Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-08-13T13:54:28Z No. of bitstreams: 1 texto completo.pdf: 1444484 bytes, checksum: b30acd53edaeef546b799098f956c6a4 (MD5) / Made available in DSpace on 2018-08-13T13:54:28Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1444484 bytes, checksum: b30acd53edaeef546b799098f956c6a4 (MD5) Previous issue date: 2017-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Chlamydomonas reinhardtii (filo Chlorophyta) é um organismo modelo amplamente utilizado para estudos de carência de nitrogênio (N), no intuito de avaliar a acumulação de lipídeos, alterações na bioenergética e a regulação da fotossíntese. As clorófitas podem incorporar N inorgânico e orgânico, e a fonte de N utilizada influencia no padrão de acúmulo de lipídeos. As fontes inorgânicas mais frequentemente disponíveis são amônio (NH 4+ ), nitrato (NO 3- ) e nitrito (NO 2- ); enquanto as fontes orgânicas mais comuns são purinas, ureia e aminoácidos. O uso de ureia por outras clorófitas usualmente promove melhoras no crescimento, na produção de lipídeos e biomassa quando comparado ao NH 4+ . Contudo, para C. reinhardtii faltam informações mais específicas de como o seu metabolismo responde à assimilação de ureia. Assim, neste trabalho estudou-se o metabolismo e o crescimento de C. reinhardtii CC125 em ureia como única fonte nitrogenada, bem como diferentes proporções de ureia combinada com NH 4+ . Alíquotas de C. reinhardtii CC125 foram mantidas em crescimento mixotrófico em meio TAP (Tris- Acetato-Fosfato), sob temperatura de 24±2 ° C, fotoperíodo de 16:8 h (luz: escuro), 90 μmol fótons m -2 s -1 e constante agitação a 110 rpm. Cinco tratamentos foram testados: (i) 100% NH 4+ (0,4 g L -1 NH 4 Cl); (ii) 25% ureia (0,3 g L -1 NH 4 Cl e 0,11 g L -1 ureia); (iii) 50% ureia (0,2 g L -1 NH 4 Cl e 0,21 g L -1 ureia); (iv) 75% ureia (0,1 g L -1 NH 4 Cl e 0,32 g L -1 ureia); e (v) 100% ureia (0,42 g L -1 ureia). Os tratamentos foram avaliados na fase logarítmica (LOG) após 50 h de cultivo e na fase estacionária (STA), após 240 h de crescimento. O número de células, área celular, biomassa livre de cinzas, os conteúdos de clorofila a e b, aminoácidos, amido, proteínas, carboidratos e lipídeos foram determinados nas fases LOG e STA. O perfil metabólico e de ácidos graxos foram determinados na fase STA. O crescimento observado nos meios com ureia foi similar ao 100% NH 4+ , quando comparadas as curvas e os parâmetros de crescimento. O número de células foi superior nos tratamentos com menos ureia, tanto na fase LOG quanto STA. Na fase LOG não houve diferença nos totais de clorofila a e b, aminoácidos e proteínas. Na fase STA os níveis de clorofila total bem como clorofila a foram maiores nos tratamentos 75% e 100% ureia. Os totais de clorofila b e proteínas solúveis totais aumentaram com o aumento de ureia. Os níveis de carboidratos, na fase LOG, foram maiores no tratamento 100% ureia. Na fase STA, os maiores valores obtidos foram dos tratamentos 100% ureia e 100% NH 4+ . O tratamento 100% ureia produziu maior quantidade de lipídeos nas duas fases de crescimento analisadas. A quantificação de açúcares indicou que alguns dissacarídeos aumentaram nos tratamentos com mais de 75% de ureia. Dos 14 ácidos orgânicos quantificados, 11 diminuíram no tratamento com 25% de ureia, assim como muitos aminoácidos. Quatro intermediários do ciclo TCA (citrato, isocitrato, succinato e malato) aumentaram no tratamento 100% ureia. O perfil de ácidos graxos foi modificado pelas concentrações de NH 4+ e ureia em C. reinhardtii: O total de ácidos graxos saturados (ΣSFA) aumentou com o aumento de ureia, porém o total de ácidos graxos monoinsaturados diminuiu com o aumento de ureia. O ácido graxo mais abundante foi o ácido palmítico (C16:0) e apresentou uma tendência ao aumento com o aumento de ureia no meio. A porcentagem de ácido oleico (C18:1 w8) decresceu com o amento de ureia, enquanto as porcentagens do ácido linoleico (C18:2 w6) dobrou nos tratamentos que continha ureia. Logo, nossos dados indicam que quanto maior a disponibilidade de ureia, maiores são as mudanças no metabolismo de Carbono (C) e N, sem, contudo, promover drásticas mudanças no crescimento. Além disso, nossos resultados sugerem que a ureia pode fornecer C adicional para a biossíntese, alterando a razão C:N no meio e promovendo mudanças no total de lipídeos e no perfil de ácidos graxos produzidos. / Chlamydomonas reinhardtii (phylum Chlorophyta) as a model organism for nitrogen (N) starvation studies in order to evaluate lipid accumulation, changes in bioenergetics and the regulation of photosynthesis. Chlorophytes can incorporate N in inorganic or organic form and the source of N influences the accumulation of lipids. The most frequently available inorganic sources are Ammonium (NH 4+ ), Nitrate (NO 3- ) and Nitrite (NO 2- ) and the most common organic sources are purines, urea and amino acids. Use of urea in others chlorophytes usually promotes enhancement in both growth, lipid production and biomass when compared to NH 4+ . However, for C. reinhardtii there is a lack of more specific information on how their metabolism responds to the assimilation of N organic sources. Thus, in this work we studied the metabolism and growth of C. reinhardtii CC125 in urea as the only N source as well as combined with NH 4+ . Aliquots of C. reinhardtii CC125 were maintained in mixotrophic growth in TAP (Tris-Acetate-Phosphate) medium under temperature between 24±2 ° C, photoperiod of 16:8 h (light: dark), 90 μmol photons m -2 s -1 and constant shaking of 110 rpm. Five treatments were carried out: (i) 100% NH 4+ (0,4 g L -1 NH 4 Cl); (ii) 25% urea (0,3 g L -1 NH 4 Cl and 0,11 g L -1 urea); (iii) 50% urea (0,2 g L -1 NH 4 Cl and 0,21 g L -1 urea); (iv) 75% urea (0,1 g L -1 NH 4 Cl and 0,32 g L -1 urea); and (v) 100% urea (0,42 g L -1 urea). The treatments were evaluated in the logarithmic phase (LOG), after 50 hours of growth and in the stationary phase (STA), after 240 hours of growth. The number of cells, cell area, ash-free dry weight, chlorophyll a and b contents, amino acids, starch, proteins, total carbohydrates and lipids were determined at LOG and STA phases. The metabolic profile and fatty acids profile was determined at STA phase. The growth observed in medium with urea was similar to 100% NH 4+ by comparing growth curves and kinetic growth. The number of cells after in LOG and STA phase was higher in treatments with lower percentage of urea. Determination of total chlorophyll a and b, free amino acids and proteins showed no differences between treatments in the LOG phase. At STA phase level of total chlorophyll as well as chlorophyll a were higher in 75 and 100% urea. The levels of chlorophyll b and total soluble proteins increased with increasing urea. The levels of carbohydrates, in the LOG phase, were higher in 100% urea treatment. In the STA phase, the highest values were observed for 100% urea and 100% NH 4+ treatments. 100% urea treatment produced more lipids than other treatments in the two growth phases. Quantification of sugars indicated that disaccharides increased in treatments with more than 75% urea. Out of 14 quantified organic acids, 11 decreased in the treatment with 25% of urea as well many amino acids. Four intermediates of TCA cycle (citrate, isocitrate, succinate and malate) increased in the treatment with 100% urea. The FAMEs profile of C. reinhardtii was altered by concentration of NH 4+ and urea: Total saturated fatty acids (ΣSFA) increase with amount of urea; however, total monounsaturated fatty acids (ΣMUFA) decrease with amount of urea. The most abundant fatty acid observed was palmitic acid (C16:0) which there is a tendency to increased with the amount of urea in the medium. The percentage of oleic acid (C18:1 w8) decreased with amount of urea, while percentages of linoleic acid (C18:2 w6) doubled in treatments containing urea. Thus, our data indicate that higher the availability of urea, higher are the Carbon (C) and N metabolism changes, without, however, promoting drastic changes in growth. In addition, our results suggest that urea might also provide additional C, altering C:N ratio in medium and lead changes in lipids and total fatty acid production and profile.

