Sulfadiazine selection enables high-efficiency transformation in Nannochloropsis and Microchloropsis

Nannochloropsis and Microchloropsis are important as eustigmatophyte model organisms and the production of high-value lipids. Efficient selectable markers are indispensable for genetic engineering to study and improve production yield of these species. Here, we establish sulfadiazine resistance mediated by the bacterial sul1 gene as an efficient and robust selection system. Growth assays identified 300 μg·mL^-1 sulfadiazine as an effective selection concentration. Mitochondrial targeting of Sul results in transformation efficiencies and false-positive rates superior to commonly used markers aphVII and Sh ble. Sulfadiazine-resistant transformants exhibit stable growth under selection and no cross-resistance to hygromycin B, Zeocin, blasticidin S, G418, or nourseothricin. Expression of Chromochloris zofingiensis β-carotene ketolase using the Sul selectable marker in Nannochloropsis oceanica and Microchloropsis gaditana resulted in accumulation of the ketocarotenoids canthaxanthin and adonirubin, producing a distinct brown phenotype. Mitochondrial-targeted Sul thus provides a robust, high-efficiency selectable marker for transformation and genetic engineering in Nannochloropsis and Microchloropsis.