The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

By applying the principles of Darwinian natural selection in the laboratory, directed evolution has become a powerful practical approach to study enzymes and optimize them to catalyze industrially relevant transformations. In this thesis, I applied this strategy to the engineering of amino acid dehydrogenases for biocatalysis of chiral amines, focusing on two crucial features for successful directed evolution experiments. A first key aspect is the development of technologies allowing the screening of large libraries of enzyme variants to explore sequence space efficiently. Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsions has recently led to the development of ultrahigh-throughput screening platforms that allow sorting of more than 106 variants per hour. So far, these microfluidic droplet sorters have relied exclusively on fluorescent readouts. To further extend the range of applications toward enzymes for which no fluorescent assays are available, I successfully developed a sorting module based on absorbance detection. Using this new module, microdroplets could be sorted based on an absorbance readout at rates of up to 1 million droplets per hour. To demonstrate the utility of this module for protein engineering, three rounds of directed evolution were performed to improve a poorly stable NAD+ dependent phenylalanine dehydrogenase (PheDH) toward its native substrate. Five hits showed increased activity (improved up to 10-fold in lysate; kcat increased >3.5-fold), soluble protein expression levels (>2.5-fold) and thermostability (Tm, 8 °C higher). To increase the sensitivity of the device (3–4 orders of magnitude lower than fluorescence assays) for detection of enzymes with limited stability and low turnovers, an extra step of growth in droplets from single cell encapsulation, followed by piconinection of substrates and lysis agents was implemented. As a result, a fivefold signal enhancement over background was achieved, for an amine dehydrogenase (AmDH) reaction shown to be undetectable in a droplet single cell assay. Second, I investigated how mutational robustness may correlate with protein stability and lead to successful hits after mutagenesis and screening. To examine this issue, I initially investigated various approaches (including ancestral resurrection and computational design) to identify stabilized PheDH variants. One such variant (dubbed Pross 4) showed increased expression levels (>3.3-fold) and thermostability (Tm, 13 °C higher) compared to the wild-type PheDH. I further compared the mutational tolerance and the hit rate between PheDH and Pross 4 by generating variant libraries focused on key active site residues and screening them for improved AmDH activity. The Pross 4 background generated 6.4 times more active variants than the PheDH background, the best hits displaying increased activity (up to 2.5-fold in lysate; kcat/KM increased up to 8-fold) compared to previously engineered AmDHs with the PheDH scaffold. In conclusion, this work highlights how directed evolution experiments could be designed for increased success rates, by combining reliable high-throughput screens with careful choice of evolutionary robust starting points.

Protein-based materials are an important area of research for various reasons. Natural protein materials such as spider silk have mechanical properties which compare favourably to artificial or inorganic materials, and in addition are biodegradable and can be produced from easily available feedstocks. It is also possible to produce materials that incorporate the functionality of a natural protein, such as ligand-binding or catalysis of reactions, thus allowing this functionality to be used in the solid rather than solution phase. Two particularly interesting components for protein-based materials are amyloid fibrils and tandem repeat proteins. Amyloid fibrils are exceptionally strong, tough, highly-ordered structures that self-assemble from a wide range of simple building blocks. Meanwhile, tandem repeat proteins are a class of proteins that act as scaffolds to mediate protein-protein interactions and are known to act as elastic springs. Unlike globular proteins, tandem repeat proteins can be designed to bind specific ligands, and their ligand-binding properties and stability can be tuned separately. This work details the synthesis and characterisation of repeat protein and amyloid fibril components for a “smart” hydrogel, the production of these gels, and their characterisation using a microfluidic method that I developed. Although amyloid fibrils have previously been decorated with functional proteins, hitherto, this has usually been done by assembling the fibrils from already-functionalised components. This approach limits the functionality to species that can survive the harsh conditions of amyloid aggregation and do not disturb fibril assembly. Therefore, a method was developed to produce amyloid fibrils that displayed an alkyne functionality on their surface to allow functional proteins or other species to be attached after assembly. This involved the design and synthesis (using solid-phase peptide chemistry) of a peptide based on the previously known TTR105-115 peptide (derived from the amyloidogenic Transthyretin protein). These fibrils were characterised by AFM and TEM and it was then shown that the assembled fibrils could be functionalised using an azide-alkyne “click” reaction. The reaction was shown to work with a variety of ligands including proteins, which were found to retain their structure and function after crosslinking to the fibril. The fibrils with ligands attached were characterised by a variety of methods including LCMS (liquid chromatography-mass spectrometry) and super-resolution optical microscopy. Next, repeat proteins were produced recombinantly containing non-natural azido amino acids at their termini. Incorporation of non-natural amino acids was carried out using a number of different methods including amber codon suppression and methionine replacement. Micron-sized hydrogels were then formed from microfluidic-generated droplets by covalently crosslinking the alkyne-functionalised fibrils with the azide-functionalised repeat proteins. The initial experiments to show proof of principle were carried out with consensus-designed repeat proteins, but repeat proteins based on natural sequences were also used to make hydrogels that could later be tested for potential uptake of peptides known to bind these proteins. These hydrogels could potentially be used for drug delivery or other applications in which a chemical response to a mechanical stimulus is desired. The mechanical properties of the hydrogels were measured using novel microfluidic devices, which were designed and fabricated using standard PDMS-based soft lithography.

