Transgenic Chlamydomonas Research Articles

PhbZIP2 regulates photosynthesis-related genes in an intertidal macroalgae, Pyropia haitanensis, under stress.

Intertidal macroalgae are important research subjects in stress biology. Basic region-leucine zipper transcription factors (bZIPs) play an important regulatory role in the expression of target genes under abiotic stress. We herein identified a bZIP2 gene PhbZIP2 to regulate abiotic stress tolerance in Pyropia haitanensis, a representative intertidal macroalgal species. Cloning and sequencing of the cDNA characterized a BRLZ structure and an α coiled-coil structure between amino acids and Expression of PhbZIP2 was detected to upregulate under both high temperature and salt stresses. A DAP-seq analysis revealed the PhbZIP2-binding motifs of (T/C)TCCA(C/G) and A (A/G)AAA (G/A), which differed from the conserved motifs in plants. Overexpression of PhbZIP2 was indicative of a high temperature and salt stress tolerances in transgenic Chlamydomonas reinhardtii. It was suggested that PhbZIP2 was probably involved in regulating expression of the photosynthetic-related genes and the response to the abiotic stresses in P. haitanensis, which provide new insights for elucidating efficient adaptation strategies of intertidal macroalgae.

Effect of marker-free transgenic Chlamydomonas on the control of Aedes mosquito population and on plankton

BackgroundMore than half of the world’s population suffers from epidemic diseases that are spread by mosquitoes. The primary strategy used to stop the spread of mosquito-borne diseases is vector control. Interference RNA (RNAi) is a powerful tool for controlling insect populations and may be less susceptible to insect resistance than other strategies. However, public concerns have been raised because of the transfer of antibiotic resistance marker genes to environmental microorganisms after integration into the recipient genome, thus allowing the pathogen to acquire resistance. Therefore, in the present study, we modified the 3-hydroxykynurenine transaminase (3hkt) and hormone receptor 3 (hr3) RNAi vectors to remove antibiotic resistance marker genes and retain the expression cassette of the inverse repeat sequence of the 3hkt/hr3 target gene. This recombinant microalgal marker-free RNAi insecticide was subsequently added to the suburban water in a simulated-field trial to test its ability to control mosquito population.MethodsThe expression cassette of the 3hkt/hr3 inverted repeat sequence and a DNA fragment of the argininosuccinate lyase gene without the ampicillin resistance gene were obtained using restriction enzyme digestion and recovery. After the cotransformation of Chlamydomonas, the recombinant algae was then employed to feed Aedes albopictus larvae. Ten and 300 larvae were used in small- and large-scale laboratory Ae.albopictus feeding trials, respectively. Simulated field trials were conducted using Meishe River water that was complemented with recombinant Chlamydomonas. Moreover, the impact of recombinant microalgae on phytoplankton and zooplankton in the released water was explored via high-throughput sequencing.ResultsThe marker-free RNAi-recombinant Chlamydomonas effectively silenced the 3hkt/hr3 target gene, resulting in the inhibition of Ae. albopictus development and also in the high rate of Ae. albopictus larvae mortality in the laboratory and simulated field trials. In addition, the results confirmed that the effect of recombinant Chlamydomonas on plankton in the released water was similar to that of the nontransgenic Chlamydomonas, which could reduce the abundance and species of plankton.ConclusionsThe marker-free RNAi-recombinant Chlamydomonas are highly lethal to the Ae. albopictus mosquito, and their effect on plankton in released water is similar to that of the nontransgenic algal strains, which reduces the abundance and species of plankton. Thus, marker-free recombinant Chlamydomonas can be used for mosquito biorational control and mosquito-borne disease prevention.Graphical

Efficiency of transgenic Chlamydomonas reinhardtii for removing of toxicity of cyanide compound

Efficiency of transgenic Chlamydomonas reinhardtii for removing of toxicity of cyanide compound

The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae.

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis.

Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system

The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes.

Development of an RNAi-based microalgal larvicide for the control of Aedes aegypti

