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

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

   Lin, Myat T.; Stone, William D.; Chaudhari, Vishalsingh; Hanson, Maureen R. (September 2020, Nature Plants)

   Rubisco catalyses the first step in carbon fixation and is a strategic target to improve photosynthetic efficiency. In plants, Rubisco is composed of eight large and eight small subunits and its biogenesis requires multiple chaperones. We optimised a system to produce tobacco Rubisco in Escherichia coli by co-expressing chaperones in auto-induction 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 very similar to that of the native Rubisco. Tobacco Rubisco assembled with a recently discovered trichome small subunit has a higher catalytic rate and a lower CO2 affinity than those assembled with other small subunits. Our E. coli expression system will allow probing of features of both subunits of Rubisco that affect its kinetic properties. 

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2. Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes

   https://doi.org/10.1073/pnas.2523360123  

   Chaudhari, Vishalsingh R; Lin, Myat T; Hines, Kevin M; Hanson, Maureen R (January 2026, Proceedings of the National Academy of Sciences)

   Improving the photosynthetic enzyme Rubisco is a key target for enhancing C₃crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-nullNicotiana tabacumplatform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (k_cat,air/K_c,air) under ambient conditions, driven by a significantly faster turnover rate (k_cat,air). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C₃crops. 

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3. Prospects for Engineering Biophysical CO2 Concentrating Mechanisms into Land Plants to Enhance Yields

   https://doi.org/10.1146/annurev-arplant-081519-040100  

   Hennacy, Jessica H; Jonikas, Martin C (March 2020, Annual review of plant biology)

   Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO2 fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO2-fixing enzyme Rubisco using biophysical CO2 concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO2 in a compartment that houses Rubisco (in contrast with other CCMs that concentrate CO2 via an organic intermediate, such as malate in the case of C4 CCMs). This activity provides Rubisco with a high concentration of its substrate, thereby increasing its reaction rate. The genetic engineering of a biophysical CCM into land plants is being pursued as a strategy to increase crop yields. This review focuses on the progress toward understanding the molecular components of cyanobacterial and algal CCMs, as well as recent advances toward engineering these components into land plants. 

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4. Modelling the pyrenoid-based CO2-concentrating mechanism provides insights into its operating principles and a roadmap for its engineering into crops

   https://doi.org/10.1038/s41477-022-01153-7  

   Fei C, Wilson AT (May 2022, Nature plants)

   Many eukaryotic photosynthetic organisms enhance their carbon uptake by supplying concentrated CO2 to the CO2-fixing enzyme Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer this pyrenoid-based CO2-concentrating mechanism (PCCM) into crops to increase yields. Here we develop a computational model for a PCCM on the basis of the postulated mechanism in the green alga Chlamydomonas reinhardtii. Our model recapitulates all Chlamydomonas PCCM-deficient mutant phenotypes and yields general biophysical principles underlying the PCCM. We show that an effective and energetically efficient PCCM requires a physical barrier to reduce pyrenoid CO2 leakage, as well as proper enzyme localization to reduce futile cycling between CO2 and HCO3−. Importantly, our model demonstrates the feasibility of a purely passive CO2 uptake strategy at air-level CO2, while active HCO3− uptake proves advantageous at lower CO2 levels. We propose a four-step engineering path to increase the rate of CO2 fixation in the plant chloroplast up to threefold at a theoretical cost of only 1.3 ATP per CO2 fixed, thereby offering a framework to guide the engineering of a PCCM into land plants. 

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5. New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields

   https://doi.org/10.1093/plphys/kiac373  

   Adler, Liat; Díaz-Ramos, Aranzazú; Mao, Yuwei; Pukacz, Krzysztof Robin; Fei, Chenyi; McCormick, Alistair J (August 2022, Plant Physiology)

   Abstract Many photosynthetic species have evolved CO2-concentrating mechanisms (CCMs) to improve the efficiency of CO2 assimilation by Rubisco and reduce the negative impacts of photorespiration. However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities. 

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