Growth Enhancement Facilitated by Gaseous CO2 through Heterologous Expression of Reductive Tricarboxylic Acid Cycle Genes in Escherichia coli

Shou-Chen Lo,Tien-Tsai Su,Jian-Hau Peng,Ya-Tang Yang,Chu-Han Huang,Si-Yu Li,En-Pei Isabel Chiang,Shang-Yieng Tsai,Chieh-Chen Huang,Dong-Yan Wu,Chia-Hua Yu,Kenji Tsuge

doi:10.3390/fermentation7020098

Abstract

The enzymatic mechanisms of carbon fixation by autotrophs, such as the reductive tricarboxylic acid cycle (rTCA), have inspired biotechnological approaches to producing bio-based chemicals directly through CO2. To explore the possibility of constructing an rTCA cycle in Escherichia coli and to investigate their potential for CO2 assimilation, a total of ten genes encoding the key rTCA cycle enzymes, including α-ketoglutarate:ferredoxin oxidoreductase, ATP-dependent citrate lyase, and fumarate reductase/succinate dehydrogenase, were cloned into E. coli. The transgenic E. coli strain exhibited enhanced growth and the ability to assimilate external inorganic carbon with a gaseous CO2 supply. Further experiments conducted in sugar-free medium containing hydrogen as the electron donor and dimethyl sulfoxide (DMSO) as the electron acceptor proved that the strain is able to undergo anaerobic respiration, using CO2 as the major carbon source. The transgenic stain demonstrated CO2-enhanced growth, whereas the genes involved in chemotaxis, flagellar assembly, and acid-resistance were upregulated under the anaerobic respiration. Furthermore, metabolomic analysis demonstrated that the total concentrations of ATP, ADP, and AMP in the transgenic strain were higher than those in the vector control strain and these results coincided with the enhanced growth. Our approach offers a novel strategy to engineer E. coli for assimilating external gaseous CO2.

Highlights

Global climate change as a consequence of anthropogenic carbon dioxide emissions has motivated approaches to reducing those emissions through the recycling of carbon dioxide directly into desirable metabolites or existing platform chemicals [1,2,3]
(CT2040 to CT2042, total 3.6 kb and CT2266 to CT2268, total 3.4 kb), these genes were amplified from C. tepidum TLS cellular DNA as gene cassettes with designed OGAB primer pairs (Extended Data Table 1)
The designed DNA cassettes were cloned into plasmid pGETS118 to form pGETS-KAFS (K, α-ketoglutarate:ferredoxin oxidoreductase; A, ATP-dependent citrate lyase; F, fumarate reductase; S, succinate dehydrogenase) in B

Summary

Introduction

Global climate change as a consequence of anthropogenic carbon dioxide emissions has motivated approaches to reducing those emissions through the recycling of carbon dioxide directly into desirable metabolites or existing platform chemicals [1,2,3]. The rTCA cycle is considered to be an ancient core metabolic process in the reducing environment of the early Earth that provided precursors of organic compounds for the synthesis of all major classes of biomolecules [5]. It is basically the reverse of the oxidative tricarboxylic acid cycle and leads to fixation of CO2 for autotrophic growth [6]. The oxidative TCA cycle mostly occurs among heterotrophic organisms, and the metabolic pathway has already been driven to replenish metabolites in the cycle through reactions known as anaplerotic reactions [9] These characteristics give the machinery of the reductive TCA cycle great potential for carbon fixation in heterotrophic host cells

Methods

Results

Discussion

Conclusion