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Small subunits can determine enzyme kinetics of tobacco Rubisco expressed in Escherichia coli


Ribulose-1,5-bisphosphate carboxylase–oxygenase (Rubisco) catalyses the first step in carbon fixation and is a strategic target for improving photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits, and its biogenesis requires multiple chaperones. Here, we optimized a system to produce tobacco Rubisco in Escherichia coli by coexpressing chaperones in autoinduction medium. We successfully assembled tobacco Rubisco in E. coli with each small subunit that is normally encoded by the nuclear genome. Even though each enzyme carries only a single type of small subunit in E. coli, the enzymes exhibit carboxylation kinetics that are very similar to the carboxylation kinetics of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO2 affinity compared with Rubisco complexes that are assembled with other small subunits. Our E. coli expression system will enable the analysis of features of both subunits of Rubisco that affect its kinetic properties.

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Fig. 1: Gene arrangements in pET and pCDF E. coli expression vectors created in this study.
Fig. 2: Native PAGE analysis of BL21 Star (DE3) E. coli soluble extracts with tobacco Rubisco subunits expressed from either a PBAD or PT7 promoter.
Fig. 3: Comparison of Rubisco yields from E. coli under different expression conditions.
Fig. 4: Survey of the RbcSs in tobacco.
Fig. 5: Native PAGE immunoblots of tobacco Rubisco expressed in BL21 Star (DE3) E. coli with different small subunits.
Fig. 6: RuBP carboxylation rates in 42 μM [CO2] for tobacco Rubisco with individual small subunits expressed in E. coli and induced with either IPTG in LB medium or autoinduced in ZYP-5052 medium.
Fig. 7: The enzyme kinetics of tobacco Rubisco expressed in E. coli with different small subunits compared with the native tobacco Rubisco.

Data availability

A list of the accession numbers of proteins expressed in this study is provided in Extended Data Fig. 4 and is publicly available at or The tobacco transcriptomic data are available at NCBI under Bioproject accession PRJNA208209. Source data are provided with this paper.


  1. 1.

    Whitney, S. M., Houtz, R. L. & Alonso, H. Advancing our understanding and capacity to engineer nature’s CO2-sequestering enzyme, Rubisco. Plant Physiol. 155, 27–35 (2011).

    CAS  PubMed  Google Scholar 

  2. 2.

    Bauwe, H., Hagemann, M., Kern, R. & Timm, S. Photorespiration has a dual origin and manifold links to central metabolism. Curr. Opin. Plant Biol. 15, 269–275 (2012).

    CAS  PubMed  Google Scholar 

  3. 3.

    Walker, B. J., VanLoocke, A., Bernacchi, C. J. & Ort, D. R. The costs of photorespiration to food production now and in the future. Annu. Rev. Plant. Biol. 67, 107–129 (2016).

    CAS  PubMed  Google Scholar 

  4. 4.

    Davidi, D. et al. Highly active rubiscos discovered by systematic interrogation of natural sequence diversity. EMBO J. (2020).

  5. 5.

    Jordan, D. B. & Ogren, W. L. A sensitive assay procedure for simultaneous determination of ribulose-1,5-bisphosphate carboxylase and oxygenase activities. Plant Physiol. 67, 237–245 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Flamholz, A. I. et al. Revisiting trade-offs between Rubisco kinetic parameters. Biochemistry 58, 3365–3376 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Tcherkez, G. G. B., Farquhar, G. D. & Andrews, T. J. Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc. Natl Acad. Sci. USA 103, 7246–7251 (2006).

    CAS  PubMed  Google Scholar 

  8. 8.

    Iniguez, C. et al. Evolutionary trends in Rubisco kinetics and their co-evolution with CO2 concentrating mechanisms. Plant J. 101, 897–918 (2020).

    CAS  PubMed  Google Scholar 

  9. 9.

    Evans, J. R. Photosynthesis and nitrogen relationships in leaves of C3 plants. Oecologia 78, 9–19 (1989).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Young, J. N. et al. Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms. J. Exp. Bot. 67, 3445–3456 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Wilson, R. H. & Hayer-Hartl, M. Complex chaperone dependence of Rubisco biogenesis. Biochemistry 57, 3210–3216 (2018).

    CAS  PubMed  Google Scholar 

  12. 12.

    Gatenby, A. A., van der Vies, S. M. & Bradley, D. Assembly in E. coli of a functional multi-subunit ribulose bisphosphate carboxylase from a blue-green alga. Nature 314, 617–620 (1985).

