Transgenic Algae Research Articles

Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%-3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.

BIOSYNTHESIS OF POLY-3-HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGACHLAMYDOMONAS REINHARDTII

A cell-wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC-849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris-acetate-phosphate media containing 10 μg · mL−1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly-3-hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min−1 · mg protein−1 to 126 nmol · min−1 · mg protein−1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.

Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii

An ORF cDNA fragment of one of leghemoglobin genes, lba was cloned from Glycine max and transferred into chloroplasts of Chlamydomonas reinhardtii. More rapidly O 2 consumption, lower O 2 content and higher H 2 output were monitored in the transgenic algal cultures than those in WT cultures either in S-free or S-containing medium. Maximum expression of lba in the transgenic algae consisted with the time when minimal O 2 contents and maximal H 2 evolution occurred. The highest H 2 production achieved in sulfur-free medium for both algal cultures. When restoring sulfate in the medium, H 2 production in the transgenic algal cultures kept steadily around 130–145 μl per bottle while that in WT cultures decreased gradually from 98 μl per bottle at 12.5 μM sulfate to 40 μl per bottle at 100 μM sulfate. The results indicated that heteroexpression of lehemoglaobin genes in chloroplasts of green algae improved H 2 yield by decreasing O 2 content in the medium. This protein had potential to be used in improvement of H 2 production in green algae.

Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection

While 15 million deaths per year are caused by communicable pathogens worldwide, health care authorities emphasize the considerable impact of poverty on the incidence of infectious diseases. The emergence of antigen-expressing plant tissues (e.g. rice, tomato, potato) has indicated the potential of land plants for low-cost vaccines in oral immunization programs. In this study, we engineered the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii for the stable expression of the D2 fibronectin-binding domain of Staphylococcus aureus fused with the cholera toxin B subunit (CTB), under the control of rbcL UTRs. Analysis of sera and faeces of mice, fed for 5 weeks with transgenic algae grown in confined Wave Bioreactor™, revealed the induction of specific mucosal and systemic immune responses. Algae-based vaccination significantly reduced the pathogen load in the spleen and the intestine of treated mice and protected 80% of them against lethal doses of S. aureus. Importantly, the alga vaccine was stable for more than 1.5 years at room temperature. These results indicate that C. reinhardtii may play an important role in molecular pharming, as it combines the beneficial features of land plant vaccines, while offering unmatched ease of growth compared to other members of the plant kingdom.

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

BackgroundHuman glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga Chlamydomonas reinhardtii may offer a potential solution.ResultsA DNA cassette encoding full-length hGAD65, under the control of the C. reinhardtii chloroplast rbcL promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of C. reinhardtii by particle bombardment. Integration of hGAD65 DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of hGAD65 was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein.ConclusionOur results demonstrate the potential value of C. reinhardtii chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract

Antibiotics are commonly employed in most fish aquacultures to prevent disease. One major risk in this practice is that antibiotic-resistant pathogens may be selected. Therefore, we wanted to examine the feasibility of producing an economical, non-antibiotic alternative. The microalga Nannochloropsis oculata is an essential phytoplankton used as live feed for fish larvae. We attempted to culture N. oculata in a way that would provide an organism against bacterial pathogenic infection. To test this idea, we constructed an algae-codon-optimized bovine lactoferricin (LFB) fused with a red fluorescent protein (DsRed) driven by a heat-inducible promoter, which is a heat shock protein 70A promoter combined with a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2′ promoter from Chlamydomonas reinhardtii. After electroporation, we examined 491 microalgal clones and generated two stable transgenic lines, each expressing a stable transgene inheritance for at least 26 months. This was confirmed by the positive detection of the mRNA transcript and the protein of LFB-DsRed produced by the transgenic microalgae. To test the efficacy of the antimicrobial peptide LFB, medaka fish ( Oryzias latipes) were adapted from freshwater to seawater and were fed with the transgenic algae by oral-in-tube delivery method. Bacterial infection with 1 × 10 5 Vibrio parahaemolyticus per fish was induced 6 h thereafter by oral-in-tube delivery as well. For medaka fish fed with 1 × 10 8 transgenic algae per fish, the average survival rate after a 24-h period of infection was much higher than that of medaka fed with wild-type algae (85 ± 7.1% versus 5 ± 7.1%). This result suggests that medaka fish fed with the LFB-containing transgenic microalgae will have bactericidal defense against V. parahaemolyticus infection in its digestive tract.

