Characterization, metagenomic screening and engineering of bacterial nitroreductases for biomedical research applications

Bacterial nitroreductases are NAD(P)H-dependent oxidoreductases (generally homodimeric and FMN-binding) that can catalyse the 4- or 6-electron reduction of nitro groups on aromatic rings. This results in a profound electronic shift that can dramatically alter the properties of the molecule as a whole, e.g. activating latent cytotoxins, or detoxifying certain pollutants or antibiotics. We have exploited these properties and the characteristic promiscuity of these enzymes to develop useful tools for biomedical research and therapy, in particular the anticancer strategy gene-directed enzyme prodrug therapy, and targeted cellular ablation in zebrafish models of degenerative disease. We have used directed evolution to improve desirable activities and are also investigating the use of dual positive and negative selection strategies to tailor reaction specificity and to better understand how the evolution of promiscuous enzymes is modulated by in vivo constraints. A further application of our positive selection capabilities has been the recovery of novel nitroreductases from libraries of uncharacterised environmental DNA

High-level expression, high-throughput screening and direct recovery of nitroreductase enzymes from metagenome libraries

We have developed generally applicable library generation methods to maximize expression of cloned environmental genes, enabling screening for weak phenotypes in metagenome libraries. Our method also permits direct recovery of the encoded enzymes, providing rapid access to an almost unlimited diversity of previously unexplored biocatalysts. We have exemplified this for nitroreductases, members of a diverse family of oxidoreductase enzymes that can catalyze the bioreductive activation of nitroaromatic prodrugs such as metronidazole. These capabilities have diverse applications in medicine and research, including anti-cancer gene therapy and targeted ablation of nitroreductase-expressing tissues in transgenic animal models. However, research in these fields has largely been focused on the canonical nitroreductase NfsB from Escherichia coli, which exhibits sub-optimal levels of metronidazole activity. In previous work we have investigated alternative nitroreductase enzymes, sourced from genome-sequenced bacteria. To complement this work we have now turned to the discovery of novel nitroreductases from metagenomic DNA fragments, derived from the uncultivable bacteria present in New Zealand soil and lichen species

Engineering the biosynthesis of non-ribosomal peptides

Non-ribosomal peptides are a class of natural product that exhibit diverse properties and function as toxins, antibiotics, siderophores, and pigments. Their range of activity means they have roles in medicine, agriculture and bioremediation. Non-ribosomal peptides are biosynthesised by linking monomers together via peptide bonds. They are assembled from a pool of hundreds of monomers, and often contain cyclisation or other modifications not found in ribosomally-synthesised peptides. Their structural diversity means they can be expensive and/or difficult to synthesise. Consequently, many non-ribosomal peptides are produced using fermentation and then modified to generate compounds suitable for medical or industrial applications. Modifying the biosynthetic pathways could provide a cheap and scalable source of new compounds but attempts to engineer them have previously had a low success rate. Using pyoverdine as a model system, this study investigated how to rationally engineer non-ribosomal peptide biosynthesis and generated modified pyoverdines in 6/9 cases. The results of modifying pyoverdine were then used to engineer a second pathway to make dipeptides with a 3/5 success rate. The high success rate and similar results using two biosynthetic pathways suggest this approach is highly transferable and will be valuable for engineering other pathways

Metagenomic domain substitution for the high-throughput creation of non-ribosomal peptide analogues

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Assessing the evolutionary potential of novel resistance elements to the candidate antibacterial, niclosamide

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Directed evolution of the non-ribosomal peptide synthetase BpsA to enable recognition by the human Sfp-like PPTase

Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that have an assembly line architecture and synthesise a diverse range of compounds such as antibiotics, siderophores and immunosuppressants. Within the assembly line, the peptidyl carrier protein (PCP) domain has a crucial role in shuttling substrates between the different catalytic domains. The PCP domain is a small four-helix bundle that requires a phosphopantetheinyl moiety to be attached to a conserved serine on the second alpha helix for functionality. This post-translational modification is catalysed by a family of enzymes called the phosphopantetheinyl transferases (PPTases). Due to their central role in activating enzymes involved in both primary (e.g., fatty acid synthetases) and secondary metabolism (e.g., NRPSs), PPTases have been identified as a promising antibiotic target in bacterial species such as Mycobacterium tuberculosis. We have previously developed a high-throughput enzymatic screen for PPTase inhibitors based on co-incubation of a target PPTase with a blue-pigment synthesising NRPS, BpsA. As part of the development of a complete screening platform, we also wanted to be able to rapidly counter-screen inhibitors for cross-inhibition of the endogenous human PPTase, as this is a potential source of toxicity. We found we were unable to use the native BpsA enzyme for this, as the human PPTase is incapable of recognising the PCP domain of BpsA. To improve with the ability of BpsA to be activated by the human PPTase, a directed evolution campaign was undertaken. Firstly, error-prone PCR was used to introduce mutations into the PCP domain of BpsA. Approximately 200,000 variants were screened using a high-throughput plate-based assay. Forty ‘hits’ were then characterized in a semi-quantitative liquid assay. Based on the pattern of amino acid substitutions in the most active variants, specific combinations of substitutions were rationally introduced into BpsA. The top variant identified was now capable of being rapidly phosphopantetheinylated by the human PPTase and we have shown this can be used to quickly screen bacterial PPTase inhibitors for cross-reactivity with the human PPTase. This work illustrates the flexible nature of the PCP domain and provides further evidence that only a few point mutations may be sufficient to dramatically change the specificity of PCP domains for different PPTases

Development of a selection to recover improved DNA ligase enzymes during directed evolution

DNA ligases are essential enzymes used in many molecular biology applications. Of particular note, they are important enzymes in next generation sequencing (NGS) technologies. The improved speed, efficiency, and affordability of NGS over Sanger sequencing has greatly expanded the applications of DNA sequencing. In most NGS technologies ligase enzymes play a crucial role, for instance in ligating adaptors onto sequence fragments during sample preparation. This key step requires a blunt-ended ligation reaction, with highly efficient ligases required in order to create a sample library of high quality. The current go-to enzyme is T4 DNA ligase, which has not evolved in Nature to perform blunt ended ligations, and as such has relatively poor levels of activity when compared to other substrates. There is therefore potential to improve upon this enzyme and engineer a ligase that is more efficient with blunt-ended substrates. We have developed a novel function-based directed evolution selection to evolve blunt-ended ligases that have greater catalytic efficiency. The basis for this approach is the over-expression of a ligase enzyme variant which is then incubated with a linearised plasmid encoding for that same ligase variant. More efficient ligases will ligate the plasmid encoding for their own gene variant more efficiently (in a blunt-ended ligation), and so greater numbers of the circularised plasmid will be produced. Through successive rounds of transformation, amplification and ligation the most improved enzyme variants are enriched. This selection approach is being used to evaluate a panel of ligase variants in order to identify the best ligases for blunt-ended ligation applications. Please click Additional Files below to see the full abstract

Engineering bacterial nitroreductases for anticancer gene therapy and targeted cell ablation

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