Study of molecular mechanisms to increase carbon use efficiency in microalgae

In order to better understand alga\u2019s biology and allow to design biotechnological approaches to improve biomass yield, in this PhD thesis we investigated the molecular mechanism involved in the microalgae carbon use efficiency. In the Chapter 1 we studied the model algae C. reinhardtii. In section A Photosystem II assembly were investigated. Indeed, no detailed studies of the assembly factors of PSII have been performed. In this work we focus on a putative assembly factor of the CP43 subunit, called LPA2 (low PSII accumulation 2), previously identified in A. thaliana. A candidate lpa2 gene in C. reinhardtii was identified by homology and its role was studied in vivo thank to a CRISPR-cas9 mutant. The data collected demonstrated that LPA2 protein is involved in both de novo biogenesis and repair of PSII. In the section B the relationship between chloroplast and mitochondrion metabolism was explored studying a mutant of C. reinhardtii knockout for a mitochondrial transcription factor. Previous studies demonstrated that the mutation affect the mitochondrial respiration and resulted in a light-sensitive phenotype. In this work we investigated how a mutation affecting the mitochondrial respiration perturbed light acclimation of the strain. Chapter 2 regards two species of Chlorella genus. In the section A we elucidated the molecular basis of the improved growth and biomass yield in mixotrophic condition, where the cross-talk between chloroplast and mitochondria metabolism is essential for efficient biomass production. C. sorokiniana is able to combine an autotrophic metabolism with the utilization of reduced carbon source (mixotrophic condition). The de novo assembly transcriptome allowed to identify the regulation of several genes involved in control of carbon flux. In section B genetic basis of the highly productive phenotype of C. vulgaris in low light vs. high light condition was examined. Nuclear and organelle genomes were obtained combining short-reads Illumina, long PacBio reads and Bionano optical mapping, allowing to assembly a near-chromosome scale genome of 14 scaffolds and the two complete circular organelle genomes. All the genes encoding for photosynthetic subunit, as well as, genes involved in the key metabolic pathway were identified. In section C the two Chlorella species was compared for their adaptation to high CO2 level. In C. sorokiniana in 3% CO2 were observed several reorganizations of the photosynthetic machinery leading to an improved carbon fixation, while mitochondrial respiration was essentially unaffected. Instead, in C. vulgaris the 3% CO2 induced an improved uptake of reducing power by chloroplast leading to a reduced mitochondrial respiration. Chapter 3 is focused on the marine algae. In the section A was isolated a chemical mutant of N. gaditana with a reduction chlorophyll content per cell combined with increased lipids productivity. The mutant did not show an increased biomass accumulation but induced an increased lipid content, a class of macromolecules with a higher energy content per gram. This is in any case an indication of improved light energy conversion in line with an improved light penetration in the photobioreactor and more homogenous light availability due to the reduced chlorophyll content per cell in the mutant. Moreover, thank to Illumina sequencing, we found putative genes responsible of the observed phenotype. In the section B cells of T. weissflogii were grown together with an artificial cyanine molecular antenna (Cy5) that extends the absorbance range of the photosynthetic apparatus exploiting light energy in the orange spectral region. The dye was incorporate in the algae increasing light dependent growth, oxygen and biomass production. Time-resolved spectroscopy data indicates that a Cy5-chlorophyll a energy transfer mechanism happen, compatible with a FRET process

Biomass from microalgae: The potential of domestication towards sustainable biofactories

