Phosphonoalamides Reveal the Biosynthetic Origin of Phosphonoalanine Natural Products and a Convergent Pathway for Their Diversification.

Phosphonate natural products, with their potent inhibitory activity, have found widespread use across multiple industries. Their success has inspired development of genome mining approaches that continue to reveal previously unknown bioactive scaffolds and biosynthetic insights. However, a greater understanding of phosphonate metabolism is required to enable prediction of compounds and their bioactivities from sequence information alone.  Here, we expand our knowledge of this natural product class by reporting the complete biosynthesis of the phosphonoalamides, antimicrobial tripeptides with a conserved N-terminal l-phosphonoalanine (PnAla) residue produced by Streptomyces. The phosphonoalamides result from the convergence of PnAla biosynthesis and peptide ligation pathways. We elucidate the biochemistry underlying the transamination of phosphonopyruvate to PnAla, a new early branchpoint in phosphonate biosynthesis catalyzed by an aminotransferase with evolved specificity for phosphonate metabolism. Peptide formation is catalyzed by two ATP-grasp ligases, the first of which produces dipeptides, and a second which ligates dipeptides to PnAla to produce phosphonoalamides. Substrate specificity profiling revealed a dramatic expansion of dipeptide and tripeptide products, while finding PnaC to be the most promiscuous dipeptide ligase reported thus far. Our findings highlight previously unknown transformations in natural product biosynthesis, promising enzyme biocatalysts, and unveil insights into the diversity of phosphonopeptide natural products.

A Novel Pathway for Biosynthesis of the Herbicidal Phosphonate Natural Product Phosphonothrixin Is Widespread in Actinobacteria.

Phosphonothrixin is an herbicidal phosphonate natural product with an unusual, branched carbon skeleton. Bioinformatic analyses of the ftx gene cluster, which is responsible for synthesis of the compound, suggest that early steps of the biosynthetic pathway, up to production of the intermediate 2,3-dihydroxypropylphosphonic acid (DHPPA) are identical to those of the unrelated phosphonate natural product valinophos. This conclusion was strongly supported by the observation of biosynthetic intermediates from the shared pathway in spent media from two phosphonothrixin producing strains. Biochemical characterization of ftx-encoded proteins confirmed these early steps, as well as subsequent steps involving the oxidation of DHPPA to 3-hydroxy-2-oxopropylphosphonate and its conversion to phosphonothrixin by the combined action of an unusual heterodimeric, thiamine-pyrophosphate (TPP)-dependent ketotransferase and a TPP-dependent acetolactate synthase. The frequent observation of ftx-like gene clusters within actinobacteria suggests that production of compounds related to phosphonothrixin is common within these bacteria. IMPORTANCE Phosphonic acid natural products, such as phosphonothrixin, have great potential for biomedical and agricultural applications; however, discovery and development of these compounds requires detailed knowledge of the metabolism involved in their biosynthesis. The studies reported here reveal the biochemical pathway phosphonothrixin production, which enhances our ability to design strains that overproduce this potentially useful herbicide. This knowledge also improves our ability to predict the products of related biosynthetic gene clusters and the functions of homologous enzymes.

Discovery of Antimicrobial Phosphonopeptide Natural Products from Bacillus velezensis by Genome Mining.