BackgroundMosquito-borne diseases affect over half of the human population globally. Multiple studies have shown that chemical insecticides are ineffective because of resistance. Therefore, environmentally safe mosquito population control tools need to be developed. Ribonucleic acid interference (RNAi) is a reverse genetic mechanism recently introduced as a new pest control tool. This technique represents a new class of biorational technology that could combat the increased global incidence of insecticide resistance. The technique has the potential of becoming a critical component of integrated vector control programs.MethodsA 3-hydroxykynurenine transaminase (3-HKT) RNAi expression plasmid was constructed, generated and transformed into Chlamydomonas and Chlorella algae. The transgenic algae were then used to feed Ae. aegypti mosquito larvae. The feeding experiments were conducted on a small and large scale with 10 and about 300 larvae, respectively. The mortality rate of the larvae was calculated over 30 days. In addition, histological examination of the insect tissues was performed to examine the extent of tissue damage.ResultsThe integumentary system and midguts of larvae fed with transgenic Chlamydomonas were severely damaged. The mortality rate of the larvae fed with transgenic Chlamydomonas ranged from 60 to 100% in small-scale tests. The survival rate of adult mosquitoes was 0.0% in a large-scale feeding experiment when the larvae were fed with transgenic Chlamydomonas. Moreover, when the larvae were fed with transgenic Chlorella, the mortality rate ranged from 6.7% to 43% compared to that fed wild-type Chlorella.Conclusions3HKT RNAi transgenic algae are in some scales lethal to Ae. aegypti. The findings of this study indicate that technology based on microalgae RNAi may provide a new way to control mosquito populations.Graphical

A RING type ubiquitin ligase PhCUL4 is involved in thermotolerance of Pyropia haitanensis

High temperatures stress (HTS) is one of main factors limiting the sustainable improvement of the Pyropia haitanensis aquaculture industry. Our previous study suggested that protein quality control mediated by the ubiquitin-proteasome system is crucial for the resistance of P. haitanensis to HTS. In the current study, we cloned and characterized a P. haitanensis RING-type ubiquitin ligase gene, PhCUL4. The putative protein contained cullin and Nedd8 domains and a transmembrane domain, but no signal peptide. The expression of PhCUL4 was upregulated in P. haitanensis thalli under HTS. Yeast two-hybrid analyses suggested that the interaction between PhCUL4 and an IST1-like protein (IST1) might regulate the autophagic degradation of misfolded/denatured proteins and damaged organelles in P. haitanensis under HTS. To further investigate the function of PhCUL4 in response to HTS, we generated transgenic Chlamydomonas reinhardtii overexpressing PhCUL4. Compared with wild type, the transgenic lines had higher photosynthesis and energy metabolism under HTS, which provided efficient energy and carbohydrates to maintain redox and protein homeostasis via antioxidant and heat shock protein systems, respectively. These results strongly suggest that PhCUL4 is a good candidate for genetic engineering of Pyropia/Porphyra species to improve their resistance to HTS.

Molecular mechanism underlying Pyropia haitanensis PhHsp22-mediated increase in the high-temperature tolerance of Chlamydomonas reinhardtii

Global warming is one of the key limiting factors affecting the cultivation of Pyropia haitanensis which is an economically important macroalgae species grown in southern China. However, the mechanism underlying the high-temperature tolerance of P. haitanensis remains largely unknown. In a previous study, we showed that the expression of the small heat shock protein 22 gene (Hsp22) is upregulated in P. haitanensis in response to high-temperature stress, but the associated regulatory mechanism was not fully elucidated. In this study, a transgenic Chlamydomonas reinhardtii expression system was used to functionally characterize P. haitanensis Hsp22. Our analyses indicated that the C-terminal of PhHsp22 is highly conserved and contains an A-crystal structure domain. A phylogenetic analysis revealed PhHsp22 is not closely related to small heat shock protein genes in other species. Additionally, PhHsp22 expression significantly increased at 3 and 6 h after initiating 33 °C treatment, which improved the survival rate of transgenic C. reinhardtii during the early stage of high-temperature treatment. The further transcriptome analysis revealed that PhHsp22 expression can promote pathways related to energy metabolism, metabolites metabolism, and protein homeostasis in transgenic C. reinhardtii cells exposed to high temperatures. Therefore, PhHsp22 may be crucial for the response of Pyropia species to high-temperature stress. Furthermore, this gene may be useful for breeding new high-temperature algal strains.

Efficiency of transgenic Chlamydomonas reinhardtii for removing of toxicity of cyanide compound.

Efficiency of transgenic Chlamydomonas reinhardtii for removing of toxicity of cyanide compound.

Transgenic Chlamydomonas Expressing Human Transient Receptor Potential Ankyrin 1 (TRPA1) Channels to Assess the Effect of Agonists and Antagonists.

