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Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth


Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis GmRubisco. To address this, we determined the crystal structure of GmRubisco to 1.7 Å. Three structurally divergent domains were identified relative to the red-type bacterial Rhodobacter sphaeroides RsRubisco that, unlike GmRubisco, are expressed in Escherichia coli and plants. Kinetic comparison of 11 RsRubisco chimaeras revealed that incorporating C329A and A332V substitutions from GmRubisco Loop 6 (corresponding to plant residues 328 and 331) into RsRubisco increased the carboxylation rate (kcatc) by 60%, the carboxylation efficiency in air by 22% and the CO2/O2 specificity (Sc/o) by 7%. Plastome transformation of this RsRubisco Loop 6 mutant into tobacco enhanced photosynthesis and growth up to twofold over tobacco producing wild-type RsRubisco. Our findings demonstrate the utility of RsRubisco for the identification and in planta testing of amino acid grafts from algal Rubisco that can enhance the enzyme’s carboxylase potential.

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Fig. 1: Three-dimensional crystal structure of G. monilis Rubisco.
Fig. 2: Structural comparison between Form IB SoRubisco, Form IC RsRubisco and Form ID GmRubisco.
Fig. 3: Phylogenetic grafting of GmRubisco into RsRubisco to enhance catalysis.
Fig. 4: The simulated improvement of tobacco photosynthesis by RsRubiscoChi11.
Fig. 5: The improved carboxylation properties of RsRubisco-chi11 translate to improvements in the photosynthesis and growth of tobacco.

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Data availability

Atomic coordinates and structure factors for G. monilis Rubisco are accessible via Protein Data Bank ( accession number 8BDB. All other data are available online as source files and via Source data are provided with this paper.


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This research was supported by the Australian Government through the Australian Research Council Centre of Excellence for Translational Photosynthesis CE140100015 and through the Formas Future Research Leaders grant 2017-00963. The work was also supported by the project ‘Structural dynamics of biomolecular systems (ELIBIO)’ (NO. CZ.02.1.01/0.0/0.0/15_003/0000447) from the European Regional Development Fund. We thank Diamond Light Source for access to beamline I02 (proposal MX11171-5) and B. Li for technical assistance.

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Authors and Affiliations



Y.Z., L.H.G. and S.M.W. conceived the project and all authors wrote the paper; L.H.G. and I.A. determined the crystal structure; L.H.G. and S.M.W. generated and analysed all enzymes; Y.Z. and R.B. generated the transgenic tobacco lines, performed the molecular and protein analyses, and measured photosynthesis and growth.

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Correspondence to Spencer M. Whitney.

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Nature Plants thanks Cong-Zhao Zhou and Thomas Sharkey for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Interactions of the GmRbcS βE-βF hairpin loop.

Interactions between residues within the βE-βF sheet (hairpin loop) of adjacent GmRbcS subunits (coloured teal, residue labeling in red and blue correspond to GmRbcS subunits B and C in PDB 8BDB, respectively) and residues in neighboring RbcL subunits C and A (coloured grey, labels in black). Residue contacts are indicated by dashed lines.

Extended Data Fig. 2 Quantifying Rubisco Ko.

(A) Linear response of Kc to increasing [O2] for E. coli produced RsRubisco, (B) RsRubisco-chi11 and (C) Rubisco from tobacco leaves. The Kc is derived from the y-axis (that is where [O2] = 0) and Ko calculated as Kc/slope of the linear fit. Fits were made to the mean Kc values (±S.E) derived from Michaelis-Menten fits to 14CO2 fixation assays measured using n = 3 or 4 independent biological samples per Rubisco isoform (each symbol representing a differing enzyme preparation). See Table 1 for Ko values.

Source data

Extended Data Fig. 3 Generation of the tobRr::X transforming master-line and tobRsL11S.

(A) Schematic summary of the Rubisco genetics and transformation process used by20 to generate the tobRsLS::X genotype from which a homozygous line coding a single cbbX allele was generated and used to pollinate the Rhodospirillum rubrum L2 RrRubisco tobRr genotype26 to produce (B) the heterozygous RsCbbX (RsRca) producing tobRr::X line with all the progeny (C) kanamycin resistant (see pBinTP-cbbX plasmid in panel A showing co-transformation of the nptII and cbbX alleles). (D) The RsRubisco bioengineering tobRr::X master-line was plastome transformed with pLEVRsL11S that contained plastome flanking sequence (see dashed lines with numbering relative to the corresponding tobacco plastome GenBank accession NC_001879.2 sequence) that directed the replacement of the rbcM gene with the Rs-rbcL-rbcS-aadA operon coding RsRubisco-chi11 that differs by 3 nucleotides (as shown) to the RsRbcL in the tobRsLS::X line produced by20. Shown are the location of the 5UTR-probe and the BglII sites and fragment size it hybridizes to. (E) 32P-5UTR-probe labelled genomic DNA-blot screen of 6 independent tobRsL11S::X lines following 2 rounds of spectinomycin selection, a tobRsLS::X line (positive control) and tobRr::X (negative control). Lines #2 and #5 were deemed homoplasmic as no rbcM containing plastome copies (5.5 kb fragment) were evident. (F) Subsequent native-PAGE screen of soluble protein from three tobacco (wt), three tobRsLS::X plants and three of the six T0 tobRsL11S::X lines grown to maturity for seed. The seed (T1 progeny) from tobRsL11S line #1::X was used in subsequent growth comparisons (Fig. 5).

Source data

Extended Data Fig. 4 Comparative Loop 6 interactions.

Comparative interactions around the GmRubisco Loop 6 site 1 (A331) and site 2 (V334) residues relative to (A, C) Rs and (B, D) SoRubisco. Residue contacts are indicated by dashed lines.

Supplementary information

Supplementary Information

Supplementary Protocols, Figs.1 and 2, Tables 1–3 and References.

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Source data

Source Data Fig. 3

Unprocessed Coomassie-stained native PAGE.

Source Data for Figs. 3 and 5, and Extended Data Fig. 2

Spreadsheet-based data for Table 1, Figs. 3c and 5c–g, and Extended Data Fig. 2 combined into a workbook with multiple tabs.

Source Data Extended Fig. 3e

Unprocessed DNA blot (only low-resolution source image available) for Extended Data Fig. 3e and unprocessed Coomassie-stained native PAGE for Extended Data Fig. 3f.

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Zhou, Y., Gunn, L.H., Birch, R. et al. Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth. Nat. Plants 9, 978–986 (2023).

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