Sulfadiazine selection enables high‐efficiency transformation inand
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
SUMMARY andare 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 bacterialgene as an efficient and robust selection system. Growth assays identified 300 μg·mLsulfadiazine 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‐carotene ketolase using the Sul selectable marker inandresulted 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 inand. Significance Statement Genetic engineering in the marine eustigmatophytesandis constrained by intrinsic antibiotic resistance and reduced marker efficacy at high salinity. We show that mitochondrial‐targeted bacterial Sul provides a robust, cost‐effective selectable marker, enabling sulfadiazine selection with minimal background. This system supports stable transformant growth, avoids cross‐resistance, and is suitable for stable g
Abstract
SUMMARY andare 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 bacterialgene as an efficient and robust selection system. Growth assays identified 300 μg·mLsulfadiazine 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‐carotene ketolase using the Sul selectable marker inandresulted 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 inand. Significance Statement Genetic engineering in the marine eustigmatophytesandis constrained by intrinsic antibiotic resistance and reduced marker efficacy at high salinity. We show that mitochondrial‐targeted bacterial Sul provides a robust, cost‐effective selectable marker, enabling sulfadiazine selection with minimal background. This system supports stable transformant growth, avoids cross‐resistance, and is suitable for stable gene expression. short