Thesis advisor: Abhishek Chatterjee / Thesis advisor: Jia Niu / Directed Evolution has emerged as an invaluable tool for advancing protein functions in both research and industry. Our lab has pioneered a directed evolution platform in mammalian cells, utilizing an AAV delivery vector to package a DNA library and linking the biomolecule of interest to AAV production. During my tenure in Prof. Chatterjee's lab, I focused on harnessing our lab’s directed evolution platform, known as Virus-Assisted Directed Evolution of tRNA (VADER), to develop highly efficient tRNAs for genetic code expansion. Additionally, I contributed to the development of the AAV-based selection platform, termed Virus-Assisted Mutational Profiling (VAMP), as a profiling tool. Through the utilization of VAMP, I conducted comprehensive profiling of tRNA and RNA polymerase III promoter sequences. This enabled me to gain insights into regions of flexibility and evolution, ultimately leading to the construction of improved constructs with enhanced activity relative to the starting sequence. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The goals of this research were 1) to produce hydrogen in high yields from cellulosic materials and water by synthetic pathway biotranformation (SyPaB), and 2) to increase the hydrogen production rate to a level comparable to microbe-based methods (~ 5 mmol H2/L/h). Cell-free SyPaB is a new biocatalysis technology that integrates a number of enzymatic reactions from four different metabolic pathways, e.g. glucan phosphorylation, pentose phosphate pathway, gluconeogenesis, and hydrogenase-catalyzed hydrogen production, so as to release 12 mol hydrogen per mol glucose equivalent. To ensure the artificial enzymatic pathway would work for hydrogen production, thermodynamic analysis was firstly conducted, suggesting that the artificial enzymatic pathway would spontaneously release hydrogen from cellulosic materials. A kinetic model was constructed to assess the rate-limited step(s) through metabolic control analysis. Three phosphorylases, i.e. α-glucan phosphorylase, cellobiose phosphorylase, and cellodextrin phosphorylase, were cloned from a thermophile Clostridium thermocellum, and heterologously expressed in Escherichia coli, purified and characterized in detail. Finally, up to 93% of hydrogen was produced from cellulosic materials (11.2 mol H2/mol glucose equivalent). A nearly 20-fold enhancement in hydrogen production rates has been achieved by increasing the rate-limiting hydrogenase concentration, increasing the substrate loading, and elevating the reaction temperature slightly from 30 to 32°C. The hydrogen production rates were higher than those of photobiological systems and comparable to the rates reported in dark fermentations. Now the hydrogen production is limited by the low stabilities and low activities of various phosphorylases. Therefore, non-biologically based methods have been applied to prolong the stability of α-glucan phosphorylases. The catalytic potential of cellodextrin phosphorylase has been improved to degrade insoluble cellulose by fusion of a carbohydrate-binding module (CBM) family 9 from Thermotoga maritima Xyn10A. The inactivation halftime of C. thermocellum cellobiose phosphorylase has been enhanced by three-fold at 70°C via a combination of rational design and directed evolution. The phosphorylases with improved properties would work as building blocks for SyPaB and enabled large-scale enzymatic production of cellulosic hydrogen. / Ph. D.

