Negli ultimi anni, si è assistito ad un significativo aumento di studi riguardanti diverse specie di alghe, importanti dal punto di vista delle conoscenze fisiologiche e genetiche, ma anche per scopi applicativi. La possibilità di ottenere alte rese in termini di produzione di biomassa, rispetto alle piante superiori, rende difatti questi organismi particolarmente interessanti per applicazioni biotecnologiche. Diversi ceppi algali sono attualmente studiati per la produzione di biocarburanti, come bio-diesel o bio-idrogeno, grazie alla loro capacità di crescere usando anidride carbonica come substrato attraverso il processo fotosintetico, con un’elevata efficienza. In particolare, tra le alghe verdi, Chlamydomonas reinhardtii rappresenta l’organismo modello grazie al completo sequenziamento del genoma e alla disponibilità di efficienti tecniche di trasformazione sia nucleare che cloroplastica. Chlamydomonas reinhardtii possiede l’idrogenasi, un enzima che permette la sintesi di idrogeno utilizzando i protoni e il potere riducente generati dal processo fotosintetico, ossia in ultima analisi acqua e luce. Tuttavia, l’utilizzo di colture algali in fotobioreattore per produrre bio-idrogeno presenta alcune importanti limitazioni. Infatti, le alghe possiedono sistemi antenna deputati alla raccolta della luce aventi grandi dimensioni, che costituiscono un vantaggio evolutivo nel loro ambiente naturale, ma che influenzano la penetrazione e distribuzione della luce all’interno del fotobioreattore. Questo determina un eccessivo assorbimento energetico negli strati più esterni esposti ad alta luce, con conseguente aumento della dissipazione termica, e allo stesso tempo un ombreggiamento degli strati più interni, con riduzione dell’efficienza fotosintetica complessiva. Inoltre, l’attività dell’idrogenasi è fortemente inibita dall’ossigeno molecolare, anche a basse concentrazioni, evoluto durante la fotosintesi. Infine, alcuni processi che influenzano le dinamiche dei flussi di elettroni all’interno della cellula possono avere un effetto competitivo nei confronti dell’idrogenasi, diminuendo la resa di produzione di idrogeno. Tenendo in considerazione tutti questi elementi, nel presente lavoro di tesi è stato adottato un approccio in vivo, per identificare mutanti con migliorate caratteristiche di accumulo della biomassa in prospettiva della produzione di bio-idrogeno. E’ stata quindi creata una libreria di mutanti inserzionali in C. reinhardtii, poi analizzata tramite diverse strategie, come descritto nel capitolo 2, per selezionare ceppi con migliori caratteristiche fenotipiche. Inoltre, è stata avviata l’analisi genetica di questi mutanti per identificare i geni responsabili dei fenotipi osservati. Una volta individuati, questi geni possono offrire l’opportunità di regolare vari meccanismi tramite sistemi di espressione inducibile. Lo screening della libreria ha permesso inoltre di isolare mutanti che, pur non essendo adatti alla produzione di biomassa, risultano di particolare interesse per ricerche di base. E’ il caso del mutante gun4, finora mai caratterizzato in Chlamydomonas; la proteina Gun4 è stata identificata in piante e cianobatteri come fattore di regolazione nella via di biosintesi della clorofilla e nella trasduzione del segnale dal cloroplasto al nucleo. E’ stata quindi avviata un’analisi più approfondita di questo mutante, che potrà fornire informazioni su questi interessanti aspetti biologici, come discusso nel capitolo 3. Infine, il capitolo 4 presenta un approccio in vitro per la caratterizzazione funzionale di tutte le proteine Lhca che formano i complessi di raccolta della luce del fotosistema I in C. reinhardtii. Le differenti subunità di questi complessi non sono funzionalmente equivalenti, ed è quindi importante analizzare il loro ruolo specifico nella raccolta della luce e nella fotoprotezione. La loro caratterizzazione costituisce la base per procedere a una delezione selettiva delle diverse proteine Lhc, allo scopo di migliorare l’assorbimento della luce senza influenzare i meccanismi fotoprotettivi, incrementando così l’efficienza fotosintetica complessiva.