Interest in bulk biomass from microalgae, for the extraction of high-value nutraceuticals, bio-products, animal feed and as a source of renewable fuels, is high. Advantages of microalgal vs. plant biomass production include higher yield, use of non-arable land, recovery of nutrients from wastewater, efficient carbon capture and faster development of new domesticated strains. Moreover, adaptation to a wide range of environmental conditions evolved a great genetic diversity within this polyphyletic group, making microalgae a rich source of interesting and useful metabolites. Microalgae have the potential to satisfy many global demands; however, realization of this potential requires a decrease of the current production costs. Average productivity of the most common industrial strains is far lower than maximal theoretical estimations, suggesting that identification of factors limiting biomass yield and removing bottlenecks are pivotal in domestication strategies aimed to make algal-derived bio-products profitable on the industrial scale. In particular, the light-to-biomass conversion efficiency represents a major constraint to finally fill the gap between theoretical and industrial productivity. In this respect, recent results suggest that significant yield enhancement is feasible. Full realization of this potential requires further advances in cultivation techniques, together with genetic manipulation of both algal physiology and metabolic networks, to maximize the efficiency with which solar energy is converted into biomass and bio-products. In this review, we draft the molecular events of photosynthesis which regulate the conversion of light into biomass, and discuss how these can be targeted to enhance productivity through mutagenesis, strain selection or genetic engineering. We outline major successes reached, and promising strategies to achieving significant contributions to future microalgae-based biotechnology

Identification and study of lipid metabolism genes by qPCR in Rubisco mutants of Chlamydomonas reinhardtii

