A broadly applicable artificial selection system for biomolecule evolution

Biocatalysis offers an attractive alternative to traditional chemical catalysis. However, it is often found that an enzyme with the optimal properties for a specific application is not available within the natural repertoire of enzymes. It is then desirable to obtain an improved variant by altering the sequence of a known enzyme, in a process known as protein engineering. Directed evolution is one of the most powerful tools for protein engineering. In directed evolution, the process of natural evolution is mimicked in the laboratory at a much shorter timescale and selecting for properties that make the enzyme (or any other type of biomolecule) more suitable for an application of human interest. The main bottleneck of directed evolution is the identification of the desired variants amongst a majority of variants without the sought altered or improved property. Selection approaches link the desired activity to an increased survival rate or improved growth. While in principle such methodologies allow for ultra high-throughput analysis of libraries, most selection techniques have a limited scope, and can only be applied to a relatively reduced set of biomolecules or properties. This thesis presents the most broadly-applicable artificial selection system for the evolution of biomolecules ever reported. The selection platform is based on an engineered E. coli strain with impaired regeneration of NAD+, causing a conditional growth defect during anaerobic fermentation. By directly or indirectly linking the activity of the biomolecules of interest to the oxidation of NADH, cells can be rescued from this growth defect. The efficacy of such selection system has been demonstrated by using it to select alcohol dehydrogenase, imine reductase and nitroreductase variants with altered or enhanced catalytic properties, as well as an isopropanol-producing metabolic pathway with optimised regulatory elements leading to a maximised yield of isopropanol. These results confirm the wide scope of the developed selection system, which can replace conventional screening currently used in many cases of direct relevance for industrial processes. Increasing the throughput of the variant search process by many orders of magnitude will lead to the discovery of novel biomolecules and accelerate the implementation of biocatalysis.Open Acces

Engineering & molecular biology approaches to improving trypsin-based bioprocesses.

This project was designed, in partnership with Eli Lilly, Fegersheim, with the aim of developing a series of methodologies for the mutation and characterisation of trypsin variants with extended substrate specificities for certain amino acid combinations. This library of modified enzymes with enhanced specificity towards 4-aa 1 sequence motifs could then be used as efficient and 'clean' biocatalytic agents. The goal was thus to explore the tailoring of industrial bioprocess enzymes to better suit process criteria, thus lowering the overall cost. It was a principle aim of the project to design a practical method of directed evolution for the breeding of variants with novel or enhanced substrate specificities. The organism on which this work was to be based was the Eli Lilly commercial recombinant bovine trypsinogen production strain, termed ELTRP-1. In order to devise a high-throughput assay suitable for screening a mutant library of 103+ variants, it was essential to fully characterise a method of microwell fermentation, and also to engineer the recombinant protein for solubility, i.e render it available for assaying. A high-throughput screen such as this would have to involve the minimum number of process steps and be entirely microplate-based. A microplate-scale solubilisation and refolding protocol for the r-trypsinogen inclusion bodies was developed that was successful for commercial enzyme but was impractical for use on recombinant inclusion bodies as the nature of the screen disallowed extensive purification, and thus the highly heterologous nature of the solutions appeared to inhibit refolding. Two variants of the enzyme, one with the prosequence removed, were cloned into a pET26b vector, behind a pelB leader, and all available fermentation parameters were experimented with. The construct plasmid was then subjected to a range of mutation rates using the XL1-Red mutator strain and variants were screened for increases in solubility. A series of mutants were obtained which demonstrated over 100 g/ml soluble mature enzyme after microplate fermentation, and enzyme translocation was monitored over 5ml and 100ml scale-up. Observed plasmid instability was due, in part, to the autotoxicity of the expressed enzyme and compounded by the fact that the T7 induction control mechanism of the strain appeared to have been disabled. These problems only manifested when the fermentation was scaled-up above microplate volumes and thus it appeared that the screening of the library for soluble mutant enzymes had been entirely successful by its own specific criteria, i.e. the isolated variants performed as required only when in well-culture

Interrogation and engineering of RAD51:BRC repeat interactions

The maintenance of genetic information is a fundamental function of every organism. The DNA in every human cell endures thousands of lesions per day, and eukaryotes have developed a sophisticated response to tackle this damage. Double strand breaks (DSBs) are the most toxic type of DNA damage, potentially leading to chromosomal rearrangements and tumorigenesis. Homologous recombination (HR) is a repair pathway that uses a homologous template to faithfully repair the DSBs. Central to HR is a recombinase protein RAD51, which forms a nucleoprotein filament with the broken ends, and allows efficient homology search. RAD51 is a promising therapeutic target in oncology. BRCA2 is a crucial mediator of RAD51 function and interacts with RAD51 through a set of 8 BRC repeats. A BRC repeat consists of two modules, an FxxA and an LFDE module, each containing hot-spot residues that mediate binding. In this work, I set out to utilise these for the pharmacological targeting of human RAD51. First, using biophysical methods, I investigated a novel, high-affinity chimeric repeat BRC8-2 composed of FxxA and LFDE modules from different natural repeats. I determined the X-ray crystallographic structure of its complex, which allowed me to uncover the determinants of high affinity binding. I applied these findings to the design of novel macrocyclic peptide inhibitors of RAD51. A method for cysteine stapling of recombinantly produced peptides was optimised to yield the correct product of high purity. The optimised stapling methodology provides a promising general approach for recombinant production and evaluation of cysteine-stapled peptides. Peptides were rationally designed in a structure-guided manner, and a variety of stapling architectures were evaluated for binding. The resulting purified molecules exhibit high potency and are stable in serum. Further crystallographic studies shed light on how the stapling moiety affects the peptide binding mode. The derived peptides serve as novel modalities for targeting Rad51 protein-protein interactions and can inform subsequent development of therapeutic drugs. In addition to the human proteins, I investigated the BRC:RAD51 interactions in orthologs from Leishmania infantum, the causative agent of leishmaniasis. Using crystallography and biophysical methods, I uncovered novel features of BRC repeat binding. The presented work expands our understanding of the structural determinants of homologous recombination.MRC DT

Intra-genomic variation in symbiotic dinoflagellates: Recent divergence or natural hybridization?

