Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves

Abstract

Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes’ complex chloroplast biogenesis requirements. New Synbio approaches however now allow the production of some plant Rubisco isoforms in E. coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimise and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome transformed tobacco we show the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low cost screening tool that can accurately emulate chloroplast expression.

Cell-free expression of RuBisCO for ATP production in the synthetic cells

“…These advantages make it possible to simultaneously examine various RuBisCO expressions and reaction conditions. The limitation of our results is that we could not detect the activity of S. elongatus RuBisCO, which could be resolved by the addition of chaperones or RuBisCO activase that contribute to the folding of RuBisCO, as in previous studies ( 34 , 37 , 38 ).…”

A carboxysome‐based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead

“…While studies examining Rubisco assembly and evolution have utilised E. coli expression systems (Aigner et al., 2017; Lin et al., 2020; Prywes et al., 2023; Rydzy et al., 2023; Zhou & Whitney, 2019), a separate study has demonstrated that Rubisco assembly in E. coli did not always closely correlate with Rubisco assembly in plants (Buck et al., 2023). We envision this will be the subject of future research, to ensure results from E. coli will have direct translational outcomes for optimal carboxysomal Rubisco assembly in plants.…”

Engineering Rubisco to enhance CO2 utilization

“…Further investigations have demonstrated that this strategy is comparably efficacious for Rubisco sourced from potatoes, carrots, strawberries, and tobacco. While assembly is achievable within E. coli , the corresponding enzymatic activity has not yet been reported [ 126 ]. A robust Golden Gate cloning E. coli expression system has been developed, which can effectively predict high-level Rubisco production in chloroplasts.…”

“…A robust Golden Gate cloning E. coli expression system has been developed, which can effectively predict high-level Rubisco production in chloroplasts. Despite its limitations in accurately predicting the biogenesis potential of isoforms with impaired production in planta, this study also contributes to the development of a truly efficient and low-cost RDE screening tool that can accurately emulate chloroplast expression [ 126 ].…”