Exploring protein fitness landscapes with new high-throughput technologies

The concept of a protein’s fitness landscape – an abstract space in which related sequences are close together and matched with their fitness – is a useful tool to visualize core principles of protein evolution. Acquiring a new function, for example the laboratory evolution of an enzyme to convert an industrially relevant substrate, can be understood as a stepwise climb through a fitness landscape, reaching higher fitness (or activity) with each step (or mutation). The valleys of such a space relate to the starting points of protein engineering campaigns. Understanding this area could enlighten principles of how proteins quickly adapt in nature and help to identify starting points with a high potential for evolution, a high ‘evolvability’, speeding up protein engineering. In this study, high-throughput technologies will be developed that enable the read-out of directed evolution on a large scale, tracking the exploration of the valley of a fitness landscape: the conversion of an amino acid- to amine dehydrogenase will be investigated as a model of enzyme evolvability with a drastic change of substrate specificity. A sensitive high-throughput screening assay as well as a comprehensive sequencing read-out will be required to establish the identity of selected variants during evolution. I will first generate and characterize three different but related starting points and test their initial evolvability. Stabilizing the starting point results in increased mutational robustness, broadening the range of accepted mutations. However, increased initial stability does not necessarily correlate to higher functional improvement, hinting at a nuanced view of evolvability. A sensitive high-throughput assay is necessary to verify the full potential of the starting points and study the early steps of evolution comprehensively. Broadly applicable ultrahigh-throughput assays of enzyme function, such as absorbance-activated droplet sorting, currently lack the sensitivity of more specific fluorescence-based or low-throughput counterparts. A universal approach to increase detectability in single cell-lysate microfluidic enzyme assays is established by amplifying the enzyme content per droplet more than 10-fold via homogeneous clonal cell growth. Clonal amplification enables the sensitive and precise detection of newly introduced amine dehydrogenase activities, a feat restricted in conventional assays by low initial activity and stability. To generate a truly complete view of directed evolution in a fitness landscape, however, an equally powerful sequencing read-out is necessary to identify all selected variants. Here, unique molecular identifiers are used to increase the accuracy of nanopore sequencing to levels that can reliably distinguish point mutations. I establish an inexpensive and straightforward long read amplicon sequencing workflow which is then applied to map the trajectories of two comparative long-term directed evolution campaigns. In the parallel evolution campaigns, initial beneficial mutations are exclusive to each starting point and lead to incompatible trajectories. Beneficial mutations are scarce and large improvements are unavailable until recombination occurs and a jump through the fitness landscape is realized. The recombined variant holds high evolvability and quickly evolves to take over the population and form the most successful lineages, indicating the power of recombination as a means to innovation in protein evolution. The tools established in this thesis can help protein engineers explore fitness landscapes more economically and comprehensively. Their application to mapping full trajectories of early adaptation uncovers differences in the evolvability of homologs, potentially aiding the identification of evolvable starting points as well as strategies to increase evolvability for efficient protein engineering in the future

Halogenation effects in Intramolecular Furan Diels-Alder reactions:broad scope synthetic and computational studies

For the first time a comprehensive synthetic and computational study of the effect of halogen substitution on both furan and dienophile for the intramolecular Furan Diels-Alder (IMDAF) reaction has been undertaken. Contrary to our initial expectations, halogen substitution on the dienophile was found to have a significant effect, making the reactions slower and less thermodynamically favourable. However, careful choice of the site of furan halogenation could be used to overcome dienophile halogen substitution, leading to highly functionalised cycloadducts. These reactions are thought to be controlled by the interplay of three factors: positive charge stabilisation in the transition state and product, steric effects and a dipolar interaction term identified by high level calculations. Frontier orbital effects do not appear to make a major contribution in determining the viability of these reactions, which is consistent with our analysis of calculated transition state structural data

Phenomenology of infrared smooth warped extra dimensions

We study the effect of the infrared (IR) geometry on the phenomenology of warped extra dimensions with gauge and fermion fields in the bulk. We focus in particular on a "mass gap'' metric which is AdS in the ultraviolet, but asymptotes to flat space in the IR, breaking conformal symmetry. These metrics can be dialed to approximate well the geometries arising in certain classes of warped string compactifications. We find, similar to our earlier results on the Kaluza-Klein (KK) graviton, that these metrics give rise to phenomenologically significant shifts in the separation of KK gauge modes in the mass spectrum (up to factors ~ 2) and their couplings to IR localized fields (up to factors ~ 5−10 increase). We find that, despite shifts in the spectra, the constrain

Chaos and Quantum-Classical Correspondence via Phase Space Distribution Functions

Quantum-classical correspondence in conservative chaotic Hamiltonian systems is examined using a uniform structure measure for quantal and classical phase space distribution functions. The similarities and differences between quantum and classical time-evolving distribution functions are exposed by both analytical and numerical means. The quantum-classical correspondence of low-order statistical moments is also studied. The results shed considerable light on quantum-classical correspondence.Comment: 16 pages, 5 figures, to appear in Physical Review

Quantitative Trait Locus Mapping Reveals Regions of the Maize Genome Controlling Root System Architecture.

<p>Root system architecture (RSA) is the spatial distribution of roots of individual plants. As part of a collaborative effort I adapted a gellan gum based system for imaging and phenotyping of root systems in maize. This system was first used to perform a survey of 26 distinct maize varieties of the Nested Association Mapping (NAM) population. The analysis of these data showed a large amount of variation between different RSA, in particular demonstrating tradeoffs between architectures favoring sparse, but far reaching, root networks versus those favoring small but dense root networks. To study this further I imaged and phenotyped the B73 (compact) x Ki3 (exploratory) mapping population. These data were used to map 102 quantitative trait loci (QTL). A large portion of these QTL had large, ranging from 5.48% to 23.8%. Majority of these QTLs were grouped into 9 clusters across the genome, with each cluster favoring either the compact of exploratory RSA. In summary, our study demonstrates the power of the gellan based system to locate loci controlling root system architecture of maize, by combining rapid and highly detailed imaging techniques with semi-automated computation phenotyping.</p>Dissertatio

Growth amplification in ultrahigh-throughput microdroplet screening increases sensitivity of clonal enzyme assays and minimizes phenotypic variation.

Microfluidic ultrahigh-throughput screening of enzyme activities provides information on libraries with millions of variants in a day. Each individual library member is represented by a recombinant single cell, compartmentalised in an emulsion droplet, in which an activity assay is carried out. Key to the success of this approach is the precision and sensitivity of the assay. Assay quality is most profoundly challenged when initially weak, promiscuous activities are to be enhanced in early rounds of directed evolution or when entirely novel catalysts are to be identified from metagenomic sources. Implementation of measures to widen the dynamic range of clonal assays would increase the chances of finding and generating new biocatalysts. Here, we demonstrate that the assay sensitivity and DNA recovery can be improved by orders of magnitude by growth of initially singly compartmentalised cells in microdroplets. Homogeneous cell growth is achieved by continuous oxygenation and recombinant protein expression is regulated by diffusion of an inducer from the oil phase. Reaction conditions are adjusted by directed droplet coalescence to enable full control of buffer composition and kinetic incubation time, creating level playing field conditions for library selections. The clonal amplification multiplies the product readout because more enzyme is produced per compartment. At the same time, phenotypic variation is reduced by measuring monoclonal populations rather than single cells and recovery efficiency is increased. Consequently, this workflow increases the efficiency of lysate-based microfluidic enzyme assays and will make it easier for protein engineers to identify or evolve new enzymes for applications in synthetic and chemical biology.EPSRC, H2020, ER

Phenomenology of infrared smooth warped extra dimensions

We study the effect of the infrared (IR) geometry on the phenomenology of warped extra dimensions with gauge and fermion fields in the bulk. We focus in particular on a "mass gap'' metric which is AdS in the ultraviolet, but asymptotes to flat space in the IR, breaking conformal symmetry. These metrics can be dialed to approximate well the geometries arising in certain classes of warped string compactifications. We find, similar to our earlier results on the Kaluza-Klein (KK) graviton, that these metrics give rise to phenomenologically significant shifts in the separation of KK gauge modes in the mass spectrum (up to factors ~ 2) and their couplings to IR localized fields (up to factors ~ 5−10 increase). We find that, despite shifts in the spectra, the constrain