The perpetuity of coral reefs will ultimately depend on the ability of corals to adapt to changing conditions. Inter-specific hybridization can provide the raw genetic material necessary for adaptation, and stimulate macro-evolutionary leaps during periods of environmental upheaval. Though well-documented in corals, hybridization has yet to be identified in their dinoflagellate symbionts (genus Symbiodinium), despite growing evidence of sexual reproduction in this genus. The integral roles that these symbiotic algae play in coral productivity, reef accretion and ‘coral bleaching’ emphasize the need to better understand their short-term evolutionary potential. In this thesis, I develop new molecular and statistical methodology, and combine lab- and field-based analysis to explore the potential for hybridization between divergent Symbiodinium taxa. To screen for putative Symbiodinium hybrids, intra-genomic variation was examined within individual symbionts isolated from the reef-building coral Pocillopora damicornis at Lord Howe Island (Australia). A nested quantitative PCR (qPCR) assay was developed to quantify polymorphic internal transcribed spacer 2 (ITS2) sequences within the genome of each symbiont cell. Three genetically distinct Symbiodinium populations were detected co-existing within the symbiont consortium of P. damicornis. Mixed populations of ‘pure’ Symbiodinium types C100 and C109 coexisted with a population of cells hosting co-dominant C100 and C109 ITS2 repeats. Genetically heterogeneous Symbiodinium cells were more common than homogeneous symbionts in four of the six colonies analysed, with a maximum proportional abundance of 89%. Morphological, functional and ecological attributes of heterogeneous Symbiodinium cells were characterized to assess their candidacy as putative hybrids. The proportional abundance of genetically heterogeneous symbionts was spatially and temporally conserved within colonies, indicating a lack of competition between Symbiodinium populations. However, this abundance ratio varied considerably between colonies separated by metres to tens of metres, and to a greater extent between sites isolated by hundreds to thousands of metres. The local thermal maximum emerged as a significant predictor of the proportional abundance of genetically heterogeneous Symbiodinium cells, suggesting that the distribution of these ‘putative hybrids’ is influenced by a reduced affinity for thermal stress. Genetically heterogeneous Symbiodinium cells were around 50% larger (by volume) than homogeneous cells, occupied tissue of the coral host at reduced densities, and showed relatively poor light-harvesting efficiency. Colonies hosting a higher proportion of these symbionts suffered a reduction in overall photosynthetic performance (maximum gross photosynthesis normalised to respiration; P:R) at the ambient temperature of 25 °C. This disparity was maintained when the temperature was elevated to simulate the maximum experienced within the LHI lagoon (29 °C). Under these stressful conditions, colonies dominated by putative Symbiodinium hybrids were only marginally capable of net oxygen production. The influence of putative Symbiodinium hybrids on the growth and survival of P. damicornis was tested by reciprocally transplanting coral colonies between reef sites featuring distinct temperature regimes. Neither calcification nor mortality was influenced by the proportional abundance of genetically heterogeneous cells in the symbiont consortium. This uncoupling of symbiont performance and host fitness may be explained by stochastic events such as predation and disease, which substantially increase variation in growth and mortality in field experiments. Alternatively, it may represent some unknown benefit associated with hosting hybrid symbionts, belying their relatively poor photosynthetic performance, and explaining the widespread abundance of these heterogeneous Symbiodinium cells on the Lord Howe Island reef. Our inability to maintain many clade C Symbiodinium types in culture prevents direct observations of hybridization between C100 and C109. Unequivocal evidence of this phenomenon will therefore likely remain elusive until high-resolution, single-copy nuclear markers can be developed, since the incomplete displacement of ancestral polymorphisms can leave a similar genomic signature to that of hybridization. However, this study serves to provide an initial proof-of-principle for hybridization between divergent Symbiodinium taxa. In doing so, it highlights the need to better understand the evolutionary processes underpinning coral- and symbiont-adaptation in a changing climate

Understanding and visualising the variation in HLA

The HLA region is a segment of the human genome containing immune system genes, which orchestrate the defence against infection. A key aspect distinguishing the HLA genes from other genes in the human genome is their extensive levels of variation. This variation increases the depth and breadth of the weaponry used against pathogen infections; and helps impede the spread of infection within families and communities. Analysing and understanding these high levels of variation gives an insight into how the human immune system has developed and the breadth of variation seen in the human population This is possible using data generated over the last twenty years, from the sequencing of millions of individuals across the world, and these sequences deposited in public databases. This gives an unprecedented opportunity to compare more than 15,000 sequences and distinguish aspects of the variation that are important for immune functions, from those that are not. This thesis examines methods used to assess variation and develops new methods to both catalogue and visualise the data. Initial analysis focusses on the antigen recognition domain of the HLA class I genes and is expanded to analyse the full sequence of both the HLA class I and II genes. The analysis of non-human primate orthologs to HLA is also investigated. The analysis reveals the extensive levels of variation at both the nucleotide and protein levels and identifies mechanisms responsible for generating this variation. This allows evolutionary lineages to be identified, and the identification of a minimum set of 42 core HLA class I alleles and 47 HLA class II alleles. This also allows estimates of the total numbers of HLA alleles in the worldwide population, with the potential for 2-3 million alleles of a single gene in HLA class I and up to 1.7 million in HLA class II

