An improved Escherichia coli screen for Rubisco identifies a protein–protein interface that can enhance CO2-fixation kinetics

Abstract

An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity, global food security, and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts. Here we refashion metabolism by expressing a phosphoribulokinase-neomycin phosphotransferase fusion protein to produce a high-fidelity, high-throughput Rubisco-directed evolution (RDE2) screen that negates false-positive selection. Successive evolution rounds using the plant-like-Rubisco from the cyanobacterium BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate, efficiency, and specificity. Structural analysis revealed the amino acids clustered in an unexplored subunit interface of the holoenzyme. To study its effect on plant growth, the-Rubisco was transformed into tobacco by chloroplast transformation. As previously seen for PCC6301 Rubisco, the specialized folding and assembly requirements of-Rubisco hinder its heterologous expression in leaf chloroplasts. Our findings suggest that the ongoing efforts to improve crop photosynthesis by integrating components of a cyanobacteria CO-concentrating mechanism will necessitate co-introduction of the ancillary molecular components required for Rubisco biogenesis.

Improved recombinant expression and purification of functional plant Rubisco

“…As the pET28 and pET11a plasmids (for expression of Rubisco and chaperonins, respectively) share the pBR322 origin of replication, the long‐term coexistence of these plasmids may result in variation of copy number and thus fluctuations in expression levels . Thus, applications sensitive to fluctuations of Rubisco expression, such as directed evolution screening or metabolic engineering , may require the use of compatible plasmids.…”

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

“…To obtain the crystal structure of BSD2 bound to the RbcL 8 core, we used the thermostable RbcL 8 from the cyanobacterium Thermosynechococcus elongatus BP-1. Mutations F345I and P415A [TeRbcL(IA)] were introduced to further increase TeRbcL stability (33,34). (Fig.…”

Applications of Protein Engineering and Directed Evolution in Plant Research

“…One issue in these screens is that false positives are obtained at high frequencies (Greene et al, 2007;Cai et al, 2014) due to natural transposon-mediated silencing of phosphoribulokinase (Wilson and Whitney, 2017). In a clever approach, the problem of false positives was combated by expressing a phosphoribulokinaseneomycin phosphotransferase fusion protein and including the additional selection pressure of antibiotic resistance (Wilson et al, 2018). In an approach similar to the one taken in E. coli, the soil bacterium Ralstonia eutropha has also been developed for in vivo screening of Rubisco variants (Satagopan and Tabita, 2016).…”