A chimeric hydrolase-PTXD transgene enables chloroplast-based heterologous protein expression and non-sterile cultivation of Chlamydomonas reinhardtii

Photosynthetic microalgae hold great potential as light-driven heterologous protein expression hosts. In particular, the algal chloroplast is an ideal sub-cellular site for the compartmentalized synthesis and accumulation of high-value recombinant proteins. However, full integration of transplastomic algal biotechnology in the large-scale production of biocatalysts still suffers from major bottlenecks, such as genetic instability and pest contamination. To enhance the reliability of plastid-based algal expression platforms we developed a self-reinforcing genetic system in Chlamydomonas reinhardtii. We transformed the plastome with a bifunctional transgene encoding an in vivo cleavable fusion polypeptide composed of a hyperthermophilic cellulase and the phosphite dehydrogenase PTXD. The dual use of phosphite as a low-cost, environmentally friendly selective agent and fertilizer afforded axenic algal cultivation via mixotrophic metabolism and efficient expression of the hydrolytic enzyme. This study provides an example of chloroplast genetic engineering in which biosafety is integrated in the sustainable management of microalgal monocultures to produce enzymes with industrial applications.