(M0930-08-47) Microalgae as Cell Factories for Antiviral Protein Production

Purpose: In the context of the recurrent emergence of new virus and the increased risks of epidemic outbreaks, part of the drug development is focused on natural products, as for instance lectins from algae which have shown potent antibacterial and antiviral activities against HIV, herpes, MERS-CoV, SARS-CoV viruses. Lectins are ubiquitous proteins capable of recognizing and binding specifically and reversibly to free carbohydrates or glycoconjugates of cell membranes. They are involved in many biological processes, such as host-pathogen interactions, cell targeting and communication, induction of apoptosis, cancer metastasis and differentiation. For all these reasons, lectins find a lot of industrial and pharmaceutical applications as antiviral agents, cancer therapeutics, or diagnostics. For large-scale production, the purification of lectins from their natural sources is not suitable in order to preserve biodiversity and because of the low yield obtained, the lack of reproducibility and the risks of the presence of polluents. Therefore, the production of recombinant lectins by genetic engineering is absolutely required. Engineered microalgae are novel and promising recombinant system because of their exceptional properties. Indeed they are able to produce recombinant bioactive peptides to large or complex proteins with the possibility to form endogenous disulfide bonds (essential for the stability and activity of numerous proteins) and obtained in the soluble fraction without insoluble aggregates, in contrast to recombinant bacteria. Moreover, microalgae are safer cell factories without infectious agent to humans (no virus, no endo- nor exotoxins, no prions). They have already shown to be industrial robust and cost effective chassis, grown either in photobioreactors or in fermenters. In the present study, we demonstrate that microalgae engineered by our disruptive technology in synthetic biology are able to produce active antiviral compounds, as for instance the mannose-specific lectin Griffithsin (GRFT) the red macroalga Griffithsia sp. and the fucose-specific lectin HML from the red marine alga Hypnea musciformis. Both lectins was already shown to exhibit antiviral activities. HML has the particularity to be a small protein difficult to produce in recombinant Escherichia coli because of the presence of 7 disulfide bound indispensable for its activity.

Methods: In order to engineer the chloroplast genome of Chlamydomonas reinhardtii, the transformation vectors contained two expression cassettes for the expression of the genes encoding the selectable marker (gos) or the recombinant lectin (goi) (either HML or GRFT) under the control of regulatory elements from C. reinhardtii. Each goi were optimized with the codon usage of C. reinhardtii chloroplast genome. The selection cassette contained the selectable marker aadA gene coding aminoglycoside 3''-adenylyltransferase and conferring the resistance to spectinomycin and streptomycin. These two expression cassettes were flanked by a left (LHRR) and right (RHRR) endogenous homologous recombination sequences which were identical to those surrounding the targeted integration site into the C. reinhardtii chloroplast genome. Microalgae cells were transformed using the helium gun bombardment technique of gold microprojectiles complexed with transforming DNA. After transformation, algae colonies growing onto restrictive solid medium plates were expected to have the antibiotic resistant gene incorporated the transgene(s) into their genome. In order to identify stable integration of the recombinant genes encoding fusion protein into the chloroplast algal genome, spectinomycin resistant colonies were screened by PCR analysis. PCR positive clones were grown in liquid media in order to perform further biochemical analysis on total soluble protein extracts. The proper folding and activity of HML was demonstrated by hemagglutination assay using total soluble protein extracts.

Results: Antibiotic resistant transformants containing the goi inserted inside the chloroplast genome of C. reinhardtii were identified by PCR analysis. Western blot analysis were performed on total soluble protein extracts from C. reinhardtii transformants and showed the production of HML or GRFT in engineered C. reinhardtii. Preliminary results of hemagglutination assay demonstrate that HLM produced in C. reinhardtii agglutinates erythrocytes and thus that HML is active and properly folded thanks to the formation of all its disulfide bounds.

Conclusion: Our disruptive technology in microalgae synthetic biology and platform therefore provide innovative and sustainable solutions for the industrial production of bioactive compounds, such as antiviral molecules, and can allow the discovery and the development of a wide array of molecules with various therapeutic and pharmaceuticals.