Regulating light use efficiency by genetic engineering of Chlamydomonas reinhardtii

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.