Microalgae are a diverse group of single-cell photosynthetic organisms that include cyanobacteria and a wide range of eukaryotic algae. A number of microalgae contain high-value compounds such as oils, colorants, and polysaccharides, which are used by the food additive, oil, and cosmetic industries, among others. They offer the potential for rapid growth under photoautotrophic conditions, and they can grow in a wide range of habitats. More recently, the development of genetic tools means that a number of species can be transformed and hence used as cell factories for the production of high-value chemicals or recombinant proteins. In this article, we review exploitation use of microalgae with a special emphasis on genetic engineering approaches to develop cell factories, and the use of synthetic ecology approaches to maximize productivity. We discuss the success stories in these areas, the hurdles that need to be overcome, and the potential for expanding the industry in general.

RNA interference (RNAi) is an effective way of combating shrimp viruses by using sequence-specific double-stranded (dsRNA) designed to knock down key viral genes. The aim of this study was to use microalgae expressing antiviral dsRNA as a sustainable feed supplement for shrimp offering viral protection. In this proof of concept, we engineered the chloroplast genome of the green microalga Chlamydomonas reinhardtii for the expression of a dsRNA cassette targeting a shrimp yellow head viral gene. We used a previously described chloroplast transformation approach that allows for the generation of stable, marker-free C. reinhardtii transformants without the supplementation of antibiotics. The generated dsRNA-expressing microalgal strain was then used in a shrimp feeding trial to evaluate the efficiency of the algal RNAi-based vaccine against the virus. Shrimps treated with dsRNA-expressed algal cells prior to YHV infection had 50% survival at 8 day-post infection (dpi), whereas 84.1% mortality was observed in control groups exposed to the YHV virus. RT-PCR using viral specific primers revealed a lower infection rate in dsRNA-expressing algae treated shrimp (55.6 ± 11.1%) compared to control groups (88.9 ± 11.1% and 100.0 ± 0.0%, respectively). Our results are promising for using microalgae as a novel, sustainable alternative as a nutritious, anti-viral protective feedstock in shrimp aquaculture.

The unicellular green alga Chlamydomonas reinhardtii has potential as a cell factory for the production of recombinant proteins and other compounds, but mainstream adoption has been hindered by a scarcity of genetic tools and a need to identify products that can be generated in a cost-effective manner. A promising strategy is to use algal chloroplasts as a site for synthesis of high value bioactive compounds such as diterpenoids since these are derived from metabolic building blocks that occur naturally within the organelle. However, synthesis of these complex plant metabolites requires the introduction of membrane-associated enzymes including cytochrome P450 enzymes (P450s). Here, we show that a gene (CYP79A1) encoding a model P450 can be introduced into the C. reinhardtii chloroplast genome using a simple transformation system. The gene is stably expressed and the P450 is efficiently targeted into chloroplast membranes by means of its endogenous N-terminal anchor domain, where it is active and accounts for 0.4% of total cell protein. These results provide proof of concept for the introduction of diterpenoid synthesis pathways into the chloroplast of C. reinhardtii.

Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO2, light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca. Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L−1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria. We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.