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017Chlamydomonas reinhardtii é uma microalga modelo tendo sido usada para estudos da fotossíntese e do metabolismo energético dos lípidos. É o organismo eucariótico em que pela primeira vez foi possível modificar os genes das subunidades da enzima chave da fotossíntese, a ribulose-1,5-bisfosfato carboxilase/oxigenase (Rubisco). A enzima, além de assimilar o CO2 atmosférico, também funciona como oxigenase, catalisando a primeira reação da via fotorrespiratória, o que a torna um ponto fundamental do metabolismo do carbono. A Rubisco é uma das mais abundantes enzimas na natureza, com uma massa molecular total de 560 kDa. É constituída por 16 subunidades, 8 subunidades grandes (LSU, 55 kDa) e 8 subunidades pequenas (SSU, 15 kDa). Após a formação da enzima, as 8 subunidades grandes estão dispostas em dímeros em torno de um canal central que influencia a eficiência do centro catalítico, a especificidade CO2 / O2 e a estabilidade de toda a enzima. Contudo, pouco se sabe sobre a expressão génica e metabolismo energético dos lípidos em organismos com uma alteração profunda no canal central da Rubisco. A enzima também está presente no ciclo de Calvin-Benson, onde catalisa a primeira reação da via fotorrespiratória, o que o torna um ponto fundamental do metabolismo fotossintético do carbono. A síntese de Rubisco consome uma parcela substancial de recursos nutricionais de plantas e a sua degradação afeta a redistribuição de nutrientes dentro do organismo, o que significa que a Rubisco pode ter uma função de armazenamento em condições fisiológicas específicas, como falta de enxofre (S) ou azoto (N). Um dos focos principais para o uso desta alga tem sido a sua capacidade de produzir H2 e biodiesel, que ocorre especificamente em condições de stress ambiental, como por exemplo falta de azoto. Uma das consequências deste tipo de stress em C. reinhardtii é a síntese e acumulação de triacilglicerol (TAG), que é um precursor do biodiesel. Além disso outros processos como a fotossíntese e a produção/concentração de proteínas e clorofilas também sofrem alterações. Tendo tudo isto em conta, o foco desta investigação foi a caracterização fenotípica de um mutante de C. reinhardtii, I58W3, que na cadeia polipeptídica da subunidade pequena da Rubisco poSSUi uma mutação, três triptofanos (W) substituem uma isoleucina (I) na posição do resíduo aminoácido 58. Pelos estudos de cristalografia da Rubisco de Chlamydomonas o resíduo 58 localiza-se na entrada do canal central da estrutura da holoenzima. No presente foram realizados vários testes ao longo do crescimento das culturas controlo e I58W3 que visaram caracterizar este mutante a nível fotossintético, através de leituras da concentração de O2 nas células e respetivas taxas fotossintéticas e de respiração, assim como leituras acerca da eficiência do aparelho fotossintético através de PAM; foram determinadas as concentrações de clorofilas e de proteínas ao longo do tempo das culturas em células mutantes e no respetivo controlo, bem como a monitorização do peso seco das culturas e do número de células ao longo dos cinco dias de crescimento; foi feita com detalhe uma análise da composição lipídica através de cromatografia gasosa e cromatografia por camada fina, assim como leituras por espetrofotómetro de amostras das culturas coradas com Red Nile. Os níveis de Rubisco em I58W3 foram comparados com o controlo por eletroforese de proteínas e immunoblotting. Finalmente, alguns genes de interesse foram estudados, tentando comparar a sua expressão em células da cultura controlo com células da cultura I58W3. Adicionou-se mais um fator de comparação a este estudo, a carência de azoto no meio de cultura, por ser indutor da síntese de lípidos de reserva em microalgas. Em situação de deficiência de azoto tanto o mutante I58W3 como o controlo diminuíram o teor de clorofila e de proteína. A nível fotossintético, comparando o controlo com o mutante I58W3, as taxas de fotossíntese foram menores para o mutante, o que indica alguma dificuldade na assimilação de CO2 pela Rubisco, estando em linha com a mutação destas células. A análise por PAM também indica possíveis danos no aparelho fotossintético nesta estirpe mutante, o que também contribuirá para uma menor eficiência na produtividade. O mutante I58W3 mostra uma menor capacidade de absorção de energia quando comparado com o controlo, assim como valores inferiores de eficácia no transporte de eletrões e menos centros de reação. Além disso também apresenta valores mais acentuados de dissipação de energia sob forma de calor. A análise dos níveis da Rubisco mostram ser menores no mutante I58W3 do que no controlo, o que pode indicar também que esta estirpe não só tem dificuldades em assimilar eficientemente CO2, como este fator é agravado pelo teor baixo da enzima Rubisco no cloroplasto das células de C. reinhardtii. Uma análise da composição lipídica leva-nos a crer que, como resposta das células a uma fotossíntese deficiente, ocorre uma alteração metabólica que provoca uma maior síntese e acumulação de lípidos, principalmente TAG. A análise genética, por qPCR, de genes relacionados com a síntese lipídica parece indicar que estes estão a ser expressos mais frequentemente na estirpe I58W3 do que em células controlo. Além disso, a expressão de um gene envolvido na fotossíntese também parece corroborar a hipótese de que este processo não é tão eficaz em I58W3. Uma análise genética feita a amostras controlo e I58W3 a vários genes indica que as alterações a nível de acumulação de lípidos podem estar relacionadas com a síntese de novo destes, ou com uma menor síntese de dessaturases de ácidos gordos. O mutante I58W3 apresenta maior expressão de DGAT1, uma proteína envolvida na síntese de TAG, que o controlo, sendo que esse aumento é mais acentuado em condições de deficiência de azoto. Este mutante também apresenta valores de expressão de CrDES inferiores ao controlo, o que nos indica que existe uma acumulação de ácido linoleico em detrimento de ácido pinolénico em I58W3. A proteína cytb6f encontra-se mais expressa em I58W3, embora a eficiência fotossintética deste seja menor que a do controlo. A síntese da hidrogenase HydA1 não varia significativamente em I58W3 independentemente da presença ou ausência de azoto. Os resultados obtidos permitem-nos concluir que, dos genes estudados, existem diferenças a nível da sua expressão devido à mutação em conjunto com a deficiência de azoto. Por outro lado a síntese de proteínas como a hidrogenase HydA1 ou a galactolipase PGD1 parecem não mudar com a mutação. O fato de que esta estirpe consegue acumular lípidos neutros como resposta às consequências da sua mutação, assim como à deficiência de azoto, é fulcral no âmbito da temática proposta. A produção de biodiesel seria então possível utilizando estas algas em conjunto com um meio cuja característica principal seria a falta de azoto. A união destes dois fatores poderá tornar possível no futuro culturas cujo objetivo é a produção de biodiesel.Chlamydomonas reinhardtii is a unicellular, soil-dwelling green alga that has been the focus of several studies regarding photosynthesis and biodiesel production. When grown in specific conditions, such as sulphur or nitrogen (N) deficiency, this organism increases its H2 production as well as triacylglycerol (TAG) synthesis, which occurs as a response to lower photosynthetic rates. In this investigation we characterize the phenotype of a C. reinhardtii mutant that performs a deficient CO2 assimilation. This mutant, named I58W3, has three tryptophan amino acids replacing one isoleucine amino acid in Rubisco small subunit, close to Rubisco central channel. Phenotypic characterization involved the growth of C. reinhardtii cultures under standard growth condition (N-replete) and N deprivation in TAP medium for 5 days. During the growth period cell number, dry weight, protein and chlorophyll contents and photosynthetic rates were measured. Lipid composition of C. reinhardtii cell lines was assessed through gas chromatography and thin layer chromatography, as well as Red Nile staining and subsequent spectrophotometry. Photosynthetic apparatus efficiency and integrity was also evaluated by pulse amplitude modulation (PAM) analysis. In order to compare Rubisco enzyme levels in control and I58W3 mutants, protein gel electrophoresis and immunoblotting were performed. Finally, quantitative PCR of several genes related to lipid metabolism and photosynthesis was performed in order to investigate transcriptional changes between I58W3 mutants and the control strain, under N-replete and N deprivation conditions. Both protein and chlorophyll levels were affected under nitrogen deprivation as their concentration is lower in both control and mutant cells. Mutant cells appear to have a decreased photosynthetic efficiency and their photosynthetic apparatus does not function the same way as it does in control cells. Rubisco enzyme levels decrease in I58W3 cells and the expression of several genes, such as FAD or ω-13, is also lower in I58W3 cells in response to nitrogen deprivation. Overall, I58W3 cells show a promising role in subsequent biodiesel production, since they show an increase in TAG lipid accumulation and decreased photosynthetic rates. Further genetic analyses of other genes, regarding different photosynthetic pathways, should be made in order to guarantee a thorough research and a complete database on I58W3 mutants

