Computational method to reduce the search space for directed protein evolution

We introduce a computational method to optimize the in vitro evolution of proteins. Simulating evolution with a simple model that statistically describes the fitness landscape, we find that beneficial mutations tend to occur at amino acid positions that are tolerant to substitutions, in the limit of small libraries and low mutation rates. We transform this observation into a design strategy by applying mean-field theory to a structure-based computational model to calculate each residue's structural tolerance. Thermostabilizing and activity-increasing mutations accumulated during the experimental directed evolution of subtilisin E and T4 lysozyme are strongly directed to sites identified by using this computational approach. This method can be used to predict positions where mutations are likely to lead to improvement of specific protein properties.

Molding a peptide into an RNA site by in vivo peptide evolution

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial α-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an “RNA world.”

Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the Goldfinger isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The selective-shotgun approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional linker-scanning deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.

An agent-based evolutionary approach for manufacturing system layout design

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Electrotécnica e de Computadores

Organization of pre-Variscan basement areas at the north-Gondwanan margin

Pre-Variscan basement elements of Central Europe appear in polymetamorphic domains juxtaposed through Variscan and/or Alpine tectonic events. Consequently, nomenclatures and zonations applied to Variscan and Alpine structures, respectively, cannot be valid for pre-Variscan structures. Comparing pre-Variscan relics hidden in the Variscan basement areas of Central Europe, the Alps included, large parallels between the evolution of basement areas of future Avalonia and its former peri-Gondwanan eastern prolongations (e.g. Cadomia, Intra-Alpine Terrane) become evident. Their plate-tectonic evolution from the Late Proterozoic to the Late Ordovician is interpreted as a continuous Gondwana-directed evolution. Cadomian basement, late Cadomian granitoids, late Proterozoic detrital sediments and active margin settings characterize the pre-Cambrian evolution of most of the Gondwana-derived microcontinental pieces. Also the Rheic ocean, separating Avalonia from Gondwana, should have had, at its early stages, a lateral continuation in the former eastern prolongation of peri-Gondwanan microcontinents (e.g. Cadomia, Intra-Alpine Terrane). Subduction of oceanic ridge (Proto-Tethys) triggered the break-off of Avalonia, whereas in the eastern prolongation, the presence of the ridge may have triggered the amalgamation of volcanic arcs and continental ribbons with Gondwana (Ordovician orogenic event). Renewed Gondwana-directed subduction led to the opening of Palaeo-Tethys.

Large-scale evolutionary trends of Acrochordiceratidae Arthaber, 1911 (Ammonoidea, Middle Triassic) and Cope's rule

Directed evolution of life through millions of years, such as increasing adult body size, is one of the most intriguing patterns displayed by fossil lineages. Processes and causes of such evolutionary trends are still poorly understood. Ammonoids (externally shelled marine cephalopods) are well known to have experienced repetitive morphological evolutionary trends of their adult size, shell geometry and ornamentation. This study analyses the evolutionary trends of the family Acrochordiceratidae Arthaber, 1911 from the Early to Middle Triassic (251228 Ma). Exceptionally large and bed-rock-controlled collections of this ammonoid family were obtained from strata of Anisian age (Middle Triassic) in north-west Nevada and north-east British Columbia. They enable quantitative and statistical analyses of its morphological evolutionary trends. This study demonstrates that the monophyletic clade Acrochordiceratidae underwent the classical evolute to involute evolutionary trend (i.e. increasing coiling of the shell), an increase in its shell adult size (conch diameter) and an increase in the indentation of its shell suture shape. These evolutionary trends are statistically robust and seem more or less gradual. Furthermore, they are nonrandom with the sustained shift in the mean, the minimum and the maximum of studied shell characters. These results can be classically interpreted as being constrained by the persistence and common selection pressure on this mostly anagenetic lineage characterized by relatively moderate evolutionary rates. Increasing involution of ammonites is traditionally interpreted by increasing adaptation mostly in terms of improved hydrodynamics. However, this trend in ammonoid geometry can also be explained as a case of Copes rule (increasing adult body size) instead of functional explanation of coiling, because both shell diameter and shell involution are two possible paths for ammonoids to accommodate size increase.

