Effect of lhcsr gene dosage on oxidative stress and light use efficiency by Chlamydomonas reinhardtii cultures

Unicellular green algae, a promising source for renewable biofuels, produce lipid-rich biomass from light and CO2. Productivity in photo-bioreactors is affected by inhomogeneous light distribution from high cell pigment causing heat dissipation of light energy absorbed in excess and shading of the deep layers. Contrasting reports have been published on the relation between photoprotective energy dissipation and productivity. Here, we have re-investigated the relation between energy quenching (qE) activity, photodamage and light use efficiency by comparing WT and two Chlamydomonas reinhardtii strains differing for their complement in LHCSR proteins, which catalyse dissipation of excitation energy in excess (qE). Strains were analysed for ROS production, protein composition, rate of photodamage and productivity assessed under wide light and CO2 conditions.The strain lacking LHCSR1 and knocked down in LHCSR3, thus depleted in qE, produced O-2 at significantly higher rate under high light, accompanied by enhanced singlet oxygen release and PSII photodamage. However, biomass productivity of WT was delayed in respect for mutant strains under intermittent light conditions only, implying that PSII activity was not the limiting factor under excess light. Contrary to previous proposals, domestication of Chlamydomonas for carbon assimilation rate in photo-bioreactors by down-regulation of photoprotective energy dissipation was ineffective in increasing algal biomass productivity

Effect of lhcsr gene dosage on oxidative stress and light use efficiency by Chlamydomonas reinhardtii cultures

Unicellular green algae, a promising source for renewable biofuels, produce lipid-rich biomass from light and CO2. Productivity in photo-bioreactors is affected by inhomogeneous light distribution from high cell pigment causing heat dissipation of light energy absorbed in excess and shading of the deep layers. Contrasting reports have been published on the relation between photoprotective energy dissipation and productivity. Here, we have re-investigated the relation between energy quenching (qE) activity, photodamage and light use efficiency by comparing WT and two Chlamydomonas reinhardtii strains differing for their complement in LHCSR proteins, which catalyse dissipation of excitation energy in excess (qE). Strains were analysed for ROS production, protein composition, rate of photodamage and productivity assessed under wide light and CO2 conditions. The strain lacking LHCSR1 and knocked down in LHCSR3, thus depleted in qE, produced O2 at significantly higher rate under high light, accompanied by enhanced singlet oxygen release and PSII photodamage. However, biomass productivity of WT was delayed in respect for mutant strains under intermittent light conditions only, implying that PSII activity was not the limiting factor under excess light. Contrary to previous proposals, domestication of Chlamydomonas for carbon assimilation rate in photo-bioreactors by down-regulation of photoprotective energy dissipation was ineffective in increasing algal biomass productivity

Potential and challenges of improving photosynthesis in algae

Sunlight energy largely exceeds the energy required by anthropic activities, and therefore its exploitation represents a major target in the field of renewable energies. The interest in the mass cultivation of green microalgae has grown in the last decades, as algal biomass could be employed to cover a significant portion of global energy demand. Advantages of microalgal vs. plant biomass production include higher light‐use efficiency, efficient carbon capture and the valorization of marginal lands and wastewaters. Realization of this potential requires a decrease of the current production costs, which can be obtained by increasing the productivity of the most common industrial strains, by the identification of factors limiting biomass yield, and by removing bottlenecks, namely through domestication strategies aimed to fill the gap between the theoretical and real productivity of algal cultures. In particular, the light‐to‐biomass conversion efficiency represents one of the major constraints for achieving a significant improvement of algal cell lines. This review outlines the molecular events of photosynthesis, which regulate the conversion of light into biomass, and discusses how these can be targeted to enhance productivity through mutagenesis, strain selection or genetic engineering. This review highlights the most recent results in the manipulation of the fundamental mechanisms of algal photosynthesis, which revealed that a significant yield enhancement is feasible. Moreover, metabolic engineering of microalgae, focused upon the development of renewable fuel biorefineries, has also drawn attention and resulted in efforts for enhancing productivity of oil or isoprenoids

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

High carotenoid mutants of Chlorella vulgaris show enhanced biomass yield under high irradiance

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photo-bioreactors. However, wild-type strains show biological limitations making algal bioproducts ex-pensive compared to those extracted from other feedstocks. Such constraints include inhomoge-neous light distribution due to high optical density of the culture, and photoinhibition of the sur-face-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobiore-actors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent

A phosphite dehydrogenase variant with promiscuous access to nicotinamide cofactor pools sustains fast phosphite-dependent growth of transplastomic chlamydomonas reinhardtii

Heterologous expression of the NAD+-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD+/NADP+ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP+ and NAD+ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance

Combined resistance to oxidative stress and reduced antenna size enhance light-to-biomass conversion efficiency in Chlorella vulgaris cultures

Background: Microalgae are efficient producers of lipid-rich biomass, making them a key component in developing a sustainable energy source, and an alternative to fossil fuels. Chlorella species are of special interest because of their fast growth rate in photobioreactors. However, biological constraints still cast a significant gap between the high cost of biofuel and cheap oil, thus hampering perspective of producing CO2-neutral biofuels. A key issue is the inefficient use of light caused by its uneven distribution in the culture that generates photoinhibition of the surface-exposed cells and darkening of the inner layers. Efficient biofuel production, thus, requires domestication, including traits which reduce optical density of cultures and enhance photoprotection. Results: We applied two steps of mutagenesis and phenotypic selection to the microalga Chlorella vulgaris. First, a pale-green mutant (PG-14) was selected, with a 50% reduction of both chlorophyll content per cell and LHCII complement per PSII, with respect to WT. PG-14 showed a 30% increased photon conversion into biomass efficiency vs. WT. A second step of mutagenesis of PG-14, followed by selection for higher tolerance to Rose Bengal, led to the isolation of pale-green genotypes, exhibiting higher resistance to singlet oxygen (strains SOR). Growth in photobioreactors under high light conditions showed an enhanced biomass production of SOR strains with respect to PG-14. When compared to WT strain, biomass yield of the pale green+ sor genotype was enhanced by 68%. Conclusions: Domestication of microalgae like Chlorella vulgaris, by optimizing both light distribution and ROS resistance, yielded an enhanced carbon assimilation rate in photobioreactor

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

High-temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β-glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non-axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline-treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by-products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose-derived sugars by C. vulgaris under mixotrophic growth