Transient receptor potential ankyrin 1 (TRPA1) channel is an ion channel whose gating is controlled by agonists, such as allyl isothiocyanate (AITC), and temperature. Since TRPA1 is associated with various disease symptoms and chemotherapeutic side effects, it is a frequent target of drug development. To facilitate the screening of TRPA1 agonists and antagonists, this study aimed to develop a simple bioassay for TRPA1 activity. To this end, transgenic Chlamydomonas reinhardtii expressing human TRPA1 was constructed. The transformants exhibited positive phototaxis at high temperatures (≥20°C) but negative phototaxis at low temperatures (≤15°C); wild-type cells showed positive phototaxis at all temperatures examined. In the transgenic cells, negative phototaxis was inhibited by TRPA1 antagonists, such as HC030031, A-967079, and AP18, at low temperatures. Negative phototaxis was induced by TRPA1 agonists, such as icilin and AITC, at high temperatures. The effects of these agonists were blocked by TRPA1 antagonists. In wild-type cells, none of these substances had any effects on phototaxis. These results indicate that the action of TRPA1 agonists and antagonists can be readily assessed using the behavior of C. reinhardtii expressing human TRPA1 as an assessment tool.

An oral delivery system for controlling white spot syndrome virus infection in shrimp using transgenic microalgae

White spot disease (WSD) is a longstanding and serious viral disease of various shrimp species that has caused high mortality rates for many decades. Currently, there is no practical method to control this disease. Therefore, we have explored the development of a novel vaccine-based method to control this disease using transgenic algae. During infection by white spot syndrome virus (WSSV), the interaction between viral envelope proteins and cell surface protein receptors on target cells is the key step of viral entry and replication. Hence, transgenic lines of the green microalga Chlamydomonas reinhardtii harbouring a WSSV VP28 viral envelope protein were created as an oral delivery system for vaccinating shrimp. Two separate transplastomic lines containing wild-type and codon optimized gene sequences for VP28 were evaluated for recombinant protein levels. Only the codon optimized line gave rise to detectable VP28 in western blot analysis, which demonstrated that optimization for chloroplast codon bias improved the efficiency of expression and that the gene design produced a favourable RNA secondary structure with suitable free energy for translation. In addition, bile salt and acid tolerance tests demonstrated that this transgenic Chlamydomonas can tolerate mildly acidic (pH 5.0) conditions and 0.30% bile salts. These features indicated that algal cells are suitable for delivering viral antigens through a shrimp's digestive system. In WSSV infection experiments, the highest survival rate (87%) was recorded in shrimps fed with the codon optimized VP28 line mixed into their feed, indicating that this line could be employed in the control of WSSV spread in shrimp populations. This algal strategy offers a new, efficient, fast and less labour-intensive method for the control of other diseases in aquatic animals through oral delivery.

Transgenic and cell wall-deficient Chlamydomonas reinhardtii food affects life history of Daphnia magna

The economical interest in the use of genetically modified microalgae (GMM) increases because of their potential for low-cost industrial mass cultivation, high productivity (e.g., biomass, lipids, and recombinant proteins), and short generation times. However, the unintentional release of GMMs to the environment might cause harmful biotic interactions affecting ecosystems. GMMs could invade natural ecosystems and could affect ecosystem services by biotic interactions with other species from the same trophic level, as well as organisms from higher trophic levels. So far, potential negative influences of GMMs on natural ecosystems have mainly been hypothesized and empirical studies are rare and usually consider only direct short-term effects on adult organisms. In analyzing life history effects on Daphnia magna fed with transgenic Chlamydomonas reinhardtii synthesizing and secreting recombinant human vascular endothelial growth factor VEGF-165 (VEGF), we found that neonate size, fecundity, and fitness were significantly influenced by the different C. reinhardtii food sources. Clear GMM specific effects on Daphnia resulted in lower fecundity and neonates were small in size but higher in numbers. These, especially, might be consequences of changes in behavior or metabolism in Daphnia induced by the growth-enhancing protein VEGF. The results indicate a measurable response of Daphnia life history, which can result in further effects on higher trophic levels such as planktivorous invertebrates or fish.

Insight into transketolase of Pyropia haitanensis under desiccation stress based on integrative analysis of omics and transformation

BackgroundPyropia haitanensis, distributes in the intertidal zone, can tolerate water losses exceeding 90%. However, the mechanisms enabling P. haitanensis to survive harsh conditions remain uncharacterized. To elucidate the mechanism underlying P. haitanensis desiccation tolerance, we completed an integrated analysis of its transcriptome and proteome as well as transgenic Chlamydomonas reinhardtii carrying a P. haitanensis gene.ResultsP. haitanensis rapidly adjusted its physiological activities to compensate for water losses up to 60%, after which, photosynthesis, antioxidant systems, chaperones, and cytoskeleton were activated to response to severe desiccation stress. The integrative analysis suggested that transketolase (TKL) was affected by all desiccation treatments. Transgenic C. reinhardtii cells overexpressed PhTKL grew better than the wild-type cells in response to osmotic stress.ConclusionP. haitanensis quickly establishes acclimatory homeostasis regarding its transcriptome and proteome to ensure its thalli can recover after being rehydrated. Additionally, PhTKL is vital for P. haitanensis desiccation tolerance. The present data may provide new insights for the breeding of algae and plants exhibiting enhanced desiccation tolerance.

