Stenotrophomonas goyi sp. nov., a novel bacterium associated with the alga Chlamydomonas reinhardtii.

Background: A culture of the green algae Chlamydomonas reinhardtii was accidentally contaminated with three different bacteria in our laboratory facilities. This contaminated alga culture showed increased algal biohydrogen production. These three bacteria were independently isolated. Methods: The chromosomic DNA of one of the isolated bacteria was extracted and sequenced using PacBio technology. Tentative genome annotation (RAST server) and phylogenetic trees analysis (TYGS server) were conducted. Diverse growth tests were assayed for the bacterium and for the alga-bacterium consortium. Results: Phylogenetic analysis indicates that the bacterium is a novel member of the Stenotrophomonas genus that has been termed in this work as S. goyi sp. nov. A fully sequenced genome (4,487,389 base pairs) and its tentative annotation (4,147 genes) are provided. The genome information suggests that S. goyi sp. nov. is unable to use sulfate and nitrate as sulfur and nitrogen sources, respectively. Growth tests have confirmed the dependence on the sulfur-containing amino acids methionine and cysteine. S. goyi sp. nov. and Chlamydomonas reinhardtii can establish a mutualistic relationship when cocultured together. Conclusions: S. goyi sp. nov. could be of interest for the design of biotechnological approaches based on the use of artificial microalgae-bacteria multispecies consortia that take advantage of the complementary metabolic capacities of their different microorganisms.

Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as "gene loading" and "culture optimizing" were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae.

Overexpression of native ORANGE (OR) and OR mutant protein in Chlamydomonas reinhardtii enhances carotenoid and ABA accumulation and increases resistance to abiotic stress.

The 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, a strong light-inducible promoter was used to overexpress either wild type CrOR (CrORWT) or a mutated CrOR (CrORHis) containing a single histidine substitution for a conserved arginine in the microalgae Chlamydomonas reinhardtii. Overexpression of CrORWT and CrORHis roughly doubled and tripled, respectively, the accumulation of several different carotenoids, including ß-carotene, α-carotene, lutein and violaxanthin in C. reinhardtii and upregulated the transcript abundance of nearly all 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 salt and paraquat. Together these results offer new insights regarding the role of the native OR protein in regulating carotenoid biosynthesis and the accumulation of several carotenoids in microalgae, and establish a new functional role for OR to modulate oxidative stress tolerance potentially mediated by ABA.

Truncated hemoglobin 2 modulates phosphorus deficiency response by controlling of gene expression in nitric oxide-dependent pathway in Chlamydomonas reinhardtii.

MAIN CONCLUSION: Truncated hemoglobin 2 is involved in fine-tuning of PSR1-regulated gene expression during phosphorus deprivation. Truncated hemoglobins form a large family found in all domains of life. However, a majority of physiological functions of these proteins remain to be elucidated. In the model alga Chlamydomonas reinhardtii, macro-nutritional deprivation is known to elevate truncated hemoglobin 2 (THB2). This study investigated the role of THB2 in the regulation of a subset of phosphorus (P) limitation-responsive genes in cells suffering from P-deficiency. Underexpression of THB2 in amiTHB2 strains resulted in downregulation of a suite of P deprivation-induced genes encoding proteins with different subcellular location and functions (e.g., PHOX, LHCSR3.1, LHCSR3.2, PTB2, and PTB5). Moreover, our results provided primary evidence that the soluble guanylate cyclase 12 gene (CYG12) is a component of the P deprivation regulation. Furthermore, the transcription of PSR1 gene for the most critical regulator in the acclimation process under P restriction was repressed by nitric oxide (NO). Collectively, the results indicated a tight regulatory link between the THB2-controlled NO levels and PSR1-dependent induction of several P deprivation responsive genes with various roles in cells during P-limitation.

Responses of Chlamydomonas reinhardtii during the transition from P-deficient to P-sufficient growth (the P-overplus response): The roles of the vacuolar transport chaperones and polyphosphate synthesis.

Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO43- ; 10 mg P·L-1 ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C. reinhardtii assimilated PO43- and stored polyP within minutes of adding PO43- to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO43- . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO43- assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C. reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO43- assimilation in C. reinhardtii. RNA-sequencing was carried out on C. reinhardtii cells that were either P-depleted (control) or supplied with PO43- following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24 h after the addition of PO43- to the cells, although polyP granules were continuously synthesized during this 24 h period. These results suggest that the VTC complex remains active for at least 24 h after supplying the cells with PO43- . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.

Loss of two families of SPX domain-containing proteins required for vacuolar polyphosphate accumulation coincides with the transition to phosphate storage in green plants.