Phosphonate natural products are renowned for inhibitory activities which underly their development as antibiotics and pesticides. Although most phosphonate natural products have been isolated from Streptomyces, bioinformatic surveys suggest that many other bacterial genera are replete with similar biosynthetic potential. While mining actinobacterial genomes, we encountered a contaminated Mycobacteroides data set which included a biosynthetic gene cluster predicted to produce novel phosphonate compounds. Sequence deconvolution revealed that the contig containing this cluster, as well as many others, belonged to a contaminating Bacillus and is broadly conserved among multiple species, including the epiphyte Bacillus velezensis. Isolation and structure elucidation revealed a new di- and tripeptide composed of l-alanine and a C-terminal l-phosphonoalanine which we name phosphonoalamides E and F. These compounds exhibit broad-spectrum antibacterial activity, including strong inhibition against the agricultural pests responsible for vegetable soft rot (Erwinia rhapontici), onion rot (Pantoea ananatis), and American foulbrood (Paenibacillus larvae). This work expands our knowledge of phosphonate metabolism and underscores the importance of including underexplored microbial taxa in natural product discovery. IMPORTANCE Phosphonate natural products produced by bacteria have been a rich source of clinical antibiotics and commercial pesticides. Here, we describe the discovery of two new phosphonopeptides produced by B. velezensis with antibacterial activity against human and plant pathogens, including those responsible for widespread soft rot in crops and American foulbrood. Our results provide new insight on the natural chemical diversity of phosphonates and suggest that these compounds could be developed as effective antibiotics for use in medicine or agriculture.

Biosynthesis of Argolaphos Illuminates the Unusual Biochemical Origins of Aminomethylphosphonate and Nε-Hydroxyarginine Containing Natural Products.

Phosphonate natural products have a history of successful application in medicine and biotechnology due to their ability to inhibit essential cellular pathways. This has inspired efforts to discover phosphonate natural products by prioritizing microbial strains whose genomes encode uncharacterized biosynthetic gene clusters (BGCs). Thus, success in genome mining is dependent on establishing the fundamental principles underlying the biosynthesis of inhibitory chemical moieties to facilitate accurate prediction of BGCs and the bioactivities of their products. Here, we report the complete biosynthetic pathway for the argolaphos phosphonopeptides. We uncovered the biochemical origins of aminomethylphosphonate (AMPn) and Nε-hydroxyarginine, two noncanonical amino acids integral to the antimicrobial function of argolaphos. Critical to this pathway were dehydrogenase and transaminase enzymes dedicated to the conversion of hydroxymethylphosphonate to AMPn. The interconnected activities of both enzymes provided a solution to overcome unfavorable energetics, empower cofactor regeneration, and mediate intermediate toxicity during these transformations. Sequential ligation of l-arginine and l-valine was afforded by two GCN5-related N-acetyltransferases in a tRNA-dependent manner. AglA was revealed to be an unusual heme-dependent monooxygenase that hydroxylated the Nε position of AMPn-Arg. As the first biochemically characterized member of the YqcI/YcgG protein family, AglA enlightens the potential functions of this elusive group, which remains biochemically distinct from the well-established P450 monooxygenases. The widespread distribution of AMPn and YqcI/YcgG genes among actinobacterial genomes suggests their involvement in diverse metabolic pathways and cellular functions. Our findings illuminate new paradigms in natural product biosynthesis and realize a significant trove of AmPn and Nε-hydroxyarginine natural products that await discovery.

Valinophos Reveals a New Route in Microbial Phosphonate Biosynthesis That Is Broadly Conserved in Nature.

Phosphonate natural products are potent inhibitors of cellular metabolism with an established record of commercialization in medicine and biotechnology. Although genome mining has emerged as an accelerated method for the discovery of new phosphonates, a robust framework of their metabolism is needed to identify the pathways most likely to yield compounds with desired activities. Here we expand our understanding of these natural products by reporting the complete biosynthetic pathway for valinophos, a phosphonopeptide natural product containing the unusual (R)-2,3-dihydroxypropylphosphonate (DHPPA) scaffold. The pathway was defined by several enzymatic transformations and intermediates previously unknown to phosphonate natural products. A dedicated dehydrogenase served as a new phosphoenolpyruvate mutase coupling enzyme. Notably, its reduction of phosphonopyruvate to phosphonolactate defined a new early branchpoint in phosphonate biosynthesis. Functionally interconnected kinase and reductase enzymes catalyzed reactions reminiscent of glycolysis and arginine biosynthesis to produce a transient, but essential, phosphonolactaldehyde intermediate. We demonstrate esterification of l-valine onto DHPPA as a new biochemical activity for ATP-Grasp ligase enzymes. Unexpectedly, a second amino acid ligase then adjoined additional amino acids at the valinyl moiety to produce a suite of DHPPA-dipeptides. The genes for DHPPA biosynthesis were discovered among genomes of bacteria from wide-ranging habitats, suggesting a wealth of unknown compounds that may originate from this core pathway. Our findings establish new biosynthetic principles for natural products and provide definition to unexplored avenues for bioactive phosphonate genome mining.

Fosmidomycin biosynthesis diverges from related phosphonate natural products.

Fosmidomycin and related molecules comprise a family of phosphonate natural products with potent antibacterial, antimalarial and herbicidal activities. To understand the biosynthesis of these compounds, we characterized the fosmidomycin producer, Streptomyces lavendulae, using biochemical and genetic approaches. We were unable to elicit production of fosmidomycin, instead observing the unsaturated derivative dehydrofosmidomycin, which we showed potently inhibits 1-deoxy-D-xylulose-5-phosphate reductoisomerase and has bioactivity against a number of bacteria. The genes required for dehydrofosmidomycin biosynthesis were established by heterologous expression experiments. Bioinformatics analyses, characterization of intermediates and in vitro biochemistry show that the biosynthetic pathway involves conversion of a two-carbon phosphonate precursor into the unsaturated three-carbon product via a highly unusual rearrangement reaction, catalyzed by the 2-oxoglutarate dependent dioxygenase DfmD. The required genes and biosynthetic pathway for dehydrofosmidomycin differ substantially from that of the related natural product FR-900098, suggesting that the ability to produce these bioactive molecules arose via convergent evolution.

PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in phosphonocystoximic acid biosynthesis in Streptomyces.

Several oxime-containing small molecules have useful properties, including antimicrobial, insecticidal, anticancer, and immunosuppressive activities. Phosphonocystoximate and its hydroxylated congener, hydroxyphosphonocystoximate, are recently discovered oxime-containing natural products produced by Streptomyces sp. NRRL S-481 and Streptomyces regensis NRRL WC-3744, respectively. The biosynthetic pathways for these two compounds are proposed to diverge at an early step in which 2-aminoethylphosphonate (2AEPn) is converted to (S)-1-hydroxy-2-aminoethylphosphonate ((S)-1H2AEPn) in S. regensis but not in Streptomyces sp. NRRL S-481). Subsequent installation of the oxime moiety into either 2AEPn or (S)-1H2AEPn is predicted to be catalyzed by PcxL or HpxL from Streptomyces sp. NRRL S-481 and S. regensis NRRL WC-3744, respectively, whose sequence and predicted structural characteristics suggest they are unusual N-oxidases. Here, we show that recombinant PcxL and HpxL catalyze the FAD- and NADPH-dependent oxidation of 2AEPn and 1H2AEPn, producing a mixture of the respective aldoximes and nitrosylated phosphonic acid products. Measurements of catalytic efficiency indicated that PcxL has almost an equal preference for 2AEPn and (R)-1H2AEPn. 2AEPn was turned over at a 10-fold higher rate than (R)-1H2AEPn under saturating conditions, resulting in a similar but slightly lower kcat/Km We observed that (S)-1H2AEPn is a relatively poor substrate for PcxL but is clearly the preferred substrate for HpxL, consistent with the proposed biosynthetic pathway in S. regensis. HpxL also used both 2AEPn and (R)-1H2AEPn, with the latter inhibiting HpxL at high concentrations. Bioinformatic analysis indicated that PcxL and HpxL are members of a new class of oxime-forming N-oxidases that are broadly dispersed among bacteria.

Ent-homocyclopiamine B, a Prenylated Indole Alkaloid of Biogenetic Interest from the Endophytic Fungus Penicillium concentricum.

Ent-homocyclopiamine B (1), a new prenylated indole alkaloid bearing an alicyclic nitro group along with 2-methylbutane-1,2,4-triol (2) were isolated from an endophytic fungus Penicillium concentricum of the liverwort Trichocolea tomentella (Trichocoleaceae). The structure of 1 was elucidated through extensive spectroscopic analyses and comparison with data reported for a structurally related nitro-bearing Penicillium metabolite, clopiamine C (3), which contain an indolizidine ring instead of the quinolizine ring in 1. The new compound, ent-homocyclopiamine B, exhibited slight growth inhibition against Gram-positive bacteria. Based on the reported biosynthesis of related compounds and the isolation of the mevalonic acid derived compound 2-methyl-1,2,4-butanetriol (2), we proposed that ent-homocylopiamine B (1) was biosynthesized from lysine and prenyl group-producing mevalonic pathway.

New Kid on the Block: LmbU Expands the Repertoire of Specialized Metabolic Regulators in Streptomyces.

Streptomyces has an extensive natural product repertoire, including most of the naturally derived antibiotics. Understanding the control of natural product biosynthesis is central to antibiotic discovery and production optimization. Here, Hou et al. (J. Bacteriol. 200:00447-17, 2018, https://doi.org/10.1128/JB.00447-17) report the identification and characterization of a novel regulator-LmbU-that functions primarily as an activator of lincomycin production in Streptomyces lincolnensis Importantly, members of this new regulator family are associated with natural product biosynthetic clusters throughout the streptomycetes and their actinomycete relatives.

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

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 N(5)-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.

Phylogenetic relationships in the family Streptomycetaceae using multi-locus sequence analysis.

The family Streptomycetaceae, notably species in the genus Streptomyces, have long been the subject of investigation due to their well-known ability to produce secondary metabolites. The emergence of drug resistant pathogens and the relative ease of producing genome sequences has renewed the importance of Streptomyces as producers of new natural products and resulted in revived efforts in isolating and describing strains from novel environments. A previous large study of the phylogeny in the Streptomycetaceae based on 16S rRNA gene sequences provided a useful framework for the relationships among species, but did not always have sufficient resolution to provide definitive identification. Multi-locus sequence analysis of 5 house-keeping genes has been shown to provide improved taxonomic resolution of Streptomyces species in a number of previous reports so a comprehensive study was undertaken to evaluate evolutionary relationships among species within the family Streptomycetaceae where type strains are available in the ARS Culture Collection or genome sequences are available in GenBank. The results of the analysis supported the distinctiveness of Kitasatospora and Streptacidiphilus as validly named genera since they cluster outside of the phylogenetic radiation of the genus Streptomyces. There is also support for the transfer of a number of Streptomyces species to the genus Kitasatospora as well for reducing at least 31 species clusters to a single taxon. The multi-locus sequence database resulting from the study is a useful tool for identification of new isolates and the phylogenetic analysis presented also provides a road map for planning future genome sequencing efforts in the Streptomycetaceae.

Metabolomic data suggest regulation of black howler monkey (Alouatta pigra) diet composition at the molecular level.

In addition to macronutrients, foods consist of a complex set of chemical compounds that can influence dietary selectivity and consumer physiology. Metabolomics allow us to describe this complexity by quantifying all small molecules, or metabolites, in a food item. In this study we use GC-MS based metabolomics to describe the metabolite profiles of foods consumed by one population of Mexican black howler monkeys (Alouatta pigra) over a 10-month period. Our data indicate that each food exhibited a distinct metabolite profile, and the average weekly intake of metabolites such as neochlorogenic acid and serotonin (5-hydroxytryptamine) was correlated with the consumption of certain plant parts. We speculate that these patterns result in temporal changes in howler monkey physiology such as food retention time. In contrast, variation in the weekly intake of metabolites such as oxalic acid was 70% less than variation in the concentration of the same metabolites across food items, suggesting that howler monkeys regulated the intake of these metabolites, possibly to avoid physiological consequences such as kidney stone formation. Finally, seasonal variation in the consumption of individual nutrient and non-nutrient metabolites were correlated with changes in the relative abundances of associated gut microbial taxa, implying indirect effects of food item metabolites on howler monkey nutritional ecology that likely drive foraging decisions. While additional research is needed to validate these findings, the patterns we report serve as important baseline data for understanding the effects of plant metabolites on the food choice in primates.

A roadmap for natural product discovery based on large-scale genomics and metabolomics.

Actinobacteria encode a wealth of natural product biosynthetic gene clusters, whose systematic study is complicated by numerous repetitive motifs. By combining several metrics, we developed a method for the global classification of these gene clusters into families (GCFs) and analyzed the biosynthetic capacity of Actinobacteria in 830 genome sequences, including 344 obtained for this project. The GCF network, comprising 11,422 gene clusters grouped into 4,122 GCFs, was validated in hundreds of strains by correlating confident mass spectrometric detection of known small molecules with the presence or absence of their established biosynthetic gene clusters. The method also linked previously unassigned GCFs to known natural products, an approach that will enable de novo, bioassay-free discovery of new natural products using large data sets. Extrapolation from the 830-genome data set reveals that Actinobacteria encode hundreds of thousands of future drug leads, and the strong correlation between phylogeny and GCFs frames a roadmap to efficiently access them.

Taxonomic evaluation of species in the Streptomyces hirsutus clade using multi-locus sequence analysis and proposals to reclassify several species in this clade.

Previous phylogenetic analysis of species of the genus Streptomyces based on 16S rRNA gene sequences resulted in a statistically well-supported clade (100 % bootstrap value) containing eight species that exhibited very similar gross morphology in producing open looped (Retinaculum-Apertum) to spiral (Spira) chains of spiny- to hairysurfaced, dark green spores on their aerial mycelium. The type strains of the species in this clade, specifically Streptomyces bambergiensis, Streptomyces cyanoalbus, Streptomyces emeiensis, Streptomyces hirsutus, Streptomyces prasinopilosus and Streptomyces prasinus, were subjected to multi-locus sequence analysis (MLSA) utilizing partial sequences of the housekeeping genes atpD, gyrB, recA, rpoB and trpB to clarify their taxonomic status. The type strains of several recently described species with similar gross morphology, including Streptomyces chlorus, Streptomyces herbaceus, Streptomyces incanus, Streptomyces pratens and Streptomyces viridis, were also studied along with six unidentified green-spored Streptomyces strains from the ARS Culture Collection. The MLSAs suggest that three of the species under study (S. bambergiensis, S. cyanoalbus and S. emeiensis) represent synonyms of other previously described species (S. prasinus, S. hirsutus and S. prasinopilosus, respectively). These relationships were confirmed through determination of in silico DNA-DNA hybridization estimates based on draft genome sequences. The five recently described species appear to be phylogenetically distinct but the unidentified strains from the ARS Culture Collection could be identified as representatives of S. hirsutus, S. prasinopilosus or S. prasinus.

Selection for growth on 3-nitrotoluene by 2-nitrotoluene-utilizing Acidovorax sp. strain JS42 identifies nitroarene dioxygenases with altered specificities.

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.

Cyanohydrin phosphonate natural product from Streptomyces regensis.

Streptomyces regensis strain WC-3744 was identified as a potential phosphonic acid producer in a large-scale screen of microorganisms for the presence of the pepM gene, which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate phosphonomutase. (31)P NMR revealed the presence of several unidentified phosphonates in spent medium after growth of S. regensis. These compounds were purified and structurally characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric analyses. Three new phosphonic acid metabolites, whose structures were confirmed by comparison to chemically synthesized standards, were observed: (2-acetamidoethyl)phosphonic acid (1), (2-acetamido-1-hydroxyethyl)phosphonic (3), and a novel cyanohydrin-containing phosphonate, (cyano(hydroxy)methyl)phosphonic acid (4). The gene cluster responsible for synthesis of these molecules was also identified from the draft genome sequence of S. regensis, laying the groundwork for future investigations into the metabolic pathway leading to this unusual natural product.

Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways.

Phosphonate natural products have proven to be a rich source of useful pharmaceutical, agricultural, and biotechnology products, whereas study of their biosynthetic pathways has revealed numerous intriguing enzymes that catalyze unprecedented biochemistry. Here we review the history of phosphonate natural product discovery, highlighting technological advances that have played a key role in the recent advances in their discovery. Central to these developments has been the application of genomics, which allowed discovery and development of a global phosphonate metabolic framework to guide research efforts. This framework suggests that the future of phosphonate natural products remains bright, with many new compounds and pathways yet to be discovered.

Use of a phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster.

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for "stable isotope labeling of phosphonates in extract" (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S-adenosyl methionine. Mass-guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.

Biosynthesis of Bacillus Phosphonoalamides Reveals Highly Specific Amino Acid Ligation.

Phosphonate natural products have a history of commercial success across numerous industries due to their potent inhibition of metabolic processes. Over the past decade, genome mining approaches have successfully led to the discovery of numerous bioactive phosphonates. However, continued success is dependent upon a greater understanding of phosphonate metabolism, which will enable the prioritization and prediction of biosynthetic gene clusters for targeted isolation. Here, we report the complete biosynthetic pathway for phosphonoalamides E and F, antimicrobial phosphonopeptides with a conserved C-terminal l-phosphonoalanine (PnAla) residue. These peptides, produced by Bacillus, are the direct result of PnAla biosynthesis and serial ligation by two ATP-grasp ligases. A critical step of this pathway was the reversible transamination of phosphonopyruvate to PnAla by a dedicated transaminase with preference for the forward reaction. The dipeptide ligase PnfA was shown to ligate alanine to PnAla to afford phosphonoalamide E, which was subsequently ligated to alanine by PnfB to form phosphonoalamide F. Specificity profiling of both ligases found each to be highly specific, although the limited acceptance of noncanonical substrates by PnfA allowed for in vitro formation of products incorporating alternative pharmacophores. Our findings further establish the transaminative branch of phosphonate metabolism, unveil insights into the specificity of ATP-grasp ligation, and highlight the biocatalytic potential of biosynthetic enzymes.

The genome of Bacillus tequilensis EA-CB0015 sheds light into its epiphytic lifestyle and potential as a biocontrol agent.

Different Bacillus species have successfully been used as biopesticides against a broad range of plant pathogens. Among these, Bacillus tequilensis EA-CB0015 has shown to efficiently control Black sigatoka disease in banana plants, presumably by mechanisms of adaptation that involve modifying the phyllosphere environment. Here, we report the complete genome of strain EA-CB0015, its precise taxonomic identity, and determined key genetic features that may contribute to its effective biocontrol of plant pathogens. We found that B. tequilensis EA-CB0015 harbors a singular 4 Mb circular chromosome, with 3,951 protein-coding sequences. Multi-locus sequence analysis (MLSA) and average nucleotide identity (ANI) analysis classified strain EA-CB0015 as B. tequilensis. Encoded within its genome are biosynthetic gene clusters (BGCs) for surfactin, iturin, plipastatin, bacillibactin, bacilysin, subtilosin A, sporulation killing factor, and other natural products that may facilitate inter-microbial warfare. Genes for indole-acetic acid (IAA) synthesis, the use of diverse carbon sources, and a multicellular lifestyle involving motility, biofilm formation, quorum sensing, competence, and sporulation suggest EA-CB0015 is adept at colonizing plant surfaces. Defensive mechanisms to survive invading viral infections and preserve genome integrity include putative type I and type II restriction modification (RM) and toxin/antitoxin (TA) systems. The presence of bacteriophage sequences, genomic islands, transposable elements, virulence factors, and antibiotic resistance genes indicate prior occurrences of genetic exchange. Altogether, the genome of EA-CB0015 supports its function as a biocontrol agent against phytopathogens and suggest it has adapted to thrive within phyllosphere environments.