    CAS  Google Scholar 

  13. 13.

    Lin, M. T., Occhialini, A., Andralojc, P. J., Parry, M. A. & Hanson, M. R. A faster Rubisco with potential to increase photosynthesis in crops. Nature 513, 547–550 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Long, B. M. et al. Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat. Commun. 9, 3570 (2018).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Tabita, F. R. & Small, C. L. Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits. Proc. Natl Acad. Sci. USA 82, 6100–6103 (1985).

    CAS  PubMed  Google Scholar 

  16. 16.

    Whitney, S. M., Birch, R., Kelso, C., Beck, J. L. & Kapralov, M. V. Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone. Proc. Natl Acad. Sci. USA 112, 3564–3569 (2015).

    CAS  PubMed  Google Scholar 

  17. 17.

    Sharwood, R. E., Ghannoum, O., Kapralov, M. V., Gunn, L. H. & Whitney, S. M. Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis. Nat. Plants 2, 16186 (2016).

    CAS  PubMed  Google Scholar 

  18. 18.

    Lin, M. T. & Hanson, M. R. Red algal Rubisco fails to accumulate in transplastomic tobacco expressing Griffithsia monilis RbcL and RbcS genes. Plant Direct 2, e00045 (2018).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Whitney, S. M., Baldet, P., Hudson, G. S. & Andrews, T. J. Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant J. 26, 535–547 (2001).

    CAS  PubMed  Google Scholar 

  20. 20.

    Brutnell, T. P., Sawers, R. J., Mant, A. & Langdale, J. A. BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize. Plant Cell 11, 849–864 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Feiz, L. et al. A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants. Plant J. 80, 862–869 (2014).

    CAS  PubMed  Google Scholar 

  22. 22.

    Feiz, L. et al. Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize. Plant Cell 24, 3435–3446 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Fristedt, R. et al. RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana. Plant J. 94, 146–156 (2018).

    CAS  PubMed  Google Scholar 

  24. 24.

    Aigner, H. et al. Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2. Science 358, 1272–1278 (2017).

    CAS  PubMed  Google Scholar 

  25. 25.

    Hauser, T. et al. Structure and mechanism of the Rubisco-assembly chaperone Raf1. Nat. Struct. Mol. Biol. 22, 720–728 (2015).

    CAS  PubMed  Google Scholar 

  26. 26.

    Liu, C. et al. Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature 463, 197–202 (2010).

    CAS  PubMed  Google Scholar 

  27. 27.

    Kolesinski, P. et al. Insights into eukaryotic Rubisco assembly—crystal structures of RbcX chaperones from Arabidopsis thaliana. Biochim. Biophys. Acta 1830, 2899–2906 (2013).

    CAS  PubMed  Google Scholar 

  28. 28.

    Schmidt, J. A., McGrath, J. M., Hanson, M. R., Long, S. P. & Ahner, B. A. Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts. Nat. Plants 5, 715–721 (2019).

    CAS  PubMed  Google Scholar 

  29. 29.

    South, P. F., Cavanagh, A. P., Liu, H. W. & Ort, D. R. Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field. Science 363, eaat9077 (2019).

    CAS  PubMed  Google Scholar 

  30. 30.

    Laterre, R., Pottier, M., Remacle, C. & Boutry, M. Photosynthetic trichomes contain a specific Rubisco with a modified pH-dependent activity. Plant Physiol. 173, 2110–2120 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Wilson, R. H., Thieulin-Pardo, G., Hartl, F. U. & Hayer-Hartl, M. Improved recombinant expression and purification of functional plant Rubisco. FEBS Lett. 593, 611–621 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    CAS  PubMed  Google Scholar 

  33. 33.

    Gong, L., Olson, M. & Wendel, J. F. Cytonuclear evolution of Rubisco in four allopolyploid lineages. Mol. Biol. Evol. 31, 2624–2636 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Sierro, N. et al. The tobacco genome sequence and its comparison with those of tomato and potato. Nat. Commun. 5, 3833 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Morita, K., Hatanaka, T., Misoo, S. & Fukayama, H. Identification and expression analysis of non-photosynthetic Rubisco small subunit, OsRbcS1-like genes in plants. Plant Gene 8, 26–31 (2016).

    CAS  Google Scholar 

  36. 36.

    Pottier, M., Gilis, D. & Boutry, M. The hidden face of rubisco. Trends Plant Sci. 23, 382–392 (2018).

    CAS  PubMed  Google Scholar 

  37. 37.

