Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

Dual conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High yield expression of recombinant products and maternal inheritance of plastid encoded traits make plastids attractive sites for modification. Here we describe the cloning and expression of a dao gene encoding D-amino acid oxidase from Schizosaccharomyces pombe in Nicotiana tabacum plastids. The results provide genetic evidence for the uptake of D-amino acids into plastids, which contain a target that is inhibited by D-alanine. Importantly, this non-antibiotic based selection system allows the use of cheap and widely available D-amino acids, which are relatively non-toxic to animals and microbes, to either select against (D-valine) or for (D-alanine) cells containing transgenic plastids. Positive/negative selection with D-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment where it can be used to: provide tolerance against potential D-alanine based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine based methods to manage the spread of transgenic plastids tagged with dao.