“Green microalgae biohydrogen production”

Nowadays, renewable energy is one of the most discussed issues by the international scientific community. The unrestrained use of fossil fuels has raised relevant questions about sustainability and effects on the environment. Hydrogen (H2) is considered a promising fuel due to its thermodynamical properties and CO2-free combustion. Nevertheless, environmental problems arise when H2 is produced using energy deriving from fossil sources: only “green hydrogen” identifies a production 100% based on renewable energy. Currently, several microorganisms are known for their ability to produce H2 as a metabolic feature. Regarding microalgae, most of the information comes from Chlamydomonas reinhardtii. In this green microalgae, two different photosynthetic production pathways and one fermentative-like metabolism have been described concerning transitory H2 production. To extend H2 production one requirement is the creation of a hypoxic environment. This occurs when photosynthetic activity slows down or if there is an increase in mitochondrial respiration rates. Moreover, the electron flow should be directed preferentially towards the H2 evolution enzyme, hydrogenase. Concerning physiological conditioning, one of the most promising strategies for H2 production is sulphur deprivation from cultivation medium: within three days, anaerobiosis develops under saturated light. Chlamydomonas has also represented an excellent example for the development of different molecular strategies, that allow to overcome some limitations and extend the H2 production. One of the limitations is related to sunlight saturation and dissipation that also affect H2 production. Mutants with the truncated light-harvesting antenna (tla) in the chloroplast are subject to fewer phenomena of photoinhibition and light saturation. Another limitation is linked to the competitive pathways that remove electrons from hydrogenase. Pgrl1 (protein gradient regulation like 1) mutant showed improved H2 production reducing this phenomenon. Another relevant issue is the oxygen sensitivity of the hydrogenase enzyme. The use of an O2-tolerant clostridial [FeFe]-hydrogenase, expressed in C. reinhardtii, showed better enzymatic rate, as the bacterial hydrogenase had a lower inactivation rate in aerobiosis. The work carried out over the past three years was aimed primarily at the isolation and characterization of microalgal species in the Basilicata region for the identification of new biohydrogen producers. Particular attention has been given to the search for strains with good growth rates and able to use different carbon sources. Secondly, the physiological behaviour of single and double mutants of Chlamydomonas was analyzed concerning H2 production by modulating light condition without resorting to stress application, such as sulphur deprivation. Freshwater samples collected in different villages of the Basilicata region were used to isolate microalgae with different morphologies. Microscopical observations and molecular identification made it possible to identify the genus of the isolated pure colonies. The growth of the various strains was followed by different methods: absorbance and chlorophyll content proved to be effective and fast for monitoring cell growth over the days. This made it possible to evaluate the growth rates of the species under examination. Various tests were carried out to detect the production of H2. Bioreactors were kept in dark, limited light (12 PAR) or sulfur deprivation (with intense light, 100 PAR). All the experiments considered different carbon source too. The levels of H2 gas produced were daily assessed by gas chromatography by taking a sample of the airspace in contact with the liquid culture in the airtight bioreactors. Desmodesmus sp. and Haematococcus sp. strains demonstrated production of H2 similar to wild type Chlamydomonas (5-10 ml/litre of culture). Furthermore, the same production occurred similarly using acetate or glucose. For Chlamydomonas mutants, the experiments were conducted in collaboration with the University of Córdoba (Spain). Investigated mutants were tla3, pgrl1, and one engineered with Clostridium bacterial hydrogenase (clostr) and the relative combinations tla3 + pgrl1 and clostr + pgrl1 from genetic cross. In this case, the wild type, single and double mutant strains were subjected to different lighting conditions (12, 50, 100, 450 PAR). In particular, the combination tla3 + pgrl proved to be the best as it is capable of producing H2 even at light intensities that are generally less tolerated, opening up new application scenarios. The single mutant Clostr showed instead a fast hydrogenase activity in a replete media also proportionally with the increase of light. In conclusion, the algae isolated during the PhD project have shown interesting implications for the production of H2 such as the metabolic versatility regarding the use of the different carbon sources. This leads to the need to carry out a more in-depth investigation of the mechanisms underlying the metabolism of these microalgae both from a physiological and a molecular point of view. Regarding single and double Chlamydomonas mutants, knowledge about their behaviour in different light conditions and the feasibility of H2 production has been expande

