Production of recombinant enzymes in the marine alga Dunaliella tertiolecta

Photosynthetic marine algae are attractive targets for the production of biofuels and bio-products because they have the ability to capture and fix carbon dioxide using solar energy and they grow in seawater, thereby minimizing fresh water usage. Algae are a large and diverse group and transformation of algal chloroplasts has been limited to very few examples, mainly the model freshwater alga, Chlamydomonas reinhardtii. However, the potential for metabolic engineering and recombinant protein production using algal chloroplasts has been well demonstrated in this model species. Here we report the transformation of the chloroplast of the marine green alga Dunaliella tertiolecta. D. tertiolecta is an ideal species for biofuel production because it can maintain relatively high growth rates in a wide range of pH and salt concentrations, and because it contains relatively high lipid content. Here we show that transformation of the chloroplast of D. tertiolecta can occur by homologous recombination and selection for resistance to the antibiotic erythromycin using the erythromycin esterase gene, ereB. We successfully produce measurable quantities of five different classes of recombinant enzymes; xylanase, alpha-galactosidase, phytase, phosphate anhydrolase, and beta-mannanase, in the plastids of D. tertiolecta or C reinhardtii. This was achieved by transforming the plastid of D. tertiolecta via particle bombardment using a D. tertiolecta psbD promoter with 5' UTR and psbA terminator with 3' UTR to drive stable expression of codon optimized transgenes. Similar strategies should allow for recombinant protein production in many species of marine algae. (C) 2012 Elsevier B.V. All rights reserved.