ORCID Profile
0000-0002-6188-9902
Current Organisation
Victoria University of Wellington
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Biochemistry and cell biology | Natural products and bioactive compounds | Synthetic biology | Structural biology (incl. macromolecular modelling) |
Publisher: Research Square Platform LLC
Date: 12-06-2023
DOI: 10.21203/RS.3.RS-3011833/V1
Abstract: Non-ribosomal peptides are a erse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale ersification and screening. By adopting a metagenomic approach of lifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of in idual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with erse biological activities.
Publisher: Wiley
Date: 26-10-2017
DOI: 10.1111/MMI.13855
Abstract: Alternative sigma (σ) factors govern expression of bacterial genes in response to erse environmental signals. In Pseudomonas aeruginosa σ
Publisher: Cold Spring Harbor Laboratory
Date: 06-2023
DOI: 10.1101/2023.05.31.543161
Abstract: Non-ribosomal peptides are a erse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale ersification and screening. By adopting a metagenomic approach of lifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of in idual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with erse biological activities.
Publisher: American Chemical Society (ACS)
Date: 26-01-2021
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D0CB00228C
Abstract: Heterologous expression of an aromatic polyketide biosynthetic gene cluster recovered from a New Zealand soil metagenome library resulted in the discovery of new bioactive aureolic acids.
Publisher: American Chemical Society (ACS)
Date: 15-07-2019
DOI: 10.1021/ACS.JNATPROD.9B00493
Abstract: Six new lamellarin sulfates (
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8NP90037J
Abstract: The Natural Product Reports themed issue on Understanding biosynthetic protein–protein interactions is introduced by the Guest Editors, David Ackerley, Gregory Challis and Max Cryle.
Publisher: American Society for Microbiology
Date: 28-04-2020
Abstract: Mycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli . Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among in iduals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.
Publisher: Elsevier BV
Date: 07-2004
Publisher: Springer Science and Business Media LLC
Date: 30-11-2014
Publisher: Springer Science and Business Media LLC
Date: 20-07-2023
Publisher: American Society for Microbiology
Date: 05-2003
DOI: 10.1128/JB.185.9.2848-2855.2003
Abstract: Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdine PAO , which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli , and the protein product was purified. l -Threonine, one of the amino acid residues in pyoverdine PAO , was an effective substrate for the recombinant protein in ATP-PP i exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including d -threonine, l -serine, and l - allo -threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases.
Publisher: American Chemical Society (ACS)
Date: 07-09-2021
DOI: 10.1021/ACS.JNATPROD.1C00547
Abstract: The skyllamycins are a class of heavily modified, non-ribosomal peptides, first isolated from
Publisher: American Society for Microbiology
Date: 15-09-2014
DOI: 10.1128/AEM.01453-14
Abstract: 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.
Publisher: Cold Spring Harbor Laboratory
Date: 25-04-2020
DOI: 10.1101/2020.04.23.058875
Abstract: One avenue to combat multidrug-resistant Gram-negative bacteria is the co-administration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using erse biochemical and genetic assays, we reveal the molecular mechanisms of niclosamide, a clinically-approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress and reducing ATP production to cause cell death. These insights guided the identification of erse compounds that synergized with salicylanilides when co-administered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development and stewardship of novel combination therapies. There is a critical need for more effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We reveal the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide, and subsequently exploit this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a erse range of recalcitrant Gram-negative clinical isolates, and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide, but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.
Publisher: Elsevier BV
Date: 02-2023
DOI: 10.1016/J.ENZMICTEC.2022.110153
Abstract: DNA ligases are widely used in molecular biology to generate recombinant DNA. However, having evolved for nick-sealing, they are inefficient at catalysing the blunt-ended ligations that are critical to many biotechnological applications, including next-generation sequencing. To facilitate engineering of superior blunt-ended DNA ligases, we have developed and validated a compartmentalised self-replication protocol that can select for the most effective ligases from a library of variants. Parallel cultures of Escherichia coli cells expressing different plasmid-encoded variants act as both a source of template DNA for discrete whole-plasmid PCR reactions, and a source of expressed ligase to circularise the corresponding PCR licons. The most efficient ligases generate the greatest number of self-encoding plasmids, and are thereby selected over successive rounds of transformation, lification and ligation. By in idually optimising critical steps, we arrived at a coherent protocol that, over five rounds of selection, consistently enriched for cells expressing the more efficient of two recombinant DNA ligases.
Publisher: American Society for Microbiology
Date: 27-10-2020
Abstract: There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a erse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.
Publisher: Elsevier
Date: 2020
Start Date: 2023
End Date: 12-2025
Amount: $507,300.00
Funder: Australian Research Council
View Funded Activity