A systems biology approach for the characterization of metabolic bottlenecks in recombinant protein production processes

Tese de doutoramento em Engenharia Química e BiológicaThe main purpose of this thesis is to investigate the influence of recombinant protein production in the reorganization of the metabolic activities and the resulting stress-induced responses in the bacterium Escherichia coli. More specifically, the focus is on the RelA-mediated stringent response, a stress response that is triggered by the sudden lack of intracellular amino acids and that has been associated with the metabolic burden imposed by recombinant processes. To identify the main metabolic bottlenecks in recombinant biosynthetic processes, which include maintenance of recombinant DNA and expression of heterologous genes, a systematic modelling approach is proposed, capable of predicting the amino acid shortages caused by recombinant processes and the consequent activation of the RelA-dependent guanosine pentaphosphate (ppGpp) synthesis. The view of ppGpp as a primarily regulator of gene transcription has been expanded and it is now clear that the response controlled by ppGpp is crucial for cell survival during the adaptation to stressful conditions. Major advances have been achieved to understand this regulatory system governing gene expression in response to environmental growth perturbations, but so far mainly transcriptome and proteome analyses that have been applied to elucidate the stringent control mediated by ppGpp. Metabolomics analysis can provide substantial information on the impact of this stress response at the biochemical level, in particular during recombinant bioprocesses. Therefore, two metabolomics-based approaches were applied: metabolic profiling to evaluate the intracellular metabolic profiles and metabolic footprinting to estimate the profiles of extracellular metabolites. In these metabolomics studies two E. coli strains (E. coli W3110 and the isogenic ΔrelA mutant) were used to investigate the influence of recombinant processes on the host cells’ metabolism, as well as the main metabolic activities affected by the RelA activity under different growth conditions. The mutant strain presented a “relaxed” phenotype that characterized this bacterial system by an acute delay in most metabolic adaptations to transient growth conditions. Most importantly, it was shown that these mutant cells lack metabolic adjustments that are often observed after metabolic burden phenomena. Nevertheless, this cellular system presented major advantages in terms of biomass yield and productivity, which imply a remarkable improvement in recombinant bioprocesses. Thus, alleviating stress responses can be beneficial if they impair the desired quality and quantity of the recombinant product. However, it must be pointed out that this may be an alternative as long as recombinant bioprocesses are designed to achieve a finer balance between strain improvement strategies and culturing conditions.O trabalho realizado no âmbito desta tese teve como principal finalidade a avaliação das alterações metabólicas relacionadas com a produção de proteínas recombinantes em células bacterianas de Escherichia coli e a consequente activação de respostas de stress. Foi evidenciada a resposta restringente promovida pela actividade da enzima RelA, dado ser uma das principais respostas de stress induzidas pelo decréscimo da quantidade de aminoácidos disponíveis no meio intracelular como consequência da expressão de proteínas recombinantes. As diferenças na composição em aminoácidos entre as proteínas da biomassa e recombinantes, têm sido apontadas como principais causas para o desequilíbrio metabólico que conduz à exaustão de alguns metabolitos, nomeadamente de aminoácidos. De modo a explorar estes fenómenos e avaliar o impacto dos processos recombinantes no metabolismo das células hospedeiras, foi proposto um modelo matemático capaz de identificar pontos de estrangulamento na rede metabólica. Estes locais correspondem a vias metabólicas que apresentam limitações na capacidade catalítica e que serão essenciais para compensar o consumo desproporcionado de aminoácidos levado a cabo pela síntese de proteínas recombinantes. Associado a este fenómeno foi considerada a descrição da síntese de nucleótidos guanosina pentafosfato (ppGpp) induzida pela escassez de aminoácidos no meio intracelular. O reconhecimento deste nucleótido como um regulador fundamental na transcrição da informação genética tem sido amplamente descrito e tornou-se evidente que as respostas celulares controladas pelo ppGpp são determinantes para a sobrevivência e adaptação dos organismos a condições adversas. Neste sentido, vários estudos foram elaborados para elucidar o papel do ppGpp no controlo destas respostas de stress e nas alterações fisiológicas que advêm destes processos, nomeadamente ao nível do metabolismo. A análise do metaboloma, em comparação com o transcriptoma ou o proteoma, é capaz de capturar de forma mais directa a relação entre as actividades metabólicas e a fisiologia dos organismos, designadamente em sistema recombinantes. Neste trabalho foram elaborados alguns estudos em que se aplicaram duas abordagens de análise metabolómica distintas: profiling metabólico, que se refere à análise do perfil de metabolitos intracelulares; e footprinting metabólico, que se refere à análise do perfil de metabolitos extracelulares. Nestes estudos foram usadas duas estirpes de E. coli (W3110 e a estirpe isogénica com mutação no gene relA) clonadas com um vector de expressão pTRC-His- AcGP1 que codifica a proteína verde fluorescente AcGFP1, derivada da proteína AcGFP da Aequorea coerulescens. Foram avaliadas as principais alterações metabólicas provocadas pela indução da produção de proteína recombinante e pela actividade catalítica da enzima RelA em diversas condições de crescimento. Comparando os perfis metabólicos das duas estirpes, foram estimadas várias diferenças significativas que se podem revelar críticas durante processos recombinantes. A estirpe mutante revelou um comportamento típico de um fenótipo “relaxado”, que é caracterizado por um retardamento significativo na adaptação do metabolismo a alterações nas condições de crescimento. Não obstante, a estirpe mutante exibiu melhores resultados em termos de rendimento em biomassa e produtividade, o que representa uma vantagem notável para a aplicação destes sistemas bacterianos recombinantes ao nível industrial. Em resumo, a restrição de respostas de stress pode trazer benefícios se a qualidade e quantidade do produto estiverem em causa, mas deve salientar-se que não é uma solução absoluta, sendo que as condições de processamento devem ser também levadas em consideração na implementação destes bioprocessos

