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1. Bacterial Secondary Metabolite Biosynthetic Potential in Soil Varies with Phylum, Depth, and Vegetation Type

   https://doi.org/10.1128/mBio.00416-20  

   Sharrar, Allison M. ; Crits-Christoph, Alexander ; Méheust, Raphaël ; Diamond, Spencer ; Starr, Evan P. ; Banfield, Jillian F. ( June 2020 , mBio)

   Davies, Julian E. (Ed.)

   ABSTRACT Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils and whether the genetic potential for production of these metabolites differs with soil depth and vegetation type within a geographic region. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with various vegetation and bedrock characteristics and reconstructed 1,334 metagenome-assembled genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria , Chloroflexi , and candidate phylum “ Candidatus Dormibacteraeota.” Surprisingly, one genome of a candidate phyla radiation (CPR) bacterium coded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy. IMPORTANCE Microbes produce specializedmore »compounds to compete or communicate with one another and their environment. Some of these compounds, such as antibiotics, are also useful in medicine and biotechnology. Historically, most antibiotics have come from soil bacteria which can be isolated and grown in the lab. Though the vast majority of soil bacteria cannot be isolated, we can extract their genetic information and search it for genes which produce these specialized compounds. These understudied soil bacteria offer a wealth of potential for the discovery of new and important microbial products. Here, we identified the ability to produce these specialized compounds in diverse and novel bacteria in a range of soil environments. This information will be useful to other researchers who wish to isolate certain products. Beyond their use to humans, understanding the distribution and function of microbial products is key to understanding microbial communities and their effects on biogeochemical cycles.« less
2. The expanding spectrum of diketopiperazine natural product biosynthetic pathways containing cyclodipeptide synthases

   https://doi.org/10.1039/C8OB03063D  

   Borgman, Paul ; Lopez, Ryan D. ; Lane, Amy L. ( February 2019 , Organic & Biomolecular Chemistry)

   Microorganisms are remarkable chemists, with enzymes as their tools for executing multi-step syntheses to yield myriad natural products. Microbial synthetic aptitudes are illustrated by the structurally diverse 2,5-diketopiperazine (DKP) family of bioactive nonribosomal peptide natural products. Nonribosomal peptide synthetases (NRPSs) have long been recognized as catalysts for formation of DKP scaffolds from two amino acid substrates. Cyclodipeptide synthases (CDPSs) are more recently recognized catalysts of DKP assembly, employing two aminoacyl-tRNAs (aa-tRNAs) as substrates. CDPS-encoding genes are typically found in genomic neighbourhoods with genes encoding additional biosynthetic enzymes. These include oxidoreductases, cytochrome P450s, prenyltransferases, methyltransferases, and cyclases, which equip the DKP scaffold with groups that diversify chemical structures and confer biological activity. These tailoring enzymes have been characterized from nine CDPS-containing biosynthetic pathways to date, including four during the last year. In this review, we highlight these nine DKP pathways, emphasizing recently characterized tailoring reactions and connecting new developments to earlier findings. Featured pathways encompass a broad spectrum of chemistry, including the formation of challenging C–C and C–O bonds, regioselective methylation, a unique indole alkaloid DKP prenylation strategy, and unprecedented peptide-nucleobase bond formation. These CDPS-containing pathways also provide intriguing models of metabolic pathway evolution across related and divergent microorganisms, and openmore »doors to synthetic biology approaches for generation of DKP combinatorial libraries. Further, bioinformatics analyses support that much unique genetically encoded DKP tailoring potential remains unexplored, suggesting opportunities for further expansion of Nature's biosynthetic spectrum. Together, recent studies of DKP pathways demonstrate the chemical ingenuity of microorganisms, highlight the wealth of unique enzymology provided by bacterial biosynthetic pathways, and suggest an abundance of untapped biosynthetic potential for future exploration.« less
3. An updated catalogue of diverse type II polyketide synthase biosynthetic gene clusters captured from large-scale nucleotide databases

   https://doi.org/10.1099/mgen.0.000965  

   McBride, Christina M. ; Miller, Eric L. ; Charkoudian, Louise K. ( March 2023 , Microbial Genomics)