The extent to which evolution is repeatable has important implications. If evolution is highly repeatable, the trajectories and outcomes of evolution in different lineages will always be the same. On the other hand, if evolution is not repeatable, then trajectories and outcomes will be diverse. Thus, the repeatability of evolution affects our understanding of the nature of biodiversity and can inform the extent to which evolutionary theory can be used to make predictions. The repeatability of evolution depends on the relative contribution of selection, chance, and history. To determine what factors affect the importance of chance and history during evolution, I propagated replicated populations of the unicellular green alga Chlamydomonas reinhardtii in controlled environments. I measured the change in fitness after a few hundred generations and determined how much variation had arisen among replicate populations and among populations with different histories. I applied a similar approach to study the importance of history in extinctions, and measured rates of extinction in populations with different histories. I found that evolution is much less repeatable in small than in large populations because history is more constraining and selection less efficient in small than in large populations. There is also a significant effect of sex and recombination on the repeatability of evolution at the fitness level, but this effect is highly dependent on the environment of selection. Sex can increase the importance of chance or history in some environments, but lower their importance in others, thereby leading to convergence or divergence depending on the environment. Thirdly, I found that the importance of history during evolution does not appear to come from the accumulation of past evolutionary selection pressures, but rather comes from only the most recent selection pressure as it determines genetic correlations for growth between different environments and the amount of genetic variance. Finally, I found that extinction risks are extremely high during continuous environmental deterioration, although a history of sexual reproduction and phenotypic plasticity play an important role in adaptation. By focusing not solely on the effect of treatments on mean trait values, but also on the variance that arises in our evolution experiments, we can gain a better understanding of the contribution that chance and history make to evolution. The repeatability of evolution can therefore inform us about the adaptive vs. stochastic nature of the diversity we see today, and about the specificity or generality of evolutionary outcomes.

As the worldwide demand for recombinant proteins and valuable metabolites continues to grow, and as the biological toolset at our disposal continues to expand, the development of novel, robust, and effective platforms for the production of these bioproducts represents an area of ever-increasing interest. Although many such bioprocesses are currently economically viable, many more, though holding considerable promise, remain uncompetitive. The development of novel, more productive systems increases the versatility and industrial applications of bioprocesses. The work described in this thesis explores several aspects of bioprocessing, both on the upstream side, concerned with the development of novel recombinant protein expression platforms or the isolation of novel genes with products possessing characteristics of interest, and on the downstream side, through the improvement of fermentation-based bioprocesses. Thirty-six homoplasmic recombinant strains of the microalga Chlamydomonas reinhardtii were developed having integrated genes for phytase or xylanase under the control of psbA and psbD promoters, codon optimized using novel algorithms, at two different genetic loci, in chloroplasts, to be used as novel animal feed additives. Enzyme production was characterized, and results, when compared to other published work in this field, may provide insight into the factors impacting recombinant protein production in microalgae. Using a “bio-prospecting approach”, the microflora of the digestive tract of a Canadian beaver was screened for cellulase-producing microorganisms. Although the screening approach did successfully identify a novel β-glucosidase gene from an isolated strain of Bacillus thuringiensis, the sequence was not significantly different from those already characterized. Two bioprocessing studies were performed to improve recombinant protein production in Pichia pastoris. In the first, the composition of standard Basal Salt Medium (BSM) was systematically optimized for the production of recombinant phytase, and the optimized media produced significantly more enzyme than the standard one, while also containing significantly reduced concentrations of KH2PO4 and MgSO4·7H2O (27.9 g/l and 4.8 g/l respectively), lowering the price of process inputs. The second was based on the screening of unconventional carbon sources for candidates that could sustain the growth and enzyme production using the same P. pastoris strain. Fructose and ethanol have shown to be viable alternatives to glucose or glycerol as sole carbon sources, and provide flexibility in terms of process design.