En los últimos veinte años el número de genomas completos que han sido secuenciados y depositados en bancos de datos  ha crecido dramáticamente. Esta abundancia de información de secuencias ha servido de base para la creación de una disciplina llamada paleogenética. En este artículo, sin ahondar en algoritmos complejos, presentamos algunos conceptos clave para comprender cómo las proteínas han evolucionado con el tiempo. Luego ilustraremos como la paleogenética es utilizada en biotecnología. Estos ejemplos resaltan la conexión entre la química y la biología, dos disciplinas que quizás veinte años atrás parecían ser mucho más distintas que lo que parecen ser hoy. / In the last twenty years, the number of complete genomes that have been sequenced and deposited in data banks has grown dramatically. This abundance in sequence information has supported the creation of the discipline known as  paleogenetics. In this article, without going into complex algorithms, we present some key concepts for understanding how proteins have evolved in time. We then illustrate how paleogenetic analysis can be used in biotechnology. These examples highlight the connection between chemistry and biology, two disciplines that twenty years ago seemed to be more different than what they seem to be today.

The increased demand for an alternative form of fuel has raised a great interest towards exploring various metabolic pathways and enzymes in several microbial species for hydrocarbon production. In recent years, cyanobacteria have emerged as an attractive microbial host and cyanobacterial metabolic pathways were targeted for engineering to produce "drop in" fuels such as propane and butane. Whilst appealing, practicalities for producing biofuels in cyanobacteria remain challenging, requiring the identification and engineering of natural biocatalysts and their integration into metabolic processes. Cyanobacterial hydrocarbon biosynthesis arises from fatty acid metabolism involving a potential enzyme, aldehyde deformylating oxygenase (ADO), which catalyses the decarbonylation of long-chain fatty aldehydes to alkanes, mainly in the conversion of octadecanal (C17H35CHO) to heptadecane (C17H36) and formate. The substrate specificity and preferences for long-chain aldehyde by ADO necessitates a detailed kinetic and structural characterisation in order to optimise/engineer this enzyme for future biotechnological applications. Thus, the main objective was to identify a potential ADO enzyme that can be optimised for shorter chain alkane production. By studying the substrate specificity and reaction kinetics of different ADO enzymes, it was found that ADO from Prochlorococcus marinus MIT 9313 (PmADO) is a potential target for short chain alkane production. The crystal structural of PmADO was solved and further GC-MS analysis was carried out to identify the chemical origin of a mixture of long-chain fatty acid in the active site, originated from E. coli cells during recombinant over-expression and purification. It was suggested that the structure-guided protein engineering for short-chain alkane production should be carried out along with the removal of this adventitious ligand from the active site in order to increase the alkane production. Four important residues present at the entrance of the ligand-binding cavity were targeted and saturated mutagenesis was performed on PmADO to identify variants that excluded the long fatty acid ligands from the active site but have specificity and higher conversion rates for shorter chain aldehydes. This identified two variants, V41Y and A134F, with the A134F variant that not only exhibiting an improved activity and turnover value of PmADO by four-fold but also improved binding affinity for butyraldehyde by 2 times. Finally the improved variants were incorporated in a host organism (E. coli) and the possibilities for the development of a microbial platform for renewable propane synthesis based on a fermentative clostridial butanol pathway were explored. Four pathways were designed namely atoB-adhE2, atoB-TPC7, nphT7-adhE2 and nphT7-TPC7 routes, which utilise CoA intermediates selected to incorporate ADO as the terminal enzyme. When PmADO was co-expressed with these pathways, the engineered E. coli host produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed A134F variant ofPmADO with an enhanced specificity towards short-chain substrates, and (iii) including a ferredoxin-based electron supply system, the propane titre was increased up to 3.40 ± 0.19 mg/L. It was also shown that the best propane producing pathways are scalable in a 250 mL flask and in a large-scale (up to 30 L) fermentor setup. This thesis focuses on the detailed kinetics and structure-guided characterisation and engineering studies on the ADO enzyme for the development of a renewable microbial biofuel platform.

abstract: One of the greatest problems facing society today is the development of a sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they lack the critical secondary-shell interactions that enable the active site of enzymes to outperform their organometallic counterparts. To address these challenges, my work utilizes bio-hybrid systems in which artificial proteins are used to modulate the properties of organometallic catalysts. This approach couples the diversity of organometallic function with the robust nature of protein biochemistry, aiming to utilize the protein scaffold to not only enhance rates of reaction, but also to control catalytic cycles and reaction outcomes. To this end, I have used chemical biology techniques to modify natural protein structures and augment the H2 producing ability of a cobalt-catalyst by a factor of five through simple mutagenesis. Concurrently I have designed and characterized a de novo peptide that incorporates various iron sulfur clusters at discrete distances from one another, facilitating electron transfer between the two. Finally, using computational methodologies I have engineered proteins to alter the specificity of a CO2 reduction reaction. The proteins systems developed herein allow for study of protein secondary-shell interactions during catalysis, and enable structure-function relationships to be built. The complete system will be interfaced with a solar fuel cell, accepting electrons from a photosensitized dye and storing energy in chemical bonds, such as H2 or methanol. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2016

