Bradley S. Evans

Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean

Ocean Methane Is methylphosphonate a natural product? If it is, then its catabolism by microrganisms seeking phosphorus might explain the ubiquity of methane in the oceans. Anaerobic Archaea, so far, are the only significant source of methane known. Thus, Metcalf et al. (p. 1104) looked for evidence of methylphosphonate biochemistry in the widespread archaeon Nitrosopumilus maritimus. The organism can synthesize C-P bonds using phosphoenolpyruvate mutase. By using its gene, ppm, as a marker, a possible phosphonate biosynthesis gene cluster was identified, and adjacent to this a gene for a cupin was found. Together with Fe(II) and O2 the cupin catalyzed the production of methylphosphonate and formate. Homologs of the cupin gene are found in a wide range of common marine microbes, including Pelagibacter ubique and Prochlorococcus. The archaeon Nitrosopumilus maritimus makes what may be a major source of ocean methane. Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic data sets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox.

Discovery of phosphonic acid natural products by mining the genomes of 10,000 actinomycetes

Significance The discovery of natural products, an important source of human medicines, is critical for the development of new therapeutics against health threats, including cancer and multidrug-resistant pathogens. Yet, in recent years, industrial development of pharmaceuticals from natural products has been stymied due to a variety of reasons, including the repeated discovery of previously known compounds. Here, we demonstrate large-scale genomics as one potential solution to this problem by mining a collection of 10,000 actinomycetes for novel phosphonic acids, an important class of natural products with antimicrobial, antiviral, antimalarial, and herbicidal activities. The framework described here provides a foundation for rapid, large-scale discovery of other classes of natural products and their use as lead compounds in the pharmaceutical industry. Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed “genome mining” as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the discovery of a new archetypical pathway for phosphonate biosynthesis, the first (to our knowledge) dedicated pathway for H-phosphinates, and 11 previously undescribed phosphonic acid natural products. Among these compounds are argolaphos, a broad-spectrum antibacterial phosphonopeptide composed of aminomethylphosphonate in peptide linkage to a rare amino acid N5-hydroxyarginine; valinophos, an N-acetyl l-Val ester of 2,3-dihydroxypropylphosphonate; and phosphonocystoximate, an unusual thiohydroximate-containing molecule representing a new chemotype of sulfur-containing phosphonate natural products. Analysis of the genome sequences from the remaining strains suggests that the majority of the phosphonate biosynthetic repertoire of Actinobacteria has been captured at the gene level. This dereplicated strain collection now provides a reservoir of numerous, as yet undiscovered, phosphonate natural products.

Structure Determination and Interception of Biosynthetic Intermediates for the Plantazolicin Class of Highly Discriminating Antibiotics

The soil-dwelling, plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42 is a prolific producer of complex natural products. Recently, a new FZB42 metabolite, plantazolicin (PZN), has been described as a member of the growing thiazole/oxazole-modified microcin (TOMM) family. TOMMs are biosynthesized from inactive, ribosomal peptides and undergo a series of cyclodehydrations, dehydrogenations, and other modifications to become bioactive natural products. Using high-resolution mass spectrometry, chemoselective modification, genetic interruptions, and other spectroscopic tools, we have determined the molecular structure of PZN. In addition to two conjugated polyazole moieties, the amino-terminus of PZN has been modified to N(α),N(α)-dimethylarginine. PZN exhibited a highly selective antibiotic activity toward Bacillus anthracis, but no other tested human pathogen. By altering oxygenation levels during fermentation, PZN analogues were produced that bear variability in their heterocycle content, which yielded insight into the order of biosynthetic events. Lastly, genome-mining has revealed the existence of four additional PZN-like biosynthetic gene clusters. Given their structural uniqueness and intriguing antimicrobial specificity, the PZN class of antibiotics may hold pharmacological value.

A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis

Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto-NMR, proteomics, and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein (RLP) from Rhodospirillum rubrum. Our studies unravelled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine (SAM)-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

Proteomics-Based Discovery of Koranimine, a Cyclic-Imine Natural Product

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzymes responsible for the biosynthesis of medically and ecologically important secondary metabolites. In a previous report, we described a proteomics approach to screen for expressed NRPSs or PKSs from bacteria with or without sequenced genomes. Here we used this proteome mining approach to discover a novel natural product arising from rare adenylation (A) and reductase (Red) domains in its biosynthetic machinery. We also cloned the entire gene cluster and elucidated the biosynthesis of the new compound, which is produced by an unsequenced Bacillus sp. isolated from soil collected in Koran, Louisiana.

Surveys of non-ribosomal peptide and polyketide assembly lines in fungi and prospects for their analysis in vitro and in vivo.

With many bioactive non-ribosomal peptides and polyketides produced in fungi, studies of their biosyntheses are an active area of research. Practical limitations of working with mega-dalton synthetases including cell lysis and protein extraction to recombinant gene and pathway expression has slowed understanding of many secondary metabolic processes relative to bacterial counterparts. Recent advances in accessing fungal biosynthetic machinery are beginning to change this. Here we describe the successes of some studies of thiotemplate biosynthesis in fungal systems, along with very recent advances in chemical tagging and mass spectrometric strategies to selectively study biosynthetic conveyer belts in isolation, and within a few years, in endogenous fungal proteomes.

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

ABSTRACT Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

Mechanistic investigation of methylphosphonate synthase, a non-heme iron-dependent oxygenase.

Methylphosphonate synthase is a non-heme iron-dependent oxygenase that converts 2-hydroxyethylphosphonate (2-HEP) to methylphosphonate. On the basis of experiments with two enantiomers of a substrate analog, 2-hydroxypropylphosphonate, catalysis is proposed to commence with stereospecific abstraction of the pro-S hydrogen on C2 of the substrate. Experiments with isotopologues of 2-HEP indicate stereospecific hydrogen transfer of the pro-R hydrogen at C2 of the substrate to the methyl group of methylphosphonate. Kinetic studies with these substrate isotopologues reveal that neither hydrogen transfer is rate limiting under saturating substrate conditions. A mechanism is proposed that is consistent with the available data.

In Vitro Characterization of a Heterologously Expressed Nonribosomal Peptide Synthetase Involved in Phosphinothricin Tripeptide Biosynthesis

The late stages of biosynthesis of phosphinothricin tripeptide (PTT) involve peptide formation and methylation on phosphorus. The exact timing of these transformations is not known. To provide insight into this question, we developed a heterologous expression system for PhsA, one of three NRPS proteins in PTT biosynthesis. The apparent kcat/Km value for ATP−pyrophosphate exchange activity for d,l-N-acetylphosphinothricin was 3.5 μM−1 min−1, whereas the kcat/Km,app for l-N-acetyldemethylphosphinothricin was 0.5 μM−1 min−1, suggesting the former might be the physiological substrate. Each substrate could be loaded onto the phosphopantetheine arm of the thiolation domain as observed by Fourier transform mass spectrometry (FTMS).

Discovery of the antibiotic phosacetamycin via a new mass spectrometry-based method for phosphonic acid detection.

Naturally occurring phosphonates such as phosphinothricin (Glufosinate, a commercially used herbicide) and fosfomycin (Monurol, a clinically used antibiotic) have proved to be potent and useful biocides. Yet this class of natural products is still an under explored family of secondary metabolites. Discovery of the biosynthetic pathways responsible for the production of these compounds has been simplified by using gene based screening approaches, but detection and identification of the natural products the genes produce have been hampered by a lack of high-throughput methods for screening potential producers under various culture conditions. Here, we present an efficient mass-spectrometric method for the selective detection of natural products containing phosphonate and phosphinate functional groups. We have used this method to identify a new phosphonate metabolite, phosacetamycin, whose structure, biological activity, and biosynthetic gene cluster are reported.