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  1. Rubisco catalyses the first step in carbon fixation and is a strategic target to improve photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits and its biogenesis requires multiple chaperones. We optimised a system to produce tobacco Rubisco in Escherichia coli by co-expressing chaperones in auto-induction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics very similar to that of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO2 affinity than those assembled with other small subunits. Our E. coli expression system will allow probing of features of both subunits of Rubisco that affect its kinetic properties. 
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    Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast‐encoded Rubisco large subunitrbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation ofrbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations intorbcL within the model plantNicotiana tabacumfacilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild‐typeN. tabacumwith stable point mutations withinrbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco functionin plantawithin tobacco and modification ofrbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast‐encoded genes.

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