Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation

Abstract

BackgroundProduction of fuels from the abundant and wasteful CO2 is a promising approach to reduce carbon emission and consumption of fossil fuels. Autotrophic microbes naturally assimilate CO2 using energy from light, hydrogen, and/or sulfur. However, their slow growth rates call for investigation of the possibility of heterotrophic CO2 fixation. Although preliminary research has suggested that CO2 fixation in heterotrophic microbes is feasible after incorporation of a CO2-fixing bypass into the central carbon metabolic pathway, it remains unclear how much and how efficient that CO2 can be fixed by a heterotrophic microbe.ResultsA simple metabolic flux index was developed to indicate the relative strength of the CO2-fixation flux. When two sequential enzymes of the cyanobacterial Calvin cycle were incorporated into an E. coli strain, the flux of the CO2-fixing bypass pathway accounts for 13 % of that of the central carbon metabolic pathway. The value was increased to 17 % when the carbonic anhydrase involved in the cyanobacterial carbon concentrating mechanism was introduced, indicating that low intracellular CO2 concentration is one limiting factor for CO2 fixation in E. coli. The engineered CO2-fixing E. coli with carbonic anhydrase was able to fix CO2 at a rate of 19.6 mg CO2 L−1 h−1 or the specific rate of 22.5 mg CO2 g DCW−1 h−1. This CO2-fixation rate is comparable with the reported rates of 14 autotrophic cyanobacteria and algae (10.5–147.0 mg CO2 L−1 h−1 or the specific rates of 3.5–23.7 mg CO2 g DCW−1 h−1).ConclusionsThe ability of CO2 fixation was created and improved in E. coli by incorporating partial cyanobacterial Calvin cycle and carbon concentrating mechanism, respectively. Quantitative analysis revealed that the CO2-fixation rate of this strain is comparable with that of the autotrophic cyanobacteria and algae, demonstrating great potential of heterotrophic CO2 fixation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0268-1) contains supplementary material, which is available to authorized users.

Improving Succinate Productivity by Engineering a Cyanobacterial CO2 Concentrating System (CCM) in Escherichia coli

“…In order to increase the intracellular CO 2 concentration for PCK‐catalyzed carboxylation reaction, carbonic anhydrase (CA) gene from Synechococcus sp. PCC7002 was amplified from pET‐RBC‐PRK‐BT‐CA and cloned into pTrc99A‐M‐M1‐46 to obtain plasmid pCA. This plasmid was then transformed into Suc‐T110, resulting in strain Suc‐T110(pCA) (Table S2, Supporting Information).…”

“…Plasmid backbone was amplified using primer set CPEC‐99AM46‐F/CPEC‐99AM46‐R and was further digested with DpnI to exclude original plasmid DNA of pTrc99A‐M‐M1‐46. Carbonic anhydrase (CA) gene and bicarbonate transporter (BT) gene were both amplified from the plasmid of pET‐RBC‐PRK‐BT‐CA, a gift from Professor Yin Li (Institute of Microbiology, