Cell-free expression of RuBisCO for ATP production in the synthetic cells

Shugo Sugii; Katsumi Hagino; Ryo Mizuuchi; Norikazu Ichihashi

doi:10.1093/synbio/ysad016

Cell-free expression of RuBisCO for ATP production in the synthetic cells

Synth Biol (Oxf). 2023 Dec 20;8(1):ysad016. doi: 10.1093/synbio/ysad016. eCollection 2023.

Authors

Shugo Sugii, Katsumi Hagino¹, Ryo Mizuuchi^{2

3}, Norikazu Ichihashi^{1

4

5

6

7}

Affiliations

¹ Department of Life Science, Graduate School of Arts and Science, The University of Tokyo, Meguro, Tokyo 153-8902, Japan.
² Department of Electrical Engineering and Bioscience, Faculty of Science and Engineering, Waseda University, Shinjuku, Tokyo 162-8480, Japan.
³ JST FOREST, Kawaguchi, Saitama 332-0012, Japan.
⁴ Komaba Institute for Science, The University of Tokyo, Meguro, Tokyo 153-8902, Japan.
⁵ Universal Biology Institute, The University of Tokyo, Meguro, Tokyo 153-8902, Japan.
⁶ College of Arts and Science, the University of Tokyo, Meguro, Tokyo 153-8902, Japan.
⁷ Department of Medicine, the University of Tokyo, Bunkyo, Tokyo 113-8654, Japan.

Abstract

Recent advances in bottom-up synthetic biology have made it possible to reconstitute cellular systems from non-living components, yielding artificial cells with potential applications in industry, medicine and basic research. Although a variety of cellular functions and components have been reconstituted in previous studies, sustained biological energy production remains a challenge. ATP synthesis via ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), a central enzyme in biological CO₂ fixation, holds potential as an energy production system, but its feasibility in a cell-free expression system has not yet been tested. In this study, we test RuBisCO expression and its activity-mediated ATP synthesis in a reconstituted Escherichia coli-based cell-free translation system. We then construct a system in which ATP is synthesized by RuBisCO activity in giant vesicles and used as energy for translation reactions. These results represent an advance toward independent energy production in artificial cells. Graphical Abstract.

Keywords: ATP production; CO2 fixation; RuBisCO; artificial cell; in vitro synthetic biology.