The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth.

Plant photosynthesis and growth are often limited by the activity of the CO-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from (Rubisco)- red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobLS lines expressing a codon optimized - operon, the messenger RNA (mRNA) abundance was ∼25% of transcript and Rubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of Rubisco in tobLS (∼23% sites active under ambient CO), the metabolic repair protein Rca (-activase) was introduced via nuclear transformation. Rca production in the tobLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m) and enhanced Rubisco activation to 75% under elevated CO (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobLS::X lines were improved >twofold relative to tobLS. Other tobacco lines producing Rubisco containing alternate diatom and red algae S-subunits were nonviable as CO-fixation rates () were reduced >95% and CO/O specificity impaired 30-50%. We show differences in hybrid and WT Rubisco biogenesis in tobacco correlated with assembly in advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered Rubisco, an isoform amenable to directed evolution.