Abstract
Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii . However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum . Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.