Engineering of An Endogenous Phytoene Desaturase Gene As a Dominant Selectable Marker for Chlamydomonas Reinhardtii Transformation and Enhanced Biosynthesis of Carotenoids

There have been several markers used for Chlamydomonas reinhardtii transformation, consisting mainly of bacterial antibiotic-resistant genes such as the phleomycin resistance gene and an aminoglycoside 3'-phosphotransferase type VIII gene. These exogenous genes undergo likely epigenetic silencing and proper endogenous promoters and/or enhancers (e.g., intron) are commonly required for stable expression in algae. Engineering of an endogenous gene as the selectable marker may be ideal. C. reinhardtii is highly sensitive to the herbicide norflurazon that specifically targets the phytoene desaturase (PDS), a carotenogenic enzyme which synthesizes the first colored carotenoid ζ-carotene. In the present study, a PDS gene from Chlamydomonas reinhardtiiwas cloned and characterized. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery disclosed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and also possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.