Coenzyme nicotinamide adenine dinucleotide (NAD, including the oxidized form-- NAD+ and reduced form--NADH) and the phosphorylated form--nicotinamide adenine dinucleotide phosphate (NADP, including NADP+ and NADPH) are two of the most important biological electron carriers. Most NAD(P) dependent redox enzymes show a preference of either NADP or NAD as an electron acceptor or donor depending on their unique metabolic roles. In biocatalysis, the low enzymatic activities with unnatural coenzymes have made it difficult to replace costly NADP with economically advantageous NAD or other biomimetic coenzyme for catalysis. This is a significant challenge that must be addressed should in vitro biocatalysis be a viable option for the practical production of low-value biocommodities (i.e., biohydrogen). There is a significant need to first address the coenzyme selectivity of the NADP-dependent dehydrogenases and evolve mutated enzymes that accept biomimetic coenzymes. This is a major focus of this dissertation. Establishment of efficient screening methods to identify beneficial mutants from an enzymatic library is the most challenging task of coenzyme engineering of dehydrogenases. To fine tune the coenzyme preference of dehydrogenases to allow economical hydrogen production, we developed a double-layer Petri-dish based screening method to identify positive mutant of the Moorella thermoacetica 6PGDH (Moth6PGDH) with a more than 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This method was also used to screen the thermostable mutant of a highly active glucose 6-phosphate dehydrogenase from the mesophilic host Zymomonas mobilis. The resulting best mutant Mut 4-1 showed a more than 124-fold improvement of half-life times at 60oC without compromising the specific activity. The screening method was further upgraded for the coenzyme engineering of Thermotaga maritima 6PGDH (Tm6PGDH) on the biomimetic coenzyme NMN+. Through six-rounds of directed evolution and screening, the best mutant showed a more than 50-fold improvement in catalytic efficiency on NMN+ and a more than 6-fold increased hydrogen productivity rate from 6-phosphogluconate and NMN+ compared to those of wild-type enzyme. Together, these results demonstrated the effectiveness of screening methods developed in this research for coenzyme engineering of NAD(P) dependent dehydrogenase and efficient use of the less costly coenzyme in ivSB based hydrogen production. / Ph. D.

La L-asparaginase est une enzyme d’intérêt thérapeutique pour le traitement des leucémies aigües lymphoblastiques participant à l’hydrolyse de son substrat naturel L-asparagine conduisant à l’apoptose des cellules cancéreuses. À ce jour, la L-asparaginase d’origine bactérienne fait partie intégrante des formulations car possédant des activités catalytiques élevées mais provoquant de nombreux effets secondaires liés à une immunogénicité. Trois enzymes avec une activité Lasparaginase produites chez l’homme ont été découvertes récemment mais possèdent des activités catalytiques qui sont 1000 à 2000 fois inférieures aux enzymes d’origine bactérienne. Augmenter l’activité catalytique de ces enzymes par évolution dirigée pourraient permettre leurs utilisations en thérapeutique en plus de potentiellement participer à la réduction de l’immunogénicité chez les patients. Ces travaux de doctorat décrivent le développement d’outils pour l’expression etla détection de l’activité L-asparaginase à l’échelle d’une cellule individuelle. La L-asparaginase d’E. coli, utilisée en thérapeutique, a servi de référence et a permis de démontrer que le test AUR est le plus adapté pour la mesure de l’activité en microfluidique. L’expression de l’enzyme à partir de différents vecteurs d’expression a montré que l’expression périplasmique semble la plus adaptée pour le ciblage permettant un bon rendement et une bonne accessibilité pour le substrat. La viabilité des cellules à l’issu des mesures a été aussi démontrée. Ces outils pourront être directement utilisés pour le criblage de banques de mutants de L-asparaginase d’origine humaine en microfluidique. Les propriétés de la L-asparaginase ont aussi été utilisées pour démontrer la potentielle utilisation de billes de silice en tant que biocatalyseurs où sont confinées des bactéries. Ces billes sont des excellents supports pour la croissance de microorganismes qui peuvent rester viables au-delà d’une semaine. L’expression d’enzymes peut être induite et l’activité catalytique être aisément contrôlée en faisant varier la concentration bactérienne au sein du matériau. La combinaison de différentes populations bactériennes offre la possibilité d’effectuer des réactions en cascade. Le recyclage de ces billes pour différents cycles de réactions a également été démontré. Ces matériaux bioactifs peuvent avoir de nombreuses applications dans le domaine des biotechnologies. / L-asparaginase is an enzyme of therapeutic value for the treatment of acute lymphoblastic leukemia. Ths enzyme