Aline O. Santos

Biosurfactants are amphiphilic microbial metabolites with potential to reduce surface and interfacial tension and to promote emulsification. This study investigated the biosynthetic potential, emulsifying activity, and metabolomic profile of Bacillus subtilis subsp. subtilis INPA R296 cultivated in media supplemented with residual soybean oil. Phylogenomic analyses supported the taxonomic placement of the strain close to Bacillus subtilis subsp. subtilis 168 ^T . Genome mining predicted 11 biosynthetic gene clusters (BGCs), including NRPS clusters related to surfactin and fengycin biosynthesis. Synteny analyses indicated conservation of these clusters among phylogenetically related Bacillus strains. Functional assays showed that culture-medium composition influenced cell growth, emulsification indices, and emulsifying activity. UHPLC-HRMS/MS combined with molecular networking enabled the putative annotation of metabolites belonging to several chemical classes, including cyclic lipopeptides, N -acyl amino acids, and oxylipins. The main contribution of this study is the integrated evaluation of genomic biosynthetic potential, emulsifying activity, and putative metabolite annotations in strain INPA R296 cultivated with a low-cost agro-industrial residue. Because no biosurfactant yield, surface/interfacial tension, authentic-standard validation, or structural isolation was performed, the results demonstrate emulsifying potential and the occurrence of putatively annotated amphiphilic metabolites rather than confirmed biosurfactant production. These findings identify INPA R296 as a target for further investigation of amphiphilic metabolites with potential biotechnological applications.

Oosporein (OOS) is the major specialized metabolite produced by the fungus Arcopilus amazonicus, an endophyte from Paullinia cupana. Genes related to the biosynthesis of this molecule in A. amazonicus diverge from those found in Beauveria bassiana. Furthermore, high-resolution liquid chromatography coupled with mass spectrometry combined with molecular networking, revealed the production of analogous compounds with varying degrees of oxygenation. Based on these analytical data, the analogues were annotated as phoenicin diquinol (2), phoenicin quinol (4) and phoenicin (5). By combining mass spectrometry and genome mining, a comprehensive biosynthetic pathway is proposed for these compounds, including the feasibility of a novel putative biphenol oxidation pathway mediated by the monooxygenase gene AAm15, which is exclusive to A. amazonicus. Additionally, the production of OOS, 2, 4 and 5 by a single fungal species is reported for the first time.

Trichoderma is recognized as a prolific producer of nonribosomal peptides (NRPs) known as peptaibols, which have remarkable biological properties, such as antimicrobial and anticancer activities, as well as the ability to promote systemic resistance in plants against pathogens. In this study, the sequencing of 11-, 14- and 15-res peptaibols produced by a marine strain of Trichoderma isolated from the ascidian Botrylloides giganteus was performed via liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Identification, based on multilocus phylogeny, revealed that our isolate belongs to the species T. endophyticum, which has never been reported in marine environments. Through genome sequencing and genome mining, 53 biosynthetic gene clusters (BGCs) were identified as being related to bioactive natural products, including two NRP-synthetases: one responsible for the biosynthesis of 11- and 14-res peptaibols, and another for the biosynthesis of 15-res. Substrate prediction, based on phylogeny of the adenylation domains in combination with molecular networking, permitted extensive annotation of the mass spectra related to two new series of 15-res peptaibols, which are referred to herein as “endophytins”. The analyses of synteny revealed that the origin of the 15-module peptaibol synthetase is related to 18, 19 and 20-module peptaibol synthetases, and suggests that the loss of modules may be a mechanism used by Trichoderma species for peptaibol diversification. This study demonstrates the importance of combining genome mining techniques, mass spectrometry analysis and molecular networks for the discovery of new natural products.

Arcopilus is a genus recently proposed after the taxonomic restructuring of Chaetomium with only seven described species so far. During the characterization of the microbiome of the Amazonian guarana plant (Paullinia cupana var. sorbilis), nine red pigment-producing strains of fungi were isolated. These strains were identified through morphological characterization and phylogenetic analysis based on the internal transcribed spacer (ITS), 28S large subunit (LSU), partial second largest subunit of RNA polymerase II (RPB2) and β-tubulin (TUB2) regions. Moreover, chemical profiles were obtained via high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS), in which the isolated strains were able to produce significant amounts of the polyketide oosporein. The obtained results indicate the occurrence of a new fungal species, A. amazonicus. Chemical analyses showed that oosporein is overproduced by these strains under laboratory culture conditions.