    Conlan, B. et al. BSD2 is a Rubisco-specific assembly chaperone, forms intermediary hetero-oligomeric complexes, and is nonlimiting to growth in tobacco. Plant Cell Environ. 42, 1287–1301 (2019).

    CAS  PubMed  Google Scholar 

  38. 38.

    Houtz, R. L., Magnani, R., Nayak, N. R. & Dirk, L. M. Co- and post-translational modifications in Rubisco: unanswered questions. J. Exp. Bot. 59, 1635–1645 (2008).

    CAS  PubMed  Google Scholar 

  39. 39.

    Morita, K., Hatanaka, T., Misoo, S. & Fukayama, H. Unusual small subunit that is not expressed in photosynthetic cells alters the catalytic properties of rubisco in rice. Plant Physiol. 164, 69–79 (2014).

    CAS  PubMed  Google Scholar 

  40. 40.

    Spreitzer, R. J. Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase. Arch. Biochem. Biophys. 414, 141–149 (2003).

    CAS  PubMed  Google Scholar 

  41. 41.

    Genkov, T. & Spreitzer, R. J. Highly conserved small subunit residues influence rubisco large subunit catalysis. J. Biol. Chem. 284, 30105–30112 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Spreitzer, R. J., Peddi, S. R. & Satagopan, S. Phylogenetic engineering at an interface between large and small subunits imparts land-plant kinetic properties to algal Rubisco. Proc. Natl Acad. Sci. USA 102, 17225–17230 (2005).

    CAS  PubMed  Google Scholar 

  43. 43.

    Ishikawa, C., Hatanaka, T., Misoo, S., Miyake, C. & Fukayama, H. Functional incorporation of sorghum small subunit increases the catalytic turnover rate of Rubisco in transgenic rice. Plant Physiol. 156, 1603–1611 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Andersson, I. & Backlund, A. Structure and function of Rubisco. Plant Physiol. Biochem. 46, 275–291 (2008).

    CAS  PubMed  Google Scholar 

  45. 45.

    Kapralov, M. V., Kubien, D. S., Andersson, I. & Filatov, D. A. Changes in Rubisco kinetics during the evolution of C4 photosynthesis in Flaveria (Asteraceae) are associated with positive selection on genes encoding the enzyme. Mol. Biol. Evol. 28, 1491–1503 (2011).

    CAS  PubMed  Google Scholar 

  46. 46.

    Yamada, K., Davydov, I. I., Besnard, G. & Salamin, N. Duplication history and molecular evolution of the rbcS multigene family in angiosperms. J. Exp. Bot. 70, 6127–6139 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Smith, S. A. & Tabita, F. R. Positive and negative selection of mutant forms of prokaryotic (cyanobacterial) ribulose-1,5-bisphosphate carboxylase/oxygenase. J. Mol. Biol. 331, 557–569 (2003).

    CAS  PubMed  Google Scholar 

  48. 48.

    Mueller-Cajar, O., Morell, M. & Whitney, S. M. Directed evolution of rubisco in Escherichia coli reveals a specificity-determining hydrogen bond in the form II enzyme. Biochemistry 46, 14067–14074 (2007).

    CAS  PubMed  Google Scholar 

  49. 49.

    Wilson, R. H., Alonso, H. & Whitney, S. M. Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth. Sci. Rep. 6, 22284 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Zhou, Y. & Whitney, S. Directed evolution of an improved Rubisco; in vitro analyses to decipher fact from fiction. Int. J. Mol. Sci. 20, 5019 (2019).

    CAS  PubMed Central  Google Scholar 

  51. 51.

    Hanson, M. R., Gray, B. N. & Ahner, B. A. Chloroplast transformation for engineering of photosynthesis. J. Exp. Bot. 64, 731–742 (2013).

    CAS  PubMed  Google Scholar 

  52. 52.

    Orr, D. J. & Carmo-Silva, E. Extraction of RuBisCO to determine catalytic constants. Methods Mol. Biol. 1770, 229–238 (2018).

    CAS  PubMed  Google Scholar 

  53. 53.

    Whitney, S. M. & Sharwood, R. E. Plastid transformation for Rubisco engineering and protocols for assessing expression. Methods Mol. Biol. 1132, 245–262 (2014).

    CAS  PubMed  Google Scholar 

  54. 54.

    Kubien, D. S., Brown, C. M. & Kane, H. J. Quantifying the amount and activity of Rubisco in leaves. Methods Mol. Biol. 684, 349–362 (2011).

    CAS  PubMed  Google Scholar 

  55. 55.

    Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).