Phosphorus is an essential nutrient for plants. It is stored as inorganic phosphate (Pi) in the vacuoles of land plants but as inorganic polyphosphate (polyP) in chlorophyte algae. Although it is recognized that the SPX-Major Facilitator Superfamily (MFS) and VPE proteins are responsible for Pi influx and efflux, respectively, across the tonoplast in land plants, the mechanisms that underlie polyP homeostasis and the transition of phosphorus storage forms during the evolution of green plants remain unclear. In this study, we showed that CrPTC1, encoding a protein with both SPX and SLC (permease solute carrier 13) domains for Pi transport, and CrVTC4, encoding a protein with both SPX and vacuolar transporter chaperone (VTC) domains for polyP synthesis, are required for vacuolar polyP accumulation in the chlorophyte Chlamydomonas reinhardtii. Phylogenetic analysis showed that the SPX-SLC, SPX-VTC, and SPX-MFS proteins were present in the common ancestor of green plants (Viridiplantae). The SPX-SLC and SPX-VTC proteins are conserved among species that store phosphorus as vacuolar polyP and absent from genomes of plants that store phosphorus as vacuolar Pi. By contrast, SPX-MFS genes are present in the genomes of streptophytes that store phosphorus as Pi in the vacuoles. These results suggest that loss of SPX-SLC and SPX-VTC genes and functional conservation of SPX-MFS proteins during the evolution of streptophytes accompanied the change from ancestral polyP storage to Pi storage.

Selenium(â £) alleviates chromium(â ¥)-induced toxicity in the green alga Chlamydomonas reinhardtii.

The wide range of industrial applications of chromium (Cr) has led to an increasing risk of water contamination by Cr(Ⅵ). However, efficient methods to remove or decrease the toxicity of Cr(Ⅵ) in situ are lacking. The main aim of this study was to investigate the mechanisms by which selenite alleviates chromium(Ⅵ)-induced toxicity in Chlamydomonas reinhardtii. Our results showed that K2Cr2O7 had toxic effects on both the structure and physiology of C. reinhardtii in a dose-dependent manner. Adding selenite significantly alleviated chromium accumulation and toxicity in cells. RNA-seq data showed that the expression level of selenoproteins such as SELENOH was significantly increased. Both SELENOH-amiRNA knockdown mutants and selenoh insertional mutant produced more reactive oxygen species (ROS) and grew slower than the wild type, suggesting that SELENOH can reduce chromium toxicity by decreasing the levels of ROS produced by Cr(Ⅵ). We also demonstrated that selenite can reduce the absorption of Cr(Ⅵ) by cells but does not affect the process of Cr(Ⅵ) adsorption and efflux. This information on the molecular mechanism by which selenite alleviates Cr(Ⅵ) toxicity can be used to increase the bioremediation capacity of algae and reduce the human health risks associated with Cr(Ⅵ) toxicity.

Phosphorus Availability Regulates TORC1 Signaling via LST8 in Chlamydomonas.

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas (Chlamydomonas reinhardtii) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas.

Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil.

The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

Lipid remodeling regulator 1 (LRL1) is differently involved in the phosphorus-depletion response from PSR1 in Chlamydomonas reinhardtii.

The elucidation of lipid metabolism in microalgae has attracted broad interest, as their storage lipid, triacylglycerol (TAG), can be readily converted into biofuel via transesterification. TAG accumulates in the form of oil droplets, especially when cells undergo nutrient deprivation, such as for nitrogen (N), phosphorus (P), or sulfur (S). TAG biosynthesis under N-deprivation has been comprehensively studied in the model microalga Chlamydomonas reinhardtii, during which TAG accumulates dramatically. However, the resulting rapid breakdown of chlorophyll restricts overall oil yield productivity and causes cessation of cell growth. In contrast, P-deprivation results in oil accumulation without disrupting chloroplast integrity. We used a reverse genetics approach based on co-expression analysis to identify a transcription factor (TF) that is upregulated under P-depleted conditions. Transcriptomic analysis revealed that the mutants showed repression of genes typically associated with lipid remodeling under P-depleted conditions, such as sulfoquinovosyl diacylglycerol 2 (SQD2), diacylglycerol acyltransferase (DGTT1), and major lipid droplet protein (MLDP). As accumulation of sulfoquinovosyl diacylglycerol and TAG were suppressed in P-depleted mutants, we designated the protein as lipid remodeling regulator 1 (LRL1). LRL1 mutants showed slower growth under P-depletion. Moreover, cell size in the mutant was significantly reduced, and TAG and starch accumulation per cell were decreased. Transcriptomic analysis also suggested the repression of several genes typically upregulated in adaptation to P-depletion that are associated with the cell cycle and P and lipid metabolism. Thus, our analysis of LRL1 provides insights into P-allocation and lipid remodeling under P-depleted conditions in C. reinhardtii. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The sequencing data were made publicly available under the BioProject Accession number PRJDB6733 and an accession number LC488724 at the DNA Data Bank of Japan (DDBJ). The data is available at https://trace.ddbj.nig.ac.jp/BPSearch/bioproject?acc=PRJDB6733; http://getentry.ddbj.nig.ac.jp/getentry/na/LC488724. The metabolome data were made publicly available and can be accessed at http://metabolonote.kazusa.or.jp/SE195:/; http://webs2.kazusa.or.jp/data/nur/.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.

