Improving the efficiency of Rubisco by resurrecting its ancestors in the family Solanaceae

Abstract

Plants and photosynthetic organisms have a remarkably inefficient enzyme named Rubisco that fixes atmospheric CO 2 into organic compounds. Understanding how Rubisco has evolved in response to past climate change is important for attempts to adjust plants to future conditions. In this study, we developed a computational workflow to assemble de novo both large and small subunits of Rubisco enzymes from transcriptomics data. Next, we predicted sequences for ancestral Rubiscos of the (nightshade) family Solanaceae and characterized their kinetics after coexpressing them in Escherichia coli . Predicted ancestors of C 3 Rubiscos were identified that have superior kinetics and excellent potential to help plants adapt to anthropogenic climate change. Our findings also advance understanding of the evolution of Rubisco’s catalytic traits.

The small subunit of Rubisco and its potential as an engineering target

“…Nevertheless, producing Rubiscos from plants in E. coli still remains challenging due to inefficient RbcL processing and the accumulation of assembly intermediates (Ng et al, 2020), low functional Rubisco yields, and limited current knowledge of the chaperones required for Rubiscos from different plant species. Rubisco biogenesis in E. coli is also currently not a reliable predictor for assembly in chloroplasts (Wilson et al, 2018), while the catalytic parameters of plant Rubiscos assembled in E. coli are similar but not identical to those observed in planta (Lin et al, 2020(Lin et al, , 2022. However, significant improvements have already been made (Zhou and Whitney, 2019), and the increasing availability of genomic data could assist with identifying homologues or additional ancillary chaperones required for different plant species (Kress et al, 2022).…”

“…the native Rubiscosome). Secondly, engineering efforts have screened predicted ancestral Rubiscos and produced Rubisco variants that deviate from the ‘canonical’ catalytic trade-offs between k cat c , K c , and S c/o ( Wilson et al , 2018 ; Zhou and Whitney, 2019 ; Martin-Avila et al , 2020 ; Lin et al , 2022 ). Together, these studies suggest a wider scope for engineering improvements in the catalytic parameters of Rubisco, and that furthering our understanding of the underlying structural basis that confers the catalytic properties of Rubisco is still critically important ( Valegård et al , 2018 ).…”

“…clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide (Cas)] have now made it possible to edit partial or entire RbcS gene families ( Donovan et al , 2020 ; Martin-Avila et al , 2020 ; Matsumura et al , 2020 ). Furthermore, the capacity to express plant Rubiscos in Escherichia coli has opened up new opportunities to screen RbcS families, ancestral forms of RbcS, and synthetic RbcS variants ( Aigner et al , 2017 ; Lin et al , 2020 , 2022 ). Although progress is being made, chloroplast engineering is still only well established in a small number of plant species ( Ruf et al , 2019 ; Yu et al , 2020 ).…”

Nitrogen fertilization coupled with foliar application of iron and molybdenum improves shade tolerance of soybean under maize-soybean intercropping

“…On the other hand, Fe is a key component of the ribulose 1,5 bisphosphate carboxylase/oxygenase, which is a key enzyme involved in plant photosynthesis ( Wang et al., 2017 ) with Mo being directly or indirectly involved in the chlorophyll biosynthesis, chloroplast ultrastructure and rubisco enzymes ( Imran et al., 2019 ). Rubisco is the predominant key enzyme protein involved in plant photosynthesis, contributing up to 20–30% of total leaf nitrogen and 50% of the total soluble leaf proteins (1 st primary source) ( Lin et al., 2022 ). However, under environmental stress, plants showed a decrease in the rubisco enzymes, which causes a decline in the photosynthetic activities and soluble protein in plant leaves, thereby decreasing its yield ( Feller, 2016 ; Shao et al., 2021 ; Rahimi et al., 2022 ; Taylor et al., 2022 ).…”

Strategies for manipulating Rubisco and creating photorespiratory bypass to boost C3 photosynthesis: Prospects on modern crop improvement

“…In addition, the high activity of Rubisco will be screened and evaluated in E. coli by direction evolution (Lin et al, 2020). Computational technologies had been used to predict favourable sequences of Rubisco subunits in different species at different evolutionary stages, thereby predicting the ideal Rubisco subunit coding sequence with superior carboxylation efficiency (Lin et al, 2022). Artificial intelligence technology with new algorithms will help to unearth more effective Rubisco.…”