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

• Main Authors: Shou-Chen Lo, En-Pei Isabel Chiang, Ya-Tang Yang, Si-Yu Li, Jian-Hau Peng, Shang-Yieng Tsai, Dong-Yan Wu, Chia-Hua Yu, Chu-Han Huang, Tien-Tsai Su, Kenji Tsuge, Chieh-Chen Huang
• Format: Article
• Language: English
• Published: MDPI AG 2021-06-01
• Series: Fermentation
• Subjects: <i>Escherichia coli</i>; reductive tricarboxylic acid cycle; α-ketoglutarate:ferredoxin oxidoreductase; transcriptome
• Online Access: https://www.mdpi.com/2311-5637/7/2/98

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 <i>Escherichia coli</i> 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 <i>E. coli.</i> The transgenic <i>E. coli</i> 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 <i>E. coli</i> for assimilating external gaseous CO₂.