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

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.

Conversion of Escherichia coli into Mixotrophic CO2 Assimilation with Malate and Hydrogen Based on Recombinant Expression of 2-Oxoglutarate:Ferredoxin Oxidoreductase Using Adaptive Laboratory Evolution

“…The enzyme has three subunits, and the corresponding genes (citD, citE, and citF) are known to exist in E. coli from genomic analysis, and the computed Gibbs free energy is ΔG′° = 4.4 kJ/mol at pH = 7 and ion strength of 0.1 M [ 33 ]. (The enzyme activities of citrate lyase were also detected in our previous study [ 23 ].) In addition, we hypothesize that the reaction catalyzed by isocitrate dehydrogenase is the flux bottleneck for CO 2 assimilation, which is supported by the concentration of the mutation points on this specific enzyme.…”

“…The 2-oxoglutarate:ferredoxin oxidoreductase were cloned from an anaerobic phototrophic bacterium Chlorobaculum tepidum strain TLS. The OGOR enzyme activity assays were performed using a method described in a previous report with modifications ( Table 2 ) [ 23 ]. After the transformation of the strain, the strain did not assimilate CO 2 , and we used serine and malate as auxiliary carbon sources to establish basal-level growth for evolution.…”

“…The 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) was cloned from an anaerobic phototrophic bacterium Chlorobaculum tepidum strain TLS genome (GenBank accession number: AE006470) [ 23 ]. To construct an expression vector containing the genes of two sub-units of OGOR, the korAB (CT0163 and CT0162 gene symbol in GenBank, total 2.9 kb) were amplified from plasmid pGETS-KAFS [ 23 ] as a gene cassette with a designed primer pair by Ordered Gene Assembly in Bacillus subtilis (OGAB) method [ 25 ]. The primer pair used in cloning experiments is shown in Table 1 .…”

“…The plasmid was extracted and transformed into the regular E. coli cloning host strain BW25113 via heat-shock transformation. The plasmid was also sequenced before and after evolution experiments with next-generation sequencing, and the sequences of korAB genes were the same as pGETS-KAFS with a frameshift mutation (717 deleted A) in korA [ 23 ].…”

“…Previously, we have pointed out the pivotal role of OGOR in establishing in silico hydrogen-powered autotrophy in E. coli based on constraint-based modeling with flux balance analysis [ 22 ]. Moreover, the CO 2 assimilation is also confirmed with E. coli overexpressing ATP citrate lyase and OGOR with glucose as both the carbon source and the energy source [ 23 ]. Here, we would like to establish mixotrophical CO 2 assimilation solely based on heterologous OGOR ( Figure 1 a) using hydrogen as an energy source in E. coli .…”