Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) is a vital enzyme in plants, algae, and phototrophic bacteria. In the primary reaction of Calvin-Benson-Bassham Cycle, RuBisCo facilitates the fixation of atmospheric CO2 through the carboxylation of ribulose-1,5-bisphosphate (Bhat et al., 2017). This initial carboxylation reaction produces two molecules of 3-phosphoglycerate(3pG) which can later be used as substrate in several anabolic pathways (Fig. 1). RuBisCo is also considered to be the most abundant enzyme on the planet, and is estimated to be responsible for fixing 250 billion tons of CO2 per year (Bracher et al.., 2017). Life on earth is contingent on the ability of photosynthetic organisms to absorb atmosphere inorganic CO2 and process it into organic carbon usable by other organisms of the biosphere via the Calvin-Benson-Bassham pathway(Andersson et a.l, 2017). Figure 1. Calvin–Benson–Bassham Cycle: Schematic depicting CO2 fixation, as well as the side-reaction of photorespiration. Ribulose-1,5-bisphosphate(RuBP); 3-phosphoglycerate(3PG); Glyceraldehyde-3-phosphate(G3P) (Bhat et al., 2017). However, with a catalytic turnover rate of about 5 molecules of CO2 fixed per second, RuBisCo is considered to be a relatively inefficient enzyme (Feller et al., 2017). Exacerbating this issue is RuBisCo’s ability to use oxygen as a substrate instead of CO2 (Whitney et al.., 2011). This pathway produces only one molecule of 3PG as well as a by-product which is toxic for chloroplast (Fig. 1)(Bhat et al., 2017). This inability to discriminate between CO2 and oxygen results in photorespiration being energetically wasteful. To compensate for these deficiencies, phototrophic organisms need to produce a large quantity of RuBisCo to remain viable. Therefore, there is a desire to improve RuBisCo’s catalytic efficiency in order to develop crops which are more robust, and have higher yields. However, until recently, it has not been possible to express plant RuBisCo in bacteria. This has been thought to be to due, at least in part, for the need to coordinate subunit synthesis, and the presence of highly specific plant chaperones (Bhat et al., 2017). This lack of understanding has made it difficult, to induce the expression of RuBisCO in the lab, as well as limiting the generation of alternate, and more efficient forms of enzyme. Then, in December 2017, Aigner et al. published their findings on the successful expression of plant RuBisCo in E. coli. Their assembly utilizes the coexpression of several chloroplast chaperones which are vital for proper RuBisCo processing(Fig. 2). This work not only provided a system to express RuBisCo in bacteria but also elucidated the functions and characteristics of multiple chaperone process. Figure 2. E. coli RuBisCO Assembly Assay: Diagram depicting the coexpression of the chloroplast chaperones which are vital for proper RuBisCo processing in E. coli (Yeats et al., 2017).Form I RuBisCo, which is the most abundant form, is commonly found in plants, algae, cyanobacteria, and phototrophic bacteria (?lesak et al., 2017). This enzyme is a complex of eight RbcL(large) and eight RbcS(small) subunits, forming the RbcL8S8 holoenzyme (Whitney et al., 2011). The large subunits form a tetrameric core of antiparallel RbcL dimers (Fig. 3). The two active sites are located at the interface of each antiparallel RbcL dimer. Four Rbcs subunits then form caps on the top and bottom of the complex (Hayer-Hartl, 2017)Figure 3. RuBisCo Holoenzyme structures: Form I Rubisco from spinach (PDB: 1RCX). RbcL subunits(white), RbcS subunits (yellow). Antiparallel RbcL dimer(blue and green) with the RbcS subunits shown as ribbon in the side-view (Hayer-Hartl, 2017).It was discovered that chaperonin 60-subunit ?1 (Cpn60?), Cpn60?, 20-kDa chaperonin (Cpn20), RuBisCO assembly factor 1 (RAF1), RAF2, ribulose bisphosphate carboxylase factor X (RbcX), and bundle-sheath defective-2 (BSD2), were essential for plant RuBisCO biosynthesis and assembly in E. coli. Specifically Raf1, Raf2, and BSD2 were essential for recombinant RuBisCo biogenesis, while RbcX was mainly needed to maintain efficiency (Aigner et al., 2017).The Cpn60 complex is composed of Cpn60? and Cpn60? and is responsible for repairing misfolded proteins, with the assistance of Cpn10 or Cpn20. Aigner et al. realised that plant Cpn60? and Cpn60? are required for proper protein processing and cannot be replaced for their E. coli homologs. It was also found that, more highly specialized chaperones are required to assemble fully functional RuBisCO. It was also found that RbcX cannot compensate for the absence of RAF1 in E. coli. Finally it was discovered that eight RbcL and BSD2 subunits formed a stable intermediate. It was through the replacement of the BSD2 subunits by RbcS That the final and fully functional protein was made. However the role of Raf2 is still not fully characterized (Fig. 4)Figure 4. Model of Plant RuBisCo chaperone-assisted folding and assembly: The seven auxiliary proteins (Cpn60?, Cpn60?, and Cpn20 as well as the auxiliary factors Raf1, Raf2, RbcX, and BSD2) to express the functional RuBisCo in E. coli. (?) denotes enzymes who’s structures or characteristics are not fully known (Aigner et al., 2017). Although the method developed by Aigner et al did not provide structural and mechanistic data for all chaperones involved, they were able to develop an E. coli RuBisCO assembly assay. This lays the foundation to recombinantly produce plant RuBisCo and engineer a more effective or favorable enzyme through mutational analysis. Practically a more effective enzyme can lead to the development of plants which are more weather resistant, water efficient, and overall produce a higher crop yield (Alcântara et al., 2017). There is also the potential to decrease atmospheric CO2 levels with the hope of undoing some of the damage which has lead to climate change.