Discovery of a lagriamide polyketide by integrated genome mining, isotopic labeling, and untargeted metabolomics.

Microorganisms from the order Burkholderiales have been the source of a number of important classes of natural products in recent years. For example, study of the beetle-associated symbiont Burkholderia gladioli led to the discovery of the antifungal polyketide lagriamide; an important molecule from the perspectives of both biotechnology and chemical ecology. As part of a wider project to sequence Burkholderiales genomes from our in-house Burkholderiales library we identified a strain containing a biosynthetic gene cluster (BGC) similar to the original lagriamide BGC. Structure prediction failed to identify any candidate masses for the products of this BGC from untargeted metabolomics mass spectrometry data. However, genome mining from publicly available databases identified fragments of this BGC from a culture collection strain of Paraburkholderia. Whole genome sequencing of this strain revealed the presence of a homologue of this BGC with very high sequence identity. Stable isotope feeding of the two strains in parallel using our newly developed IsoAnalyst platform identified the product of this lagriamide-like BGC directly from the crude fermentation extracts, affording a culturable supply of this interesting compound class. Using a combination of bioinformatic, computational and spectroscopic methods we defined the absolute configurations for all 11 chiral centers in this new metabolite, which we named lagriamide B. Biological testing of lagriamide B against a panel of 21 bacterial and fungal pathogens revealed antifungal activity against the opportunistic human pathogen Aspergillus niger, while image-based Cell Painting analysis indicated that lagriamide B also causes actin filament disruption in U2-OS osteosarcoma cells.

The Natural Products Atlas 2.0: a database of microbially-derived natural products.

Within the natural products field there is an increasing emphasis on the study of compounds from microbial sources. This has been fuelled by interest in the central role that microorganisms play in mediating both interspecies interactions and host-microbe relationships. To support the study of natural products chemistry produced by microorganisms we released the Natural Products Atlas, a database of known microbial natural products structures, in 2019. This paper reports the release of a new version of the database which includes a full RESTful application programming interface (API), a new website framework, and an expanded database that includes 8128 new compounds, bringing the total to 32 552. In addition to these structural and content changes we have added full taxonomic descriptions for all microbial taxa and have added chemical ontology terms from both NP Classifier and ClassyFire. We have also performed manual curation to review all entries with incomplete configurational assignments and have integrated data from external resources, including CyanoMetDB. Finally, we have improved the user experience by updating the Overview dashboard and creating a dashboard for taxonomic origin. The database can be accessed via the new interactive website at https://www.npatlas.org.

Custom Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometric Database for Identification of Environmental Isolates of the Genus Burkholderia and Related Genera.

Success of discovery programs for microbial natural products is dependent on quick and concise discrimination between isolates from diverse environments. However, laboratory isolation and identification of priority genera using current 16S rRNA PCR-based methods are both challenging and time-consuming. An emerging strategy for rapid isolate discrimination is protein fingerprinting via matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Using our in-house environmental isolate repository, we have created a main spectral (MSP) library for the Bruker Biotyper MALDI mass spectrometer that contains 95 entries, including Burkholderia, Caballeronia, Paraburkholderia, and other environmentally related genera. The library creation required the acquisition of over 2,250 mass spectra, which were manually reviewed for quality control and consolidated into a single reference library using a commercial software platform. We tested the effectiveness of the reference library by analyzing 49 environmental isolate strains using two different sample preparation methods. Overall, this approach correctly identified all strains to the genus level provided that suitable reference spectra were present in the MSP library. In this study, we present a fast, accurate method for taxonomic assignment of environmentally derived bacteria from the order Burkholderiales, providing a valuable alternative to traditional PCR-based methods. The MSP library described in the manuscript is available for use.IMPORTANCE The Gram-negative proteobacterial order Burkholderiales has emerged as a promising source of novel natural products in recent years. This order includes the genus Burkholderia and the newly defined genera Caballeronia and Paraburkholderia However, development of this resource has been hampered by difficulties with rapid and selective isolation of Burkholderiales strains from the environment. Environmental metagenome sequencing has revealed that the potential for natural products is not evenly distributed throughout the microbial world. Thus, large but targeted microbial isolate libraries are needed to effectively explore the chemical potential of natural products. To study these organisms efficiently, methods to quickly identify isolates to the genus level are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is already used in clinical settings to reliably identify unknown bacterial pathogens. We have adapted similar methodology using the MALDI Biotyper instrument to rapidly identify environmental isolates of Burkholderia, Caballeronia, and Paraburkholderia for downstream natural product discovery.