catalyzes the hydrolysis of L-asparagine conducting to apoptosis of cancer cells. To date, L-asparaginase of bacterial origine is used in the treatments due to high catalytic activities but causing a number of side effects linked with an immunogenicity. The human produces three enzymes with L-asparaginase activity but their catalytic activities are 1000 to 2000 times lower than the bacterial enzymes. Increase the catalytic activity of these enzymes by directed evolution could allow their uses in therapeutic in addition to potentially reduce immunogenicity in patients. This PhD work describes the development of tools for expression and detection of L-asparaginase at the single cell level for their applications in the screening of human L-asparaginase libraries in microfluidic. E. coli L-asparaginase, used in therapy, served as a reference and allowed to demonstrate that AUR assay is most suitable for measuring activity in microfluidic. Expression of the enzyme from different expression vectors showed that the periplasmic expression seems to be the most successful for screening enabling a good yield and good accessibility for the substrate. The viability of the cells following the measures has been shown. These tools might be used for the screening of mutants libraries of human L-asparaginases in microfluidic. The properties of L-asparaginase were also used to demonstrate the potential use of silica beads as biocatalysts in which bacteria are confined. These beads are excellent supports for the growth of microorganisms which may remain viable beyond one week. The expression of the enzymes may be induced and the catalytic activity can be reliably controlled by varying the concentration of bacteria within the material. The combination of various bacterial populations provides the possibility to carry out cascades reactions. The recycling of these beads for several cycles of reactions was also demonstrated. These bioactive materials have many potential applications in the field of biotechnologies.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes. / Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions.

La Chimie Verte s’engage entre autres à mettre au point des procédés plus respectueux de l’environnement et à émanciper de la pétrochimie les filières industrielles de fabrication de produits. Dans ce contexte, les enzymes représentent des alternatives de choix pour réaliser des réactions de synthèse de molécules écoresponsable à partir de la biomasse végétale. - L’endoglycocéramidase II de Rhodoccoccus sp. M-777, une glycoside-hydrolase, a été la cible d’un travail d'ingénierie du site actif afin de réorienter son activité hydrolytique vers la synthèse de polyglucosides d’alkyles, de potentiels biosurfactants. Une transglycosylase permettant d’atteindre des rendements de production de plus de 70% a été obtenue. La modélisation de la mutation permet de proposer des pistes sur les raisons de cette inversion du ratio hydrolyse/transglycosylation.- Une stratégie d'évolution dirigée a été appliquée à la féruloyle-estérase A d’Aspergillus niger pour la rendre plus résistante aux chocs thermiques et à la présence de solvants, deux propriétés requises pour utiliser cette enzyme pour des réactions de transfert dans des conditions thermodynamiquement favorables. Un catalogue d’enzymes améliorées, pour les deux propriétés, a été obtenu. L'accumulation de ces connaissances permettra de pouvoir plus efficacement rationaliser le design de biocatalyseur pour la synthèse de molécules, en accord avec les attentes de la chimie verte. / Green chemistry promotes the development of more environmentally friendly processes and the ending of polluting petrochemical industries by promoting the use of renewable resources. In this context, enzymes represent interesting alternatives catalysts for chemical transformations. Notably, they constitute tools of choice for synthesis of organic molecules from plant biomass.- Endoglycoceramidase II from Rhodococcus sp. M-777, a retaining glycoside hydrolase, was subjected to active-site remodelling in order to reorient its activity towards the synthesis of alkyl-polyglucosides, molecules with potential biosurfactant properties. Thus, an efficient transglycosylase able to reach production yield of more than 70% of alkyl-cellobiosides was obtained. A modelling study help to identify the determinants of this complete reversion of the transfer / hydrolysis ratio.- A directed evolution strategy was applied to Aspergillus niger feruloyl-esterase A, in order to make it more resistant to heat shocks and to the presence of solvents, two prerequisites to use this enzyme for transfer reactions under thermodynamically favourable conditions. This led to the establishment of a catalog of optimized enzymes for their thermostability, their solvent resistance, or both properties.These results will pave the way towards a more efficient way to rationally design biocatalysts meeting the expectations of green chemistry.