Antigen and superantigen presentation as defined by the MHCII-accessory proteins and associated-peptides

MHCII molecules expose a weave of antigens, which send survival or activation signals to T lymphocytes. The ongoing process of peptide binding to the MHC class II groove implicates three accessory molecules: the invariant chain, DM and DO. The invariant chain folds and directs the MHCII molecules to the endosomal pathway. Then, DM exchanges the CLIP peptide, which is a remnant of the degraded invariant chain, for peptides of better affinity. Expressed in highly specialized antigen presenting cells, DO competes with MHCII molecules for DM binding and favors the presentation of receptor-internalized antigens. Altogether, these molecules exhibit potential immunomodulatory properties that can be exploited to increase the potency of peptide vaccines. DO requires DM for maturation and to exit the ER. Interestingly, it is possible to monitor this interaction through a conformation change on DOβ that is recognized by the Mags.DO5 monoclonal antibody. Using Mags.DO5, we showed that DM stabilizes the interactions between the DO α1 and β1 chains and that DM influences DO folding in the ER. Thus, the Mags.DO5+ conformation correlates with DO egress from the ER. To further evaluate this conformation change, directed evolution was applied to DO. Of the 41 unique mutants obtained, 25% were localized at the DM-DO binding interface and 12% are at the solvent-exposed β1 domain, which is thought to be the Mags.DO5 epitope. In addition, I used the library to test the ability of HLA-DO to inhibit HLA-DM and sorted for the amount of CLIP. Interestingly, most of the mutants showed a decrease inhibitory effect, supporting the notion that the intrinsic instability of DO is a required for its function. Finally, these results support the model in which DO competes against classical MHCII molecules by sequestering DM chaperone’s function. MHCII molecules are also characterized by their ability to present superantigens, a group of bacterial or viral toxins that coerces MHCII-TCR binding in a less promiscuous fashion than what is observed in a canonical setting. While the mechanism of how bacterial superantigens form trimeric complexes with TCR and MHCII is well understood, the mouse mammary tumor virus superantigens (vSAG) are poorly defined. In the absence of a crystal structure, I chose a functional approach to examine the relation between vSAG, MHCII and TCR with the goal of uncovering the overall trimolecular architecture. I showed that TCR concomitantly binds both the MHCII α chain and the vSAG and that TCR-MHCII docking is almost canonical when coerced by vSAGs. Because many peptides may be tolerated in the MHCII groove, the pressure exerted by vSAG seems to tweak conventional TCR-MHCII interactions. Furthermore, my results demonstrate that vSAG binding to MHCII molecules is conformation-dependent and abrogated by the CLIP amino-terminal residues extending outside the peptide-binding groove. In addition, they also suggest that vSAGs cross-link adjacent MHCIIs and activate T cells via a TGXY motif.

Phage therapy: A software system for phage quantification and kinetic model inference.

One of the most serious problems of the modern medicine is the growing emergence of antibiotic resistance among pathogenic bacteria. In this circumstance, different and innovative approaches for treating infections caused by multidrug-resistant bacteria are imperatively required. Bacteriophage Therapy is one among the fascinating approaches to be taken into account. This consists of the use of bacteriophages, viruses that infect bacteria, in order to defeat specific bacterial pathogens. Phage therapy is not an innovative idea, indeed, it was widely used around the world in the 1930s and 1940s, in order to treat various infection diseases, and it is still used in Eastern Europe and the former Soviet Union. Nevertheless, Western scientists mostly lost interest in further use and study of phage therapy and abandoned it after the discovery and the spread of antibiotics. The advancement of scientific knowledge of the last years, together with the encouraging results from recent animal studies using phages to treat bacterial infections, and above all the urgent need for novel and effective antimicrobials, have given a prompt for additional rigorous researches in this field. In particular, in the laboratory of synthetic biology of the department of Life Sciences at the University of Warwick, a novel approach was adopted, starting from the original concept of phage therapy, in order to study a concrete alternative to antibiotics. The innovative idea of the project consists in the development of experimental methodologies, which allow to engineer a programmable synthetic phage system using a combination of directed evolution, automation and microfluidics. The main aim is to make “the therapeutics of tomorrow individualized, specific, and self-regulated” (Jaramillo, 2015). In this context, one of the most important key points is the Bacteriophage Quantification. Therefore, in this research work, a mathematical model describing complex dynamics occurring in biological systems involving continuous growth of bacteriophages, modulated by the performance of the host organisms, was implemented as algorithms into a working software using MATLAB. The developed program is able to predict different unknown concentrations of phages much faster than the classical overnight Plaque Assay. What is more, it gives a meaning and an explanation to the obtained data, making inference about the parameter set of the model, that are representative of the bacteriophage-host interaction.