Cell Engineering and Molecular Pharming for Biopharmaceuticals

Biopharmaceuticals are often produced by recombinant E. coli or mammalian cell lines. This is usually achieved by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of producer cells. Naturally, each recombinant production system has its own unique advantages and disadvantages. This paper examines the current practices, developments, and future trends in the production of biopharmaceuticals. Platform technologies for rapid screening and analyses of biosystems are reviewed. Strategies to improve productivity via metabolic and integrated engineering are also highlighted

Stringent response of Escherichia coli: revisiting the bibliome using literature mining

Understanding the mechanisms responsible for cellular responses depends on the systematic collection and analysis of information on the main biological concepts involved. Indeed, the identification of biologically relevant concepts in free text, namely genes, tRNAs, mRNAs, gene products and small molecules, is crucial to capture the structure and functioning of different responses. Results In this work, we review literature reports on the study of the stringent response in Escherichia coli. Rather than undertaking the development of a highly specialised literature mining approach, we investigate the suitability of concept recognition and statistical analysis of concept occurrence as means to highlight the concepts that are most likely to be biologically engaged during this response. The co-occurrence analysis of core concepts in this stringent response, i.e. the (p)ppGpp nucleotides with gene products was also inspected and suggest that besides the enzymes RelA and SpoT that control the basal levels of (p)ppGpp nucleotides, many other proteins have a key role in this response. Functional enrichment analysis revealed that basic cellular processes such as metabolism, transcriptional and translational regulation are central, but other stress-associated responses might be elicited during the stringent response. In addition, the identification of less annotated concepts revealed that some (p)ppGpp-induced functional activities are still overlooked in most reviews. Conclusions In this paper we applied a literature mining approach that offers a more comprehensive analysis of the stringent response in E. coli. The compilation of relevant biological entities to this stress response and the assessment of their functional roles provided a more systematic understanding of this cellular response. Overlooked regulatory entities, such as transcriptional regulators, were found to play a role in this stress response. Moreover, the involvement of other stress-associated concepts demonstrates the complexity of this cellular response

IST Austria Thesis

One of the most striking hallmarks of the eukaryotic cell is the presence of intracellular vesicles and organelles. Each of these membrane-enclosed compartments has a distinct composition of lipids and proteins, which is essential for accurate membrane traffic and homeostasis. Interestingly, their biochemical identities are achieved with the help of small GTPases of the Rab family, which cycle between GDP- and GTP-bound forms on the selected membrane surface. While this activity switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate decisive signal propagation in space and time is unclear. In my PhD thesis, I present in vitro reconstitution experiments with theoretical modeling to systematically study a minimal Rab5 activation network from bottom-up. We find that positive feedback based on known molecular interactions gives rise to bistable GTPase activity switching on system’s scale. Furthermore, we determine that collective transition near the critical point is intrinsically stochastic and provide evidence that the inactive Rab5 abundance on the membrane can shape the network response. Finally, we demonstrate that collective switching can spread on the lipid bilayer as a traveling activation wave, representing a possible emergent activity pattern in endosomal maturation. Together, our findings reveal new insights into the self-organization properties of signaling networks away from chemical equilibrium. Our work highlights the importance of systematic characterization of biochemical systems in well-defined physiological conditions. This way, we were able to answer long-standing open questions in the field and close the gap between regulatory processes on a molecular scale and emergent responses on system’s level

Directed Evolution of Stabilized Peptides with Bacterial Display

Interactions between proteins govern cellular and the body’s states, including aberrant interactions found in diseases such as in cancers and infections. Small molecule drugs are not ideal in targeting these interactions as their size generally prevents efficient blocking of contacts over large surface areas. Antibodies and related biologics have seen clinical success in the past few decades and can block large surfaces but are typically limited to extracellular targets. Intermediate-size peptides have the potential to bridge this gap, with the ability to target large surface areas inside the cell. Peptide stapling, by chemically linking two or more amino acid residues, can confer affinity improvements, resistance to degradation, and better biological transport properties. As such, stapled peptides show promise as next-generation therapeutics. Unfortunately, existing methods to screen sequence and stapling locations suffer from numerous disadvantages including limited search space, lack of real-time monitoring of selections, and difficulty in incorporating the non-canonical amino acids used for amino acid stapling. In this dissertation, I describe my research on stapled peptide discovery with bacterial incorporation of non-canonical amino acids. To screen stapled peptides of the type desired, we incorporated azidohomoalanine (AHA) into surface displayed peptides, enabling an in situ ‘click’ chemistry reaction to bridge two turns of an alpha helical (i, i+7) amino acid library for directed evolution. Using the p53-MDM2 interaction as a model target, we developed peptides that block MDM2 degradation of the tumor suppressor protein p53, an interaction that is dysregulated in a sizeable fraction of cancers. We generated and displayed a stapled peptide library on the bacterial cell surface with fixed residues for stabilization and binding requirements, while randomizing the remaining amino acids. After multiple rounds of selection, clones were sequenced and characterized. The dissociation constants of the peptide-MDM2 interaction were measured on both the bacterial cell surface by flow cytometry and in solution by bio-layer interferometry. The highest affinity variant, named SPD-M6-V1 with sequence VCDFXCYWNDLXGY (dissociation constant = 1.8 nM; X = azidohomoalanine) was selected for structural characterization by NMR spectroscopy, revealing a bicyclic disulfide and double click-constrained peptide. Sequencing showed that peptides with two cysteines were highly enriched, further suggesting that the MDM2-binding conformation was enforced with a disulfide bond. In addition, SPD-M6-V1 was the most protease-resistant peptide from the library that we tested. Next, we stapled the displayed peptide library with chemically distinct linkers and screened each library separately. We performed deep sequencing to better understand the relationship between amino acid sequence and linker identity in contributing to high affinity MDM2 binding. We found that both linker-specific and linker-agnostic (i.e. MDM2-specific) mutations were enhanced. Finally, we developed a dual-channel, sequential labeling selection strategy to discriminate between high-display, low-affinity peptides and low-display, high-affinity peptides, two categories that would ordinarily overlap in a typical one-color screen in the absence of an independent display marker. In summary, this thesis develops the chemical tools to screen libraries of stabilized peptides on the bacterial cell surface and applies these techniques to select stabilized alpha helices that disrupt the p53-MDM2 interaction.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163094/1/tejasn_1.pd