Cystic Fibrosis (CF) is a lethal autosomal recessive genetic disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR transports anions at the apical surface of epithelial membranes and functions in many areas of the body. However in CF, loss of CFTR function in the lungs is the major source of morbidity and mortality. Replacing the defective CFTR in the lungs through gene therapy has the potential to cure the disease. Recombinant adeno-associated virus (AAV) is an effective gene transfer vector and has been used extensively to deliver genes to cells in culture. A number of clinical trials using AAV have been attempted for a variety of diseases, including CF, albeit with limited success. Poor vector transduction efficiency prevents effective gene therapy. We have previously used a technique to greatly increase the transduction efficiency of AAV in human lung tissues by selecting from a library of AAVs using a directed evolution technique. However, this evolution was performed in cultured cells and did not fully represent the in vivo environment in which the AAV would be used. In 2008, a CF pig model was developed to develop a further understanding of the mechanisms of CF and CFTR function. We hypothesized that we could use directed evolution to select for a vector in vivo using the pig, allowing gene therapy studies to be conducted in a physiologically relevant model of CF. We selected a novel AAV variant, called AAV2H22, which is closely related to AAV2 but with greatly increased transduction efficiency in pig airway epithelia. AAV2H22 displayed specific tropism for pig airway epithelia and saturated cell surface receptors, indicating specific binding in those cells. We found that AAV2H22-mediated gene transfer corrected chloride and bicarbonate transport defects both in vitro and in vivo. Importantly, bicarbonate transport was sufficient to normalize pH in the airway surface liquid, resulting in increased bacterial killing likely due to increased activity of antimicrobial peptides. To investigate the mechanics of the increased transduction of AAV2H22, capsid mutants were assayed for transduction efficiency. Two of the five amino acid differences between AAV2 and AAV2H22 lie at the surface and are predicted to alter capsid binding. This is consistent with the results showing specific binding in cultured airway epithelia. This research has important implications for gene therapy and investigations using AAV2H22 will increase our understanding of the biology needed to successfully treat CF.

As a class, protein-based therapeutics offer tremendous advantages over traditional small molecule drugs. Due to their sizes and folding energies, proteins are ideal for catalyzing chemical reactions, and can bind tightly and selectively to extended target surfaces. However, due to their large size, virtually all proteins are unable to spontaneously enter cells, and as a result protein therapeutics are restricted to extracellular targets. We developed a platform for delivery of proteins to intracellular target sites by engineering the surface chemistry of a model protein, green fluorescent protein (GFP). We found that 'supercharged' cationic GFP variants (scGFPs) bind to anionic cell surface molecules and initiate endocytosis, resulting in the efficient delivery of translationally fused cargo to intracellular targets. We discovered that scGFPs, and cationic delivery reagents in general, alter endosomal trafficking in a manner proportional to both their charge and their delivery efficiency, suggesting that avoidance of endosomal maturation is a key step in the endosomal escape of delivered protein cargos. We also developed a method for encapsulation of recombinant proteins by cationic lipid delivery reagents using negatively supercharged GFP. Genetic modification technologies have matured rapidly following the discovery of protein classes with programmable DNA-binding specificities. While site-directed genetic knockout technologies are highly effective, targeted integration and repair remain comparatively inefficient. Site-specific recombinases directly catalyze strand exchange and ligation between DNA molecules, offering an approach to efficient genomic integration. However, most site-specific recombinases are not easily reprogrammable. To address this problem, we developed a genetic selection technique based on the Phage-Assisted Continuous Evolution (PACE) system, to enable the rapid evolution of recombinase proteins towards targets of interest. Using Cre recombinase as a model, the PACE system was optimized, validated, and used to evolve Cre variants with higher activity on their native loxP target site, as well as altered specificity towards a human genomic sequence within the hROSA26 locus. Finally, we developed a method for enhancing the specificity of RNA-guided nucleases by restricting activity to sites of obligate dimeric nuclease assembly. We engineered a FokI nuclease fusion to a catalytically inactivated Cas9 protein that mediates efficient modification with significantly reduced off-target activity.