Optimization of ethanol production by yeasts from lignocellulosic feedstocks

Ethanol from lignocellulosic feedstocks is not currently competitive with corn-based ethanol in terms of yields and commercial feasibility. Through optimization of the pretreatment and fermentation steps this could change. The overall goal of this study was to evaluate, characterize, and optimize ethanol production from lignocellulosic feedstocks by the yeasts Saccharomyces cerevisiae (strain Ethanol Red, ER) and Pichia stipitis CBS 6054. Through a series of fermentations and growth studies, P. stipitis CBS 6054 and S. cerevisiae (ER) were evaluated on their ability to produce ethanol from both single substrate (xylose and glucose) and mixed substrate (five sugars present in hemicellulose) fermentations. The yeasts were also evaluated on their ability to produce ethanol from dilute acid pretreated hydrolysate and enzymatic hydrolysate. Hardwood (aspen), softwood (balsam), and herbaceous (switchgrass) hydrolysates were also tested to determine the effect of the source of the feedstock. P. stipitis produced ethanol from 66-98% of the theoretical yield throughout the fermentation studies completed over the course of this work. S. cerevisiae (ER) was determined to not be ideal for dilute acid pretreated lignocellulose because it was not able to utilize all the sugars found in hemicellulose. S. cerevisiae (ER) was instead used to optimize enzymatic pretreated lignocellulose that contained only glucose monomers. It was able to produce ethanol from enzymatically pretreated hydrolysate but the sugar level was so low (>3 g/L) that it would not be commercially feasible. Two lignocellulosic degradation products, furfural and acetic acid, were evaluated for whether or not they had an inhibitory effect on biomass production, substrate utilization, and ethanol production by P. stipitis and S. cerevisiae (ER). It was determined that inhibition is directly related to the concentration of the inhibitor and the organism. The final phase for this thesis focused on adapting P. stipitis CBS 6054 to toxic compounds present in dilute acid pretreated hydrolysate through directed evolution. Cultures were transferred to increasing concentrations of dilute acid pretreated hydrolysate in the fermentation media. The adapted strains’ fermentation capabilities were tested against the unadapted parent strain at each hydrolysate concentration. The fermentation capabilities of the adapted strain were significantly improved over the unadapted parentstrain. On media containing 60% hydrolysate the adapted strain yielded 0.30 g_ethanol/g_sugar ± 0.033 (g/g) and the unadapted parent strain yielded 0.11 g/g ±0.028. The culture has been successfully adapted to growth on media containing 65%, 70%, 75%, and 80% hydrolysate but with below optimal ethanol yields (0.14-0.19 g/g). Cell recycle could be a viable option for improving ethanol yields in these cases. A study was conducted to determine the optimal media for production of ethanol from xylose and mixed substrate fermentations by P. stipitis. Growth, substrate utilization, and ethanol production were the three factors used to evaluate the media. The three media tested were Yeast Peptone (YP), Yeast Nitrogen Base (YNB), and Corn Steep Liquor (CSL). The ethanol yields (g/g) for each medium are as follows: YP - 0.40-0.42, YNB -0.28-.030, and CSL - 0.44-.051. The results show that media containing CSL result in slightly higher ethanol yields then other fermentation media. P. stipitis was successfully adapted to dilute acid pretreated aspen hydrolysate in increasing concentrations in order to produce higher ethanol yields compared to the unadapted parent strain. S. cerevisiae (ER) produced ethanol from enzymatic pretreated cellulose containing low concentrations of glucose (1-3g/L). These results show that fermentations of lignocellulosic feedstocks can be optimized based on the substrate and organism for increased ethanol yields.

Biochemical Conversions of Lignocellulosic Biomass for Sustainable Fuel-Ethanol Production in the Upper Midwest

Biofuels are an increasingly important component of worldwide energy supply. This research aims to understand the pathways and impacts of biofuels production, and to improve these processes to make them more efficient. In Chapter 2, a life cycle assessment (LCA) is presented for cellulosic ethanol production from five potential feedstocks of regional importance to the upper Midwest - hybrid poplar, hybrid willow, switchgrass, diverse prairie grasses, and logging residues - according to the requirements of Renewable Fuel Standard (RFS). Direct land use change emissions are included for the conversion of abandoned agricultural land to feedstock production, and computer models of the conversion process are used in order to determine the effect of varying biomass composition on overall life cycle impacts. All scenarios analyzed here result in greater than 60% reduction in greenhouse gas emissions relative to petroleum gasoline. Land use change effects were found to contribute significantly to the overall emissions for the first 20 years after plantation establishment. Chapter 3 is an investigation of the effects of biomass mixtures on overall sugar recovery from the combined processes of dilute acid pretreatment and enzymatic hydrolysis. Biomass mixtures studied were aspen, a hardwood species well suited to biochemical processing; balsam, a high-lignin softwood species, and switchgrass, an herbaceous energy crop with high ash content. A matrix of three different dilute acid pretreatment severities and three different enzyme loading levels was used to characterize interactions between pretreatment and enzymatic hydrolysis. Maximum glucose yield for any species was 70% oftheoretical for switchgrass, and maximum xylose yield was 99.7% of theoretical for aspen. Supplemental β-glucosidase increased glucose yield from enzymatic hydrolysis by an average of 15%, and total sugar recoveries for mixtures could be predicted to within 4% by linear interpolation of the pure species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.

Simulación de evolución dirigida de bacteriófagos en poblaciones de bacterias en 2D

The research group is currently developing a biological computing model to be implemented with Escherichia Coli bacteria and bacteriophages M13, but it has to be modelled and simulated before any experiment in order to reduce the amount of failed attempts, time and costs. The problem that gave rise to this project is that there are no software tools which are able to simulate the biological process underlying that com- putational model, so it needs to be developed before doing any experimental implementation. There are several software tools which can simulate most of the biological processes and bacterial interactions in which this model is based, so what needs to be done is to study those available simulation tools, compare them and choose the most appropriate in order to be improved adding the desired functionality for this design. Directed evolution is a method used in biotechnology to obtain proteins or nucleic acids with properties not found in nature. It consists of three steps: 1) creating a library of mutants, 2) selecting the mutants with the desired properties, 3) replicating the variants identified in the selection step. The new software tool will be verified by simulating the selection step of a process of directed evolution applied to bacteriophages. ---ABSRACT---El grupo de investigación está desarrollando un modelo de computación biolóogica para ser implementado con bacterias Escherichia Coli y bacteriofagos M13, aunque primero tiene que ser modelizado antes de realizar cualquier experimento, de forma que los intentos fallidos y por lo tanto los costes se verán reducidos. El problema que dio lugar a este proyecto es la ausencia de herramientas software capaces de simular el proceso biológico que subyace a este modelo de computación biológica, por lo que dicha herramienta tiene que ser desarrollada antes de realizar cualquier implementación real. Existen varias herramientas software capaces de simular la mayoría de los procesos biológicos y las interacciones entre bacterias en los que se basa este modelo, por lo que este trabajo consiste en realizar un estudio de dichas herramientas de simulación, compararlas y escoger aquella más apropiada para ser mejorada añadiendo la funcionalidad deseada para este diseño. La evolución dirigida es un método utilizado en biotecnología para obtener proteínas o ácidos nucleicos con propiedades que no se encuentran en la naturaleza. Este método consiste en tres pasos: 1) crear una librería de mutantes, 2) seleccionar los mutantes con las propiedades deseadas, 3) Replicar los mutantes deseados. La nueva herramienta software será verificada mediante la simulación de la selección de mutantes de un proceso de evolución dirigida aplicado a bacteriofagos.

A structural view of evolutionary divergence

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17°C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 Å, 1.6 Å, and 2.0 Å, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the α/β hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.

RNA-peptide fusions for the in vitro selection of peptides and proteins

Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded myc epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the in vitro selection and directed evolution of proteins.

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.

Exploring the potential of xenobiotic-metabolising enzymes as biocatalysts: Evolving designer catalysts from polyfunctional cytochrome P450 enzymes

1. Biological catalysts have the advantage of being able to catalyse chemical reactions with an often exquisite degree of regio- and stereospecificity in contrast with traditional methods of organic synthesis. 2. The cytochrome P450 enzymes involved in human drug metabolism are ideal starting materials for the development of designer biocatalysts by virtue of their catalytic versatility and extreme substrate diversity. Applications can be envisaged in fine chemical synthesis, such as in the pharmaceutical industry and bioremediation. 3. A variety of techniques of enzyme engineering are currently being applied to P450 enzymes to explore their catalytic potential. Although most studies to date have been performed with bacterial P450s, reports are now emerging of work with mammalian forms of the enzymes. 4. The present minireview will explore the rationale and general techniques for redesigning P450s, review the results obtained to date with xenobiotic-metabolising forms and discuss strategies to overcome some of the logistic problems limiting the full exploitation of these enzymes as industrial-scale biocatalysts.