A new approach to express transgenes in microalgae and its use to increase the flocculation ability of Chlamydomonas reinhardtii

The choice of strong efficient promoters is a critical step in the development of efficient transformation systems for microalgae; however, the physiological and genetic diversity among microalgae groups makes very difficult to develop standard universal plasmids for a wide number of microalgal species as has been achieved for higher plants. Here, we propose a new approach to express transgenes in microalgae: cotransformation with two naked promoterless genes, a selectable antibiotic-resistant gene and a gene of our interest. These genes are randomly inserted into the nuclear genome, where their transcription relies on their adequate insertion in a region adjacent to an endogenous genomic promoter or in frame with a native gene. In a high percentage of the transformants obtained, both genes are, not only adequately incorporated in the nuclear genome, but also efficiently transcribed and translated. This transformation method is validated in the model microalga Chlamydomonas reinhardtii with the bleomycin-resistant gene from Streptoalloteichus hindustanus (ShBLE) as gene of interest, and it is employed to express a flocculin gene from Saccharomyces bayanus (SbFLO5), which is responsible for the flocculation process in yeasts. Chlamydomonas reinhardtii transformants exhibited self-flocculation abilities between 2- and 3.5-fold higher than the control untransformed strain. The successful cotransformation of C. reinhardtii with two promoterless genes opens doors for the establishment of a universal transformation system based on endogenous promoters, applicable to any microalgal species.