Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides.

Thiopeptides are natural antibiotics that are fashioned from short peptides by multiple layers of post-translational modification. Their biosynthesis, in particular the pyridine synthases that form the macrocyclic antibiotic core, has attracted intensive research but is complicated by the challenges of reconstituting multiple-pathway enzymes. By combining select RiPP enzymes with cell free expression and flexizyme-based codon reprogramming, we have developed a benchtop biosynthesis of thiopeptide scaffolds. This strategy side-steps several challenges related to the investigation of thiopeptide enzymes and allows access to analytical quantities of new thiopeptide analogs. We further demonstrate that this strategy can be used to validate the activity of new pyridine synthases without the need to reconstitute the cognate prior pathway enzymes.

Correction to: Fungal Secretome Analysis Via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis.

In the article "Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis", acknowledgement of financial support was inadvertently omitted. The authors apologize for this oversight.

Exploring bioactive peptides from bacterial secretomes using PepSAVI-MS: identification and characterization of Bac-21 from Enterococcus faecalis pPD1.

As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI-MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI-MS for the discovery of bacterial-sourced bioactive peptides through identification of the bacteriocin Bac-21 from Enterococcus faecalis pPD1. Minor pipeline modifications including implementation of bacteria-infused agar diffusion assays and optional digestion of peptide libraries highlight the versatility and wide adaptability of the PepSAVI-MS pipeline. Additionally, we have experimentally validated the primary protein sequence of the active, mature Bac-21 peptide for the first time and have confirmed its identity with respect to primary sequence and post-translational processing. Successful application of PepSAVI-MS to bacterial secretomes as demonstrated herein establishes proof-of-principle for use in novel microbial bioactive peptide discovery.

Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis.

Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety. Graphical Abstract.

Linking Natural Oil Seeps from the Gulf of Mexico to Their Origin by Use of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

We report chemical characterization of natural oil seeps from the Gulf of Mexico by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and Gas Chromatography/Atmospheric Pressure Chemical Ionization Mass Spectrometry (GC/APCI-MS), to highlight how FT-ICR MS can also be employed as a means to determine petroleum connectivity, in addition to traditional GC/MS techniques. The source of petroleum is the Green Canyon (GC) 600 lease block in the Gulf of Mexico. Within GC600, two natural oil seepage zones, Mega Plume and Birthday Candles, continuously release hydrocarbons and develop persistent oil slicks at the sea surface above them. We chemically trace the petroleum from the surface oil slicks to the Mega Plume seep itself, and further to a petroleum reservoir 5 km away in lease block GC645 (Holstein Reservoir). We establish the connectivity between oil samples and confirm a common geological origin for the oil slicks, oil seep, and reservoir oil. The ratios of seven common petroleum biomarkers detected by GC/APCI-MS display clear similarity between the GC600 and GC645 samples, as well as a distinct difference from another reservoir oil collected ∼300 km away (Macondo crude oil from MC252 lease block). FT-ICR MS and principal component analysis (PCA) demonstrate further similarities between the GC600 and GC645 samples that distinctly differ from MC252. A common geographical origin is postulated for the GC600/GC645 samples, with petroleum migrating from the GC645 reservoir to the oil seeps found in GC600 and up through the water column to the sea surface as an oil slick.