    CAS  PubMed  Google Scholar 

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We thank M. Hayer-Hartl from the Max Planck Institute of Biochemistry in Martinsried, Germany, for providing us with her laboratory’s E. coli expression vectors; and D. Orr, E. Carmo-Silva and M. Parry from Lancaster University, Lancaster, UK for providing us with purified RuBP and advice on experiments to quantify Rubisco active sites and measure RuBP carboxylation kinetics. M.T.L. and W.D.S. are supported by a grant to M.R.H. from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (award no. DE-SC0020142); and M.R.H. and V.C. by the National Science Foundation (award no. MCB-1642386).

Author information




M.T.L. and M.R.H. conceived the research. All of the authors designed the experiments. M.T.L., W.D.S. and V.C. performed the experiments. All of the authors analysed the data and contributed to writing the manuscript.

Corresponding author

Correspondence to Maureen R. Hanson.

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Extended data

Extended Data Fig. 1 Native PAGE immunoblots of tobacco Rubisco expressed in Rosetta (DE3) E. coli with different small subunits.

Nt-Cpn60α, Nt-Cpn60β, GroES, Nt-RbcX, Nt-Raf1, At-Raf2 and Nt-Bsd2 were co-expressed. 9 µg of total soluble extract was loaded for each sample. 3 µg of total soluble extract from a young tobacco leaf (N) was also included as a control. The protein expressions were auto-induced in ZYP-5052 medium at 23 °C for 18-20 h. The top panel shows Coomassie blue staining, and the bottom panel shows the immunoblot using the antibody against Rubisco. The bands for L8S8 Rubisco (540 kDa) and chaperonin-RbcL complex (~720 kDa, indicated with asterisks) are marked next to each panel. The native PAGE was performed once for the same set of samples and multiple times for samples with RbcS-S1 and RbcS-T1 with similar results.

Source data

Extended Data Fig. 2 Comparison of tobacco Rubisco expressed in BL21 StarTM (DE3) E. coli using different culture media.

a, RuBP carboxylation rates at 42 μM [CO2] were measured from incorporation of 14C into RuBP in the absence of O2 at 25 °C. Rubisco active sites were quantified with bound 14C-CABP separated in size-exclusion chromatography. The error bars represent the mean values and standard deviations of measurements from three to six E. coli growth experiments for each condition. Data were analyzed with one-way ANOVA, and the P-value was obtained from F-statistics. Tukey’s honest significance test was then carried, and samples with P value > 0.05 are indicated with the same letter. b, The native PAGE immunoblot of the same samples in a using the antibody against wheat Rubisco. The three additional media tested are the buffered medium without additional carbon source (ZYP), the auto-induction medium without Mg (ZYP-5052ΔMg) and the auto-induction medium without trace metals (ZYP-5052Δmetals). The native PAGE was performed once for the same set of samples.

Source data

Extended Data Fig. 3 The enzyme kinetics of tobacco Rubisco expressed in E. coli with different small subunits compared to the native tobacco Rubisco.

RuBP carboxylation rates were measured from incorporation of 14C into RuBP in the absence of O2 at 25 °C. At-Cpn60α, At-Cpn60β, At-Cpn20, Nt-RBCX, Nt-RAF1, At-RAF2 and Nt-BSD2 were co-expressed in BL21 StarTM (DE3) either in LB or ZYP-5052 auto-induction medium at 23 °C for 18-20 h. [CO2] in the reaction mixtures ranged from 12.9 to 108.3 µM for the E. coli extracts and 8.3 to 103.7 µM for tobacco leaf extracts. Rubisco active sites were quantified with bound 14C-CABP separated in size-exclusion chromatography. The data were fitted to the Michaelis-Menten equation with nonlinear regression. The fitted models are shown as dotted lines for the E. coli samples and dashed line for the tobacco leaf samples.

Source data

Extended Data Fig. 4

Summary of the genes expressed in this study.

Extended Data Fig. 5

Summary of plasmids used in the expression of tobacco Rubisco in E. coli.

Supplementary information

Supplementary Information

Oligonucleotide sequences used in the construction of expression vectors.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed native PAGE and western blot.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Unprocessed native PAGEs and western blots.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed native PAGE and western blot.

Source Data Extended Data Fig. 2

Unprocessed western blot.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

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Lin, M.T., Stone, W.D., Chaudhari, V. et al. Small subunits can determine enzyme kinetics of tobacco Rubisco expressed in Escherichia coli. Nat. Plants 6, 1289–1299 (2020).

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