   Nature serves as a rich source of molecules with immense chemical diversity. Aptly named, these ‘natural products’ boast a wide variety of environmental, medicinal and industrial applications. Type II polyketides, in particular, confer substantial medicinal benefits, including antibacterial, antifungal, anticancer and anti-inflammatory properties. These molecules are produced by enzyme assemblies known as type II polyketide synthases (PKSs), which use domains such as the ketosynthase chain-length factor and acyl carrier protein to produce polyketides with varying lengths, cyclization patterns and oxidation states. In this work, we use a novel bioinformatic workflow to identify biosynthetic gene clusters (BGCs) that code for the core type II PKS enzymes. This method does not rely on annotation and thus was able to unearth previously ‘hidden’ type II PKS BGCs. This work led us to identify over 6000 putative type II PKS BGCs spanning a diverse set of microbial phyla, nearly double those found in most recent studies. Notably, many of these newly identified BGCs were found in non-actinobacteria, which are relatively underexplored as sources of type II polyketides. Results from this work lay an important foundation for future bioprospecting and engineering efforts that will enable sustainable access to diverse and structurally complex molecules with medicinallymore »relevant properties.« less
4. Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

   https://doi.org/https://doi.org/10.1104/pp.18.01195  

   Lucas Busta, Won Cheol ( December 2018 , Plant physiology)

   Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resultingmore »in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules. Many organisms implement specialized biochemical pathways to convert ubiquitous metabolites into bioactive chemical compounds. Since plants comprise the majority of the human diet, specialized plant metabolites play crucial roles not only in crop biology but also in human nutrition. Some asterids produce lipid compounds called polyacetylenes (for review, see Negri, 2015) that exhibit antifungal activity (Garrod et al., 1978; Kemp, 1978; Harding and Heale, 1980, 1981; Olsson and Svensson, 1996) and accumulate in response to fungal phytopathogen attack (De Wit and Kodde, 1981; Elgersma and Liem, 1989). These observations have led to the longstanding hypothesis that polyacetylenes are natural pesticides. These same lipid compounds exhibit cytotoxic activity against human cancer cell lines and slow tumor growth (Fujimoto and Satoh, 1988; Matsunaga et al., 1989, 1990; Cunsolo et al., 1993; Bernart et al., 1996; Kobaek-Larsen et al., 2005; Zidorn et al., 2005), making them important nutritional compounds. The major source of polyacetylenes in the human diet is carrot (Daucus carota L.). Carrot is one of the most important crop species in the Apiaceae, with rapidly increasing worldwide cultivation (Rubatzky et al., 1999; Dawid et al., 2015). The most common carrot polyacetylenes are C17 linear aliphatic compounds containing two conjugated carbon-carbon triple bonds, one or two carbon-carbon double bonds, and a diversity of additional in-chain oxygen-containing functional groups. In carrot, the most abundant of these compounds are falcarinol and falcarindiol (Dawid et al., 2015). Based on their structures, it has been hypothesized that these compounds (alias falcarin-type polyacetylenes) are derived from ubiquitous fatty acids. Indeed, biochemical investigations (Haigh et al., 1968; Bohlman, 1988), radio-chemical tracer studies (Barley et al., 1988), and the discovery of pathway intermediates (Jones et al., 1966; Kawazu et al., 1973) implicate a diversion of flux away from linolenate biosynthesis as the entry point into falcarin-type polyacetylene biosynthesis (for review, see Minto and Blacklock, 2008). The final steps of linolenate biosynthesis are the conversion of oleate to linoleate, mediated by fatty acid desaturase 2 (FAD2), and linoleate to linolenate, catalyzed by FAD3. Some plant species contain divergent forms of FAD2 that, instead of or in addition to converting oleate to linoleate, catalyze the installation of unusual in-chain functional groups such as hydroxyl groups, epoxy groups, conjugated double bonds, or carbon-carbon triple bonds into the acyl chain (Badami and Patil, 1980) and thus divert flux from linolenate production into the accumulation of unusual fatty acids. Previous work in parsley (Petroselinum crispum; Apiaceae) identified a divergent form of FAD2 that (1) was up-regulated in response to pathogen treatment and (2) when expressed in soybean embryos resulted in production of the monoyne crepenynate and, by the action of an unassigned enzyme, dehydrocrepenynate (Kirsch et al., 1997; Cahoon et al., 2003). The results of the parsley studies are consistent with a pathogen-responsive, divergent FAD2-mediated pathway that leads to acetylenic fatty acids. However, information regarding the branch point into acetylenic fatty acid production in agriculturally relevant carrot is still largely missing, in particular, the identification and functional characterization of enzymes that can divert carbon flux away from linolenate biosynthesis into the production of dehydrocrepenynate and ultimately falcarin-type polyacetylenes. Such genes, once identified, could be used in the future design of transgenic carrot lines with altered polyacetylene content, enabling direct testing of in planta polyacetylene function and potentially the engineering of pathogen-resistant, more nutritious carrots. These genes could also provide the foundation for further investigations of more basic aspects of plant biology, including the evolution of fatty acid-derived natural product biosynthesis pathways across the Asterid clade, as well as the role of these pathways and compounds in plant ecology and plant defense. Recently, a high-quality carrot genome assembly was released (Iorizzo et al., 2016), providing a foundation for genome-enabled studies of Apiaceous species. This study also provided publicly accessible RNA sequencing (RNA-Seq) data from diverse carrot tissues. Using these resources, this study aimed to provide a detailed gas chromatography-based quantification of polyacetylenes in carrot tissues for which RNA-Seq data are available, then combine this information with bioinformatics analysis and heterologous expression to identify and characterize biosynthetic genes that underlie the major entry point into carrot polyacetylene biosynthesis. To achieve these goals, thin-layer chromatography (TLC) was combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection to identify and quantify polyacetylenic metabolites in five different carrot tissues. Then the sequences and tissue expression profiles of potential FAD2 and FAD2-like genes annotated in the D. carota genome were compared with the metabolite data to identify candidate pathway genes, followed by biochemical functionality tests using yeast (Saccharomyces cerevisae) and Arabidopsis (Arabidopsis thaliana) as heterologous expression systems.« less
5. High Transcriptional Activity and Diverse Functional Repertoires of Hundreds of Giant Viruses in a Coastal Marine System