The eyespot of the single-celled alga Chlamydomonas aids the cell in detecting the direction of light in the environment. The complex assembly and asymmetric placement of the eyespot provides a model to ask questions about assembly and asymmetric placement of organelles. Understanding the mechanisms that underlie assembly and asymmetric placement of the eyespot can be applied more broadly to their functions in other eukaryotic organisms. This study sought to understand the role of a key protein in those processes, Channelrhodopsin-1 (ChR1). ChR1 was found to localize along the entire length of the D4 rootlet from the region around the daughter basal body to the eyespot. ChR1 was found to primarily localize to the plasma membrane side of the D4, suggesting that ChR1 was being pulled through the plasma membrane from the region around the basal bodies to the eyespot. Further, ChR1 was found to be able to localize to the eyespot even with the truncation of the large cytoplasmic C-terminal domain, suggesting that ChR1 is able to complex with another protein that is being trafficked to the eyespot. One such protein was thought to be ChR2, the other light-activated ion channel localized to the eyespot. Efforts to isolate a mutation in ChR2 were unsuccessful. Initial efforts were made in this dissertation to perform proteomic studies of ChR1 and identify its interacting partners. ChR1 is not the master regulator of either placement or assembly of the eyespot, but work in this study lays the groundwork to further investigate transport of ChR1 and interacting proteins to the eyespot and their role in assembly of the eyespot.

This thesis studies the effects of various concentrations of sodium chloride on growth and the content of secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii. The total content of phenolic compounds, flavonoids and total antioxidant capacity was analyzed by spectrophotometry. In the context of growth, also the content of photosynthetic pigments was determined. Further, the amount of selected metabolites was determined by HPLC/MS-MS. Cultivation of microalgae with increased NaCl inhibited cell growth and production of photosynthetic pigments. Conversely, higher levels of NaCl have proven to stimulate the synthesis of complete phenolic compounds and flavonoids. Similarly, the amount of phenolic acids was significantly influenced by the effect of increasing NaCl concentration, while the total antioxidant capacity of the microalgae also increased. These results confirm the involvement of phenolic compounds in the defense mechanism of unicellular algae Ch. reinhardtii against the observed stress factor.

Les Streptophyta et Chlorophyta forment le règne des plantes vertes. Les Chlorophyta se divisent en quatre classes: Prasinophyceae, Trebouxiophyceae, Ulvophyceae et Chlorophyceae. Le séquençage des génomes entiers d' organelles et l'étude de ces génomes permettent d'observer les tendances évolutives par rapport à leurs ancêtres et d' identifier les liens qui unissent les différents taxons. L' algue chlorophycéenne Chlamydomonas moewusii, membre de l'ordre des Chlamydomonadales, a un génome chloroplastique très grand et très réarrangé présentant de nombreuses séquences répétées. Quant à son génome mitochondrial, il est petit, compact et contient peu de gènes. Dans les deux cas, ces deux génomes contiennent peu de traits communs avec les génomes homologues apparentés, si ce n' est la divergence qu' ils ont par rapport avec leur ancêtre commun. La présente étude sur C. moewusii a permis de compléter et de vérifier les études précédentes, ainsi que d'apporter plus d'information sur les insertions, uniques au génome chloroplastique

La capacité des microalgues à accumuler des quantités importantes de lipides de réserve font de ces organismes de bons candidats pour envisager une production durable de biocarburants (biodiesel). Cependant, des verrous d’ordre technologique et biologique persistent avant d’atteindre une production économiquement viable. Dans le but de mieux comprendre les mécanismes et biosynthèse et d’accumulation des lipides chez les microalgues et de proposer des voies d’amélioration biotechnologiques, nous avons développé deux approches expérimentales complémentaires en utilisant la microalgue Chlamydomonas reinhardtii comme modèle. La première a été de caractériser par des techniques de protéomique et de lipidomique la composition des gouttelettes lipidiques s’accumulant en réponse à une carence en azote. Les données de protéomique nous ont permis de montrer que les gouttelettes lipidiques étaient des structures cellulaires