Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

“…We have also demonstrated that our ptxD cassette can serve as a dominant selectable marker for chloroplast transformation and have constructed suitable plasmids for targeting the cassette into a neutral site on the plastome (either at the psbH-trnE2 or the psaA-3-trnL locus). Sandoval-Vargas et al (2019) have also recently reported the use of ptxD as a selectable marker for the C. reinhardtii chloroplast, and our combined work adds a useful dominant marker to the molecular toolbox (Esland et al 2018). Importantly, this marker is not derived from a bacterial antibiotic resistance gene and therefore raises less concerns regarding GM regulation (EFSA GMO Panel 2004;Beacham et al 2017).…”

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

“…Indeed, the expression of the PTXD enzyme in the plastid serves two highly desirable purposes: it functions as a growth selector enabling transgenic microalgae to grow in non-sterile conditions reducing the risk of culture contamination, and it also represents an environmentally friendly selectable marker for plastome engineering. This approach truly represents a pioneering research field, with only a few reports having addressed this topic so far [16][17][18]. Nevertheless, the function of the PTXD enzyme in the chloroplast appears to suffer from some limitations [21], possibly connected with this peculiar cellular environment, that hinders its catalytic activity.…”

“…We next investigated whether the modified PTXD enzyme version could serve as a reliable selectable marker for the genetic transformation of the algal plastome using a phosphite metabolism-based selection strategy. To this end, we transformed, in parallel, three batches of one-week phosphorous-starved cells with the IR-vectors carrying the wild type and modified PTXD version on TA-Phi (1 mM) agar plates, as previously described [18]. As shown in Figure 6, the transformation with the Int-ptxD E175A-A176R vector resulted in the formation of colonies three weeks post-bombardment (panels A, B and C), while no colonies were visible when the wild type PTXD version was used for transformation (panels D, E and F).…”

“…Since most parasitic organisms cannot use phosphite in their metabolism, the expression of a PTXD transgene in the nucleus [15] was shown to enable the selective growth of C. reinhardtii in the presence of phosphite as the sole phosphorous source, effectively preventing the risk of culture contamination by other species competing for available resources. More recently, PTXD was successfully expressed as a plastid-targeted transgene in C. reinhardtii [16,17], emerging as a novel selectable marker for the genetic transformation of the plastome [18]. Despite being an attractive system for the chloroplast transformation of C. reinhardtii, the use of PTXD as a selectable marker suffers from some intrinsic limitations that hinder its value.…”

“…Despite being an attractive system for the chloroplast transformation of C. reinhardtii, the use of PTXD as a selectable marker suffers from some intrinsic limitations that hinder its value. Indeed, PTXD-based selection displays a far lower efficiency compared to traditional antibiotic resistance-based methods [18], mainly due to a long selection time (reported up to 60 days), which reduces the recovery efficiency of true transformation events. The activity of the PTXD enzyme could be theoretically enhanced via rational approaches that alter its catalytic properties by overcoming the factors that may restrict its functionality.…”