Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C 3 plant chloroplasts

Abstract

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacteria CO ₂ concentrating mechanism, could enhance photosynthetic CO ₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO ₂ -dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana , but the transformed plants did not have higher carbon assimilation rates or decreased CO ₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO ₂ concentrating mechanism components to improve plant photosynthesis.

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Review of the pre-print: Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts

Summary

Plants operate at a suboptimal photosynthetic efficiency for a number of reasons, one of which being that primary carbon fixation enzyme, rubisco, is relatively inefficient. Cyanobacteria also rely on rubisco for carbon fixation but are able to improve its efficiency by using a carbon concentrating mechanism (CCM) in which they import inorganic carbon to saturate rubisco active sites with CO2. There is an agricultural interest in supplying C3 crop plants with a similar CCM, however, there are several challenges to supplying crops with a cyanobacterial inorganic carbon transporter. One such challenge is correct targeting of the transporter to the inner chloroplast membrane. This has been accomplished before in plants using single-protein transporters such as BicA and SbtA, but the expression of those transporters did not improve the plant's carbon assimilation. Rottet et al chose to express the four-component ABC transporter, BCT1 (comprised of CmpABCD), which has the potential to be a more efficient transporter, but which required targeting CmpA to the inner membrane space where it could bind the HCO3-, targeting CmpB to the inner membrane to allow HCO3- transport across the membrane, and targeting CmpC and D to the stroma to bind and hydrolyze ATP to provide the necessary energy for the transport against a concentration gradient. Another challenge to heterologous expression of this carbon transporter is the presence of regulatory domains that render the transporter non-functional in a non-native context. To circumvent this issue, the authors used rational mutant design and directed evolution in an E. coli mutant strain that required active carbon transport for survival. This led to the discovery of constitutively active variants with mutations in the putative regulatory domain of CmpC. However, expression of these variants did not improve plant growth.

Although Rottet et al did not find that expression of BCT1 in plants ultimately resulted in improved carbon assimilation, despite its correct targeting and improved heterologous activity, the authors have creatively combined directed evolution and high throughput testing of candidate transporters in E. coli with their knowledge for plant targeting systems to create an efficient workflow for future efforts in engineering a CCM in C3 plants.

Major points

• Experiments orthogonal to fluorescence microscopy—for example co-immunoprecipitation assays-- in the N. benthamiana experiments would be helpful to confirm the interactions between the BCT1 subunits.

• The work in A. thaliana seemed inconclusive. It could be that the constructs are not being expressed—it should be possible to transform with the tagged versions of the constructs and perform western blots to determine if the protein is present and inactive.

Minor Points

• In the figure 2 and 3 legends it would be helpful to define the promoter and terminator abbreviations used.

• In Figure 7, it would be helpful to have diagrams of the constructs listed on the left side of panel A, similar to panel A of figure 8.

• Figure 4 and 5 could be combined so that the schematic of the constructs are easily shown next to their results in the CAFree growth assay

Competing interests

The authors declare that they have no competing interests.

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Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C ₃ plant chloroplasts

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