The use of enzymes as catalysts in organic synthesis constitutes an attractive alternative to conventional chemical catalysis. Enzymes are non-toxic and biodegradable and they can operate under mild reaction conditions. Furthermore, they often display high chemo-, regio- and stereoselectivity, enabling specific reactions with single product outcome. By the use of protein engineering, enzymes can be altered for the specific needs of the researcher. The major part of this thesis describes engineering of lipase A from Candida antarctica (CalA), for improved enantioselectivity in organic synthetic transformations. The first part of the thesis describes a highly combinatorial method for the introduction of mutation sites in an enzyme library. By the simultaneous introduction of nine mutations, we found an enzyme variant with five out of the nine possible mutations. This quintuple variant had an enlarged active site pocket and was enantioselective and active for our model substrate, an ibuprofen ester. This is a bulky substrate for which the wild-type enzyme shows no enantioselectivity and very poor activity. In the second part of the thesis, we continued our approach of combinatorial, focused enzyme libraries. This time we aimed at decreasing the alcohol pocket of CalA, in order to increase the enantioselectivity for small and medium-sized secondary alcohols. The enzyme library was bound on microtiter plates and screened by a transacylation reaction in organic solvent. This library yielded an enzyme variant with high enantioselectivity for the model substrate 1-phenyl ethanol, and high to excellent selectivity for other alcohols tested. Screening in organic solvent is advantageous since a potential hit is more synthetically useful. In the third part of the thesis, we used manipulated beads of controlled porosity glass (EziG™) for enzyme immobilization, and demonstrated the generality of this carrier for several enzyme classes. EziG™ allowed fast enzyme immobilization with simultaneous purification and yielded active biocatalysts in all cases. The last project describes the function of the proposed active site flap in CalA. In our study, we removed this motif. The engineered variant was compared to the wild-type enzyme by testing the amount of interfacial activation and the selectivity for certain alcohols. We showed that the motif is indeed controlling the entrance to the active site and that the flap is not part of the enantioselectivity determining machinery. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

An experimental study on randomly generated protein sequences can provide important insights into the origin and mechanism of secondary structure formation and protein folding. In this study we bring biophysical characterization of five protein sequences selected from the in silico generated library of random chains. The sequences were selected on the basis of bioinformatic analysis in order to find the candidates with the maximum potential to possess secondary structure. This study shows that the random polypeptide sequences form stable secondary structures and in some show the signs of tertiary structure, such as hydrophobic core formation and distinctive oligomerization pattern. While the work presented in this thesis is work in progress on a larger study, the data already demonstrate that unevolved protein sequence space provides a lot of potential for secondary and tertiary structure formation that awaits its characterization. Powered by TCPDF (www.tcpdf.org)

Abstract The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric protein of β-oxidation possessing 2-enoyl-CoA hydratase-1, Δ3-Δ 2-enoyl-CoA isomerase, and (3S)-hydroxyacyl-CoA dehydrogenase activities. The amino-terminal part of perMFE-1 shows sequence similarity to mitochondrial 2-enoyl-CoA hydratases (ECH-1) and Δ3-Δ 2-enoyl-CoA isomerases, and belongs to the hydratase/isomerase superfamily. Family members with known structures are either homotrimers or homohexamers. The purpose of this work was to elucidate the structure-function relationship of the rat perMFE-1 with special reference to the hydratase/isomerase fold. The structural adaptations required for binding of a long chain fatty acyl-CoA were studied with rat ECH-1 via co-crystallization with octanoyl-CoA. The crystal structure revealed that the long chain fatty acyl-CoA is bound in an extended conformation. This is possible because, a flexible loop moves aside and opens a tunnel, which traverses the subunit from the solvent space to the intertrimer space. Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by ECH-1. In the present work the enzymological properties of Glu144Ala and Glu164Ala variants of ECH-1 were studied. The catalytic activity of hydration was reduced about 2000-fold. It was also demonstrated that rat ECH-1 is capable of catalyzing isomerization. The replacement of Glu164 with alanine reduced the isomerase activity 1000-fold, confirming the role of Glu164 in both the hydratase and isomerase reactions. The structural factors favoring the hydratase over the isomerase reaction were addressed studying the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. These mutants had similar enzymatic properties to wild type, thus the catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on interaction with the Gln162 side chain. The perMFE-1 was divided into five functional domains based on amino acid sequence comparisons with the homologous proteins with known structures. Deletion variants of perMFE-1 showed that the folding of an enzymatically active amino-terminal hydratase/isomerase domain requires stabilizing interactions from the two carboxy-terminal domains of perMFE-1. The last carboxy-terminal domain is also required for the folding of the dehydrogenase part of perMFE-1. The dehydrogenase part of perMFE-1 was crystallized.

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.