Metagenomic domain substitution for the high-throughput modification of nonribosomal peptides.

The modular nature of nonribosomal peptide biosynthesis has driven efforts to generate peptide analogs by substituting amino acid-specifying domains within nonribosomal peptide synthetase (NRPS) enzymes. Rational NRPS engineering has increasingly focused on finding evolutionarily favored recombination sites for domain substitution. Here we present an alternative evolution-inspired approach that involves large-scale diversification and screening. By amplifying amino acid-specifying domains en masse from soil metagenomic DNA, we substitute more than 1,000 unique domains into a pyoverdine NRPS. Initial fluorescence and mass spectrometry screens followed by sequencing reveal more than 100 functional domain substitutions, collectively yielding 16 distinct pyoverdines as major products. This metagenomic approach does not require the high success rates demanded by rational NRPS engineering but instead enables the exploration of large numbers of substitutions in parallel. This opens possibilities for the discovery and production of nonribosomal peptides with diverse biological activities.

Stictamycin, an Aromatic Polyketide Antibiotic Isolated from a New Zealand Lichen-Sourced Streptomyces Species.

Here we describe the isolation and characterization of stictamycin, a new aromatic polyketide with activity against Staphylococcus aureus. Stictamycin was identified following metabolic profiling and bioactivity guided fractionation of organic extracts from Streptomyces sp. 438-3, an isolate from the New Zealand lichen Sticta felix. Comprehensive 1D and 2D NMR analyses were performed to determine the planar structure of stictamycin and relative configurations of stereo centers, with subsequent comparison of experimental and theoretical ECD spectra allowing elucidation of the absolute configuration. Whole-genome sequencing and biosynthetic gene cluster (BGC) analysis revealed that the Streptomyces sp. 438-3 strain contains an atypical type II polyketide (T2PKS) BGC capable of assembling polycyclic-aromatic ring skeletons. Cloning and knockout studies of this T2PKS BGC were used to confirm that it is responsible for the biosynthesis of stictamycin and elucidate a plausible biosynthetic scheme.

Skyllamycins D and E, Non-Ribosomal Cyclic Depsipeptides from Lichen-Sourced Streptomyces anulatus.

The skyllamycins are a class of heavily modified, non-ribosomal peptides, first isolated from Streptomyces sp. KY11784. A Streptomyces strain with potent antibiotic activity against Bacillus subtilis was isolated from a sample of the New Zealand lichen Pseudocyphellaria dissimilis. Whole genome sequencing and biosynthetic gene cluster genetic analysis coupled with GNPS LCMS/MS molecular networking revealed that this strain had the capacity to produce skyllamycins, including previously undescribed congeners, and that these were likely the source of the observed biological activity. Guided by the results of the molecular networking, we isolated the previously reported skyllamycins A-C (1-3), along with two new congeners, skyllamycins D (4) and E (5). The structures of these compounds were elucidated using comprehensive 1D and 2D NMR analyses, along with HRESIMS fragmentation experiments. Antibacterial assays revealed that skyllamycin D possessed improved activity against B. subtilis E168 compared to previously reported congeners.

Hydrated Rubrolides from the New Zealand Tunicate Synoicum kuranui.

LCMS analysis of an extract of the New Zealand tunicate Synoicum kuranui showed evidence for numerous new rubrolides. Following a mass spectrometry-guided isolation procedure, new hydrated rubrolides V and W (5 and 6), along with previously reported rubrolide G (3), were isolated and characterized using MS and NMR. The anti-bacterial and cell cytotoxic activity of the compounds were compared to the potent anti-MRSA compound rubrolide A; hydration across the C-5/C-6 bond was shown to abrogate antibacterial activity.

The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes.

OBJECTIVES: To develop a colorimetric assay for ATP based on the blue-pigment synthesising non-ribosomal peptide synthetase (NRPS) BpsA, and to demonstrate its utility in defining the substrate specificity of other NRPS enzymes. RESULTS: BpsA is able to convert two molecules of L-glutamine into the readily-detected blue pigment indigoidine, consuming two molecules of ATP in the process. We showed that the stoichiometry of this reaction is robust and that it can be performed in a microplate format to accurately quantify ATP concentrations to low micromolar levels in a variety of media, using a spectrophotometric plate-reader. We also demonstrated that the assay can be adapted to evaluate the amino acid substrate preferences of NRPS adenylation domains, by adding pyrophosphatase enzyme to drive consumption of ATP in the presence of the preferred substrate. CONCLUSIONS: The robust nature and simplicity of the reaction protocol offers advantages over existing methods for ATP quantification and NRPS substrate analysis.

Targeted Isolation of Rubrolides from the New Zealand Marine Tunicate Synoicum kuranui.

Global natural products social (GNPS) molecular networking is a useful tool to categorize chemical space within samples and streamline the discovery of new natural products. Here, we demonstrate its use in chemically profiling the extract of the marine tunicate Synoicum kuranui, comprised of many previously reported rubrolides, for new chemical entities. Within the rubrolide cluster, two masses that did not correspond to previously reported congeners were detected, and, following MS-guided fractionation, led to the isolation of new methylated rubrolides T (3) and (Z/E)-U (4). Both compounds showed strong growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis, with minimum inhibitory concentration (MIC) values of 0.41 and 0.91 µM, respectively.

Secondary metabolism in the lichen symbiosis.

Lichens, which are defined by a core symbiosis between a mycobiont (fungal partner) and a photobiont (photoautotrophic partner), are in fact complex assemblages of microorganisms that constitute a largely untapped source of bioactive secondary metabolites. Historically, compounds isolated from lichens have predominantly been those produced by the dominant fungal partner, and these continue to be of great interest for their unique chemistry and biotechnological potential. In recent years it has become apparent that many photobionts and lichen-associated bacteria also produce a range of potentially valuable molecules. There is evidence to suggest that the unique nature of the symbiosis has played a substantial role in shaping many aspects of lichen chemistry, for example driving bacteria to produce metabolites that do not bring them direct benefit but are useful to the lichen as a whole. This is most evident in studies of cyanobacterial photobionts, which produce compounds that differ from free living cyanobacteria and are unique to symbiotic organisms. The roles that these and other lichen-derived molecules may play in communication and maintaining the symbiosis are poorly understood at present. Nonetheless, advances in genomics, mass spectrometry and other analytical technologies are continuing to illuminate the wealth of biological and chemical diversity present within the lichen holobiome. Implementation of novel biodiscovery strategies such as metagenomic screening, coupled with synthetic biology approaches to reconstitute, re-engineer and heterologously express lichen-derived biosynthetic gene clusters in a cultivable host, offer a promising means for tapping into this hitherto inaccessible wealth of natural products.

Structural, functional and evolutionary perspectives on effective re-engineering of non-ribosomal peptide synthetase assembly lines.

Covering: up to May 2018 Non-ribosomal peptide synthetases (NRPSs) are mega-enzymes that form modular templates to assemble specific peptide products, independent of the ribosome. The autonomous nature of the modules in the template offers prospects for re-engineering NRPS enzymes to generate modified peptide products. Although this has clearly been a primary mechanism of natural product diversification throughout evolution, equivalent strategies have proven challenging to implement in the laboratory. In this review we examine key examples of successful and less-successful re-engineering of NRPS templates to generate novel peptides, with the aim of extracting practical guidelines to inform future efforts. We emphasise the importance of maintaining effective protein-protein interactions in recombinant NRPS templates, and identify strengths and limitations of diverse strategies for achieving different engineering outcomes.

Marine Invertebrate Natural Products that Target Microtubules.

Marine natural products as secondary metabolites are a potential major source of new drugs for treating disease. In some cases, cytotoxic marine metabolites target the microtubules of the eukaryote cytoskeleton for reasons that will be discussed. This review covers the microtubule-targeting agents reported from sponges, corals, tunicates, and molluscs and the evidence that many of these secondary metabolites are produced by bacterial symbionts. The review finishes by discussing the directions for future development and production of clinically relevant amounts of these natural products and their analogues through aquaculture, chemical synthesis, and biosynthesis by bacterial symbionts.

Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A-E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.

Chemical-biogeographic survey of secondary metabolism in soil.

In this study, we compare biosynthetic gene richness and diversity of 96 soil microbiomes from diverse environments found throughout the southwestern and northeastern regions of the United States. The 454-pyroseqencing of nonribosomal peptide adenylation (AD) and polyketide ketosynthase (KS) domain fragments amplified from these microbiomes provide a means to evaluate the variation of secondary metabolite biosynthetic diversity in different soil environments. Through soil composition and AD- and KS-amplicon richness analysis, we identify soil types with elevated biosynthetic potential. In general, arid soils show the richest observed biosynthetic diversity, whereas brackish sediments and pine forest soils show the least. By mapping individual environmental amplicon sequences to sequences derived from functionally characterized biosynthetic gene clusters, we identified conserved soil type-specific secondary metabolome enrichment patterns despite significant sample-to-sample sequence variation. These data are used to create chemical biogeographic distribution maps for biomedically valuable families of natural products in the environment that should prove useful for directing the discovery of bioactive natural products in the future.

Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products.

Complex microbial ecosystems contain large reservoirs of unexplored biosynthetic diversity. Here we provide an experimental framework and data analysis tool to facilitate the targeted discovery of natural-product biosynthetic gene clusters from the environment. Multiplex sequencing of barcoded PCR amplicons is followed by sequence similarity directed data parsing to identify sequences bearing close resemblance to biosynthetically or biomedically interesting gene clusters. Amplicons are then mapped onto arrayed metagenomic libraries to guide the recovery of targeted gene clusters. When applied to adenylation- and ketosynthase-domain amplicons derived from saturating soil DNA libraries, our analysis pipeline led to the recovery of biosynthetic clusters predicted to encode for previously uncharacterized glycopeptide- and lipopeptide-like antibiotics; thiocoraline-, azinomycin-, and bleomycin-like antitumor agents; and a rapamycin-like immunosuppressant. The utility of the approach is demonstrated by using recovered eDNA sequences to generate glycopeptide derivatives. The experiments described here constitute a systematic interrogation of a soil metagenome for gene clusters capable of encoding naturally occurring derivatives of biomedically relevant natural products. Our results show that previously undetected biosynthetic gene clusters with potential biomedical relevance are very common in the environment. This general process should permit the routine screening of environmental samples for gene clusters capable of encoding the systematic expansion of the structural diversity seen in biomedically relevant families of natural products.

Biosynthesis of novel Pyoverdines by domain substitution in a nonribosomal peptide synthetase of Pseudomonas aeruginosa.

Pyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. The Pseudomonas aeruginosa nonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate an l-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substrate-specifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine-at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than misincorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.

Directed Evolution of the BpsA Carrier Protein Domain for Recognition by Non-cognate 4'-Phosphopantetheinyl Transferases to Enable Inhibitor Screening.

4'-Phosphopantetheinyl transferases (PPTases) play an essential role in activating the carrier protein domains of mega-synthases involved in primary and secondary metabolism and have been validated as promising drug targets in multiple pathogens. Monitoring phosphopantetheinylation of the non-ribosomal peptidase synthetase BpsA, which produces blue indigoidine pigment upon activation, is a useful strategy to screen chemical collections for inhibitors of a target PPTase. However, PPTases can exhibit carrier protein specificity and some medically important PPTases do not activate BpsA. Here, we describe how to conduct a directed evolution campaign to evolve the BpsA carrier protein domain for improved recognition by a candidate PPTase, as exemplified for the human Sfp-like PPTase. This method can be applied to other non-cognate PPTases for discovery of new drug candidates or chemical probes, or to enable development of next-generation biosensors that utilize BpsA as a reporter.

A Genomic Survey of the Natural Product Biosynthetic Potential of Actinomycetes Isolated from New Zealand Lichens.

Actinomycetes are prolific producers of industrially valuable and medically important compounds. Historically, the most efficient method of obtaining compounds has been bioactivity-guided isolation and characterization of drug-like molecules from culturable soil actinomycetes. Unfortunately, this pipeline has been met with an increasing number of rediscoveries, to the point where it is no longer considered an attractive approach for drug discovery. To address this challenge and to continue finding new compounds, researchers have increasingly focused on alternative environmental niches and screening methods. Here, we report the genetic investigation of actinomycetes from an underexplored source, New Zealand lichens. In this work, we obtain draft genome sequences for 322 lichen-associated actinomycetes. We then explore this genetic resource with an emphasis on biosynthetic potential. By enumerating biosynthetic gene clusters (BGCs) in our data sets and comparing these to various reference collections, we demonstrate that actinomycetes sourced from New Zealand lichens have the genetic capacity to produce large numbers of natural products, many of which are expected to be broadly different from those identified in previous efforts predominantly based on soil samples. Our data shed light on the actinomycete assemblage in New Zealand lichens and demonstrate that lichen-sourced actinobacteria could serve as reservoirs for discovering new secondary metabolites. IMPORTANCE Lichens are home to complex and distinctive microbial cohorts that have not been extensively explored for the ability to produce novel secondary metabolites. Here, we isolate and obtain genome sequence data for 322 actinomycetes from New Zealand lichens. In doing so, we delineate at least 85 potentially undescribed species, and show that lichen associated actinomycetes have the potential to yield many new secondary metabolites, and as such, might serve as a productive starting point for drug discovery efforts.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

Rapid and flexible biochemical assays for evaluating 4'-phosphopantetheinyl transferase activity.

PPTases (phosphopantetheinyl transferases) are of great interest owing to their essential roles in activating fatty acid, polyketide and non-ribosomal peptide synthetase enzymes for both primary and secondary metabolism, as well as an increasing number of biotechnological applications. However, existing techniques for PPTase characterization and development are cumbersome and technically challenging. To address this, we have developed the indigoidine-synthesizing non-ribosomal peptide synthetase BpsA as a reporter for PPTase activity. Simple co-transformation allows rapid assessment of the ability of a PPTase candidate to activate BpsA in vivo. Kinetic parameters with respect to either CoA or BpsA as variable substrate can then be derived in vitro by continuously measuring the rate of indigoidine synthesis as the PPTase progressively converts BpsA from its apo into holo form. Subsequently, a competition assay, in which BpsA and purified carrier proteins compete for a limited pool of CoA, enables elucidation of kinetic parameters for a PPTase with those carrier proteins. We used this system to conduct a rapid characterization of three different PPTase enzymes: Sfp of Bacillus subtilis A.T.C.C.6633, PcpS of Pseudomonas aeruginosa PAO1, and the putative PPTase PP1183 of Ps. putida KT2440. We also demonstrate the utility of this system for discovery and characterization of PPTase inhibitors.

Preparation of Soil Metagenome Libraries and Screening for Gene-Specific Amplicons.

Cosmid libraries constructed from environmental metagenome samples are powerful tools for capturing the genomic diversity of complex microbial communities. The large insert size (∼35 kb) of such libraries means they are compatible with downstream expression of large biosynthetic gene clusters (BGCs). This allows the discovery of previously undescribed natural products that would be inaccessible using traditional culture-based discovery pipelines. Here we describe methods for the construction of a cosmid metagenome library from a soil sample, and the process of screening that library for individual cosmid clones containing aromatic polyketide BGCs using degenerate primers that target the ketosynthase alpha (KSα) gene.

Draft Genome Sequence of a Novel Rhizobium Species Isolated from the Marine Macroalga Codium fragile (Oyster Thief).

In the process of studying the relationship between marine macroalgae and their bacterial symbionts, we isolated a new species of Rhizobium, which we designated Rhizobium sp. nov. C1 (for "Codium 1"). Here, we report the complete genome sequence of Rhizobium sp. nov. C1.