TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation

Abstract Background Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several microalgae are resistant to some of the antibiotics associated with the mentioned resistance genes, we have developed another alternative, tetX, a NADP-requiring Oxidoreductase that hydroxylates tetracycline substrates. We provide evidence that tetX can be used to obtain nuclear transformants of Chlamydomonas reinhardtii. Results We obtained nuclear transformants harbouring the tetX gene under the control of beta 2 tubulin or HSP70ARBCS2 promoters at an efficiency of transformation of 3.28 and 6.18 colony forming units/μg DNA respectively. This is the first report of a eukaryotic cell transformed using tetracycline as a selectable marker. Conclusions We developed a protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100\ua0μg/mL. We believe tetX can be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic.Abstract Background Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several microalgae are resistant to some of the antibiotics associated with the mentioned resistance genes, we have developed another alternative, tetX, a NADP-requiring Oxidoreductase that hydroxylates tetracycline substrates. We provide evidence that tetX can be used to obtain nuclear transformants of Chlamydomonas reinhardtii. Results We obtained nuclear transformants harbouring the tetX gene under the control of beta 2 tubulin or HSP70ARBCS2 promoters at an efficiency of transformation of 3.28 and 6.18 colony forming units/μg DNA respectively. This is the first report of a eukaryotic cell transformed using tetracycline as a selectable marker. Conclusions We developed a protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100\ua0μg/mL. We believe tetX can be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic.CONACYT Beca N° 20965