Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2

Abstract

Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of RuBisCo in by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis.

BSD2 is a Rubisco‐specific assembly chaperone, forms intermediary hetero‐oligomeric complexes, and is nonlimiting to growth in tobacco

“…The oligomeric status of each plant BSD2 isoform was examined by electrospray ionization mass spectrometry (Figure S2). Under nondenaturing conditions, all BSD2 proteins were primarily monomeric, with trace amounts of dimer, trimer, tetramer, pentamer, and hexamer forms detected, matching the findings of Aigner et al, (). These results indicate the low molecular weight SEC‐separated BSD2 protein in Figure c is uncomplexed monomer.…”

“…Nt BSD2‐immunoblot analysis of the fractions showed an Nt BSD2 peak eluting earlier (Fraction 3, shaded grey in Figure c) than the L 8 S 8 peak (Fraction 4), consistent with it comprising a structurally stable Rubisco‐ Nt BSD2 intermediary complex. On the basis of the findings of Aigner et al, () where stable asymmetrical L 8 ‐ Nt BSD2 complexes comprising some S‐subunits can form, this complex was annotated L 8 (S? )‐BSD2 as the possibility that it contains bound S‐subunits in addition to Nt BSD2 cannot be discounted (Figure c).…”

“…Understanding the biochemical foundation for this diversity in expression potential has gradually advanced over the last decade through sequential discovery of the cellular components needed for Rubisco assembly and activity (Bracher et al, ; Wilson & Hayer‐Hartl, ). These studies recently culminated in the demonstration that active Arabidopsis thaliana L 8 S 8 Rubisco could be reconstituted in Escherichia coli when coexpressed with a cocktail of cognate chloroplast protein folding machinery components (Aigner et al, ). These components included the differing subunits of the chloroplast macromolecular chaperonin folding machinery (CPN60β, CPN60α, CPN21) and the structurally unrelated Rubisco assembly chaperones Rubisco accumulating factor 1 and 2 (Raf1, Raf2), bundle sheath defective 2 protein (BSD2), and RbcX.…”

“…The efficiency of L 8 S 8 production is also influenced by sequence compatibility between the L‐subunit C‐terminus and RbcX homodimers (Durao et al, ; Emlyn‐Jones, Woodger, Price, & Whitney, ). Whereas Raf1 is crucial for Rubisco biogenesis, RbcX is not critical for recombinant plant Rubisco expression in E. coli (Aigner et al, ), provides no benefit to cyanobacteria L 8 S 8 Rubisco expression in tobacco plastids (Lin et al, ), and is not required for Rubisco production in some cyanobacteria (Emlyn‐Jones et al, ), and a Rubisco assembly role in planta remains ambiguous. Questions also remain as to the multifunctional role of the ~19 kDa Raf2 chaperone within the chloroplast and other cellular locations.…”

Optimizing photorespiration for improved crop productivity

“…Recently, a reconstituted Rubisco holoenzyme was assembled in a bacterial host (Aigner et al ). Coupled with recent insights in Rubisco species‐specific structure‐function relationships (Valegård et al , ), and assembly requirements (Saschenbrecker et al ; Feiz et al ; Whitney et al ), this provides a much‐needed technological breakthrough in our ability to screen Rubisco variants (Saschenbrecker et al ; Feiz et al ; Whitney et al ; Valegård et al , ).…”