Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates

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 the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

Blue pigment synthase A (BpsA) is a single module non-ribosomal peptide synthetase (NRPS) originally isolated from the bacterium Streptomyces lavendulae. It synthesises an easily detectible blue pigment called indigoidine from two molecules of L-glutamine in an ATP powered reaction. BpsA is readily purified and amenable to in vitro assays that have a variety of useful applications. By spectrophotometrically quantifying indigoidine levels it is possible to accurately measure the amount of L-glutamine in complex biological fluids including urine, blood plasma and cell culture media. This method has several advantages over existing methods for glutamine measurement, including that it directly reports on glutamine levels. Existing commercially available enzymatic kits first convert glutamine into glutamate and then measure the level of glutamate, which requires additional sample processing and introduces complexity if glutamate may also be present in the target sample. Additionally, we have shown that BpsA can also be used to measure ATP concentrations in a similar manner. We have further developed a BpsA based assay to detect inhibitors of 4’-phosphopantetheinyl transferases (PPTases). PPTases are enzymes that attach a phosphopantetheine arm to fatty acid synthases, NRPSs and polyketide synthases, thereby switching them from an inactive apo form to an active holo form. PPTases have been validated as promising drug targets in several pathogenic bacteria including P. aeruginosa and M. tuberculosis. In order to detect PPTase inhibition, we have shown that BpsA can be purified in its inactive apo form and mixed with the target PPTase as well as a candidate inhibitor in vitro. The level of PPTase inhibition can then be calculated by measuring the rate of indigoidine production. The assay has been optimised for high throughput screening and used to identify several compounds from chemical libraries that inhibit essential PPTases of P. aeruginosa and M. tuberculosis

Using E. coli NfsA as a model to improve our understanding of enzyme engineering

There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to the difficulty for step-wise evolutionary approaches in effectively retaining mutations that are beneficial in combination with one another, but on an individual basis are neutral or deleterious (i.e., exhibit positive epistasis). We sought to determine whether a superior enzyme identified using a simultaneous mass site directed mutagenesis approach could have been identified using a step-wise approach. We conducted simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 394 million unique variants. We then applied a powerful positive selection using chloramphenicol which is toxic to E. coli but can be detoxified via nitro-reduction. This has enabled us to recover a diverse range of highly active nitroreductase variants. For two of the most active variants, we have created all possible combinations of single mutations. This allowed us to examine whether a step-wise mutagenesis pathway could have also yielded these enzymes. As anticipated, we identified complex epistatic interactions between residues in these enzyme variants. We have also investigated the “black-box” effect of enzyme engineering, examining the consequences that evolving NfsA towards one specialist activity had on the other promiscuous activities of NfsA. Variants generated in this study have also had practical applications, in particular for targeted cell ablation in zebrafish. We have identified NfsA variants that are highly active with nil-bystander prodrugs that can selectively ablate nitroreductase expressing cells without harm to adjacent cells. In ongoing work, our lead variants are being evaluated for their utility in transgenic zebrafish models of degenerative disease

Simultaneous randomisation of eight key active site residues in E. coli NfsA to generate superior nitroreductases for prodrug activation

There is a substantial gap between the levels of enzyme activity Nature can evolve and those that scientists can engineer in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. This is most likely due to an inability of step-wise evolutionary approaches to effectively retain mutations that are beneficial in combination with one another, but on an individual basis are neutral or even slightly deleterious (i.e., exhibit positive epistasis). To overcome this limitation, we are seeking to “jump” straight to an enzyme with peak activity by conducting simultaneous mass randomisation of eight key active site residues in Escherichia coli NfsA, a nitroreductase enzyme that has several diverse applications in biotechnology. Using degenerate codons, we generated a diverse library containing 425 million unique variants. We then applied a powerful selection system using either or both of two recently identified positive selection compounds, which has enabled us to recover a diverse range of highly active nitroreductase variants. These have been screened against a panel of prodrug substrates to identify variants that are improved with specific prodrug substrates of interest. A primary focus has been developing nitroreductases as tools for targeted cell ablation in zebrafish. The basic system involves co-expression of a nitroreductase and fluorescent reporter under the control of a cell type specific promoter in a transgenic fish. Expression of the nitroreductase selectively sensitises target cells to a prodrug which, following nitroreduction, yields a cytotoxic compound that causes precise targeted cell ablation. We have identified several nil-bystander prodrugs that are able to selectively ablate nitroreductase expressing cells with no harm to nearby cells, and have paired these with highly specialised NfsA variants to improve the efficacy and accuracy of cell ablation. We have also screened our mass-randomisation libraries to recover nitroreductases that have non-overlapping prodrug specificities, to be used in a multiplex cell ablation system. This expands upon the previous system, by using pairs of selective nitroreductases and two different prodrugs to facilitate independent ablation of multiple cell types. For example, we have identified a specialist NfsA variant that has activity for tinidazole and not for metronidazole, achieved by including metronidazole as a simultaneous counter-selection during the initial positive selection process. This elegant positive/negative selection eliminated activity with metronidazole, while still ensuring that some level of nitroreductase activity was retained overall

