scholarly journals Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

2019 ◽  
Author(s):  
Federico Perozeni ◽  
Stefano Cazzaniga ◽  
Thomas Baier ◽  
Francesca Zanoni ◽  
Gianni Zoccatelli ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Nuclear Genome ◽  
Biomass Productivity ◽  
Carotenoid Biosynthesis ◽  
Growth Conditions ◽  
Promising Alternative ◽  
Green Algal ◽  
Algal Host ◽  
Constitutive Overexpression

SummaryThe green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a β-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L-1 day-1. Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

scholarly journals Chromosome-level genome assembly and transcriptome of the green alga Chromochloris zofingiensis illuminates astaxanthin production

2017 ◽  
Vol 114 (21) ◽  
pp. E4296-E4305 ◽  
Author(s):  
Melissa S. Roth ◽  
Shawn J. Cokus ◽  
Sean D. Gallaher ◽  
Andreas Walter ◽  
David Lopez ◽  
...  
Keyword(s):  
Green Alga ◽  
Light Exposure ◽  
Nuclear Genome ◽  
High Light ◽  
Cytochrome P450 Enzymes ◽  
Commercial Development ◽  
Protein Coding ◽  
Green Algal ◽  
Chromochloris Zofingiensis ◽  
Chromosome Level

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga Chromochloris zofingiensis, because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that BKT1 is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.

Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

1990 ◽  
Vol 78 (2) ◽  
pp. 254-260 ◽  
Author(s):  
Anil Day ◽  
Robert Debuchy ◽  
Jeanette Dillewijn ◽  
Saul Purton ◽  
Jean-David Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Nuclear Genome ◽  
Dna Fragments

scholarly journals Structure of a C2S2M2N2-type PSII–LHCII supercomplex from the green alga Chlamydomonas reinhardtii

2019 ◽  
Vol 116 (42) ◽  
pp. 21246-21255 ◽  
Author(s):  
Liangliang Shen ◽  
Zihui Huang ◽  
Shenghai Chang ◽  
Wenda Wang ◽  
Jingfen Wang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Higher Plants ◽  
Huge Number ◽  
Higher Plant ◽  
Low Light ◽  
Green Algal ◽  
Light Harvesting Antenna ◽  
Energy Transfer Pathway

Photosystem II (PSII) in the thylakoid membranes of plants, algae, and cyanobacteria catalyzes light-induced oxidation of water by which light energy is converted to chemical energy and molecular oxygen is produced. In higher plants and most eukaryotic algae, the PSII core is surrounded by variable numbers of light-harvesting antenna complex II (LHCII), forming a PSII–LHCII supercomplex. In order to harvest energy efficiently at low–light-intensity conditions under water, a complete PSII–LHCII supercomplex (C2S2M2N2) of the green alga Chlamydomonas reinhardtii (Cr) contains more antenna subunits and pigments than the dominant PSII–LHCII supercomplex (C2S2M2) of plants. The detailed structure and energy transfer pathway of the Cr-PSII–LHCII remain unknown. Here we report a cryoelectron microscopy structure of a complete, C2S2M2N2-type PSII–LHCII supercomplex from C. reinhardtii at 3.37-Å resolution. The results show that the Cr-C2S2M2N2 supercomplex is organized as a dimer, with 3 LHCII trimers, 1 CP26, and 1 CP29 peripheral antenna subunits surrounding each PSII core. The N-LHCII trimer partially occupies the position of CP24, which is present in the higher-plant PSII–LHCII but absent in the green alga. The M trimer is rotated relative to the corresponding M trimer in plant PSII–LHCII. In addition, some unique features were found in the green algal PSII core. The arrangement of a huge number of pigments allowed us to deduce possible energy transfer pathways from the peripheral antennae to the PSII core.

Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas reinhardtii

1990 ◽  
Vol 78 (2) ◽  
pp. 254-260 ◽  
Author(s):  
Anil Day ◽  
Robert Debuchy ◽  
Jeanette van Dillewijn ◽  
Saul Purton ◽  
Jean-David Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Nuclear Genome ◽  
Dna Fragments

scholarly journals Overexpression of Orange (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress

2020 ◽  
Author(s):  
Mohammad Yazdani ◽  
Michelle G. Croen ◽  
Tara L. Fish ◽  
Theodore W. Thannhauser ◽  
Beth A. Ahner
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Regulatory Protein ◽  
Microscopic Analysis ◽  
Normal Growth ◽  
Carotenoid Biosynthesis ◽  
Transcript Abundance ◽  
Growth Conditions ◽  
Carotenoid Content ◽  
Endogenous Phytohormone

AbstractThe carotenoid content of plants can be increased by overexpression of the regulatory protein ORANGE (OR) or a mutant variant known as the ‘golden SNP’. In the present study, transgenic lines of the microalgae Chlamydomonas reinhardtii were generated to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine. Overexpression of both CrORWT and CrORHis dramatically enhanced the accumulation of several different carotenoids, including β-cartotene, α-carotene, lutein and violaxanthin, in C. reinhardtii and, in contrast to higher plants, upregulated the transcript abundance of several relevant carotenoid biosynthetic genes. In addition, microscopic analysis revealed that the OR transgenic cells were larger than control cells and exhibited larger chloroplasts with a disrupted morphology. Moreover, both CrORWT and CrORHis cell lines showed increased tolerance to salt and paraquat stress. The levels of endogenous phytohormone abscisic acid (ABA) were also increased in CrORWT and CrORHis lines, not only in normal growth conditions but also in growth medium supplemented with paraquat. Together these results offer new insights regarding the role of the OR protein in regulating carotenoid biosynthesis and accumulation in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance mediated by ABA.

Only iron-limited cells of the cyanobacterium Anabaena flos-aquae inhibit growth of the green alga Chlamydomonas reinhardtii

2004 ◽  
Vol 82 (4) ◽  
pp. 436-442 ◽  
Author(s):  
Carlyn J Matz ◽  
Michael R Christensen ◽  
Auralee D Bone ◽  
Courtney D Gress ◽  
Scott B Widenmaier ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Activated Charcoal ◽  
Algal Growth ◽  
Inhibitory Effect ◽  
The Other ◽  
Iron Limitation ◽  
Green Algal ◽  
Cyanobacterium Anabaena ◽  
Cobalt Copper

Cocultivation of iron-limited cells of the cyanobacterium Anabaena flos-aquae (Lyng.) Brèb. and the green alga Chlamydomonas reinhardtii Dangeard resulted in growth of Anabaena but not Chlamydomonas, even in the presence of excess exogenous iron. This effect was also observed during the cultivation of Chlamydomonas in a medium in which iron-limited Anabaena cells had been growing, but were removed prior to culture of Chlamydomonas. Conversely, iron-limited Chlamydomonas cells grew very well in medium from iron (nutrient)-sufficient, phosphate-limited, and nitrogen-limited Anabaena cultures. Iron-limited Anabaena cultures produced siderophores, while the other types of Anabaena cultures did not. Treatment of Anabaena iron-limited medium with activated charcoal completely removed the inhibitory effect on Chlamydomonas growth, and boiling the medium removed most of the inhibitory effect. Both the charcoal and the boiling treatments also removed siderophores from the medium. Partially purified Anabaena siderophore preparations were also inhibitory to Chlamydomonas growth. The inhibitory effect of iron-limited Anabaena medium could be partially overcome by addition of excess micronutrients (especially cobalt copper) but not by addition of iron. We suggest that Anabaena-derived siderophores, present only in iron-limited Anabaena medium, inhibit the growth of Chlamydomonas cells via a previously uncharacterized toxicity. This effect is different from previously described experiments in which cyanobacterial siderophores suppressed green algal growth via competition for limiting amounts of iron.Key words: Anabaena, Chlamydomonas, cocultivation, iron limitation, micronutrients; siderophores.

scholarly journals Novel Shuttle Markers for Nuclear Transformation of the Green Alga Chlamydomonas reinhardtii

2011 ◽  
Vol 10 (12) ◽  
pp. 1670-1678 ◽  
Author(s):  
Laurence Meslet-Cladière ◽  
Olivier Vallon
Keyword(s):  
Antibiotic Resistance ◽  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Nuclear Genome ◽  
Nuclear Transformation ◽  
Lower Efficiency ◽  
Chloroplast Targeting ◽  
Content Type ◽  
Artificial Chromosomes ◽  
Test Gene

ABSTRACTThe green algaChlamydomonas reinhardtiitoday is a premier model organism for the study of green algae and plants. Yet the efficient engineering of its nuclear genome requires development of new antibiotic resistance markers. We have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used previously for chloroplast transformation. The recoded AadA gene, placed under the control of theHSP70A-RBCS2hybrid promoter and preceded by the RbcS2 chloroplast-targeting peptide, can be integrated into the nuclear genome by electroporation, conferring resistance to spectinomycin and streptomycin. Transformation efficiency is markedly increased when vector sequences are completely eliminated from the transforming DNA. Antibiotic resistance is stable for several months in the absence of selection pressure. Shuttle markers allowing selection in bothChlamydomonasandEscherichia coliwould also be a useful asset. By placing an artificial bacterial promoter and Shine-Dalgarno sequence in frame within the AadA coding sequence, we generated such a shuttle marker. To our surprise, we found that the classical AphVIII construct already functions as a shuttle marker. Finally, we developed a method to introduce the AadA and AphVIII markers into the vector part of the bacterial artificial chromosomes (BACs) of theChlamydomonasgenomic DNA library. Our aim was to facilitate complementation studies whenever the test gene cannot be selected for directly. After transformation of apetCmutant with a modified BAC carrying the AphVIII marker along with thePETCgene in the insert, almost half of the paromomycin-resistant transformants obtained showed restoration of phototrophy, indicating successful integration of the unselected test gene. With AadA, cotransformation was also observed, but with a lower efficiency.

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