dynamiques impliquées non seulement dans le stockage, mais aussi dans la biosynthèse, la remobilisation et le « trafficking » des lipides. Les protéines identifiées au cours de cette étude nous fournissent des gènes cibles d’intérêt pour mieux comprendre les voies de biosynthèse des triacylglycérols et accroître l’accumulation d’huile. La seconde approche, de génétique formelle, a consisté à rechercher puis à caractériser des mutants isolés à partir d’une banque de mutants d’insertion de C. reinhardtii. Deux mutants d’intérêt, l’un affecté dans la composition en acides gras (crfad7) et l’autre capable d’accumuler des lipides en l’absence de stress (coa1, pour constitutive oil accumulator 1), ont été isolés. / The ability of microalgae to accumulate high amounts of reserve lipids makes these organisms good candidates for the production of sustainable biofuel (biodiesel). However, both technological and biological bottlenecks remain to be overcome before profitable production is reached. With the aim to better understand lipid metabolic pathways in microalgae and further propose new strategies for biotechnological improvement, we have developed two complementary experimental approaches in the model microalga Chlamydomonas reinhardtii. As a first approach, we performed a proteomic and lipidomic characterization of oil bodies isolated from nitrogen-deprived cells. Based on proteomic data, we have concluded that oil bodies are dynamic structures involved not only in the storage, but also in oil biosynthesis, degradation and lipid homeostasis. The proteins identified in this study should provide useful targets for genetic studies aiming at increasing our understanding of triacylglycerol synthesis and further improve intracellular oil accumulation. The second approach, based on the development of a forward genetic screen, aimed at searching and further characterizing mutants isolated from a C. reinhardtii insertion library. Two mutants of interest, one affected in the fatty acid composition (crfad7), the other (coa1, for constitutive oil accumulator 1) able to accumulate reserve lipids in the absence of stress, have been isolated. The crfad7 mutant, affected in the expression of the unique ω3 fatty acid desaturase present in the C. reinhardtii genome, has been complemented and subjected to extensive phenotypical characterization.

Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa. / This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass.

O objetivo desta tese foi explorar o sistema de produção de proteínas heterólogas em microalga com ênfase em Chlamydomonas reinhardtii por meio de: (1) desenvolvimento de um fotobiorreator tubular fechado de escala laboratorial, utilizando técnicas de manufatura digital; (2) avaliação de 7 diferentes proteínas fluorescentes (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato e mCherry), como sistema reporter de secreção de proteínas em microalga; (3) avaliação do fotobiorreator desenvolvido utilizando cultivo de cepas recombinantes; (4) desenvolvimento de novos peptídeos sinais para secreção de proteínas em C. reinhardtii; (5) avaliação da produção de um biofármaco (hialuronidase) em microalgas, por meio da expressão de duas isoenzimas codificadas pelos genes HYA1 e SPAM1 em C. reinhardtii. O fotobiorreator tubular foi avaliado quanto a sua capacidade de resistir ao processo de esterilização por autoclavação e seu desempenho por meio do cultivo de cepa recombinante secretando mCherry. A fluorescência das proteínas fluorescentes foi medida por leitor de placas de fluorescência e visualizada intracelularmente por microscopia confocal de fluorescência. A atividade de hialuronidase foi determinada através de um ensaio enzimático turbidimétrico. O desenvolvimento do fotobiorreator resultou em um sistema fechado resistente a autoclavação, com capacidade de cultivo de cepas recombinantes de C. reinhardtii. Esse fotobiorreator proporcionou uma produtividade máxima de 10 mg/L.d de mCherry da cepa recombinante em sistema fechado, com velocidade específica de crescimento máxima de 1,27 d-1 para a cepa recombinante testada. Todas as proteínas fluorescentes avaliadas apresentaram capacidade de secreção por C. reinhardtii, com diferentes níveis de interferências em sua medição, permitindo a escolha da mCherry como proteína reporter. Entre os peptídeos sinais avaliados (quatro descritos na literatura - BiP, ARS1, CAH1 e IBP1 - e seis preditos), o peptídeo predito \"SP5\" foi o que apresentou maior capacidade de secreção, determinado por níveis de fluorescência no sobrenadante. A avaliação dos peptídeos sinais constatou a necessidade de explorar o desenvolvimento de sistemas de expressão (e.g. vetores de expressão) aliados a análises computacionais, como o SignalP 4.0. Por último, os dados desse estudo mostram que C. reinhardtii transformadas com o vetor de expressão foi capaz de produzir as duas isoformas de hialuronidase em sua forma ativa, evidenciando a capacidade desse sistema para a produção de biofármacos. Portanto, nesta tese o sistema de expressão de proteínas heterólogas baseado em microalgas foi explorado, atingindo os objetivos propostos. O fotobiorreator desenvolvido tem a capacidade de esterilização em escala laboratorial (1) e em cultivo com cepa recombinante propiciou elevada produtividade (3). As proteínas vermelhas fluorescentes apresentaram-se como as proteínas com menores interferências para estudos de secreção em C. reinhardtii (2). Além disso, o peptídeo predito SP5 apresentou o melhor desempenho na secreção de proteínas (4) e o vetor de expressão empregado permitiu a identificação de cepas produtoras de biofármaco hialuronidase (5). Portanto, o sistema de produção de proteínas heterólogas por microalgas é um sistema promissor e poderá permitir, utilizando sistemas de secreção, obter proteínas de alto valor comercial a baixos custos, empregando a secreção e técnicas de cultivo como a fermentação extrativa. / In this thesis, the heterologous protein production in microalgae with emphasis on Chlamydomonas reinhardtii was explored through: (1) development of a laboratory scale closed tubular photobioreactor using digital manufacturing techniques; (2) evaluation of different fluorescent proteins (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato and mCherry) as a reporter system for protein secretion in microalgae (3) evaluation of photobioreactor developed using recombinant strains culture; (4) development of new signals peptides for protein secretion in C. reinhardtii (5) expression evaluation of a biopharmaceutical (Hyaluronidase) in microalgae, through the expression of two isoenzymes encoded by the HYA1 and SPAM1 genes in C. reinhardtii. The tubular photobioreactor was evaluated for its ability to resist sterilization process by autoclaving and its performance by culturing recombinant strain secreting mCherry. Fluorescence of fluorescent proteins was measured by fluorescence plate reader and observed intracellularly by confocal fluorescence microscopy. The hyaluronidase activity was determined by a turbidimetric enzymatic assay. The development of the photobioreactor resulted in a closed system resistant to autoclaving, capable of culturing recombinant strains of C. reinhardtii. This recombinant strain achieved a maximum productivity of 10 mg/L.day of mcherry in the closed system, with a maximum growth rate of 1.27 d-1 for the recombinant strain tested. All the fluorescent proteins evaluated had C. reinhardtii secretion capacity, with different interference levels in their measurement, allowing the selection of mCherry as a reporter protein. Among the evaluated peptides (four described in the literature - BiP, ARS1, CAH1 and IBP1 - and six predicted), the predicted peptide \"SP5\" was the one that presented greater capacity of secretion, determined by levels of fluorescence in the supernatant. The results of this study point out the need to explore the development of biological systems (i.e., expression vectors) allied to computational analysis. Finally, the data from this study showed that C. reinhardtii could produce the two isoforms of hyaluronidase in its active form, evidencing the capacity of this system to produce biopharmaceuticals. Therefore, in this thesis the heterologous protein expression system based on microalgae was explored, reaching the proposed objectives. The developed photobioreator has sterilization capabilityin laboratorial scale (1) and in culture with recombinant strain had high productivity (3). The red fluorescent proteins was found as the most suitable proteins for studies of secretion in C. reinhardtii with lower interference levels(2). In addition, the predicted SP5 peptide showed the best performance in protein secretion (4) and the expression vector employed allowed the identification of strains producing biopharmaceutical hyaluronidase (5). Therefore, the system of heterologous proteins production by microalgae is promising and will allow, using secretion systems, to obtain proteins of high commercial value at low costs, using secretion and cultivation techniques such as extractive fermentation.

Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa. / This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass.