CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase for Efficient Astaxanthin Production in the Green Microalga Chlamydomonas reinhardtii.

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

Photoautotrophic cultivation of a Chlamydomonas reinhardtii mutant with zeaxanthin as the sole xanthophyll.

BACKGROUND: Photosynthetic microalgae are known for their sustainable and eco-friendly potential to convert carbon dioxide into valuable products. Nevertheless, the challenge of self-shading due to high cell density has been identified as a drawback, hampering productivity in sustainable photoautotrophic mass cultivation. To address this issue, mutants with altered pigment composition have been proposed to allow a more efficient light diffusion but further study on the role of the different pigments is still needed to correctly engineer this process. RESULTS: We here investigated the Chlamydomonas reinhardtii Δzl mutant with zeaxanthin as the sole xanthophyll. The Δzl mutant displayed altered pigment composition, characterized by lower chlorophyll content, higher chlorophyll a/b ratio, and lower chlorophyll/carotenoid ratio compared to the wild type (Wt). The Δzl mutant also exhibited a significant decrease in the light-harvesting complex II/Photosystem II ratio (LHCII/PSII) and the absence of trimeric LHCIIs. This significantly affects the organization and stability of PSII supercomplexes. Consequently, the estimated functional antenna size of PSII in the Δzl mutant was approximately 60% smaller compared to that of Wt, and reduced PSII activity was evident in this mutant. Notably, the Δzl mutant showed impaired non-photochemical quenching. However, the Δzl mutant compensated by exhibiting enhanced cyclic electron flow compared to Wt, seemingly offsetting the impaired PSII functionality. Consequently, the Δzl mutant achieved significantly higher cell densities than Wt under high-light conditions. CONCLUSIONS: Our findings highlight significant changes in pigment content and pigment-protein complexes in the Δzl mutant compared to Wt, resulting in an advantage for high-density photoautotrophic cultivation. This advantage is attributed to the decreased chlorophyll content of the Δzl mutant, allowing better light penetration. In addition, the accumulated zeaxanthin in the mutant could serve as an antioxidant, offering protection against reactive oxygen species generated by chlorophylls.

Overexpression of PtVDL1 in Phaeodactylum tricornutum Increases Fucoxanthin Content under Red Light.

Phaeodactylum tricornutum is a model diatom with significant biotechnological applications, including enhancing biomass, biofuel, and carotenoid production. Specifically, owing to the capacity of this organism to serve as a valuable source of essential raw materials for pharmaceuticals and nutraceuticals, ongoing research is actively focused on enhancing its productivity. One of the genes involved in various stages of fucoxanthin (Fx) biosynthesis, violaxanthin de-epoxidase like 1 (VDL1), has recently been identified. To validate the intracellular function of this gene and boost Fx production through overexpression, we established and examined three transgenic P. tricornutum lines characterized by elevated P. tricortunum VDL1 ( PtVDL1) expression and evaluate their cell growth and Fx productivity. These transgenic lines exhibited substantially increased PtVDL1 mRNA and protein levels compared to the wild type (WT). Notably, the enzyme substrate violaxanthin was entirely depleted and could not be detected in the transformants, whereas it remained at constant levels in the WT. Interestingly, under standard white light conditions, Fx productivity in the transformants remained unchanged; however, but after 48 h of exposure to red light, it increased by up to 15%. These results indicate that PtVDL1-overexpressing P. tricornutum has industrial potential, particularly for enhancing Fx production under red light conditions.

Photosystem II monomeric antenna CP26 plays a key role in nonphotochemical quenching in Chlamydomonas.

Thermal dissipation of excess excitation energy, called nonphotochemical quenching (NPQ), is 1 of the main photoprotective mechanisms in oxygenic photosynthetic organisms. Here, we investigated the function of the monomeric photosystem II (PSII) antenna protein CP26 in photoprotection and light harvesting in Chlamydomonas reinhardtii, a model organism for green algae. We used CRISPR/Cas9 genome editing and complementation to generate cp26 knockout mutants (named k6#) that did not negatively affect CP29 accumulation, which differed from previous cp26 mutants, allowing us to compare mutants specifically deprived of CP26, CP29, or both. The absence of CP26 partially affected PSII activity, causing reduced growth at low or medium light but not at high irradiances. However, the main phenotype observed in k6# mutants was a more than 70% reduction of NPQ compared to the wild type (Wt). This phenotype was fully rescued by genetic complementation and complemented strains accumulating different levels of CP26, demonstrating that ∼50% of CP26 content, compared to the Wt, was sufficient to restore the NPQ capacity. Our findings demonstrate a pivotal role for CP26 in NPQ induction, while CP29 is crucial for PSII activity. The genetic engineering of these 2 proteins could be a promising strategy to regulate the photosynthetic efficiency of microalgae under different light regimes.

Genome engineering via gene editing technologies in microalgae.

CRISPR-Cas has revolutionized genetic modification with its comparative simplicity and accuracy, and it can be used even at the genomic level. Microalgae are excellent feedstocks for biofuels and nutraceuticals because they contain high levels of fatty acids, carotenoids, and other metabolites; however, genome engineering for microalgae is not yet as developed as for other model organisms. Microalgal engineering at the genetic and metabolic levels is relatively well established, and a few genomic resources are available. Their genomic information was used for a "safe harbor" site for stable transgene expression in microalgae. This review proposes further genome engineering schemes including the construction of sgRNA libraries, pan-genomic and epigenomic resources, and mini-genomes, which can together be developed into synthetic biology for carbon-based engineering in microalgae. Acetyl-CoA is at the center of carbon metabolic pathways and is further reviewed for the production of molecules including terpenoids in microalgae.

Inhibition of monogalactosyldiacylglycerol synthesis by down-regulation of MGD1 leads to membrane lipid remodeling and enhanced triacylglycerol biosynthesis in Chlamydomonas reinhardtii.

BACKGROUND: Membrane lipid remodeling involves regulating the physiochemical modification of cellular membranes against abiotic stress or senescence, and it could be a trigger to increase neutral lipid content. In algae and higher plants, monogalactosyldiacylglycerol (MGDG) constitutes the highest proportion of total membrane lipids and is highly reduced as part of the membrane lipid remodeling response under several abiotic stresses. However, genetic regulation of MGDG synthesis and its influence on lipid synthesis has not been studied in microalgae. For development of an industrial microalgae strain showing high accumulation of triacylglycerol (TAG) by promoting membrane lipid remodeling, MGDG synthase 1 (MGD1) down-regulated mutant of Chlamydomonas reinhardtii (Cr-mgd1) was generated and evaluated for its suitability for biodiesel feedstock. RESULTS: The Cr-mgd1 showed a 65% decrease in CrMGD1 gene expression level, 22% reduction in MGDG content, and 1.39 and 5.40 times increase in diacylglyceryltrimethylhomoserines (DGTS) and TAG, respectively. The expression levels of most genes related to the decomposition of MGDG (plastid galactoglycerolipid degradation1) and TAG metabolism (diacylglycerol O-acyltransferase1, phospholipid:diacylglycerol acyltransferase, and major lipid droplet protein) were increased. The imbalance of DGDG/MGDG ratio in Cr-mgd1 caused reduced photosynthetic electron transport, resulting in less light energy utilization and increased reactive oxygen species levels. In addition, endoplasmic reticulum stress was induced by increased DGTS levels. Thus, accelerated TAG accumulation in Cr-mgd1 was stimulated by increased cellular stress as well as lipid remodeling. Under high light (HL) intensity (400 µmol photons/m2/s), TAG productivity in Cr-mgd1-HL (1.99 mg/L/d) was 2.71 times higher than that in wild type (WT-HL). Moreover, under both nitrogen starvation and high light intensity, the lipid (124.55 mg/L/d), TAG (20.03 mg/L/d), and maximum neutral lipid (56.13 mg/L/d) productivity were the highest. CONCLUSIONS: By inducing lipid remodeling through the mgd1 gene expression regulation, the mutant not only showed high neutral lipid content but also reached the maximum neutral lipid productivity through cultivation under high light and nitrogen starvation conditions, thereby possessing improved biomass properties that are the most suitable for high quality biodiesel production. Thus, this mutant may help understand the role of MGD1 in lipid synthesis in Chlamydomonas and may be used to produce high amounts of TAG.

Sex-linked deubiquitinase establishes uniparental transmission of chloroplast DNA.

Most sexual organisms inherit organelles from one parent, commonly by excluding organelles from the smaller gametes. However, post-mating elimination of organelles derived from one gamete ensures uniparental inheritance, where the underlying mechanisms to distinguish organelles by their origin remain obscure. Mating in Chlamydomonas reinhardtii combines isomorphic plus and minus gametes, but chloroplast DNA from minus gametes is selectively degraded in zygotes. Here, we identify OTU2p (otubain protein 2), encoded in the plus mating-type locus MT+, as the protector of plus chloroplast. Otu2p is an otubain-like deubiquitinase, which prevents proteasome-mediated degradation of the preprotein translocase of the outer chloroplast membrane (TOC) during gametogenesis. Using OTU2p-knockouts and proteasome inhibitor treatment, we successfully redirect selective DNA degradation in chloroplasts with reduced TOC levels regardless of mating type, demonstrating that plus-specific Otu2p establishes uniparental chloroplast DNA inheritance. Our work documents that a sex-linked organelle quality control mechanism drives the uniparental organelle inheritance without dimorphic gametes.

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants.

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin-Benson-Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

The Chlamydomonas bZIP transcription factor BLZ8 confers oxidative stress tolerance by inducing the carbon-concentrating mechanism.

Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. Here, we characterized the Chlamydomonas reinhardtii basic leucine zipper (bZIP) transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.

Inhibition of 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE expression by brassinosteroid reduces carotenoid accumulation in Arabidopsis.

Unlike the indispensable function of the steroid hormone brassinosteroid (BR) in regulating plant growth and development, the metabolism of secondary metabolites regulated by BR is not well known. Here we show that BR reduces carotenoid accumulation in Arabidopsis seedlings. BR-deficient or BR-insensitive mutants accumulated higher content of carotenoids than wild-type plants, whereas BR treatment reduced carotenoid content. We demonstrated that BR transcriptionally suppresses 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) expression involved in carotenogenesis via plastoquinone production. We found that the expression of HPPD displays an oscillation pattern that is expressed more strongly in dark than in light conditions. Moreover, BR appeared to inhibit HPPD expression more strongly in darkness than in light, leading to suppression of a diurnal oscillation of HPPD expression. BR-responsive transcription factor BRASSINAZOLE RESISTANT 1 (BZR1) directly bound to the promoter of HPPD, and HPPD suppression by BR was increased in the bzr1-1D gain-of-function mutation. Interestingly, dark-induced HPPD expression did not cause carotenoid accumulation, due to down-regulation of other carotenoid biosynthetic genes in the dark. Our results suggest that BR regulates different physiological responses in dark and light through inhibition of HPPD expression.

Augmented CO2 tolerance by expressing a single H+-pump enables microalgal valorization of industrial flue gas.

Microalgae can accumulate various carbon-neutral products, but their real-world applications are hindered by their CO2 susceptibility. Herein, the transcriptomic changes in a model microalga, Chlamydomonas reinhardtii, in a high-CO2 milieu (20%) are evaluated. The primary toxicity mechanism consists of aberrantly low expression of plasma membrane H+-ATPases (PMAs) accompanied by intracellular acidification. Our results demonstrate that the expression of a universally expressible PMA in wild-type strains makes them capable of not only thriving in acidity levels that they usually cannot survive but also exhibiting 3.2-fold increased photoautotrophic production against high CO2 via maintenance of a higher cytoplasmic pH. A proof-of-concept experiment involving cultivation with toxic flue gas (13 vol% CO2, 20 ppm NOX, and 32 ppm SOX) shows that the production of CO2-based bioproducts by the strain is doubled compared with that by the wild-type, implying that this strategy potentially enables the microalgal valorization of CO2 in industrial exhaust.

LPA2 protein is involved in photosystem II assembly in Chlamydomonas reinhardtii.

Photosynthetic eukaryotes require the proper assembly of photosystem II (PSII) in order to strip electrons from water and fuel carbon fixation reactions. In Arabidopsis thaliana, one of the PSII subunits (CP43/PsbC) was suggested to be assembled into the PSII complex via its interaction with an auxiliary protein called Low PSII Accumulation 2 (LPA2). However, the original articles describing the role of LPA2 in PSII assembly have been retracted. To investigate the function of LPA2 in the model organism for green algae, Chlamydomonas reinhardtii, we generated knockout lpa2 mutants by using the CRISPR-Cas9 target-specific genome editing system. Biochemical analyses revealed the thylakoidal localization of LPA2 protein in the wild type (WT), whereas lpa2 mutants were characterized by a drastic reduction in the levels of D1, D2, CP47 and CP43 proteins. Consequently, reduced PSII supercomplex accumulation, chlorophyll content per cell, PSII quantum yield and photosynthetic oxygen evolution were measured in the lpa2 mutants, leading to the almost complete impairment of photoautotrophic growth. Pulse-chase experiments demonstrated that the absence of LPA2 protein caused reduced PSII assembly and reduced PSII turnover. Taken together, our data indicate that, in C. reinhardtii, LPA2 is required for PSII assembly and proper function.

Discovery of Post-Translational Modifications in Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

Molecular basis of ice-binding and cryopreservation activities of type III antifreeze proteins.

Antifreeze proteins (AFPs) can inhibit the freezing of body fluid at subzero temperatures to promote the survival of various organisms living in polar regions. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We determined the thermal hysteresis (TH), ice recrystallization inhibition (IRI), and cryopreservation activity of three isoforms of the notched-fin eelpout AFP and their mutant constructs and characterized their structural and dynamic features using NMR. The QAE1 isoform is the most active among the three classes of III AFP isoforms, and the mutants of inactive QAE2 and SP isoforms, QAE2ACT and SPACT, displayed the full TH and IRI activities with resepect to QAE1 isoform. Cryopreservation studies using mouse ovarian tissue revealed that the QAE1 isoform and the active mutants, QAE2ACT and SPACT, more effectively preserved intact follicle morphology and prevented DNA double-strand break damage more efficiently than the inactive isoforms. It was also found that all active AFPs, QAE1, QAE2ACT, and SPACT, formed unique H-bonds with the first 310 helix, an interaction that plays an important role in the formation of anchored clathrate water networks for efficient binding to the primary prism and pyramidal planes of ice crystals, which was disrupted in the inactive isoforms. Our studies provide valuable insights into the molecular mechanism of the TH and IRI activity, as well as the cryopreservation efficiency, of type III AFPs.

Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395.

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

Identification of distinct pH- and zeaxanthin-dependent quenching in LHCSR3 from Chlamydomonas reinhardtii.

Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called nonphotochemical quenching. In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via a pH drop and serves as a quenching site. Using a combined in vivo and in vitro approach, we investigated quenching within LHCSR3 from Chlamydomonas reinhardtii. In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH-dependent quenching was removed within a mutant LHCSR3 that lacks the residues that are protonated to sense the pH drop. Observation of quenching in zeaxanthin-enriched LHCSR3 even at neutral pH demonstrated zeaxanthin-dependent quenching, which also occurs in other light-harvesting complexes. Either pH- or zeaxanthin-dependent quenching prevented the formation of damaging reactive oxygen species, and thus the two quenching processes may together provide different induction and recovery kinetics for photoprotection in a changing environment.

Green plants and algae rely on sunlight to transform light energy into chemical energy in a process known as photosynthesis. However, too much light can damage plants. Green plants prevent this by converting the extra absorbed light into heat. Both the absorption and the dissipation of sunlight into heat occur within so called light harvesting complexes. These are protein structures that contain pigments such as chlorophyll and carotenoids. The process of photoprotection starts when the excess of absorbed light generates protons (elementary particles with a positive charge) faster than they can be used. This causes a change in the pH (a measure of the concentration of protons in a solution), which in turn, modifies the shape of proteins and the chemical identity of the carotenoids. However, it is still unclear what the exact mechanisms are. To clarify this, Troiano, Perozeni et al. engineered the light harvesting complex LHCSR3 of the green algae Chlamydomonas reinhardtii to create mutants that either could not sense changes in the pH or contained the carotenoid zeaxanthin. Zeaxanthin is one of the main carotenoids accumulated by plants and algae upon high light stress. Measurements showed that both pH detection and zeaxanthin were able to provide photoprotection independently. Troiano, Perozeni et al. further found that pH and carotenoids controlled changes to the organisation of the pigment at two separate locations within the LHCSR3, which influenced whether the protein was able to prevent photodamage. When algae were unable to change pH or carotenoids, dissipation was less effective. Instead, specific molecules were produced that damage the cellular machinery. The results shed light onto how green algae protect themselves from too much light exposure. These findings could pave the way for optimising dissipation, which could increase yields of green algae by up to 30%. This could lead to green algae becoming a viable alternative for food, biofuels and feedstock.

Bone Graft Biomineral Complex Coderived from Marine Biocalcification and Biosilicification.

Bone graft materials have been mainly developed based on inorganic materials, including calcium phosphate. However, these graft materials usually act as osteoconductive rather than osteoinductive scaffolds. To improve bone reconstruction, a combination of several materials has been proposed. However, there are still no alternatives that can completely replace the existing animal-derived bone graft materials. In this work, a marine-inspired biomineral complex was suggested as a potential bone graft material. The proposed biosilicified coccolithophore-derived coccoliths using bioengineered mussel adhesive proteins show osteopromotive ability through the synergistic effects of osteoconductivity from calcium carbonate and osteoinductivity from silica. Its possibility of use as a bone substitute was determined by evaluating the in vitro osteogenic behaviors of multipotent mesenchymal stem cells and in vivo bone regeneration in a rat calvarial defect model. Therefore, the marine-inspired biomineral complex developed in this study could be successfully used for bone tissue engineering.

The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii.

BACKGROUND: Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin. RESULTS: In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of α-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L- 1) and content (7.28 mg g- 1) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L- 1. CONCLUSIONS: A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.