest that Philippine honey produced by T. biroi is a potential nutraceutical that possesses antibiotic and antioxidant activities.

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.