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

Abstract

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 https://www.ncbi.nlm.nih.gov or https://solgenomics.net. The tobacco transcriptomic data are available at NCBI under Bioproject accession PRJNA208209. Source data are provided with this paper.

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Acknowledgements

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).

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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). https://doi.org/10.1038/s41477-020-00761-5

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