Chlamydomonas reinhardtii chloroplasts as protein factories

Protein-based therapeutics are the fastest growing sector of drug development, mainly because of the high sensitivity and specificity of these molecules. Their high specificity leads to few side effects and excellent success rates in drug development. However, the inherent complexity of these molecules restricts their synthesis to living cells, making recombinant proteins expensive to produce. In addition to therapeutic uses, recombinant proteins also have a variety of industrial applications and are important research reagents. Eukaryotic algae offer the potential to produce high yields of recombinant proteins more rapidly and at much lower cost than traditional cell culture. Additionally, transgenic algae can be grown in complete containment, reducing any risk of environmental contamination. This system might also be used for the oral delivery of therapeutic proteins, as green algae are edible and do not contain endotoxins or human viral or prion contaminants.

The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress

Plants exposed to heavy metals accumulate an array of metabolites, some to high millimolar concentrations. This review deals with N-containing metabolites frequently preferentially synthesized under heavy metal stress such as Cd, Cu, Ni, and Zn. Special focus is given to proline, but certain other amino acids and oligopeptides, as well as betaine, polyamines, and nicotianamine are also addressed. Particularly for proline a large body of data suggests significant beneficial functions under metal stress. In general, the molecules have three major functions, namely metal binding, antioxidant defence, and signalling. Strong correlative and mechanistic experimental evidence, including work with transgenic plants and algae, has been provided that indicates the involvement of metal-induced proline in metal stress defence. Histidine, other amino acids and particularly phytochelatins and glutathione play a role in metal binding, while polyamines function as signalling molecules and antioxidants. Their accumulation needs to be considered as active response and not as consequence of metabolic dys-regulation.

Insertal orientation has no influence on the expression ofgfp gene and the growth of the hostSynechococcus sp. PCC7942

In transgenic process, a foreign gene can be integrated in the host genome in two directions, which may influence its expression. In order to study the effects of insertal orientation, thegfp reporter gene was inserted in theisiAB locus ofSynechococcus sp. PCC7942 in different directions, and the GFP expression levels and the growth of the transgenic algae were compared. It was showed that thegfp gene could express in each direction, and no significant difference was detected on algal growth and GFP expression levels between the two recombinant algae.

TRANSFERRING AND EXPRESSION OF GLUCOSE OXIDASE GENE ‐ GLUC IN PORPHYRA YEZOENSIS

He, P.1, Yao, Q.2, Chen, Q.1, Guo, M., Xiong, A.2, Wu, W.1, Ma, J.1 1Shanghai Fisheries University, Shanghai 200090, PR China; 2Shanghai Agriculture Institute, Shanghai 201106, PR China; 3University of Connecticut, Connecticut 06901‐2315, USAThe disease‐resistant gene gluc, encoding glucose oxidase, has been used to increase advance crops' resistance to diseases. Now the gluc gene has been reconstructed in the plasmid pYP1203 between gus and cm+(cat) gene, a reporter gene and an antibiotics resistance labeling gene within the RB (right border) and LB (left border). With PEG and electroporation methods, the pYP1203 with gluc gene and gus, cat genes was transferred into the cells from thalli of Porphyra yezoensis. After 24‐72 hours, the GUS detection in transgenic cells of Porphyra yezoensis was positive. Cell transformation efficiency could reach as high as about 3‐4%. The expression of gus gene in the filaments regenerated from transgenic cells was also detected. Furthermore with the chloromycetin resistance screening, the transgenic cells of Porphyra survived over one month in the 50 ug/ml of chloromycetin concentration, while the wild cells could not survive in the same chloromycetin concentration. That results indicated that the gluc gene might be integrated into the chromosomes of Porphyra yezoensis. The molecular test of these transgenic algae and their disease resistance analysis are continuing.