Rubisco catalyzes the first step in carbon fixation and has been a strategic target to improve photosynthetic efficiency. In plants, Rubisco is a complex made up of eight large subunits encoded by a chloroplast gene, rbcL, and eight small subunits expressed from a nuclear gene family and targeted to chloroplast stroma. Biogenesis of Rubisco in plants requires a chaperonin system composed of Cpn60α, Cpn60β and Cpn20, which helps fold the large subunit, and multiple chaperones including RbcX, Raf1, Raf2 and BSD2, which help the dimerization of the folded large subunits and subsequent assembly with the small subunits into L8S8 holoenzymes. A recent study successfully assembled functional Arabidopsis Rubisco in Escherichia coli by co-expressing the two subunits with Arabidopsis chaperonins and chaperones (Aigner et al., 2017). In this study, we modified the expression vectors used in that study and adapted them to express tobacco Rubisco by replacing the Arabidopsis genes with tobacco ones. Next, we surveyed the small subunits present in tobacco, co-expressed each with the large subunit and successfully produced active tobacco enzymes composed of different small subunits in E. coli. These enzymes produced in E. coli have carboxylation kinetics very similar to that of the native tobacco Rubisco. We also produced tobacco Rubisco with a recently discovered trichome small subunit in E. coli and found that it has a higher catalytic rate and a lower CO2 affinity compared to the enzymes with other small subunits. Our improvements in the E. coli Rubisco expression system will allow us to probe features of both the chloroplast and nuclear-encoded subunits of Rubisco that affect its catalytic rate and CO2 specificity. Read the full article here.