Dietary supplements and functional foods are becoming increasingly popular complements to regular diets. A recurring ingredient is the essential cofactor vitamin B12 (B12). Microalgae are making their way into the dietary supplement and functional food market but do not produce B12, and their B12 content is very variable. In this study, the suitability of using the human B12-binding protein intrinsic factor (IF) to enrich bioavailable B12 using microalgae was tested. The IF protein was successfully expressed from the nuclear genome of the model microalga Chlamydomonas reinhardtii and the addition of an N-terminal ARS2 signal peptide resulted in efficient IF secretion to the medium. Co-abundance of B12 and the secreted IF suggests the algal produced IF protein is functional and B12-binding. Utilizing IF expression could be an efficient tool to generate B12-enriched microalgae in a controlled manner that is suitable for vegetarians and, potentially, more bioavailable for humans.

Grouper farming is a billion-dollar industry with increasing global demand for its organoleptic qualities and it is also a source of food security for developing countries. The high economic value of the fish has caused the rapid expansion of grouper aquaculture. Elevated temperatures in tropical and subtropical regions promote rapid growth, while fish robustness enables it to withstand high stocking densities. However, the industry still relies on wild-caught fry and fingerlings of brown-marbled grouper for stocking. Therefore,

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture.

The present work presents an improvement of microalgal antiviral dsRNA production for controlling disease in shrimp aquaculture. 307 bp of sequence targeting the VP28 gene of white spot syndrome virus (WSSV) was inserted between two convergent rrnS promoters in the novel vector p2XTRBL, which was then subcloned into the transformation vector pSS116 using Golden Gate assembly. The recombinant plasmid was transformed into the Chlamydomonas reinhardtii chloroplast, and transformants selected by the restoration of photosynthesis. The presence of the cassette and homoplasmy of the algal transformants was confirmed by PCR analysis. Transcribed sense and antisense VP28-RNA were hypothesised to form an RNA duplex in the chloroplast stroma, and quantitative RT-PCR indicated that ~100 μg dsRNA was obtained per litre of transgenic microalgae culture. This accumulation of dsRNA represents a 10,000-fold increase relative to previous reports using convergent psaA promoters. Recombinant C. reinhardtii was assessed for its ability to prevent WSSV infection in shrimp larvae by direct feeding. After WSSV challenge, the survival of shrimp treated with dsRNA-expressing C. reinhardtii was significantly enhanced (95.2%) relative to the negative control without dsRNA treatment. The study suggests that this new algal production platform for dsRNA is significantly more efficient than the previous report, and it merits further scale-up and downstream processing studies.