   https://doi.org/10.1128/mSystems.00293-21  

   Ha, Anh D. ; Moniruzzaman, Mohammad ; Aylward, Frank O. ; Etten, James Van ( January 2021 , mSystems)

   Bordenstein, Seth (Ed.)

   ABSTRACT Viruses belonging to the Nucleocytoviricota phylum are globally distributed and include members with notably large genomes and complex functional repertoires. Recent studies have shown that these viruses are particularly diverse and abundant in marine systems, but the magnitude of actively replicating Nucleocytoviricota present in ocean habitats remains unclear. In this study, we compiled a curated database of 2,431 Nucleocytoviricota genomes and used it to examine the gene expression of these viruses in a 2.5-day metatranscriptomic time-series from surface waters of the California Current. We identified 145 viral genomes with high levels of gene expression, including 90 Imitervirales and 49 Algavirales viruses. In addition to recovering high expression of core genes involved in information processing that are commonly expressed during viral infection, we also identified transcripts of diverse viral metabolic genes from pathways such as glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting that virus-mediated reprogramming of central carbon metabolism is common in oceanic surface waters. Surprisingly, we also identified viral transcripts with homology to actin, myosin, and kinesin domains, suggesting that viruses may use these gene products to manipulate host cytoskeletal dynamics during infection. We performed phylogenetic analysis on the virus-encoded myosin and kinesin proteins, which demonstratedmore »that most belong to deep-branching viral clades, but that others appear to have been acquired from eukaryotes more recently. Our results highlight a remarkable diversity of active Nucleocytoviricota in a coastal marine system and underscore the complex functional repertoires expressed by these viruses during infection. IMPORTANCE The discovery of giant viruses has transformed our understanding of viral complexity. Although viruses have traditionally been viewed as filterable infectious agents that lack metabolism, giant viruses can reach sizes rivalling cellular lineages and possess genomes encoding central metabolic processes. Recent studies have shown that giant viruses are widespread in aquatic systems, but the activity of these viruses and the extent to which they reprogram host physiology in situ remains unclear. Here, we show that numerous giant viruses consistently express central metabolic enzymes in a coastal marine system, including components of glycolysis, the TCA cycle, and other pathways involved in nutrient homeostasis. Moreover, we found expression of several viral-encoded actin, myosin, and kinesin genes, indicating viral manipulation of the host cytoskeleton during infection. Our study reveals a high activity of giant viruses in a coastal marine system and indicates they are a diverse and underappreciated component of microbial diversity in the ocean.« less