The enzymatic conversion of lignocellulosic biomass into fermentable sugars is a promising approach for producing renewable fuels and chemicals. However, the cost of the fungal enzymes usually employed in this process remains a significant bottleneck for manufacturing low value-added products from biomass. A potential route to increase hydrolysis yield, and thereby to reduce hydrolysis cost, would be to supplement the fungal enzymes with their lacking enzymatic activities, such as Beta-glucosidase. To produce such enzymes at a low cost, the bacterium Escherichia coli is a strong contender, owing to its ability to grow rapidly on simple and inexpensive media, and to achieve high levels of productivity. Nevertheless, there is hardly any techno-economic analysis of low-value protein production using E. coli in the literature, and, more generally, there are very few techno-economic analyses of low-value protein production ever reported, with the exception of cellulase production by Trichoderma reesei. In particular, the biotechnological application of recombinant E. coli platforms equipped with toxin-antitoxin systems to ensure plasmid stability remains largely unexplored, and its economic impact, unknown. As such, this work presents a comprehensive techno-economic analysis of the industrial production of a low-cost enzyme (Beta-glucosidase) using both E. coli BL21(DE3) and E. coli SE1, a modified BL21(DE3) strain equipped with a toxin-antitoxin system for plasmid maintenance. Moreover, this study describes the actual cloning and expression of a Beta-glucosidase enzyme into E. coli BL21(DE3) and E. coli SE1, and the development of a novel inoculum production scheme that exploits the features of the SE1 strain, based on repeatedly recycling a fraction of the inoculum cells. The results of the techno-economic analysis project an enzyme production cost of 316 US$/kg in the baseline scenario, which is considerably higher than the values reported in the literature for the fungal cocktails. The facility-dependent cost, which is strongly associated with the cost of equipment, accounts for roughly half of the estimated cost, while the cost of raw materials, especially IPTG and glucose, and the cost of consumables are all quite significant. However, the simulation of multiple scenarios and optimization measures suggest that the enzyme cost can be substantially reduced on many fronts, such as: substituting the carbon source for cheaper alternatives; reducing the amount of IPTG used for induction; using an E. coli strain capable of extracellular production; or eliminating the steps of concentration and stabilization of the enzyme, in the case of on-site enzyme utilization. Developing E. coli strains capable of high rEnzyme volumetric productivities can also significantly reduce the cost of the enzyme, up to approximately 135 US$/kg in the scenario of highest productivity. In addition, based on the experimental results with the E. coli SE1 system, an inoculum recycle strategy that avoids the need of an extensive seed train was simulated, resulting in a significant reduction of the enzyme cost. Finally, the combination of multiple process improvements could lead to an enzyme cost near 20 US$/kg of protein, which comes close to the cost of fungal cellulases and demonstrates the great biotechnological potential of recombinant E. coli platforms. / A conversão enzimática de biomassa lignocelulósica em açúcares fermentescíveis é uma via promissora para a produção de combustíveis e produtos químicos renováveis. No entanto, o custo das enzimas fúngicas usualmente empregadas nesse processo permanece um gargalo significativo para a fabricação de produtos de baixo valor agregado a partir de biomassa. Uma possível estratégia para aumentar o rendimento da hidrólise e, assim, reduzir seu custo, seria suplementar as enzimas fúngicas com suas atividades enzimáticas deficientes, tais como a enzima Beta-glicosidase. Para produzir tais enzimas a um baixo custo, a bactéria Escherichia coli é uma forte candidata, dada a sua capacidade de crescer rapidamente em meios simples e baratos e de alcançar altos níveis de produtividade. No entanto, na literatura quase não há análises técnico-econômicas de produção de proteínas de baixo valor agregado utilizando E. coli e, de forma mais geral, há muito poucas análises técnico-econômicas de produção de proteínas de baixo valor agregado publicadas, com exceção da produção de celulases por Trichoderma reesei. Em particular, a aplicação biotecnológica de plataformas recombinantes baseadas em E. coli dotadas de sistemas toxina-antitoxina para garantir a estabilidade plasmidial segue em larga medida inexplorada, e seu impacto econômico, desconhecido. Assim, este trabalho apresenta uma análise técnico-econômica abrangente da produção industrial de uma enzima de baixo custo (Beta-glicosidase) usando E. coli BL21 (DE3) e E. coli SE1, uma cepa de BL21 (DE3) modificada que possui um sistema toxina-antitoxina para manutenção plasmidial. Além disso, este estudo descreve a clonagem e expressão de uma Beta-glicosidase em E. coli BL21 (DE3) e E. coli SE1, assim como o desenvolvimento de um novo método de produção de inóculo que tira proveito das peculiaridades da linhagem SE1, baseado em reciclar repetidamente uma fração das células do inóculo. Os resultados da análise técnico-econômica apontam para um custo de produção da enzima de 316 