Le alghe sono definite organismi fotosintetici ossigenici, procarioti o eucarioti, con un’organizzazione da unicellulare a pluricellulare, che non sviluppano foglie o radici giustificando la classificazione di ‘piante inferiori’. Le alghe presentano diverse potenziali applicazioni commerciali, come la produzione di biomassa per l’alimentazione umana/animale o per essere usata come fertilizzante, l’estrazione di molecole ad elevato valore aggiunto con un mercato nell’industria chimica o farmaceutica, infine, anche se ancora lontano dalla commercializzazione, la produzione di bio-combustibili. Fornire un substrato per la crescita eterotrofa potrebbe essere una possibile strategia per la crescita delle alghe, tuttavia uno dei maggiori vantaggi delle alghe rispetto ad organismi non fotosintetici è la possibilità di convertire l’energia solare, l’acqua e l’anidride carbonica in biomassa, attraverso il processo fotosintetico. D’altra parte, la coltivazione attuale di ceppi selvatici prospetta rese in biomassa nettamente inferiori rispetto a stime teoriche basate su una fotosintesi ottimale. Tale risultato riflette un problema produttivo reale che dipende sostanzialmente da un utilizzo inefficiente della luce. In particolare, i ceppi selvatici sono in genere dotati di ampi complessi antenna dei fotosistemi per la raccolta della luce, un vantaggio nell’ambiente naturale dove la luce può essere limitante e le cellule crescono a bassa densità. Tale caratteristica è stata proposta diventare invece controproducente durante la coltivazione di massa. Questo perché la fotosintesi è saturata ad intensità di luce relativamente basse, con conseguente dissipazione dell’energia assorbita in eccesso, e la luce è rapidamente attenuata all’interno della coltura. Diversamente, fenotipi a ridotta capacità d’assorbimento della luce potrebbero incrementare l’efficienza di conversione della luce in biomassa. Il principale vantaggio sarebbe di saturare la fotosintesi a più alte intensità luminose, minimizzando lo smorzamento non fotochimico dell’eccitazione. D’altra parte, tali fenotipi non sopravvivrebbero nell’ambiente naturale e non potrebbero essere isolati in natura, ma devono essere generati tramite ingegneria genetica. Chlamydomonas reinhardtii è un’alga verde unicellulare che si presta alla trasformazione e di cui sono disponibili informazioni di sequenza per tutti e tre i genomi (nucleare, mitocondriale e cloroplastico). Tecniche d’ingegneria genetica possono quindi essere applicate a questo organismo modello per generare mutanti con differenti livelli di riduzione della capacità di assorbire la luce e per verificare le promesse di questi ultimi nell’ottimizzare l’efficienza di utilizzo della luce. In seguito, conoscenze acquisite dallo studio di organismi modello possono essere d’aiuto per avanzare il miglioramento genetico di altre specie algali produttive e attraenti per applicazioni commerciali. Tramite mutagenesi inserzionale casuale del genoma nucleare di C. reinhardtii, tre mutanti ‘verde pallido’, con un ridotto contenuto in clorofilla per cellula rispetto al wild type, sono stati isolati: as1, as2 e gun4. Un ceppo a ridotta antenna deve soddisfare specifici criteri di saturazione della fotosintesi ad alte intensità luminose ed elevata efficienza quantica e un fenotipo ‘verde pallido’ non corrisponde necessariamente ad una maggiore produttività fotosintetica. Ad esempio, il mutante gun4 è compromesso nella biosintesi della clorofilla, accumula una porfirina precursore ed è fotosensibile. E’ evidente che tale ceppo non può essere coltivato come produttore di biomassa. Alternativamente, modificare il targeting delle proteine e la biogenesi dei complessi coordinanti la clorofilla potrebbe essere una strategia per regolare la capacità di assorbire la luce senza compromettere la fotoprotezione, come suggerito dal mutante as1. Quest’ultimo ha una mutazione inserzionale in un gene omologo ad arsA, possibilmente coinvolto nell’importazione delle proteine nel cloroplasto mediante regolazione della biogenesi del traslocone della membrana esterna del cloroplasto. Il fenotipo ‘verde pallido’ in as1 e as2 deriva da una riduzione sia nella taglia d’antenna dei singoli fotosistemi sia nella quantità di fotosistemi. Agire solo sulla dimensione dell’antenna per fotosistema non è fattibile, considerando la devozione dei complessi antenna alla fotoprotezione oltre che alla raccolta della luce e i limiti strutturali di una dimensione minima dell’antenna che permetta l’assemblaggio e la funzionalità del fotosistema stesso. Pertanto, diminuire la densità di fotosistemi nei tilacoidi potrebbe essere una valida strategia complementare alla sola riduzione della dimensione dell’antenna per fotosistema per ottenere fenotipi a ridotto contenuto in pigmenti. D’altra parte, i complessi fotosintetici stessi costituiscono un apparato tutt’altro che rigido e l’acclimatazione a lungo termine in grado di modulare la capacità di assorbire la luce come risposta a diverse intensità luminose in C. reinhardtii include la regolazione del contenuto in clorofilla per cellula. Fattori putativamente coinvolti nella foto-acclimatazione, come LHL4, un membro della famiglia LHC, potrebbero essere ingegnerizzati, di fatto una riduzione del contenuto in clorofilla è stata osservata nelle linee sovra-esprimenti in modo costitutivo LHL4. Sebbene il gene responsabile del fenotipo osservato in as2 non sia stato tuttora identificato, la modificazione della curva di risposta alla luce della fotosintesi sembra la più promettente per incrementare la produttività ad alte intensità luminose rispetto al wild type. as2 ha infatti dimostrato rese maggiori del wild type in termini di densità cellulare sia in piccola scala sia in un fotobioreattore di 65 litri. Tuttavia, per osservare i benefici attesi di produttività fotosintetica soprattutto su larga scala, bisogna prestare attenzione alla geometria del fotobioreattore e alle condizioni di coltura. In particolare, esiste una concentrazione ottimale di clorofilla, e quindi di cellule, per avere la massima produttività fotosintetica integrata per l’intera coltura ad una determinata radiazione luminosa. Tale concentrazione ottimale permette di assorbire il più possibile dell’energia disponibile, limitando nel contempo l’attenuazione della luce all’interno della coltura la quale risulterebbe in una perdita di biomassa dovuta alla respirazione nelle zone non sufficientemente illuminate. Al di sotto della concentrazione ottimale, la produttività fotosintetica risulta ridotta da una limitazione in clorofilla nell’assorbire la luce.
Algae are defined as oxygenic photosynthetic organisms, prokaryotic or eukaryotic, with organization ranging from unicellular to multicellular, that don’t have true stems, roots and leaves thus leading to their classification as ‘lower’ plants. Algae have several potential commercial applications, such as production of biomass for human/animal feeding or to be used as fertilizer, extraction of high-value chemicals and pharmaceuticals and, although still far from being on the market, as a biofuels feedstock. Supplying a substrate for heterotrophic growth could be a possible strategy for algae-based biorefineries, however the major advantage of using algae over non photosynthetic organisms is the possibility to convert solar energy, water and carbon dioxide into biomass, through photosynthesis. Conversely, present cultivation of wild type strains yields biomass productivities that are far below theoretical estimations based on optimal photosynthesis, enlightening an existing problem that mainly relies on light utilization inefficiency. In particular, large light-harvesting antenna systems, an advantage in the wild where light could be limiting and cells grow at low density, have been proposed to be instead detrimental during mass cultivation because of photosynthesis saturation occurring at relatively low light intensities, with dissipation of excess absorbed energy, and rapid light extinction within the culture. In contrast, phenotypes