Adaptation and diversification of <i>Escherichia coli</i> K12 MC1000 in a complex environment

Complexiteit is inherent aan natuurlijke zowel als industriele habitats. Voorgaand wetenschappelijk werk heeft duidelijk het flexibele (genotypische en fenotypische) aanpassingsvermogen van microorganismen aan complexiteit laten zien. De meeste experimenten zijn echter onder relatief simpele (uniforme) omstandigheden verricht. Derhalve richtte het huidige onderzoek zich op bacteriele evolutie in complex groeimedium, waarbij de nadruk lag op de analyse van de mate van genetische / fysiologische diversifiering naar fitnessverhoging en nichedifferentiatie.De lange-termijn-aanpassingen (~1000 generaties) van E. coli K12 MC1000 in Luria-Bertani (LB) bouillon onder aerobe, wisselende en anaerobe condities werden geevalueerd. Verschillende genetische wegen resulteerden in aanpassingen en een aantal metabole routes waren geactiveerd. De veranderingen waren reproduceerbaar met betrekking tot geselecteerde functie, waarbij habitat de belangrijkste selector bleek. Een specific response werd waargenomen in de genen die betrokken waren bij het metabolisme van galactose (galR en galE). Daarbij werd een hoge mate van heterogeniteit gevonden tussen en binnen populaties. De verschillende fenotypische aanpassingen gaven ook aan dat parallele responses werden gestuurd door de verschillende genomen.De analyse van polymorfismen binnen een geevolueerde population toonde het bestaan van twee metabole and interactieve typen aan. Derhalve werd het voorkomen van additionele specifieke fenotypische eigenschappen (stress resistentie en metabole eigenschappen) bevestigd. De interactieve en stabiele coexistentie van deze vormen liet trade-offs in groei- en stress-eigenschappen tussen de vormen, en nicheverdeling, zien. De complexiteit van de habitat kan derhalve de vorming van aangepaste coexisterende vormen sturen.An inherent characteristic of natural as well as industrial environments is complexity. Scientific studies have revealed the flexible genetic and phenotypic capacities of microorganisms to cope with such complexity. However, most experiments have been conceptually simple, as they compare populations adapting to rather uniform environments. Therefore, the present work addressed bacterial evolution in a complex environment. The emphasis was on unraveling the level of diversification in respect of the genetic and physiological changes that the organism underwent, which allowed it to either acquire superior fitness or occupy a different niche.The long-term (~1000 generations) adaptive responses of E. coli K12 MC1000 in Luria-Bertani (LB) broth under aerobic, fluctuating and anaerobic conditions were evaluated. Several genetic solutions led to adaptation and a number of metabolic pathways were activated. Reproducibility of changes on genuine targets of selection was observed in parallel populations, suggesting a response triggered by medium. A specific response occurred in genes related to the metabolisms of galactose (galR and galE). Considerable heterogeneity was also found between and within populations. Differential phenotypic outcomes, suggested that parallel responses were affected by differing genomic backgrounds. Analysis of the polymorphisms in one evolved population revealed the existence of two main metabolic and interactive types. The emergence of additional specific phenotypic traits (stress resistance and metabolic properties) was confirmed. The interactive and stable coexistence of these forms revealed the presence of trade-offs and niche partitioning. The complexity of the environment has the potential to trigger the establishment of adapted and coexisting forms

Adaptation and diversification of <i>Escherichia coli</i> K12 MC1000 in a complex environment

Complexiteit is inherent aan natuurlijke zowel als industriele habitats. Voorgaand wetenschappelijk werk heeft duidelijk het flexibele (genotypische en fenotypische) aanpassingsvermogen van microorganismen aan complexiteit laten zien. De meeste experimenten zijn echter onder relatief simpele (uniforme) omstandigheden verricht. Derhalve richtte het huidige onderzoek zich op bacteriele evolutie in complex groeimedium, waarbij de nadruk lag op de analyse van de mate van genetische / fysiologische diversifiering naar fitnessverhoging en nichedifferentiatie.De lange-termijn-aanpassingen (~1000 generaties) van E. coli K12 MC1000 in Luria-Bertani (LB) bouillon onder aerobe, wisselende en anaerobe condities werden geevalueerd. Verschillende genetische wegen resulteerden in aanpassingen en een aantal metabole routes waren geactiveerd. De veranderingen waren reproduceerbaar met betrekking tot geselecteerde functie, waarbij habitat de belangrijkste selector bleek. Een specific response werd waargenomen in de genen die betrokken waren bij het metabolisme van galactose (galR en galE). Daarbij werd een hoge mate van heterogeniteit gevonden tussen en binnen populaties. De verschillende fenotypische aanpassingen gaven ook aan dat parallele responses werden gestuurd door de verschillende genomen.De analyse van polymorfismen binnen een geevolueerde population toonde het bestaan van twee metabole and interactieve typen aan. Derhalve werd het voorkomen van additionele specifieke fenotypische eigenschappen (stress resistentie en metabole eigenschappen) bevestigd. De interactieve en stabiele coexistentie van deze vormen liet trade-offs in groei- en stress-eigenschappen tussen de vormen, en nicheverdeling, zien. De complexiteit van de habitat kan derhalve de vorming van aangepaste coexisterende vormen sturen.An inherent characteristic of natural as well as industrial environments is complexity. Scientific studies have revealed the flexible genetic and phenotypic capacities of microorganisms to cope with such complexity. However, most experiments have been conceptually simple, as they compare populations adapting to rather uniform environments. Therefore, the present work addressed bacterial evolution in a complex environment. The emphasis was on unraveling the level of diversification in respect of the genetic and physiological changes that the organism underwent, which allowed it to either acquire superior fitness or occupy a different niche.The long-term (~1000 generations) adaptive responses of E. coli K12 MC1000 in Luria-Bertani (LB) broth under aerobic, fluctuating and anaerobic conditions were evaluated. Several genetic solutions led to adaptation and a number of metabolic pathways were activated. Reproducibility of changes on genuine targets of selection was observed in parallel populations, suggesting a response triggered by medium. A specific response occurred in genes related to the metabolisms of galactose (galR and galE). Considerable heterogeneity was also found between and within populations. Differential phenotypic outcomes, suggested that parallel responses were affected by differing genomic backgrounds. Analysis of the polymorphisms in one evolved population revealed the existence of two main metabolic and interactive types. The emergence of additional specific phenotypic traits (stress resistance and metabolic properties) was confirmed. The interactive and stable coexistence of these forms revealed the presence of trade-offs and niche partitioning. The complexity of the environment has the potential to trigger the establishment of adapted and coexisting forms

Control Theory for Synthetic Biology: Recent Advances in System Characterization, Control Design, and Controller Implementation for Synthetic Biology

Living organisms are differentiated by their genetic material-millions to billions of DNA bases encoding thousands of genes. These genes are translated into a vast array of proteins, many of which have functions that are still unknown. Previously, it was believed that simply knowing the genetic sequence of an organism would be the key to unlocking all understanding. However, as DNA sequencing technology has become affordable, it has become clear that living cells are governed by complex, multilayered networks of gene regulation that cannot be deduced from sequence alone. Synthetic biology as a field might best be characterized as a learn-by-building approach, in which scientists attempt to engineer molecular pathways that do not exist in nature. In doing so, they test the limits of both natural and engineered organisms