Dynamic Interactions between Autophagosomes and Lipid Droplets in Chlamydomonas reinhardtii.

Autophagy is a highly conserved catabolic process in eukaryotic cells by which waste cellular components are recycled to maintain growth in both favorable and stress conditions. Autophagy has been linked to lipid metabolism in microalgae; however, the mechanism underlying this interaction remains unclear. In this study, transgenic Chlamydomonas reinhardtii cells that stably express the red fluorescent protein (mCherry) tagged-ATG8 as an autophagy marker were established. By using this tool, we were able to follow the autophagy process in live microalgal cells under various conditions. Live-cell and transmission electron microscopy (TEM) imaging revealed physical contacts between lipid droplets and autophagic structures during the early stage of nitrogen starvation, while fusion of these two organelles was observed in prolonged nutritional deficiency, suggesting that an autophagy-related pathway might be involved in lipid droplet turnover in this alga. Our results thus shed light on the interplay between autophagy and lipid metabolism in C. reinhardtii, and this autophagy marker would be a valuable asset for further investigations on autophagic processes in microalgae.

Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii was recently been shown to be an effective bio-manufacturing platform for the production of recombinant proteins. The advantage of using C. reinhardtii is that it is fast to grow, inexpensive to culture, and relatively safe. However, the expression of foreign proteins is always low and difficult to purify in C. reinhardtii. Human kallikrein has the potential to be developed into certain drugs, like insulin. Therefore, its biosynthesis is important to drug development. In this study, we synthesized the sg gene, a signal peptide sequence of alkaline phosphatase, and inserted it into a pH124 plasmid, which contains a HSP70A-RBCS2 promoter and a RBCS2 terminator. Then, we inserted the human kallikrein gene klk1 behind the sg sequence to make a pHsgk124 vector. The pHsgk124 were transferred into a cell-wall deficient strain of C. reinhardtii, cc-503, by using the glass bead method. Southern blot analysis showed that sg and klk1 were incorporated into genes of the transgenic C. reinhardtii. RT-PCR analysis showed that it had an active transcription and its expression increased three times under heat stress. Western blot analyses of proteins inside and outside cells (in the culture medium) showed that klk1 was expressed in the cell and the resulting protein was secreted into medium. An enzyme activity assay showed that the recombinant protein had the ability to hydrolyze the specific substrate H-D-Val-Leu-Arg-Pna. In conclusion, we successfully bioengineered C. reinhardtii to produce and secrete human kallikrein protein, which has important biomedical implications.

Overproduction of recombinant E. coli malate synthase enhances Chlamydomonas reinhardtii biomass by upregulating heterotrophic metabolism

High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic Chlamydomonas reinhardtii was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (MDH4) and acetyl-CoA synthetase (ACS3), isocitrate lyase (ICL1) and malate synthase (MAS1), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic Fv/Fm was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass.

PsCYP1 of marine red alga Pyropia seriata (Bangiales, Rhodophyta) confers salt and heat tolerance in Chlamydomonas

Cyclophilins (CYPs) are ubiquitous in all subcellular compartments, possess peptidyl-prolyl cis-trans isomerase (PPIase) activity and are present in prokaryotes and eukaryotes. Their physiological functions are various such as protein folding, symbiotic relationships, disease or plant responses to abiotic stresses. Pyropia seriata (Bangiales, Rhodophyta) is a marine red alga that is cultivated as commercially valuable seaweed. By analyzing transcriptome data, we identified six full coding sequences of CYPs from P. seriata. Among them, only PsCYP1 showed upregulation of transcription in responded to high temperature stress. PsCYP1 belongs to the single domain form of cytosolic cyclophilin and contains the typical 12 conserved amino acid residues for PPIase activity. Despite no detected nuclear localization signal (NLS), a chimeric PsCYP1 protein with green fluorescent protein (GFP) was detected predominantly in nucleus. When PsCYP1 was introduced into the green alga, Chlamydomonas, the introduced PsCYP1 conferred salt and heat stress tolerance in transgenic Chlamydomonas. Especially the transgenic Chlamydomonas cells exhibited a salt-tolerant phenotype. The cyclophilin genes from P. seriata will facilitate future studies of the molecular function of cyclophilin, and the mechanisms of abiotic stress tolerance in red algae.

Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell.

Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

A C2H2 zinc finger protein FEMU2 is required for fox1 expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38) harboring an arylsulfatase (ARS) reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7), ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF) motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/T)TTGG(G/T)(G/T)T) was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.

An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas.

The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains.