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Laboratory evolution of Rubisco solubility and catalytic switches to enhance plant productivity

Nature Plants volume 11, pages 1939–1950 (2025)Cite this article

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An Author Correction to this article was published on 24 September 2025

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Abstract

A new Escherichia coli laboratory evolution screen for detecting plant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mutations with enhanced CO2-fixation capacity has identified substitutions that can enhance plant productivity. Selected were a large subunit catalytic (Met-116-Leu) mutation that increases the kcatc of varying plant Rubiscos by 25% to 40% and a solubility (Ala-242-Val) mutation that improves plant Rubisco biogenesis in E. coli 2- to 10-fold. Plastome transformation of either mutation into the tobacco plastome rbcL gene had no impact on leaf Rubisco production, photosynthesis or plant growth. However, tobacco transformed with low-abundance hybrid Arabidopsis Rubisco coding M116L improved plant exponential growth rate by ~75% relative to unmutated hybrid enzyme, with the A242V substitution increasing both hybrid Rubisco production and plant growth by ~50%. Our identification of mutations with the potential to enhance plant growth bodes well for broadening the survey of Rubisco sequence space for catalytic switches that can impart more substantive plant productivity improvements.

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Fig. 1: The biogenesis and directed evolution of plant Rubisco.
Fig. 2: Selection and kinetics of tccRDE fitness enhancing tobacco Rubisco mutants.
Fig. 3: Structural analysis of the catalytic M116L and solubility A242V mutations.
Fig. 4: The catalytic and solubility impacts of M116L and A242V across plant Rubisco.
Fig. 5: Plastome transformation and kinetics of M116L and A242V mutated tobacco and hybrid Arabidopsis Rubisco.
Fig. 6: The impact of the M116L and A242V mutations on transplastomic tobacco growth.

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The materials generated as part of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

This research was supported by Australian Research Council grants CE140100015 (S.W.) and DP210101757 (S.W.) in addition to the Czech Science Foundation (GACR project number 24-10671S, I.A.) and the Johannes Amos Comenius Operational Programme (OPJAK project number SENDISO-CZ.02.01.01/00/22_008/0004596, I.A.). We thank N. Paul for technical assistance and acknowledge the use of the facilities of the Australian Plant Phenomics Network, which is supported by the Australian Government’s National Collaborative Research Infrastructure Strategy.

Author information

Authors and Affiliations

  1. Plant Science Division, Research School of Biology, The Australian National University, Canberra, Australian Capital Territory, Australia

    Matteo Gionfriddo, Rosemary Birch, Timothy Rhodes, Sally Buck, Tanya Skinner & Spencer Whitney

  2. Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

    Inger Andersson

  3. The European Extreme Light Infrastructure, Dolní Břežany, Czech Republic

    Inger Andersson

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  1. Matteo Gionfriddo
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  7. Spencer Whitney
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Contributions

M.G. and S.W. conceived the study. M.G., T.R. and S.B. designed the directed evolution system with M.G. performing the mutant screening experiments. M.G., R.B. and S.W. generated and analysed the Rubisco mutant tobacco lines with M.G. and S.W. performing the Rubisco kinetic analyses with assistance from T.S. and T.R. Structure–function interpretations were performed by I.A., and the paper was written by M.G., I.A. and S.W. with input from all authors.

Corresponding author

Correspondence to Spencer Whitney.

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Competing interests

The authors R.B. and I.A. declare no competing interests. A provisional patent application (AU2025902586) has been filed for aspects of the work reported in this paper with S.W., M.G., T.R., S.B. and T.S. listed as inventors.

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Extended data

Extended Data Fig. 1 Tobacco rbcL-rbcS mutagenic library construction and insertion of point mutations by golden gate (GG) cloning.

(a) Error prone PCR (epPCR) using the GeneMorph II Random Mutagenesis Kit, plasmid pBP-LS and primers 1555-F and 1568-R yielded 2075-bp products containing flanking BsaI sites to facilitate GG cloning into (b) BsaI cut pET16-GFP-NtRCA to create (c) a pET16-NtL*S*RCA (P2*) plasmid library comprising random mutations (*) in the Rubisco genes. Transformed E. coli containing pET16-GFP-NtRCA are visually identified as GFP expressing colonies while those expressing Rubisco are white. T7 promoter (P) and terminator (T). See Source data Figs. 1 and 5 and Extended Data Figs. 1 and 3 for full plasmid and primer sequences. The mutant tobacco Rubisco library was generated by error prone-PCR (ep-PCR) amplification of the rbcL-rbcS genes including their independent promoter, ribosome binding site and terminator elements) with primers 1555-F and 1568-R using Mutazyme manufacturer’s recommendations (Agilent Technologies, Santa Clara, CA, USA). Following agarose gel separation, the ep-PCR product (library) was purified using a Wizard® SV Gel and PCR Clean-Up System kit (Promega), digested with BsaI and ligated into pET16 (that is Golden Gate [GG] cloning) to generate the P2 plasmid library. Following dialysis by floating Millipore disc Filter Membranes on water for 2 hours the GG reactions were transformed into XL-1Blue E. coli by electroporation (2.5 V, 0.2 mm electro-cuvettes) and plated on LB-Amp at 37 °C. Once colonies appeared they were collected by scraping and the P2 plasmid library purified before transforming into BL21Star E. coli already transformed with plasmids P1 and P3 (step 3 of Fig. 1d) and then screening under selective (RDE) conditions (Fig. 2). The Rubisco library made contained ~1.5 × 106 different mutants.