of reduced absorption cross section could improve solar-to-biomass conversion efficiency. The main advantage would be that photosynthesis saturation occurs at higher light intensities, minimizing non photochemical quenching. On the other hand, such phenotypes would not survive in the wild and could not be encountered in nature but have to be generated by genetic engineering. Chlamydomonas reinhardtii is a unicellular green alga that is suitable to transformation and whose genomes are sequenced. Techniques of genetic engineering could thus be applied in this model organism to generate mutants with different extents in absorption cross section reduction and to verify their promises of improved light use efficiency. Then, knowledge from intensively studied organisms could help advancing in genetic improvement of other productive algal species that are attractive for commercial applications. From random insertion mutagenesis of the nuclear genome of C. reinhardtii, three ‘pale green’ strains have been isolated, namely antenna size mutant 1 (as1), antenna size mutant 2 (as2) and gun4. A truncated antenna strain must meet specific criteria of high saturation light and quantum yield of photosynthesis and not all ‘pale green’ strains are truly useful mutants for improved productivity. For instance, the gun4 mutant is compromised in chlorophyll biosynthesis, accumulating a chlorophyll precursor porphyrin and displaying photosensitivity. It’s understandable that it could not be grown as a biomass producer. Alternatively, to act on protein targeting and biogenesis of chlorophyll-binding complexes could be a mean to regulate the absorption cross section of the cell without leading to photosensitivity, as suggested by mutant as1. The latter has an insertion mutation in an arsA-homolog gene possibly involved in chloroplast protein import by mediating biogenesis of the translocon of chloroplast outer membrane. Remarkably, the ‘pale green’ phenotype of as1 and as2 derives from reduction in both photosystems antenna size and amount of photosystem core complexes. Acting only on the chlorophyll antenna size per photosystem is not feasible, considering devotion of antenna systems to both light harvesting and photoprotection and structural constrains of a minimal antenna size to allow for folding and function of photosystem core complex. Reducing the density of photosystems in thylakoids could be a valuable complementary strategy as compared to the sole reduction in photosystem antenna size to obtain phenotypes of lower absorption cross section. At the other hand, photosynthetic complexes constitute themselves an apparatus that is far from rigid and long term acclimation to adjust the light harvesting capacity to changing light conditions in C. reinhardtii relies on regulating the chlorophyll content per cell. Factors possibly involved in photo-acclimation, as LHL4, a LHC-like protein, could be target for genetic engineering and constitutive up-regulation of LHL4 has led to reduction in the chlorophyll content. Although the gene responsible for the observed phenotype is still unknown in as2, modification of the light response curve of photosynthesis seems to be the most promising to improve productivity during cultivation in high light. as2 has indeed yielded higher cell densities than wild type both in a small-scale apparatus and in a 65L-photobioreactor. However, in order to observe the expected benefits on photosynthetic productivity during scale-up, attention must be paid to photobioreactor design and growth conditions. In particular, optimum chlorophyll (cell) concentration for maximal integrated net photosynthesis exists at a given irradiance value, which would be such that most of the incident light will be absorbed while avoiding too strong light attenuation that would result in biomass loss through respiration in sub-illuminated zones. Below optimum chlorophyll concentration, limitation in chlorophyll in absorbing light could restrict overall photosynthetic productivity.
Regulating light use efficiency by genetic engineering of Chlamydomonas reinhardtii
FORMIGHIERI, Cinzia
2012-01-01
Abstract
Algae are defined as oxygenic photosynthetic organisms, prokaryotic or eukaryotic, with organization ranging from unicellular to multicellular, that don’t have true stems, roots and leaves thus leading to their classification as ‘lower’ plants. Algae have several potential commercial applications, such as production of biomass for human/animal feeding or to be used as fertilizer, extraction of high-value chemicals and pharmaceuticals and, although still far from being on the market, as a biofuels feedstock. Supplying a substrate for heterotrophic growth could be a possible strategy for algae-based biorefineries, however the major advantage of using algae over non photosynthetic organisms is the possibility to convert solar energy, water and carbon dioxide into biomass, through photosynthesis. Conversely, present cultivation of wild type strains yields biomass productivities that are far below theoretical estimations based on optimal photosynthesis, enlightening an existing problem that mainly relies on light utilization inefficiency. In particular, large light-harvesting antenna systems, an advantage in the wild where light could be limiting and cells grow at low density, have been proposed to be instead detrimental during mass cultivation because of photosynthesis saturation occurring at relatively low light intensities, with dissipation of excess absorbed energy, and rapid light extinction within the culture. In contrast, phenotypes of reduced absorption cross section could improve solar-to-biomass conversion efficiency. The main advantage would be that photosynthesis saturation occurs at higher light intensities, minimizing non photochemical quenching. On the other hand, such phenotypes would not survive in the wild and could not be encountered in nature but have to be generated by genetic engineering. Chlamydomonas reinhardtii is a unicellular green alga that is suitable to transformation and whose genomes are sequenced. Techniques of genetic engineering could thus be applied in this model organism to generate mutants with different extents in absorption cross section reduction and to verify their promises of improved light use efficiency. Then, knowledge from intensively studied organisms could help advancing in genetic improvement of other productive algal species that are attractive for commercial applications. From random insertion mutagenesis of the nuclear genome of C. reinhardtii, three ‘pale green’ strains have been isolated, namely antenna size mutant 1 (as1), antenna size mutant 2 (as2) and gun4. A truncated antenna strain must meet specific criteria of high saturation light and quantum yield of photosynthesis and not all ‘pale green’ strains are truly useful mutants for improved productivity. For instance, the gun4 mutant is compromised in chlorophyll biosynthesis, accumulating a chlorophyll precursor porphyrin and displaying photosensitivity. It’s understandable that it could not be grown as a biomass producer. Alternatively, to act on protein targeting and biogenesis of chlorophyll-binding complexes could be a mean to regulate the absorption cross section of the cell without leading to photosensitivity, as suggested by mutant as1. The latter has an insertion mutation in an arsA-homolog gene possibly involved in chloroplast protein import by mediating biogenesis of the translocon of chloroplast outer membrane. Remarkably, the ‘pale green’ phenotype of as1 and as2 derives from reduction in both photosystems antenna size and amount of photosystem core complexes. Acting only on the chlorophyll antenna size per photosystem is not feasible, considering devotion of antenna systems to both light harvesting and photoprotection and structural constrains of a minimal antenna size to allow for folding and function of photosystem core complex. Reducing the density of photosystems in thylakoids could be a valuable complementary strategy as compared to the sole reduction in photosystem antenna size to obtain phenotypes of lower absorption cross section. At the other hand, photosynthetic complexes constitute themselves an apparatus that is far from rigid and long term acclimation to adjust the light harvesting capacity to changing light conditions in C. reinhardtii relies on regulating the chlorophyll content per cell. Factors possibly involved in photo-acclimation, as LHL4, a LHC-like protein, could be target for genetic engineering and constitutive up-regulation of LHL4 has led to reduction in the chlorophyll content. Although the gene responsible for the observed phenotype is still unknown in as2, modification of the light response curve of photosynthesis seems to be the most promising to improve productivity during cultivation in high light. as2 has indeed yielded higher cell densities than wild type both in a small-scale apparatus and in a 65L-photobioreactor. However, in order to observe the expected benefits on photosynthetic productivity during scale-up, attention must be paid to photobioreactor design and growth conditions. In particular, optimum chlorophyll (cell) concentration for maximal integrated net photosynthesis exists at a given irradiance value, which would be such that most of the incident light will be absorbed while avoiding too strong light attenuation that would result in biomass loss through respiration in sub-illuminated zones. Below optimum chlorophyll concentration, limitation in chlorophyll in absorbing light could restrict overall photosynthetic productivity.File | Dimensione | Formato | |
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