A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

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New tools and strategies for metabolic engineering of the algal chloroplast

Microalgae are attractive as cell factories for production of bioactive metabolites, therapeutic proteins and high-value metabolites. Of the several microalgae that have been explored as potential biotechnological platforms, the unicellular chlorophyte Chlamydomonas reinhardtii, is the most genetically tractable given its long history as a model species for molecular- genetic studies of cell biology. In particular, the chloroplast of C. reinhardtii represents a novel sub-cellular compartment for synthesis and accumulation of recombinant products. It possesses a small, genetically tractable genome and lacks any gene-silencing mechanisms. This allows stable and high-level expression of multiple transgenes. However, the exploitation of this microalgal platform requires further advances in the molecular tools available for metabolic engineering, together with new strategies for large-scale cultivation such as simple methods for ensuring ‘crop protection’ to reduce invasion and growth of contaminating species. Due to the increasing scarcity of phosphate reserve, phosphite is an alternative P source at economical cost and can reduce demand on non-renewable phosphate fertiliser. A key goal of my project was therefore to develop a strain improvement strategy based on the expression in the chloroplast of the bacterial gene ptxD encoding an NAD(P)-dependent phosphite oxidoreductase to allow utilization of phosphite as a source of phosphorus (P). The approach is based on the fact that most organisms cannot use phosphite, therefore, growing the transgenic microalga in phosphite provides a selective advantage over competing species. The ptxD gene was successfully introduced into chloroplast and shown to produce a functional enzyme that allowed growth on phosphite media. This allows engineered strains to be grown in non-sterile medium without significant spoilage by bacteria or fungi, thereby avoiding costly medium sterilization and culture management. Furthermore, it was demonstrated that ptxD can serve as a new non-antibiotic selectable marker for chloroplast transformation, allowing direct selection of transformants for their phosphite-utilising activity. This increases the repertoire of available selectable markers and reduces the use of antibiotics. Having developed these tools, the metabolic engineering of the chloroplast was attempted by introducing a synthetic gene encoding limonene synthase (LS). Limonene is a high-value terpenoid that has applications as a pharmaceutical, a flavour and a fragrance. Whilst the LS protein was successfully produced in the chloroplast, detailed GC-MS analysis failed to detect limonene synthesis suggesting issues with either functionality of the enzyme or availability of substrate. However, the work adds important new tools to the molecular toolbox for advancing C. reinhardtii chloroplast as an expression platform