Green biologics: The algal chloroplast as a platform for making biopharmaceuticals.

Most commercial production of recombinant pharmaceutical proteins involves the use of mammalian cell lines, E. coli or yeast as the expression host. However, recent work has demonstrated the potential of eukaryotic microalgae as platforms for light-driven synthesis of such proteins. Expression in the algal chloroplast is particularly attractive since this organelle contains a minimal genome suitable for rapid engineering using synthetic biology approaches; with transgenes precisely targeted to specific genomic loci and amenable to high-level, regulated and stable expression. Furthermore, proteins can be tightly contained and bio-encapsulated in the chloroplast allowing accumulation of proteins otherwise toxic to the host, and opening up possibilities for low-cost, oral delivery of biologics. In this commentary we illustrate the technology with recent examples of hormones, protein antibiotics and immunotoxins successfully produced in the algal chloroplast, and highlight possible future applications.

Recombinant Hemagglutinin of Avian Influenza Virus H5 Expressed in the Chloroplast of Chlamydomonas reinhardtii and Evaluation of Its Immunogenicity in Chickens.

Globally, avian influenza (AI) is a serious problem in poultry farming. Despite vaccination, the prevalence of AI in México highlights the need for new approaches to control AI and to reduce the economic losses associated with its occurrence in susceptible birds. Recombinant proteins from avian influenza virus (AIV) have been expressed in different organisms, such as plants. The present study investigated the feasibility of designing and expressing the HA protein of AIV in the transplastomic microalga Chlamydomonas reinhardtii as a novel approach for AIV control and taking advantage of culture conditions, its reproductive range, and safe use in consideration of the generally regarded as safe food ingredient regulatory classification. The results showed that the HA protein of AIV in C. reinhardtii presents antigenic activity by western blot test and through its application in chickens, demonstrating its feasibility as a recombinant antigen against AIV.

Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized ß-galactosidase-like gene (Cre02.g119700), which decreased total ß-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Development of phytase-expressing chlamydomonas reinhardtii for monogastric animal nutrition.

BACKGROUND: In plant-derived animal feedstuffs, nearly 80 % of the total phosphorus content is stored as phytate. However, phytate is poorly digested by monogastric animals such as poultry, swine and fish, as they lack the hydrolytic enzyme phytase; hence it is regarded as a nutritionally inactive compound from a phosphate bioavailability point of view. In addition, it also chelates important dietary minerals and essential amino acids. Therefore, dietary supplementation with bioavailable phosphate and exogenous phytases are required to achieve optimal animal growth. In order to simplify the obtaining and application processes, we developed a phytase expressing cell-wall deficient Chlamydomonas reinhardtii strain. RESULTS: In this work, we developed a transgenic microalgae expressing a fungal phytase to be used as a food supplement for monogastric animals. A codon optimized Aspergillus niger PhyA E228K phytase (mE228K) with improved performance at pH 3.5 was transformed into the plastid genome of Chlamydomonas reinhardtii in order to achieve optimal expression. We engineered a plastid-specific construction harboring the mE228K gene, which allowed us to obtain high expression level lines with measurable in vitro phytase activity. Both wild-type and cell-wall deficient strains were selected, as the latter is a suitable model for animal digestion. The enzymatic activity of the mE228K expressing lines were approximately 5 phytase units per gram of dry biomass at pH 3.5 and 37 °C, similar to physiological conditions and economically competitive for use in commercial activities. CONCLUSIONS: A reference basis for the future biotechnological application of microalgae is provided in this work. A cell-wall deficient transgenic microalgae with phytase activity at gastrointestinal pH and temperature and suitable for pellet formation was developed. Moreover, the associated microalgae biomass costs of this strain would be between US$5 and US$60 per ton of feedstuff, similar to the US$2 per ton of feedstuffs of commercially available phytases. Our data provide evidence of phytate-hydrolyzing microalgae biomass for use as a food additive without the need for protein purification.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

PSR1 Is a Global Transcriptional Regulator of Phosphorus Deficiency Responses and Carbon Storage Metabolism in Chlamydomonas reinhardtii.

Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the genetic mechanisms regulating this response are poorly understood. Here, we show that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic analysis identified specific metabolism transcripts that are induced by P starvation but misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further examine the role of PSR1 in regulating lipid and starch metabolism, PSR1 complementation lines in the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of the psr1 phenotype. PSR1 overexpression lines exhibited increased starch content and number of starch granules per cell, which correlated with a higher expression of specific starch metabolism genes but reduced neutral lipid content. Furthermore, this phenotype was consistent in the presence and absence of acetate. Together, these results identify a key transcriptional regulator in global metabolism and demonstrate transcriptional engineering in microalgae to modulate starch biosynthesis.

Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.