In the last years, a significant increase in studies concerning different algal species has been realized, with important effects for our knowledge of physiology and genetics, but also for applicative purposes. The possibility to obtain higher yield in term of biomass production, with respect to land plants, makes these organisms of particular interest for biotechnological applications. Indeed, different algal strains are currently investigated for biofuel production, as biodiesel or bio-hydrogen, thanks to their capacity to grow using CO2 as substrate through the photosynthetic process, in a highly efficient way. In particular, among green algae, Chlamydomonas reinhardtii represents the model organism, thanks to the complete genome sequencing and the availability of efficient nuclear and chloroplast transformation technologies. C. reinhardtii possesses an hydrogenase enzyme which allows light-driven hydrogen synthesis, using protons and reducing power generated by the photosynthetic process. However, the exploitation of mass algal cultures in photobioreactor with the final aim of bio-hydrogen production presents some important limiting factors. In fact, algae are equipped with a large light-harvesting antenna system, which constitutes an evolutive advantage in their natural environment, but affecting the light penetration and distribution within the photobioreactor. This causes an energy over absorption in the high light exposed superficial layers, with increased heat dissipation, and at the same time in shading of the inner layers, thus reducing the photosynthetic efficiency. Moreover, hydrogenase activity is strongly inhibited by molecular oxygen, also at very low concentration, which is evolved during the photosynthesis. Finally, some processes affecting the dynamics of electron fluxes inside the cell can compete with the hydrogenase for reducing power, thus diminishing hydrogen production yield. Considering all these elements, in this thesis we decided to adopt an in vivo approach, in order to identify strains with improved characteristics for biomass accumulation at the final aim of bio-hydrogen production. Therefore, we created a Chlamydomonas reinhardtii insertional mutant library, which has been screened by multiple strategies, as described in chapter 2, to select mutants with improved phenotypic characteristics. Moreover, the genetic analysis of these mutants has been initiated in order to identify the genes responsible of the phenotype. Once identified, these genes could offer the possibility to modulate different mechanisms by using inducible expression systems. The screening also allowed to isolate mutants particularly interesting for basic researches, although not suitable for growth in mass culture conditions. This is the case of the gun4 mutant, never characterized so far in Chlamydomonas. The Gun4 protein has been identified in plants and in cyanobacteria as a regulatory factor in the chlorophyll biosynthesis pathway and in the retrograde signaling from chloroplast to nucleus. Therefore, we started a deeper analysis of this mutant, which could provide information about these interesting biological problems, as will be discussed in chapter 3. Finally, chapter 4 presents an in vitro approach for the functional characterization of all Lhca proteins which form the PSI light-harvesting complexes in Chlamydomonas reinhardtii. The different antenna complex subunits are not all functionally equivalent, and it important to elucidate their specific role in the light-harvesting and photo-protection. Their characterization constitutes the basis for proceeding to a selective depletion of the different Lhc proteins in order to improve light-absorption without affecting the photoprotective mechanisms, thus optimising the overall photosynthetic efficiency.
Selection of Chlamydomonas reinhardtii mutantsfor improved growth in photobioreactor andperspectives for bio-hydrogen production
MANTELLI, Manuela
2009-01-01
Abstract
In the last years, a significant increase in studies concerning different algal species has been realized, with important effects for our knowledge of physiology and genetics, but also for applicative purposes. The possibility to obtain higher yield in term of biomass production, with respect to land plants, makes these organisms of particular interest for biotechnological applications. Indeed, different algal strains are currently investigated for biofuel production, as biodiesel or bio-hydrogen, thanks to their capacity to grow using CO2 as substrate through the photosynthetic process, in a highly efficient way. In particular, among green algae, Chlamydomonas reinhardtii represents the model organism, thanks to the complete genome sequencing and the availability of efficient nuclear and chloroplast transformation technologies. C. reinhardtii possesses an hydrogenase enzyme which allows light-driven hydrogen synthesis, using protons and reducing power generated by the photosynthetic process. However, the exploitation of mass algal cultures in photobioreactor with the final aim of bio-hydrogen production presents some important limiting factors. In fact, algae are equipped with a large light-harvesting antenna system, which constitutes an evolutive advantage in their natural environment, but affecting the light penetration and distribution within the photobioreactor. This causes an energy over absorption in the high light exposed superficial layers, with increased heat dissipation, and at the same time in shading of the inner layers, thus reducing the photosynthetic efficiency. Moreover, hydrogenase activity is strongly inhibited by molecular oxygen, also at very low concentration, which is evolved during the photosynthesis. Finally, some processes affecting the dynamics of electron fluxes inside the cell can compete with the hydrogenase for reducing power, thus diminishing hydrogen production yield. Considering all these elements, in this thesis we decided to adopt an in vivo approach, in order to identify strains with improved characteristics for biomass accumulation at the final aim of bio-hydrogen production. Therefore, we created a Chlamydomonas reinhardtii insertional mutant library, which has been screened by multiple strategies, as described in chapter 2, to select mutants with improved phenotypic characteristics. Moreover, the genetic analysis of these mutants has been initiated in order to identify the genes responsible of the phenotype. Once identified, these genes could offer the possibility to modulate different mechanisms by using inducible expression systems. The screening also allowed to isolate mutants particularly interesting for basic researches, although not suitable for growth in mass culture conditions. This is the case of the gun4 mutant, never characterized so far in Chlamydomonas. The Gun4 protein has been identified in plants and in cyanobacteria as a regulatory factor in the chlorophyll biosynthesis pathway and in the retrograde signaling from chloroplast to nucleus. Therefore, we started a deeper analysis of this mutant, which could provide information about these interesting biological problems, as will be discussed in chapter 3. Finally, chapter 4 presents an in vitro approach for the functional characterization of all Lhca proteins which form the PSI light-harvesting complexes in Chlamydomonas reinhardtii. The different antenna complex subunits are not all functionally equivalent, and it important to elucidate their specific role in the light-harvesting and photo-protection. Their characterization constitutes the basis for proceeding to a selective depletion of the different Lhc proteins in order to improve light-absorption without affecting the photoprotective mechanisms, thus optimising the overall photosynthetic efficiency.File | Dimensione | Formato | |
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