A cell-free self-replenishing CO2-fixing system

Shanshan Luo,Liang-Yu Nieh,Chi Chen,Paul P Lin,James C Liao,Po-Wen Tang,Guan-Bo Liao

doi:10.1038/s41929-022-00746-x

Abstract

Biological CO2 fixation is so far the most effective means for CO2 reduction at scale and accounts for most of the CO2 fixed on Earth. Through this process, carbon is fixed in cellular components and biomass during organismal growth. To uncouple CO2 fixation from growth and cellular regulation, cell-free CO2 fixation systems represent an alternative approach since the rate can be independently manipulated. Here we designed an oxygen-insensitive, self-replenishing CO2 fixation system with opto-sensing. The system comprises a synthetic reductive glyoxylate and pyruvate synthesis (rGPS) cycle and the malyl-CoA-glycerate (MCG) pathway to produce acetyl-coenzyme A (CoA), pyruvate and malate from CO2, which are also intermediates in the cycle. We solved various problems associated with the in vitro system, and implemented opto-sensing modules to control the regeneration of cofactors. We accomplished sustained operation for 6 hours with a CO2-fixing rate comparable to or greater than typical CO2 fixation rates of photosynthetic or lithoautotrophic organisms.

Highlights

Biological CO2 fixation is so far the most effective means for CO2 reduction at scale and accounts for most of the CO2 fixed on Earth
Biological CO2 fixation rate is controlled by cellular regulation that balances CO2 fixation with cell physiology and organismal growth, and typically limited by oxygen sensitivity and the carboxylase activity
While this paper was under review, the reductive pyruvate synthesis (rPS) pathway was revealed by a computational approach[23] without experimental proof

Summary

Introduction

Biological CO2 fixation is so far the most effective means for CO2 reduction at scale and accounts for most of the CO2 fixed on Earth. The lack of cellular regulation for in vitro cell-free systems presents a problem in balancing the rates of cofactor consumption and regeneration under dynamic conditions. These problems need to be addressed to assess the feasibility of in vitro CO2 fixation at scale. The Calvin–Benson– Bassham (CBB) cycle can output C3, C4, C5, C6 or C7 as a product, and still maintain a steady state, provided that the product was not drained beyond the rate of replenishing This requirement is relatively easy to achieve by controlling the output activity. We accomplished sustained operation for 6 hours with a CO2-fixing rate comparable to or greater than typical CO2 fixation rates of photosynthetic or lithoautotrophic organisms

Objectives

Methods

Conclusion