Squalene synthase (SS) is an essential enzyme in eukaryotic systems responsible for an important branch point in isoprenoid metabolism that leads to sterol formation. The mechanistic complexity of SS has made it a difficult enzyme to study. The green alga Botryococcus braunii race B possesses several squalene synthase-like (SSL) enzymes that afford a unique opportunity to study the complex mechanism of triterpene biosynthesis. SSL-1 catalyzes presqualene diphosphate (PSPP) formation, which can either be converted to squalene by SSL-2 or botryococcene by SSL-3. A rationally designed mutant study of B. braunii squalene synthase (BbSS) and SSL-3 was conducted to understand structure-function relations among these enzymes. These studies revealed two amino acid positions in SSL-3 (N171, G207) that appeared to control 1’-3 versus 1’-1 linkages. The reciprocal mutations in the corresponding positions of BbSS did not convert this enzyme into a botryococcene synthase. Next, a genetic selection was developed to evolve SSL enzymes towards a fully functional SS. Previous studies have shown that Saccharomyces cerevisiae squalene synthase (ScSS) can be knocked out and although lethal, growth can be restored by providing an exogenous source of ergosterol. Additional studies have shown that successful complementation of the ScSS knockout with a non-fungal SS is possible but requires a fungal SS carboxy- terminus region. Given these observations, proof-of-principle experiments were conducted to demonstrate that SSL-SSL fusion enzymes could complement the ScSS knockout followed by construction of a mutant SSL-SSL fusion enzyme library that was screened in the ScSS knockout yeast line. From this library, mutant SSL-SSL fusion enzymes were identified that were able to complement, which demonstrated the feasibility of this approach as a genetic selection for mutant SSL enzymes. Squalene and botryococcene have valuable industrial applications in vaccine adjuvant formations, cosmetic products, and renewable energy feedstock material. Limitations in natural sources of these molecules have made heterologous production of them an important research target. Algae represent a desirable group of organisms that could be engineered to produce these metabolites because they are photosynthetic and capable of using non-arable farmland. The feasibility, approach, and progress for engineering green algae to produce squalene and botryococcene are discussed.

The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation. In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response. / Syftet med denna avhandling har varit att förbättra förmågan hos proteinet DntR att upptäcka DNT. Det långsiktiga målet har varit att använda DntR i en biosensor för att upptäcka sprängämnet TNT, som avger DNT som en ”signaturmolekyl”. En annan aspekt har varit att bättre förstå den detaljerade mekanismen för hur DntR fungerar. DntR är ett protein som binder till en viss DNA sekvens (promotor) och reglerar hur gener intill denna promotorsekvens läses av. När en inducerande molekyl som t.ex. DNT binder till DntR förändras proteinets struktur på ett sådant sätt att DntR kan aktivera transkription av de gener som finns intill promotor-sekvensen. För att mäta hur DntR reagerar på olika inducerande molekyler har DntR uttryckts i bakterien Escherichia coli, som också innehållit promotorn som DntR binder till. Intill promotorn sitter en gen som kodar för proteinet GFP. När en inducerande molekyl binder till DntR, slås avläses gfp-genen, och det fluorescerande proteinet GFP produceras. Ju mer GFP som produceras i cellerna, desto högre fluorescens kan uppmätas när cellerna analyseras.   I de artiklar som presenteras i avhandlingen har vi undersökt hur olika substitutioner i DntR proteinet påverkar specificiten och sensitiviteten och hur dessa egenskaper kan påverkas av olika experimentella faktorer. Effekten av substitutioner har relaterats till strukturdata, där bilder av hur proteinet ser ut på molekylär nivå har tagits fram. Dessutom presenteras även en bild av hur DntR förändras beroende på om inducerande molekyler är bundna eller inte. En sådan strukturbild ökar förståelsen för de mekanismer som gör att bindning av en inducerande molekyl orsakar en förändring av formen hos DntR på så sätt att avläsning av gener kan aktiveras. Vi har också använt en metod där evolutionära processer härmats för att få fram varianter av DntR med förbättrad respons till DNT. En variant med en drastisk ökning av DNT-responsen har isolerats, och dess egenskaper har karaktäriserats. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript