Evolutionary computation for predicting optimal reaction knockouts and enzyme modulation strategies

One of the main purposes of Metabolic Engineering is the quantitative prediction of cell behaviour under selected genetic modifications. These methods can then be used to support adequate strain optimization algorithms in a outer layer. The purpose of the present study is to explore methods in which dynamical models provide for phenotype simulation methods, that will be used as a basis for strain optimization algorithms to indicate enzyme under/over expression or deletion of a few reactions as to maximize the production of compounds with industrial interest. This work details the developed optimization algorithms, based on Evolutionary Computation approaches, to enhance the production of a target metabolite by finding an adequate set of reaction deletions or by changing the levels of expression of a set of enzymes. To properly evaluate the strains, the ratio of the flux value associated with the target metabolite divided by the wild-type counterpart was employed as a fitness function. The devised algorithms were applied to the maximization of Serine production by Escherichia coli, using a dynamic kinetic model of the central carbon metabolism. In this case study, the proposed algorithms reached a set of solutions with higher quality, as compared to the ones described in the literature using distinct optimization techniques.This work is funded by National Funds through the FCT - Fundacao para a Ciencia e a Tecnologia (Portuguese Foundation for Science and Technology) within project PEst-OE/EEI/UI0752/2011. The work is also partially funded by ERDF - European Regional Development Fund through the COMPETE Programme (operational programme for competitiveness) and by National Funds through the FCT within project ref. COMPETE FCOMP-01-0124- FEDER-015079. PEs work is supported by a PhD grant FCT SFRH/BD/51016/2010 from the Portuguese FCT

Novel approaches for dynamic modelling of E. coli and their application in Metabolic Engineering

PhD thesis in BioengineeringOne of the trends of modern societies is the replacement of chemical processes by biochemical ones, with new compounds being synthesized by engineered microorganisms, while some waste products are also being degraded by biotechnological means. Biotechnology holds the promise of creating a more profitable and environmental friendly industry, with a reduced number of waste products, when contrasted with the traditional chemical industry. However, in an era in which genomes are sequenced at a faster pace than ever before, and with the advent omic measurements, this information is not directly translated into the targeted design of new microorganisms, or biological processes. These experimental data in isolation do not explain how the different cell constituents interact. Reductionist approaches that dominated science in the last century study cellular entities in isolation as separate chunks, without taking into consideration interactions with other molecules. This leads to an incomplete view of biological processes, which compromises the development of new knowledge. To overcome these hurdles, a formal systems approach to Biology has been surging in the last thirty years. Systems biology can be defined as the conjugation of different fields (such as Mathematics, Computer Science, Biology), to describe formally and non-ambiguously the behavior of the different cellular systems and their interactions, using to models and simulations. Metabolic Engineering takes advantage of these formal specifications, using mathematically based methods to derive strategies to optimize the microbial metabolism, in order to achieve a desired goal, such as the increase of the production of a relevant industrial compound. In this work, we develop a mechanistic dynamic model based on ordinary differential equations, comprised by elementary mass action descriptions of each reaction, from an existing model of Escherichia coli in the literature. We also explore different calibration processes for these reaction descriptions. We also contribute to the field of strain design by utilizing evolutionary algorithms with a new representation scheme that allows to search for enzyme modulations, in continuous or discrete scales, as well as reaction knockouts, in existing dynamic metabolic models, aiming at the maximization of product yields. In the bioprocess optimization field, we extended the Dynamic Flux Balance Analysis formulation to incorporate the possibility to simulate fed-batch bioprocesses. This formulation is also enhanced with methods that possess the capacity to design feed profiles to attain a specific goal, such as maximizing the bioprocess yield or productivity. All the developed methods involved some form of sensitivity and identifiability analysis, to identify how model outputs are affected by their parameters. All the work was constructed under a modular software framework (developed during this thesis), that permits the interaction of distinct algorithms and languages, being a flexible tool to utilize in a cluster environment. The framework is available as an open-source software package, and has appeal to systems biologists describing biological processes with ordinary differential equations.Uma das tendências na nossa sociedade actual é a substituição de processos químicos por processos bioquímicos, e a síntese de novos compostos por microrganismos, bem como a degradação de resíduos por meios biotecnológicos. A Biotecnologia tem, assim, a promessa de criar uma indústria mais rentavél e mais amiga do ambiente, com um número reduzido de resíduos, contrastando com a indústria química. No entanto, numa era em que os genomas são sequenciados a um ritmo nunca visto, assim como as medições de dados ómicos, esta informação não é diretamente traduzida no desenho de estirpes microbianas ou processos biológicos. Estes dados experimentais em isolamento não explicam como os diferentes componentes celulares interagem. As abordagens reducionistas que dominaram a ciência no século passado, estudam os constituintes celulares em isolamento, como pedaços isolados, sem tomar em consideração as interacções com outras moléculas, o que traduz uma visão incompleta do mundo, que compromete o desenvolvimento de novo conhecimento. Para superar estes obstáculos, uma nova abordagem à Biologia tem emergido nos últimos trinta anos. A Biologia de Sistemas pode ser definida como a conjugação de diferentes áreas (como a Matemática, Ciência da Computação, Biologia), para descrever formalmente e de forma não ambígua o comportamento dos diferentes sistemas celulares e as suas interações utilizando a modelação. A Engenharia Metabólica tira partido destas especificações formais, utilizando métodos matemáticos para derivar estratégias tendo em vista a optimização do metabolismo de microrganismos, de forma a atingir um objetivo definido como por exemplo o aumento da produção de um composto relevante a nível industrial. Neste trabalho, desenvolvemos um modelo dinâmico mecanístico baseado em equações diferenciais ordinárias, composto por descrições ação de massas elementares para cada reacção, partindo de um modelo já existente da Escherichia coli na literatura. Utilizamos também algoritmos evolucionários com um novo esquema de representação que permite pesquisar por modulações enzimáticas, numa escala contínua ou discreta, assim como eliminar reações em modelos metabólicos existentes de forma a maximizar o rendimento ou a produtividade. Todos os métodos desenvolvidos envolveram alguma forma de análise de sensibilidade ou identifiabilidade, de forma a verificar como as saídas do modelo são afetados pelos parâmetros. Todo o trabalho foi construído de acordo com uma plataforma de software modular (desenvolvida durante esta tese) que permite a interação de algoritmos e linguagens distintos, sendo uma ferramenta flexível para utilizar em ambientes de cluster. A plataforma encontra-se disponível como um pacote de software de código aberto e tem utilidade para biólogos de sistemas que pretendam descrever processos com equações diferencias ordinárias

Stoichiometric representation of geneproteinreaction associations leverages constraint-based analysis from reaction to gene-level phenotype prediction

Genome-scale metabolic reconstructions are currently available for hundreds of organisms. Constraint-based modeling enables the analysis of the phenotypic landscape of these organisms, predicting the response to genetic and environmental perturbations. However, since constraint-based models can only describe the metabolic phenotype at the reaction level, understanding the mechanistic link between genotype and phenotype is still hampered by the complexity of gene-protein-reaction associations. We implement a model transformation that enables constraint-based methods to be applied at the gene level by explicitly accounting for the individual fluxes of enzymes (and subunits) encoded by each gene. We show how this can be applied to different kinds of constraint-based analysis: flux distribution prediction, gene essentiality analysis, random flux sampling, elementary mode analysis, transcriptomics data integration, and rational strain design. In each case we demonstrate how this approach can lead to improved phenotype predictions and a deeper understanding of the genotype-to-phenotype link. In particular, we show that a large fraction of reaction-based designs obtained by current strain design methods are not actually feasible, and show how our approach allows using the same methods to obtain feasible gene-based designs. We also show, by extensive comparison with experimental 13C-flux data, how simple reformulations of different simulation methods with gene-wise objective functions result in improved prediction accuracy. The model transformation proposed in this work enables existing constraint-based methods to be used at the gene level without modification. This automatically leverages phenotype analysis from reaction to gene level, improving the biological insight that can be obtained from genome-scale models.DM was supported by the Portuguese Foundationfor Science and Technologythrough a post-doc fellowship (ref: SFRH/BPD/111519/ 2015). This study was supported by the PortugueseFoundationfor Science and Technology (FCT) under the scope of the strategic fundingof UID/BIO/04469/2013 unitand COMPETE2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145FEDER-000004) fundedby EuropeanRegional Development Fund under the scope of Norte2020Programa Operacional Regional do Norte. This project has received fundingfrom the European Union’s Horizon 2020 research and innovation programme under grant agreementNo 686070. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Computational methods for multi-omic models of cell metabolism and their importance for theoretical computer science

To paraphrase Stan Ulam, a Polish mathematician who became a leading figure in the Manhattan Project, in this dissertation I focus not only on how computer science can help biologists, but also on how biology can inspire computer scientists. On one hand, computer science provides powerful abstraction tools for metabolic networks. Cell metabolism is the set of chemical reactions taking place in a cell, with the aim of maintaining the living state of the cell. Due to the intrinsic complexity of metabolic networks, predicting the phenotypic traits resulting from a given genotype and metabolic structure is a challenging task. To this end, mathematical models of metabolic networks, called genome-scale metabolic models, contain all known metabolic reactions in an organism and can be analyzed with computational methods. In this dissertation, I propose a set of methods to investigate models of metabolic networks. These include multi-objective optimization, sensitivity, robustness and identifiability analysis, and are applied to a set of genome-scale models. Then, I augment the framework to predict metabolic adaptation to a changing environment. The adaptation of a microorganism to new environmental conditions involves shifts in its biochemical network and in the gene expression level. However, gene expression profiles do not provide a comprehensive understanding of the cellular behavior. Examples are the cases in which similar profiles may cause different phenotypic outcomes, while different profiles may give rise to similar behaviors. In fact, my idea is to study the metabolic response to diverse environmental conditions by predicting and analyzing changes in the internal molecular environment and in the underlying multi-omic networks. I also adapt statistical and mathematical methods (including principal component analysis and hypervolume) to evaluate short term metabolic evolution and perform comparative analysis of metabolic conditions. On the other hand, my vision is that a biomolecular system can be cast as a “biological computer”, therefore providing insights into computational processes. I therefore study how computation can be performed in a biological system by proposing a map between a biological organism and the von Neumann architecture, where metabolism executes reactions mapped to instructions of a Turing machine. A Boolean string represents the genetic knockout strategy and also the executable program stored in the “memory” of the organism. I use this framework to investigate scenarios of communication among cells, gene duplication, and lateral gene transfer. Remarkably, this mapping allows estimating the computational capability of an organism, taking into account also transmission events and communication outcomes

Key challenges in designing CHO chassis platforms

Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.This work has been supported the Federal Ministry for Digital and Economic Affairs (bmwd), the Federal Ministry for Transport, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, Government of Lower Austria and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. A.H. has been supported by the Portuguese NORTE-08-5369-FSE-000053 operation. Additional funding came from the PhD program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF Project W1224) and MIT-Portugal PhD program (Bioengineering Systems). The funding agencies had no influence on the conduct of this research. Open Access Funding by the University of Vienna.info:eu-repo/semantics/publishedVersio

Algorithms and tools for in silico design of cell factories

PhD thesis in BioengineeringThe progressive shift from chemical to biotechnological processes is one of the pillars of the 21st century industrial biotechnology. Projections from the Organization for Economic Co-operation and Development estimate that, within the next two decades, about 35% of the production of chemicals will be guaranteed by biotechnological processes. The development of efficient cell-factories, capable of outperforming current chemical processes, is vital for this leap to happen. The development of constraint-based models of metabolism and rational computational strain optimization algorithms (CSOMs) hold the promise to accelerate these e orts. Here, we aim to provide an in depth and critical review of the currently available constraint-based CSOMs, their strengths and limitations, as well as to discuss future trends in the field. Then, we cover in detail the main tasks in strain design and provide a taxonomy of the main CSOMs. These are presented in detail and their features and limitations are explored. One of the identified problems is their limited offering of trade-o solutions of biotechnological objectives (e.g. overproducing desired compounds or minimizing the cost of solutions) versus cellular objectives (e.g. maximizing biomass). To tackle this problem we developed an evolutionary multi-objective (MO) framework for strain optimization capable of finding high-quality, trade-off solutions that can be explored by metabolic engineering experts. Also, the majority of the strain optimization algorithms rely on phenotype prediction methods based on debatable biological assumptions. We verified that, for a large percentage of solutions generated by a CSOM using one phenotype prediction method, the results would not hold when simulated with an alternative method. Leveraging on the previously developed framework and driven by the MO nature of this problem, we proposed a tandem approach capable of finding strain designs that comply with the assumptions of distinct phenotype prediction methods, validating the approach with multiple case studies. Finally, all the algorithms developed during this work are made available in the form of an open and flexible software framework. This framework is a powerful tool for both common users, interested in exploring the available methods, and experienced programmers which are able to easily extend it to support new features.A conversão de processos químicos em processos biotecnológicos e um dos grandes objetivos da biotecnologia industrial para o seculo XXI. A Organização para a Cooperação e Desenvolvimento Economico estima que, nas próximas duas décadas, cerca de 35% da produção de compostos químicos sejam assegurados por processos biotecnológicos. O desenvolvimento de fabricas celulares eficientes, capazes de superar o rendimento dos atuais processos químicos, é vital para que este avanço seja possível. O desenvolvimento de modelos metabólicos e algoritmos para otimização de estirpes (AOEs), e uma das grandes esperanças para acelerar estes esforços. Neste trabalho, pretendemos efetuar uma revisão aprofundada dos AOEs atuais baseados na modelação por restrições, analisar os seus pontes fortes e limitações, e discutir temas de interesse futuro na área. De seguida, estudamos em detalhe os tipos de estratégias comuns para o desenho de estirpes e formulamos uma taxonomia para os principais AOEs. Estes são avaliados em detalhe e as suas características principais são devidamente exploradas. Um dos problemas identificados prende-se com a sua oferta limitada de soluções de compromisso entre objetivos industriais (como produzir em excesso um composto de interesse, ou reduzir o custo de implementar uma solução) e objetivos celulares (como a maximização do crescimento). Para enfrentar este problema, desenvolvemos uma plataforma para otimização de estirpes baseada em computação evolucionária multiobjectivo, capaz de encontrar soluções de compromisso de elevada qualidade, que podem ser exploradas por peritos em engenharia metabólica. Para além disso, a grande maioria dos AOEs baseia-se em métodos de previsão de fenótipos que, por sua vez, são construídos sobre assunções biológicas discutíveis. Verificamos que uma grande percentagem das soluções geradas por um AOE, usando um método de previsão de fenótipos, deixaria de ser valida quando simulada com um método alternativo. Tirando partido da plataforma desenvolvida anteriormente e motivados pela natureza multiobjectivo deste problema, propusemos uma abordagem capaz de encontrar estirpes que respeitassem as assunções de diferentes métodos de previsão de fenótipos. Esta abordagem foi validada com vários casos de estudo. Por fim, todos os algoritmos desenvolvidos ao longo deste trabalho são disponibilizados sob a forma de uma aplicação de software aberto. Esta constitui uma ferramenta poderosa, tanto para utilizadores comuns interessados em explorar os métodos disponibilizados, como para programadores experientes que podem estendê-la facilmente com novos métodos.Esta investigação foi financiada pela Fundação para a Ciência e Tecnologia através da concessão de uma bolsa de doutoramento (SFRH/BD/61465/2009), co-financiada pelo POPH – QREN – Tipologia 4.1 – Formação Avançada – e comparticipado pelo Fundo Social Europeu (FSE) e por fundos nacionais do Ministério da Ciência, Tecnologia e Ensino Superior (MCTES)

Improved differential search algorithms for metabolic network optimization

The capabilities of Escherichia coli and Zymomonas mobilis to efficiently converting substrate into valuable metabolites have caught the attention of many industries. However, the production rates of these metabolites are still below the maximum threshold. Over the years, the organism strain design was improvised through the development of metabolic network that eases the process of exploiting and manipulating organism to maximize its growth rate and to maximize metabolites production. Due to the complexity of metabolic networks and multiple objectives, it is difficult to identify near-optimal knockout reactions that can maximize both objectives. This research has developed two improved modelling-optimization methods. The first method introduces a Differential Search Algorithm and Flux Balance Analysis (DSAFBA) to identify knockout reactions that maximize the production rate of desired metabolites. The latter method develops a non-dominated searching DSAFBA (ndsDSAFBA) to investigate the trade-off relationship between production rate and its growth rate by identifying knockout reactions that maximize both objectives. These methods were assessed against three metabolic networks – E.coli core model, iAF1260 and iEM439 for production of succinic acid, acetic acid and ethanol. The results revealed that the improved methods are superior to the other state-of-the-art methods in terms of production rate, growth rate and computation time. The study has demonstrated that the two improved modelling-optimization methods could be used to identify near-optimal knockout reactions that maximize production of desired metabolites as well as the organism’s growth rate within a shorter computation time

Current Challenges in Modeling Cellular Metabolism

Mathematical and computational models play an essential role in understanding the cellular metabolism. They are used as platforms to integrate current knowledge on a biological system and to systematically test and predict the effect of manipulations to such systems. The recent advances in genome sequencing techniques have facilitated the reconstruction of genome-scale metabolic networks for a wide variety of organisms from microbes to human cells. These models have been successfully used in multiple biotechnological applications. Despite these advancements, modeling cellular metabolism still presents many challenges. The aim of this Research Topic is not only to expose and consolidate the state-of-the-art in metabolic modeling approaches, but also to push this frontier beyond the current edge through the introduction of innovative solutions. The articles presented in this e-book address some of the main challenges in the field, including the integration of different modeling formalisms, the integration of heterogeneous data sources into metabolic models, explicit representation of other biological processes during phenotype simulation, and standardization efforts in the representation of metabolic models and simulation results