Source data

Extended Data Fig. 2 PAGE analysis of tobacco Rubisco production in E. coli BL21-Star.

PAGE analysis of a single experiment examining the soluble protein from a tobacco (Nt) leaf and E. coli BL21-star expressing wildtype (WT) and mutant tobacco L8S8 Rubiscos (coded by plasmid P2, Fig. 1c) and tobacco chloroplast chaperones (plasmid P3) were separated by native-PAGE and (a) stained with Coomassie or (b) blotted onto nitrocellulose and immuno-probed with tobacco Rubisco antibody. In addition to the A242V substitution promoting L8S8 holoenzyme production, it facilitated the production of a larger RbcL intermediary complex (*), previously shown to contain RAF1 (R1) and/or BSD2 (B2) subunits (L8(R1/B2)n28. (c) Corresponding SDS PAGE separation of the same soluble (S) protein fractions and corresponding total (T) cellular protein (comprising soluble and insoluble membrane and aggregated/inclusion body proteins) showing the Cpn60 and Cpn20 produced are mostly fully soluble while only a small proportion ( < 20%) of the RbcL, RbcS and Rca produced in E. coli are soluble (+), more so in those containing the A242V mutation in RbcL (++). Asmbl refers to the expression of only P3 plasmid; EV, empty pCDF plasmid; GroE, E. coli GroELS chaperonin cages; CPN, tobacco Cpn60α/β/20 chaperonin complexes; CPN + L, CPN encaged RbcL (slightly visible in panel b). Uncropped images of the original gels and blot are provided in Source Data Fig. 2 and Extended Data Figs. 2, 4, 6 and 7.

Source data

Extended Data Fig. 3 Generating rbcL plastome transformation plasmids containing direct repeats to facilitate marker excision via homologous recombination within the plastome.

(a) The primers, PCR amplifications and plasmids required for (b) the (i) Golden Gate assembly of the (ii) 4 cloning fragments required to (iii) construct the final transformation vector pLEV-GG-rbcL that contain 405-bp direct repeats (DR) of duplicated regions of the 3’rbcL coding sequence (∆L) and 3’UTR (green T). P, tobacco rbcL promoter + 5′UTR; T, tobacco rps16 terminator; p, T7 promoter; t, T7 terminator.

Source data

Extended Data Fig. 4 Generation of gene edited rbcL tobacco plastome transformants.

(a) Plasmids pLEVNt-RbcL116/RbcL242 (see Extended Data Fig. 3 for method of synthesis) were (b) transformed into the 100 kDa L2 R. rubrum Rubisco producing TobRr35 and (c) independent spectinomycin (0.5 mg.ml−1) resistant (specR) T0 transplastomic Tob116 and Tob242 lines selected. Correctly transformed lines (that is those making L8S8 Rubisco) were (d) identified by native PAGE and three Tob116 and two Tob242 lines grown to maturity and fertilized with (e) wild-type (WT) pollen over the (f) T1 and (g) T2 generations. The seeds from each generation were screened for specR, with 12–16% white spectinomycin sensitive progeny detected in the T2 generation and assumed to be (h) aadA free lines resulting from aadA excision via recombination between the homologous direct repeat (DR) regions and thus restoring the plastome sequence to WT apart from single nucleotide gene edits in rbcL (as indicated in panel (a)). Growth of the aadA-free plants was restored once spectinomycin selection was removed and at maturity (i) fertilised with WT pollen to produce T3 plants that were all spectinomycin sensitive and used for growth analyses. (j) A DNA blot analysis of BamHI (B) digested DNA to confirm the T3 plants were aadA-free homoplasmic rbcL gene edited lines (that is contained no aadA-containing 2.7 kb plastome fragment) and the T1 plants had both aadA-containing and aadA-excised plastome copies (that is were heteroplasmic). Uncropped germination plate photos and PAGE images are provided in Source Data Fig. 2 and Extended Data Figs. 2, 4, 6 and 7.

Source data

Extended Data Fig. 5 Comparing the Ko and Kc of mutant tobacco and hybrid Arabidopsis Rubisco.

(a) Linear response in Km for CO2 (Kc) with elevating [O2] for the differing Rubiscos. Data shows a collation of measures made on n = 3 to 5 samples (each symbol indicating an independent biological sample). For comparison, the grey dashed line represents the linear fit to the Kc vs [O2] response for wild-type tobacco Rubisco from the top left panel. The mean ( ± SD) values of (b) Kc derived the y-axis (that is where [O2] = 0) and (c) Ko calculated as Kc/slope of the linear fit for each biological sample assayed (circles representing values for each sample). Shown are the mean ( ± SD) of individual values (white circles) from n = 4–6 biological replica measurements as indicated. Lower case letters indicate significant differences to p < 0.05 using a Tukey multiple comparison test with p-values and all data points provided in Source Data Figs. 4 and 5 and Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Ambient CO2 growth experiments on tobacco and the RbcL gene edited tob116 (T3, T4) and tob242 (T3) lines.

(a) Summary detail of the experimental conditions for three temperature-controlled growth experiments (GE) under ambient (410 ppm) CO2 and (b) the corresponding individual measures of height increase over with destructive measures of leaf and stem dry biomass taken at differing time points (the number of plants (n) harvested at each time point indicated). Black lines represent sigmoidal growth fits to the height measurements (circles; see Source Data Fig. 6 and Extended Data Figs. 6 and 7 for measurements of each plant) with the wild-type tob growth fit for each GE shown as a dashed line in the lower panels. Shown are the mean ( ± SD) of the linear exponential growth rates (red text and line) measured for n = 6 to 10 plants during stem elongation as indicated. (c) Representative phenotype of the plants at the time of harvest indicated by the circled numbers. Timing of A-Ci gas exchange measurements performed during GE#3 are indicated by the horizontal yellow line (that is when plants at 35 ± 3 cm in height). Uncropped images plants are provided in Source Data Fig. 2 and Extended Data Figs. 2, 4, 6 and 7.

Source data

Extended Data Fig. 7 Comparative growth of tobacco and hybrid Arabidopsis Rubisco producing tobacco lines under elevated CO2 – Growth experiment 4.

Plants were grown in a greenhouse as in growth experiment 2 (Extended Data Fig. 6) during Oct 2023 – Feb 2024 in air containing 600 ppm CO2. (a) Individual measures of height increase over time were made for n = 12 plants with destructive measures of leaf and stem dry biomass for (b) n = 6 or7 plants when 66 ± 2 cm in height (indicated by purple horizontal line in panel a) at the times indicated for each genotype (numbers circles correlating to panel a). Black lines represent sigmoidal growth fits to the height measurements (see Source Data Fig. 6 and Extended Data Figs. 6 and 7 for measurements of each plant). Shown are the mean ( ± SD) of the linear exponential growth rates (red text and line) measured for each plant during stem elongation. Timing of A-Ci gas exchange measurements are indicated by the horizontal yellow line (that is, when plants were at 35 ± 3 cm in height). Uncropped plants images are provided in Source Data Fig. 2 and Extended Data Figs. 2, 4, 6 and 7.

Source data

Extended Data Fig. 8 Contacts within van der Waals distance of residue M/L116.

Contacts (dashed red lines) within van der Waals distance of (a) the modelled M116 in wild type (PDB id 4RUB) and (b) mutant L116 along with (c) a full list of contacts around atoms Cβ and Cγ of Met116 and Leu116. The structure analysis is based on the crystal structure of Rubisco from tobacco (PDB 4RUB). The M116L and A242V substitutions were modeled in their most likely conformations from a library of rotamers53. The modelled residue conformations aligned with the corresponding residues in the structure of the highly CO2-specific non-green algae Griffithsia monilis (PDB id 8BDB)24. The effect of cavity-creating substitutions on protein structure has been discussed in detail in the literature54.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Fig. 1.

Reporting Summary

Source data

Source Data Figs. 1 and 5 and Extended Data Figs. 1 and 3

Gene, plasmid, primer and protein sequence information.

Source Data Fig. 2 and Extended Data Figs. 2, 4, 6 and 7

Unprocessed micrographs and images.

Source Data Figs. 4 and 5 and Extended Data Fig. 5

E. coli-made Rubisco kinetic and biochemical raw data values and statistics for Fig. 4, plant-made Rubisco kinetic and biochemical raw data values and statistics for Fig. 5 and raw kinetic data of plant-made Rubisco that was used to determine Ko for Exended Data Fig. 5.

Source Data Fig. 6 and Extended Data Figs. 6 and 7

Plant growth, biomass, photosynthesis and leaf Rubisco/protein raw data values and statistics for Fig. 6 and plant growth raw data values for Extended Data Figs. 6 and 7.

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Gionfriddo, M., Birch, R., Rhodes, T. et al. Laboratory evolution of Rubisco solubility and catalytic switches to enhance plant productivity. Nat. Plants 11, 1939–1950 (2025). https://doi.org/10.1038/s41477-025-02093-8

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