Neonatal BCG vaccination reduces interferon gamma responsiveness to heterologous pathogens in infants from a randomised controlled trial

Background Bacille Calmette-Guérin (BCG) vaccination has beneficial non-specific (heterologous) effects that protect against non-mycobacterial infections. We have previously reported that BCG vaccination at birth alters in vitro cytokine responses to heterologous stimulants in the neonatal period. This study investigated heterologous responses in 167 infants in the same trial seven months after randomisation. Methods A whole blood assay was used to interrogate in vitro cytokine responses to heterologous stimulants (killed pathogens) and Toll-like receptor (TLR) ligands. Results Compared to BCG-naïve infants, BCG-vaccinated infants had increased production of MIG and IFN-γ in response to mycobacterial stimulation and decreased production of IFN-γ in response to heterologous stimulation. Reduced IFN-γ responses to heterologous stimulants and TLR ligands were attributable to a decrease in the proportion of infants who mounted a detectable IFN-γ response. BCG-vaccinated infants also had increased production of MIG and IL-8, and decreased production of IL-10, MIP-1α and MIP-1ß, the pattern of which varied by stimulant. IL-1Ra responses following TLR1/2 (Pam3CYSK4) stimulation were increased in BCG-vaccinated infants. Both sex and maternal BCG vaccination status influenced the effect of neonatal BCG vaccination. Conclusions BCG vaccination leads to changes in IFN-γ responsiveness to heterologous stimulation. BCG-induced changes in other cytokine responses to heterologous stimulation varies by pathogen

A Novel Synthetic Yeast for Enzymatic Biodigester Pretreatment

Lignin, a complex organic polymer, is a major roadblock to the efficiency of biofuel conversion as it both physically blocks carbohydrate substrates and poisons biomass degrading enzymes, even if broken down to monomer units. A pretreatment process is often applied to separate the lignin from biomass prior to biofuel conversion. However, contemporary methods of pretreatment require large amounts of energy, which may be economically uncompelling or unfeasible. Taking inspiration from several genes that have been isolated from termites and fungi which translate to enzymes that degrade lignin, we want to establish a novel “enzymatic pretreatment” system where microbes secrete these enzymes to degrade lignocellulosic biomass. We incorporated the following genes into yeast vectors: laccase, lignin peroxidase, and alpha-keto-reductase from Reticulitermes flavipes; versatile peroxidase from Colletotrichum fioriniae PJ7; manganese peroxide from Heterobasidion irregulare TC 32-1; and tyrosinase from Agaricus bisporus. These vectors code for fusion proteins with yeast secretion tags at the end of each enzyme gene, fluorescent protein tags at the beginning, as well as standardized restriction sites for synthetic biology manipulation. Furthermore, we designed an additional vector to contain our genetically modified yeast using an oxygen-repressed killswitch. We expect that transformants with our construct will be able to secrete said enzymes and contribute to lignin degradation if added to a biomass slurry. Future studies may focus on constructing a prototype bioreactor system and optimizing which combination of enzymes lead to the most efficient biofuel production

Gene editing enables rapid engineering of complex antibiotic assembly lines

From Springer Nature via Jisc Publications RouterHistory: received 2021-09-09, accepted 2021-11-02, registration 2021-11-08, pub-electronic 2021-11-25, online 2021-11-25, collection 2021-12Publication status: PublishedFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/L013754/1, BB/N023536/1Abstract: Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized

Genetic and Clinical Predictors of Age of ESKD in Individuals With Autosomal Dominant Tubulointerstitial Kidney Disease Due to UMOD Mutations

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