Directed evolution of an organophosphate hydrolase : methyl parathion hydrolase

Organophosphates (OPs) are the most common pesticides used in agriculture. Although they can be broken down in nature, OPs pose a severe health hazard to human due to their inhibitory effect on acetylcholine, a major enzyme in nervous transmission. Therefore, detoxification of water and soil contaminated by OPs is important. One way of achieving this is by bioremediation of sites with OP-degrading enzymes. One such enzyme is methyl parathion hydrolase (MPH) isolated from Pseudomonas sp. WBC-3. MPH is a highly efficient enzyme that is capable of hydrolysing methyl parathion at near diffusion-limited rate. While MPH can hydrolyse a wide range of OPs, the substrate specificity of the enzyme was not well characterised. In order to study MPH, the enzyme was expressed and purified. In the process of MPH protein purification, proteolytic degradation was observed. Various methods, including protein engineering and optimising the purification, were employed to investigate and overcome the degradation. A protocol that allowed rapid purification of MPH was developed so that the proteolysis can be minimised. Due to initial suspicion of autoproteolysis, nickel affinity chromatography was also used in further investigations and autoproteolysis was eventually ruled out. Stability is one of the most important characteristics that define an enzyme's practical use in the industry. For MPH to be an effective bioremediator, it needs to be thermally and chemically stable. Unfortunately, MPH does not have exceptional thermostability and could benefit from extra thermostability. To achieve this, MPH was subjected to directed evolution for enhanced thermostability. In the course of characterising the mutants isolated, it was discovered that MPH expressed in E. coli had lower than expected metal content. It was also found that Zn2+ supplementation prior to activity and stability assay drastically increased the activity and stability of WT MPH. Since the evolution was performed without metal supplementation and the isolated mutant did not have enhanced stability with Zn2+ supplementation, we hypothesised that the mutant isolated was stabilised "metal independently". Another desired characteristic for a bioremediator is the ability to hydrolyse various OPs efficiently. The substrate profile characterisation of WT MPH revealed that while MPH is highly efficient towards methyl parathion, its activity towards other OPs varies. To alter and broaden the substrate specificity of MPH, structure-guided site saturation mutagenesis (SSM) on active site lining residues was performed to obtain mutants with enhanced activity towards ethyl paraoxon. Mutants with modest improvements were isolated and two rounds of DNA shuffling were performed to compound the mutations. The best mutant towards ethyl paraxon exhibited 98-fold increase in kcat/Km. Several other mutants exhibited interesting and respectable changes in their substrate profiles. One mutant with selective activity towards chlorpyrifos class substrates was found. These results highlighted the 'plasticity' of MPH active site that allow efficient hydrolysis of other OPs with only minor changes. In short, progress had been made in purifying MPH and in evolving it to be more stable - although further work is required in this area. Considerable progress had been made in identifying mutations that alter the substrate specificity of MPH

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date. This entomopathogenic bacterium produces different kinds of proteins whose specific toxicity has been shown against a wide range of insect orders, nematodes, mites, protozoa, and human cancer cells. Some of these proteins are accumulated in parasporal crystals during the sporulation phase (Cry and Cyt proteins), whereas other proteins are secreted in the vegetative phase of growth (Vip and Sip toxins). Currently, insecticidal proteins belonging to different groups (Cry and Vip3 proteins) are widely used to control insect pests and vectors both in formulated sprays and in transgenic crops (the so-called Bt crops). Despite the extensive use of these proteins in insect pest control, especially Cry and Vip3, their mode of action is not completely understood. The aim of this Special Issue was to gather information that could summarize (in the form of review papers) or expand (research papers) the knowledge of the structure and function of Bt proteins, as well as shed light on their mode of action, especially regarding the insect receptors. This subject has generated great interest, and this interest has been materialized into the 18 papers of important scientific value in the field (5 reviews and 13 research papers) that have been compiled in this issue

Landscape genetics and the effects of climate change on the population viability of declining avifauna in fragmented eucalypt woodlands of the West Australian wheatbelt

The Rufous Treecreeper (Climacteris rufa), Yellow-plumed honeyeater (Lichenostomus ornatus) and the Western Yellow Robin (Eopsaltria griseogularis) are focal species and were investigated to assess the impacts of climate change and severe habitat fragmentation on the genetics and viability of remaining populations. This study was located within the west Australian wheatbelt where 93% of the native vegetation, including 97% of the York gum, wandoo and salmon gum woodlands have been cleared for agriculture (Saunders, et al., 1989) and where climate modelling predicts hotter and dryer weather conditions (CSIRO, 2005, IOCI, 2002). The Dryandra woodlands contains the largest native vegetation remnants in the central wheatbelt with a combined area of 28 066 ha and provides habitat for a diverse assemblage of flora and fauna many of which are in Decline, Threatened or Specially Protected (NWC, 1991). The effects of habitat loss and fragmentation on the gene flow and population structure on the Rufous Treecreeper, was assessed within the Dryandra woodlands and across a range of fragmented habitat spanning approximately 100 km. Microsatellite and mitochondrial DNA data was applied to a spatial genetic and phylogeographic analysis. AMOVA shows genetic variation to be higher within populations (78%) than among populations (22%) and populations did not conform to Hardy Weinberg Equilibrium. This infers gene flow exceeds genetic drift across the region and the presence of migration between remnant habitats. Isolation by Distance was not found within Dryandra or across the region and infers the effective dispersal distance of the Rufous Treecreeper exceeds the geographical distance of sampling sites. However a Mantel’s Test found a correlation (r=0.316, p=0.004) with a distance of 28kms, within the Dryandra woodlands. A Spatial Autocorrelation of microsatellite DNA found a genetic structure of up to approximately 25kms (V=0.55) and beyond the Dryandra woodlands, shows genetic discontinuities where dispersal is more likely to occur. Landscape interpolation of genetic distance shows high genetic differentiation within the Dryandra woodlands and decreasing in an easterly direction where habitat size decreases and the distance between habitat increases. The Maximum Difference Delaunay Triangulation shows population boundaries of 12 populations within the woodlands including 3 central populations that are 1.3 km apart. A Bayesian Computation of microsatellites found a Continent-Island pattern of population structure across a distance of 85 km. Ritland’s Kinship Coefficient found dispersal patterns amongst populations within the Dryandra woodlands and a genetic neighbourhood size of about 1.7 km. Loiselle’s Kinship Coefficient found a unidirectional pattern of migration from the woodlands to smaller, isolated habitats with a maximum dispersal distance of 48 km. A Landscape Interpolation of male and female Rufous Treecreepers show a female bias in dispersal from Dryandra, with higher genetic divergence patterns in isolated remnants where habitat and nesting hollows are limiting. Rufous Treecreeper mitochondrial DNA (partial cytochrome b gene) data was applied to the Mantel’s Test and found no correlation in Dryandra or the surrounding area but did show a positive correlation at a distance of 500kms and infers at least 2 different bioregions within this distance for this species. Results from the Interpolation and Principal Component Analysis show genetic variation decreasing with increasing distance from Dryandra in an easterly and southerly direction. The highest divergence patterns were found in Dryandra, North Yilliminning, Wickepin and Commondine Reserve. Genetic patterns with high similarity were found in Dongolocking and Highbury sites south- east of Dryandra and are most likely remnant populations that once belonged to a larger, continuous population or gene pool. A geographical distribution of shared mitochondrial haplotypes found a historical range prior to land clearing of approximately 85kms. A genealogy study based on coalescence found the earliest ancestral haplotypes belonged to Dryandra, North Yilliminning and Wickepin populations and should be prioritised for long term conservation purposes. Also, novel sequences of partial cytochrome b gene for the Yellow-plumed Honeyeater and Control Region for the Western Yellow Robin was resolved for further research. The ecological niche and distribution of the Rufous Treecreeper was assessed using a distance based Redundancy Analysis (db-RDA) and a Habitat Suitability study. The db-RA found slope and aspect explained 29.16% (p= 0.04) of the genetic variation (phi) of mitochondrial DNA, which infers a relationship between landscape features and historical divergence patterns. Since old growth Eucalyptus wandoo trees are a critical habitat requirement for nesting hollows (Rose, 1993) a georeferenced (GIS) habitat suitability map was constructed from a vegetation survey (Coates, 1995) to show the distribution of E.wandoo and Rufous Treecreepers within Dryandra. Also using demographic information of the Rufous Treecreeper from a previous study (Luck, 2001) and RAMAS GIS (Akcakaya, 2002), it was estimated that the Dryandra contained enough suitable habitat for a maximum of 158 populations or 1 106 individuals. The impact of climate change on the Dryandra woodlands and the Rufous Treecreeper was measured by annual rainfall measurements (BOM, 2011), satellite imagery of tree foliage cover of each sampling site and mist net capture recapture data. This study found a declining trend in rainfall patterns and in 2010, the annual rainfall (277.4mm) fell below the minimum climatic range (350mm) of E.wandoo forests. Based on climate modelling (CSIRO, 2005) the predicted reduction rainfall will eventually will negatively impact these forests by inducing a permanent state of drought. A critical threshold of 7.73% foliage cover was found, where foliage cover does not appear to recover foliage cover beyond 11.53% after a reduction to 7.73% in 2003. This indicates a critical threshold of percentage tree canopy cover for the E. wandoo in Dryandra. A linear regression found a significant relationship (p = 0.036) between previous year’s rainfall and percentage foliage cover. This delayed response to rainfall is explained by the defence mechanisms of E.wandoo that provide this species with drought tolerance (Veneklaas & Manning, 2007). A logistic regression (GLM) found foliage cover within the same year to be a significant predictor (p = 0.039) of Rufous Treecreeper captures. Therefore declining rainfall patterns and tree canopy cover have a direct impact on the abundance of Rufous Treecreepers. The apparent survival rate estimate for the Rufous Treecreeper was 0.65 (SE 0.13) and 0.303 (SE 0.08) for the Yellow-plumed Honeyeater. Alternate modelling is required for the Yellow-plumed honeyeaters to account for their varied seasonal dispersal patterns and the Western Yellow Robin data could not be used for this demographic study because of small sample size. During 1997 and 1999 adult survival rates for Rufous Treecreepers within Dryandra was 0.76 (Luck (2001) and show the Rufous Treecreepers within the Dryandra woodlands are continuing to decline. A comparison of the two survival rates shows there is a reduction of 0.11 within an 8 year period (a single generation), which coincided with a 5.16% decrease in mean foliage cover during sampling times. This study concludes that climate change is negatively impacting E.wandoo forests and that tree foliage cover is not only a significant predictor in determining the presence of Rufous Treecreepers within the Dryandra woodlands, but also effects the short term survival and long term viability of this focal species

Structurally Primed Phage display Libraries

"Therapeutic monoclonal antibodies (mAbs) are one of the main drivers of revenue of the pharmaceutical market. Regardless of the origin and platform used, monoclonal antibodies generated against a given target may have room for improvement. Using in vitro affinity maturation libraries aims to surpass the throughput limitations of classical X-ray crystallography affinity maturation approaches, by providing a generalizable approach (or blind) that can be applied to many candidates. The current blind methods do not always assure that synergistic mutations are found and may not respect the structural constraints of the IgG molecule in question. Ideally, innovative affinity maturation methods should be generalizable to provide high-throughput results while maintaining a certain degree of specificity towards the antibody structure being considered. As such they require attention to be paid to specific regions, such as the ones likely to be in contact with the antigen, or regions that influence the antibodies’ structural integrity and overall developability.(...)"N/