US$/kg no cenário-base, valor consideravelmente superior àqueles relatados na literatura para os coquetéis fúngicos. Os custos de overhead da planta, que estão fortemente associados ao custo de aquisição dos equipamentos, são responsáveis por aproximadamente metade do custo total, enquanto o custo de matérias-primas, especialmente IPTG e glicose, e o custo de consumíveis são bastante significativos. Porém, a simulação de múltiplos cenários e medidas de otimização sugerem que o custo da enzima pode ser substancialmente reduzido em muitas frentes, tais como: a substituição da fonte de carbono por alternativas mais baratas; a redução da quantidade de IPTG usado para indução; a utilização de cepas capazes de produzir a enzima extracelularmente; ou a eliminação das etapas de concentração e estabilização da enzima, em caso de utilização da enzima in situ. O desenvolvimento de cepas de E. coli capazes de atingir altas produtividades volumétricas de rEnzima também pode reduzir significativamente o seu custo, chegando a US$ 135/kg no cenário de maior produtividade. Com base nos resultados experimentais com a linhagem E. coli SE1, uma estratégia de reciclagem de inóculo que evita a necessidade de um extenso trem de inoculação também foi simulada, gerando significativa diminuição do custo da enzima. Por fim, a combinação de múltiplas melhorias no processo poderia levar a um custo de enzima em torno de 20 US$/kg de proteína, valor que se aproxima do custo das celulases fúngicas e que demonstra o grande potencial biotecnológico de plataformas de expressão baseadas em E. coli recombinante.

Four type II toxin-antitoxin (TA) systems were previously identified in Burkholderia pseudomallei K96243. Type II TA toxins are able to induce cell growth arrest or death by interfering with key processes within the organism. BPSS0390-0391 is one of the TA systems previously identified and has homology to hicBA system in Acinetobacter baumannii. B. pseudomallei HicA is able to cause a reduction in the number of culturable cells after expression in E. coli. This study aimed to characterise B. pseudomallei HicA in three ways: by inducing expression of HicA in bacterial species other than E. coli, by identifying amino acids in HicA involved in toxicity and neutralisation by the antitoxin HicB and by examining the interaction of HicA with other TA antitoxins identified within B. pseudomallei genome. A broad host range plasmid encoding BPSS0390 was transformed into a range of Gram negative bacteria including Yersinia pseudotuberculosis IP32953, Vibrio vulnificus E64MW, Salmonella enterica serovar Typhimurium SL1344 and Burkholderia thailandensis E264. Expression of BPSS0390 was toxic in all bacterial species tested, despite the presence of antitoxin BPSS0391 homologues in some species. Unregulated expression in E. coli resulted in the appearance of escape mutants encoding non-toxic variants of HicA. An alanine scanning mutagenesis study of HicA identified 20 mutants where toxicity was abolished despite high levels of expression, but identified no mutants that affected TA complex formation. Finally an existing co-expression assay was modified to examine interactions between HicA and other type II TA antitoxins in B. pseudomallei. The assay revealed no interaction between HicA and non-cognate antitoxins and clarified the role of IPTG as an inhibitor of PBAD promoter on the arabinose operon.

Uma abordagem promissora para a obtenção de coquetéis enzimáticos eficientes para a etapa de hidrólise na produção de etanol de 2ª geração é o enriquecimento em termos de atividades que lhes faltam, mas podem ser obtidas através de sistemas heterólogos. O trabalho teve como objetivo avaliar o sistema toxina -antitoxina (TA) para estabilização plasmidial em E. coli na produção de expansina e endoglucanase recombinantes. Os resultados indicaram que o sistema de expressão com estabilização TA é tão eficaz quanto o dependente de antibiótico para estabilização plasmidial. Além disso, mostrou-se mais eficiente pelo fato de não permitir a sobrevivência de células sem o plasmídeo. Estudos indicaram a quantidade mínima de 0,05mM de IPTG para expressão das proteínas nesta linhagem, cerca de 20 vezes menos que a concentração usualmente aplicada no momento da indução. Sendo assim, o sistema de estabilização plasmidial TA mostrou-se uma ótima ferramenta para o desenvolvimento de uma plataforma alternativa para a produção de proteínas recombinantes. / A promising approach to obtain efficient enzyme cocktails for the hydrolysis step in the production of 2nd generation ethanol is the enrichment in terms of activities that are lacking, but can be obtained by heterologous systems. The study aimed to evaluate the toxina-antitoxin (TA) system for plasmid stabilization in E. coli in the production of recombinant expansin and endoglucanase. The results indicated that the expression system with TA stabilization is as effective as the antibiotic dependent for plasmid stabilization. Moreover, it proved to be more efficient by not allo wing the survival of cells without the plasmid. Studies have indicated the minimum amount of 0.05 mM IPTG for expression of proteins in this strain, about 20 times less than the concentration usually applied for induction. Thus, the plasmid stabilization TA system proved to be a great tool for the development of an alternative platform for producing recombinant proteins.

Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased  replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations.

Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle).<p>Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique. <p>Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Uma abordagem promissora para a obtenção de coquetéis enzimáticos eficientes para a etapa de hidrólise na produção de etanol de 2ª geração é o enriquecimento em termos de atividades que lhes faltam, mas podem ser obtidas através de sistemas heterólogos. O trabalho teve como objetivo avaliar o sistema toxina -antitoxina (TA) para estabilização plasmidial em E. coli na produção de expansina e endoglucanase recombinantes. Os resultados indicaram que o sistema de expressão com estabilização TA é tão eficaz quanto o dependente de antibiótico para estabilização plasmidial. Além disso, mostrou-se mais eficiente pelo fato de não permitir a sobrevivência de células sem o plasmídeo. Estudos indicaram a quantidade mínima de 0,05mM de IPTG para expressão das proteínas nesta linhagem, cerca de 20 vezes menos que a concentração usualmente aplicada no momento da indução. Sendo assim, o sistema de estabilização plasmidial TA mostrou-se uma ótima ferramenta para o desenvolvimento de uma plataforma alternativa para a produção de proteínas recombinantes. / A promising approach to obtain efficient enzyme cocktails for the hydrolysis step in the production of 2nd generation ethanol is the enrichment in terms of activities that are lacking, but can be obtained by heterologous systems. The study aimed to evaluate the toxina-antitoxin (TA) system for plasmid stabilization in E. coli in the production of recombinant expansin and endoglucanase. The results indicated that the expression system with TA stabilization is as effective as the antibiotic dependent for plasmid stabilization. Moreover, it proved to be more efficient by not allo wing the survival of cells without the plasmid. Studies have indicated the minimum amount of 0.05 mM IPTG for expression of proteins in this strain, about 20 times less than the concentration usually applied for induction. Thus, the plasmid stabilization TA system proved to be a great tool for the development of an alternative platform for producing recombinant proteins.

Staphylococcus aureus (S. aureus) est un pathogène humain majeur dont l’impact sur la santé publique est majoré par les phénomènes de résistance et de tolérance aux antibiotiques. Au cours de cette thèse, nous avons identifié deux nouveaux systèmes toxine-antitoxine (STA) de type I appartenant au core génome et exprimés par S. aureus nommés sprG2/SprF2, sprG3/SprF3. Ces nouveaux STA sont homologues au STA sprG1/SprF1 localisé dans un îlot de pathogénie. Nous avons montré que des interactions croisées influençant le niveau des ARN sprG et SprF pouvaient avoir lieu entre les STA homologues sprG/SprF, mais que celles-ci n’avaient pas d’impact sur la neutralisation spécifique de chaque toxine SprG par son antitoxine SprF. Nous avons démontré que les peptides encodés par sprG2 et sprG3 sont bactériostatiques, contrairement aux peptides encodés par sprG1 qui sont bactéricides. Nous avons démontré que l’expression des ARN sprG et SprF pouvait varier en réponse à des stress environnementaux comme un stress hyperosmotique ou un stress oxydatif. Pour le STA sprG1/SprF1, nous avons démontré que l’antitoxine SprF1 pourrait participer à l’entrée en persistance de S. aureus, en atténuant la traduction globale via son association aux ribosomes. Ainsi, SprF1 est le premier exemple d’une antitoxine ARN avec une double fonction de neutralisation de la toxine sprG1 via son extrémité 3’ et de fixation aux ribosomes pour atténuer la traduction de S. aureus via son extrémité 5’. Ensemble, ces travaux de thèse suggèrent que les STA sprG/SprF seraient impliqués dans l’adaptation de S. aureus au stress antibiotique ou dans l’échappement au système immunitaire. / Staphylococcus aureus (S. aureus) is a human pathogen that causes nosocomial and community-associated infections. The antibiotic resistance and tolerance of S. aureus increase its impact on public health. During my PhD thesis, we identified two novel type I toxin-antitoxin systems (TAS) located in the core genome and expressed in S. aureus named sprG2/SprF2 and sprG3/SprF3. These TAS are homologues of the sprG1/SprF1 TAS, encoded in a pathogenicity island. We showed that cross-interactions affecting sprG and SprF RNA level can occur between sprG/SprF homologous TAS, but without any impact on the specific neutralization of a sprG toxin by its SprF antitoxin. We demonstrated that overexpression of sprG2- and sprG3-encoded peptide induce bacteriostasis, as opposed to the sprG1-encoded peptides that induced S. aureus death. We showed that sprG and SprF RNA levels can be modulated by environmental triggers such as hyperosmotic and oxidative stresses. Concerning sprG1/SprF1, we demonstrated in S. aureus strain N315 that the SprF1 antitoxin could be involved in persister cells formation, by a translation attenuation mechanism via its association with the ribosomes. SprF1 is the first example of an untranslated RNA antitoxin with a dual function that neutralize sprG1 toxin and that bind to ribosome to attenuate S. aureus translation. Altogether, these thesis experiments suggest an involvement of the sprG/SprF TAS in S. aureus adaptation to antibiotic stress or in the escape of the immune system.

Les systèmes toxine-antitoxine (TA) sont de petits modules génétiques largement présents dans les génomes bactériens. Ils codent pour une petite protéine toxique et une antitoxine. Ils sont classés en six types en fonction de la nature et du mode d'action de l'antitoxine. Ce travail a porté sur l'étude du type I, pour lequel l'antitoxine est un ARN antisens qui cible l'ARNm de la toxine afin de réprimer son expression. Au cours de cette thèse, nous avons étudié le système aapA3/IsoA3, codé sur le chromosome du pathogène gastrique humain Helicobacter pylori. À ce jour, la plupart des systèmes TA ont été étudiés à l'aide de systèmes d'expression artificiels, qui ne permettent pas de caractériser la régulation transcriptionnelle ou post-transcriptionnelle. En utilisant la létalité induite par l’expression chromosomique de la toxine obtenue en absence d’antitoxine, nous avons développé une sélection génétique de mutants suppresseurs révélés par séquençage haut-débit. Cette approche, appelée FASTBAC-Seq, nous a permis de cartographier une myriade de déterminants de toxicité localisés dans les régions codantes et non codantes du gène de la toxine AapA3. En particulier, certaines de ces mutations ont révélé l'existence de tige-boucles ARN transitoires qui agissent de manière co-transcriptionnelle pour empêcher l'initiation de la traduction pendant la synthèse de l'ARNm codant pour la toxine. Ces structures ARN métastables fonctionnelles sont nécessaires pour découpler les processus de transcription et de traduction et permettent la présence de ces gènes toxiques sur le chromosome bactérien. Bien que les ARNm non traduits deviennent rapidement instables, nos travaux ont également révélé l'existence de deux tige-boucles protectrices situées aux deux extrémités de l'ARNm. Ces structures secondaires empêchent des activités exonucléolytiques agissant en 5' et 3'. Dans l’ensemble, notre travail met en évidence les conséquences de la forte pression de sélection pour limiter l'expression des toxines sous laquelle évoluent les systèmes TA. Cela nous a permis de mieux comprendre l’influence du repliement secondaire des ARNm, non seulement lors de la régulation posttranscriptionnelle, mais aussi co-transcriptionnelle de l’expression de cette famille particulière de gènes. Ces caractéristiques de régulation basées sur l'ARN peuvent être exploitées à l'avenir pour des applications biotechnologiques (p. ex., production accrue de protéines par stabilisation d'ARNm) ou biomédicales (p.ex., développement de stratégies antimicrobiennes alternatives pour l'activation de la synthèse de toxines). / Toxin-antitoxin (TA) systems are small genetic modules widely present in bacterial genomes. They usually code for a small toxic protein and its cognate antitoxin and can be classified into six types depending on the nature and mode of action of the antitoxin. This work focuses on the study of type I, for which the antitoxin is an antisense RNA that targets the toxin mRNA to inhibit its expression. We characterized the aapA3/IsoA3 system, encoded on the chromosome of the human gastric pathogen Helicobacter pylori. To date, most TAs have been studied using artificial expression systems, which do not allow the characterization of transcriptional or post-transcriptional regulation. Taking advantage of the lethality induced by the toxin chromosomal expression in the absence of antitoxin, we developed a high-throughput genetic selection of suppressor mutations revealed by Next-Generation Sequencing. This approach, named FASTBAC-Seq, allowed us to map a myriad of toxicity determinants located in both, coding and noncoding regions, of the aapA3 toxic gene. More precisely, some suppressor mutations revealed the existence of transient RNA hairpins that act co-transcriptionally to prevent translation initiation while the toxinencoding mRNA is being made. Such functional RNA metastable structures are essential to uncouple the transcription and translation processes and allow the presence of these toxic genes on bacterial chromosomes. Although untranslated mRNAs become rapidly unstable, our work also revealed the presence of two protective stem-loops located at both mRNA ends that prevent from both, 5’ and 3’ exonucleolytic activity. Altogether, our work evidenced the consequences of the strong selection pressure to silence toxin expression under which the TAs evolve, and highlighted the key role of mRNA folding in the co- and post-transcriptional regulation of this family of genes. These RNA-based regulatory mechanisms may be exploited in the future for biotechnological (e.g., increased protein production through mRNA stabilization) or biomedical (e.g., development of alternative antimicrobial strategies aiming at the activation of toxin synthesis) applications.