Engineering the Unicellular Alga Phaeodactylum tricornutum for Enhancing Carotenoid Production.

Microalgae represent a promising resource for the production of beneficial natural compounds due to their richness in secondary metabolites and easy cultivation. Carotenoids feature among distinctive compounds of many microalgae, including diatoms, which owe their golden color to the xanthophyll fucoxanthin. Carotenoids have antioxidant, anti-obesity and anti-inflammatory properties, and there is a considerable market demand for these compounds. Here, with the aim to increase the carotenoid content in the model diatom Phaeodactylum tricornutum, we exploited genetic transformation to overexpress genes involved in the carotenoid biosynthetic pathway. We produced transgenic lines over-expressing simultaneously one, two or three carotenoid biosynthetic genes, and evaluated changes in pigment content with high-performance liquid chromatography. Two triple transformants over-expressing the genes Violaxanthin de-epoxidase (Vde), Vde-related (Vdr) and Zeaxanthin epoxidase 3 (Zep3) showed an accumulation of carotenoids, with an increase in the fucoxanthin content up to four fold. Vde, Vdr and Zep3 mRNA and protein levels in the triple transformants were coherently increased. The exact role of these enzymes in the diatom carotenoid biosynthetic pathway is not completely elucidated nevertheless our strategy successfully modulated the carotenoid metabolism leading to an accumulation of valuable compounds, leading the way toward improved utilization of microalgae in the field of antioxidants.

Dynamic Changes between Two LHCX-Related Energy Quenching Sites Control Diatom Photoacclimation.

Marine diatoms are prominent phytoplankton organisms that perform photosynthesis in extremely variable environments. Diatoms possess a strong ability to dissipate excess absorbed energy as heat via nonphotochemical quenching (NPQ). This process relies on changes in carotenoid pigment composition (xanthophyll cycle) and on specific members of the light-harvesting complex family specialized in photoprotection (LHCXs), which potentially act as NPQ effectors. However, the link between light stress, NPQ, and the existence of different LHCX isoforms is not understood in these organisms. Using picosecond fluorescence analysis, we observed two types of NPQ in the pennate diatom Phaeodactylum tricornutum that were dependent on light conditions. Short exposure of low-light-acclimated cells to high light triggers the onset of energy quenching close to the core of photosystem II, while prolonged light stress activates NPQ in the antenna. Biochemical analysis indicated a link between the changes in the NPQ site/mechanism and the induction of different LHCX isoforms, which accumulate either in the antenna complexes or in the core complex. By comparing the responses of wild-type cells and transgenic lines with a reduced expression of the major LHCX isoform, LHCX1, we conclude that core complex-associated NPQ is more effective in photoprotection than is the antenna complex. Overall, our data clarify the complex molecular scenario of light responses in diatoms and provide a rationale for the existence of a degenerate family of LHCX proteins in these algae.

LHCSR Expression under HSP70/RBCS2 Promoter as a Strategy to Increase Productivity in Microalgae.

Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, limited biomass productivity is commonly experienced in their cultivating system despite their high potential. One of the reasons for this limitation is the high thermal dissipation of the light absorbed by the outer layers of the cultures exposed to high light caused by the activation of a photoprotective mechanism called non-photochemical quenching (NPQ). In the model organism for green algae Chlamydomonas reinhardtii, NPQ is triggered by pigment binding proteins called light-harvesting-complexes-stress-related (LHCSRs), which are over-accumulated in high light. It was recently reported that biomass productivity can be increased both in microalgae and higher plants by properly tuning NPQ induction. In this work increased light use efficiency is reported by introducing in C. reinhardtii a LHCSR3 gene under the control of Heat Shock Protein 70/RUBISCO small chain 2 promoter in a npq4 lhcsr1 background, a mutant strain knockout for all LHCSR genes. This complementation strategy leads to a low expression of LHCSR3, causing a strong reduction of NPQ induction but is still capable of protecting from photodamage at high irradiance, resulting in an improved photosynthetic efficiency and higher biomass accumulation.

A systems-wide understanding of photosynthetic acclimation in algae and higher plants.

The ability of phototrophs to colonise different environments relies on robust protection against oxidative stress, a critical requirement for the successful evolutionary transition from water to land. Photosynthetic organisms have developed numerous strategies to adapt their photosynthetic apparatus to changing light conditions in order to optimise their photosynthetic yield, which is crucial for life on Earth to exist. Photosynthetic acclimation is an excellent example of the complexity of biological systems, where highly diverse processes, ranging from electron excitation over protein protonation to enzymatic processes coupling ion gradients with biosynthetic activity, interact on drastically different timescales from picoseconds to hours. Efficient functioning of the photosynthetic apparatus and its protection is paramount for efficient downstream processes, including metabolism and growth. Modern experimental techniques can be successfully integrated with theoretical and mathematical models to promote our understanding of underlying mechanisms and principles. This review aims to provide a retrospective analysis of multidisciplinary photosynthetic acclimation research carried out by members of the Marie Curie Initial Training Project, AccliPhot, placing the results in a wider context. The review also highlights the applicability of photosynthetic organisms for industry, particularly with regards to the cultivation of microalgae. It intends to demonstrate how theoretical concepts can successfully complement experimental studies broadening our knowledge of common principles in acclimation processes in photosynthetic organisms, as well as in the field of applied microalgal biotechnology.

Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum.

Diatoms are phytoplanktonic organisms that grow successfully in the ocean where light conditions are highly variable. Studies of the molecular mechanisms of light acclimation in the marine diatom Phaeodactylum tricornutum show that carotenoid de-epoxidation enzymes and LHCX1, a member of the light-harvesting protein family, both contribute to dissipate excess light energy through non-photochemical quenching (NPQ). In this study, we investigate the role of the other members of the LHCX family in diatom stress responses. Our analysis of available genomic data shows that the presence of multiple LHCX genes is a conserved feature of diatom species living in different ecological niches. Moreover, an analysis of the levels of four P. tricornutum LHCX transcripts in relation to protein expression and photosynthetic activity indicates that LHCXs are differentially regulated under different light intensities and nutrient starvation, mostly modulating NPQ capacity. We conclude that multiple abiotic stress signals converge to regulate the LHCX content of cells, providing a way to fine-tune light harvesting and photoprotection. Moreover, our data indicate that the expansion of the LHCX gene family reflects functional diversification of its members which could benefit cells responding to highly variable ocean environments.