Saccharification of Brown Macroalgae Using an Arsenal of Recombinant Alginate Lyases: Potential Application in the Biorefinery Process.

Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that 30°C with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.

Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas reinhardtii.

Oxygenic photosynthesis provides the energy to produce all food and most of the fuel on this planet. Photosystem II (PSII) is an essential and rate-limiting component of this process. Understanding and modifying PSII function could provide an opportunity for optimizing photosynthetic biomass production, particularly under specific environmental conditions. PSII is a complex multisubunit enzyme with strong interdependence among its components. In this work, we have deleted the six core genes of PSII in the eukaryotic alga Chlamydomonas reinhardtii and refactored them in a single DNA construct. Complementation of the knockout strain with the core PSII synthetic module from three different green algae resulted in reconstitution of photosynthetic activity to 85, 55, and 53% of that of the wild-type, demonstrating that the PSII core can be exchanged between algae species and retain function. The strains, synthetic cassettes, and refactoring strategy developed for this study demonstrate the potential of synthetic biology approaches for tailoring oxygenic photosynthesis and provide a powerful tool for unraveling PSII structure-function relationships.

In Metabolic Engineering of Eukaryotic Microalgae: Potential and Challenges Come with Great Diversity.

The great phylogenetic diversity of microalgae is corresponded by a wide arrange of interesting and useful metabolites. Nonetheless metabolic engineering in microalgae has been limited, since specific transformation tools must be developed for each species for either the nuclear or chloroplast genomes. Microalgae as production platforms for metabolites offer several advantages over plants and other microorganisms, like the ability of GMO containment and reduced costs in culture media, respectively. Currently, microalgae have proved particularly well suited for the commercial production of omega-3 fatty acids and carotenoids. Therefore most metabolic engineering strategies have been developed for these metabolites. Microalgal biofuels have also drawn great attention recently, resulting in efforts for improving the production of hydrogen and photosynthates, particularly triacylglycerides. Metabolic pathways of microalgae have also been manipulated in order to improve photosynthetic growth under specific conditions and for achieving trophic conversion. Although these pathways are not strictly related to secondary metabolites, the synthetic biology approaches could potentially be translated to this field and will also be discussed.

Production of recombinant proteins in microalgae at pilot greenhouse scale.

Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae.

Advances in microalgae engineering and synthetic biology applications for biofuel production.

Among the technologies being examined to produce renewable fuels, microalgae are viewed by many in the scientific community as having the greatest potential to become economically viable. Algae are capable of producing greater than 50,000 kg/acre/year of biomass [1]. Additionally, most algae naturally accumulate energy-dense oils that can easily be converted into transportation fuels. To reach economic parity with fossil fuels there are still several challenges. These include identifying crop protection strategies, improving harvesting and oil extraction processes, and increasing biomass productivity and oil content. All of these challenges can be impacted by genetic, molecular, and ultimately synthetic biology techniques, and all of these technologies are being deployed to enable algal biofuels to become economically competitive with fossil fuels.

Analysis of heterologous regulatory and coding regions in algal chloroplasts.

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II (psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5' UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5' UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts.