Marker‐free genetic engineering of the chloroplast in the green microalga <i><scp>C</scp>hlamydomonas reinhardtii</i>

Summary The work applied a transgene expression method based on the replacement of an inactive rbc L gene as the selection marker in C hlamydomonas reinhardtii chloroplasts. The native rbc L gene in strain CC 2653 has a point mutation that causes early translation termination, thus resulting in a photosynthesis mutant. Recovery of rbc L function for selection is offered along with the heterologous expression of the alcohol dehydrogenase ADH 1 gene from S accharomyces cerevisiae in the C hlamydomonas chloroplast. The C r C p ADH 1 gene was inserted via double homologous recombination in the psa B ‐rbc L chloroplast intergenic region of recipient strain CC 2653, using the psa B and rbc L gene sequences for the double homologous recombination. This transformation conferred a functional rbc L gene and expression of the C r C p ADH 1 transgene in the recipient strain. This method alleviated the need to use antibiotics for selection, resulting in a negligible number of false positives during screening, and attaining a transformation efficiency greater than 90%. The approach also ensured segregation of chloroplast DNA copies, so as to achieve homoplasmy of the transformant chloroplast DNA , with a concomitant elimination of recipient strain Cp DNA . High levels of steady‐state C r C p ADH 1 transcripts were detected in the homoplasmic transformants. However, C r C p ADH 1 protein levels were attenuated under continuous illumination growth conditions due to oxygen accumulation in the cells. Under conditions of low oxygen partial pressure, or anoxia, accumulation of C r C p ADH 1 protein in the cells and ethanol in the growth medium was observed. A metabolic pathway for ethanol production is proposed in C hlamydomonas, mediated by the chloroplast‐localized C r C p ADH 1 transgenic enzyme.