An Optimized Version\nof the Small Synthetic Genome\n(Mini-Synplastome) for Plastid Metabolic Engineering in <i>Solanum\ntuberosum</i> (Potato)

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