An Optimized Version of the Small Synthetic Genome (Mini-Synplastome) for Plastid Metabolic Engineering in <i>Solanum tuberosum</i> (Potato)

Plastids represent promising targets in plant genetic engineering for many biotech applications, ranging from their use as bioreactors for the overproduction of valuable molecules to the installation of transgenes for improving plant traits. For over 30 years, routine methods of plastid transformation have relied on homologous recombination integrating vectors. However, nonintegrating episomal plasmids have recently received more attention as an innovative tool for the plastid genetic engineering of plant cells. One of these novel technologies is the mini-synplastome, an episomal plasmid with a chloroplast-specific origin of replication (<i>ori</i>) used to express transgenes in plastids. In order to improve episome sequence stability overtime by reducing the frequency of spurious recombination events, an optimized version of mini-synplastome (Gen3) was designed. The innovation in the Gen3 design was to substantially reduce the size of the plastomic sequence containing <i>oris</i> to include only domains involved in replication and to reduce the sequence homology of the whole episome with the endogenous plastome. In this work, we have demonstrated that Gen3 can be used to install a multigene pathway in <i>Solanum tuberosum</i> (potato) chloroplasts, and the episome is stable in a full-length circular form at high copy number throughout all plant developmental stages to anthesis in plants with normal phenotypic parameters. It is anticipated that in the next decade the mini-synplastome will be a valuable tool for installing complex genetic circuits in plastids.