Inmaculada Couso

Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta) by Improvement of Culture Conditions and Random Mutagenesis

Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.

Overexpression of an exogenous phytoene synthase gene in the unicellular alga Chlamydomonas reinhardtii leads to an increase in the content of carotenoids.

Phytoene synthase (PSY) catalyses the first step in the production of carotenoids, which has been described as a key regulatory step in the carotenoids biosynthetic pathway. PSY gene from Dunaliella salina was constitutively expressed in Chlamydomonas reinhardtii under the control of the RBCS2 and HSP70A promoters and targeted to the chloroplast by the RBCS2 transit peptide. DsPSY overexpression resulted in a stable increase in the corresponding PSY transcript level and in the content of carotenoids such as violaxanthin, lutein, and β‐carotene, reaching between 125 and 260% the levels in control untransformed cells.

ISOLATION AND CHARACTERIZATION OF A LYCOPENE β‐CYCLASE GENE FROM THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

The isolation, characterization, and regulation by light and nitrogen of the lycopene β‐cyclase gene from Chlorella zofingiensis Dönz (CzlcyB), involved in the biosynthesis of astaxanthin and lutein, have been performed in this work. These carotenoids are of high commercial value as dyes in food and as nutraceuticals. The open reading frame (ORF) of CzlcyB encoded a polypeptide of 546 amino acids. A single copy of CzlcyB has been found in C. zofingiensis. The chararacteristic Rossmann or dinucleotide binding fold, present in most lycopene cyclases, has been also identified in the LCYb of C. zofingiensis (CzLCYb). Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to cycle both lycopene and δ‐carotene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the lycopene β‐cyclases (LCYb) of the chlorophycean microalgae studied, being very closely related to LCYb of plants. Transcript levels of CzlcyB were increased under nitrogen deprivation, but no increase was observed under high‐light conditions. However, high irradiance triggered astaxanthin synthesis, while nitrogen deprivation by itself could not induce it. The combination of high irradiance and nitrogen deprivation led to a significant enhancement of the astaxathin accumulation.

Isolation and Characterization of a Lycopene ε-Cyclase Gene of Chlorella (Chromochloris) zofingiensis. Regulation of the Carotenogenic Pathway by Nitrogen and Light

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii

The carotenoid biosynthesis pathway catalyses the synthesis of essential pigments that are crucial for light harvesting and photoprotection in photosynthetic organisms. It allows the production of several commercially important compounds and is the target of many herbicides. In the present work we studied the influence of light on the carotenoid composition and the expression of genes encoding the main steps of the pathway in the freshwater microalga Chlamydomonas reinhardtii. We observed that there is an activation of the xanthophyll cycle in response to high light, but also in response to other stress conditions, such as nitrogen starvation, which has not been reported previously. We analysed the expression level of (1) genes encoding the two first enzymes of the pathway, phytoene synthase and phytoene desaturase; (2) the enzymes responsible for the cyclization of lycopene, lycopene β-cyclase and lycopene ε-cyclase; (3) zeaxanthin epoxidase, which catalyses the epoxidation of zeaxanthin; and (4) the three known carotene hydroxylases, directly involved in the synthesis of xanthophylls from α and β-carotene. Measurements of carotenoid content in the presence of inhibitors of protein and carotenoid synthesis suggest that only one of the two possible routes for the synthesis of zeaxanthin upon transference to high light, either the de novo synthesis of carotenoids or the interconversion of violaxanthin and zeaxanthin, is dependent on protein synthesis. The high increase in the transcript levels of the cytochrome-dependent carotene β- and ε-hydroxylases in response to high light suggests an important role of these enzymes in regulation of xanthophyll synthesis upon light stress. These conclusions may be of high interest if efficient engineering of the pathway is to be accomplished.

Synergism between inositol polyphosphates and TOR kinase signaling in nutrient sensing, growth control and lipid metabolism in Chlamydomonas

A mutation in a Chlamydomonas inositol kinase sensitizes cells to inhibition of TOR kinase signaling in a carbon source-dependent manner and uncouples lipid accumulation from starvation signals. The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP6) to produce the low abundance signaling molecules InsP7 and InsP8. Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP7 and InsP8, both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation.

Efficient heterologous transformation of Chlamydomonas reinhardtii npq2 mutant with the zeaxanthin epoxidase gene isolated and characterized from Chlorella zofingiensis.

In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

The TOR Signaling Network in the Model Unicellular Green Alga Chlamydomonas reinhardtii

Cell growth is tightly coupled to nutrient availability. The target of rapamycin (TOR) kinase transmits nutritional and environmental cues to the cellular growth machinery. TOR functions in two distinct multiprotein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). While the structure and functions of TORC1 are highly conserved in all eukaryotes, including algae and plants, TORC2 core proteins seem to be missing in photosynthetic organisms. TORC1 controls cell growth by promoting anabolic processes, including protein synthesis and ribosome biogenesis, and inhibiting catabolic processes such as autophagy. Recent studies identified rapamycin-sensitive TORC1 signaling regulating cell growth, autophagy, lipid metabolism, and central metabolic pathways in the model unicellular green alga Chlamydomonas reinhardtii. The central role that microalgae play in global biomass production, together with the high biotechnological potential of these organisms in biofuel production, has drawn attention to the study of proteins that regulate cell growth such as the TOR kinase. In this review we discuss the recent progress on TOR signaling in algae.