UC San Diego

Recombinant proteins are used for many industrial purposes including food processing, pharmaceuticals, and nutraceuticals. The use of Chlamydomonas reinhardtii, a type of microalgae, as a molecular farming platform has piqued research interests due to their edibility, photosynthetic capabilities, and eukaryotic cellular machinery, attributes that contribute to their potential to produce bioactive recombinant proteins in an economic and scalable way. However, several challenges remain before algae can be considered an industrial organism for expression of recombinant proteins. One area that will require additional improvement is the low product yield in transgenic lines, brought about by a lack of molecular tolls as well as efficient screening methods. To overcome these challenges, we developed a multi-cistronic nuclear transformation vector that utilizes an intracellular fluorescent protein reporter to facilitate the identification of transgenic strains with high expression levels, in a high-throughput fashion. This was made possible by establishing a linkage between an upstream fluorescent reporter gene and a downstream recombinant protein gene: a self-cleaving viral peptide was transcriptional fused between the two recombinant protein genes, allowing the fluorescent reporter to be produced separately from recombinant protein of interest. In this study we show the utility of this polycistronic vector and high-throughput screening method by first showing that both the upstream reporter and downstream protein of interest and produced in equal amounts over a range of expression levels. We then demonstrate that we can use the cassette and fluorescence-activated cell sorting to identify transgenic lines with increasing quantities of secreted recombinant proteins. We also show that mutagenesis can be used to increase the co-expression of FP reporter and RP from our vector after it is successfully transformed in C. reinhardtii.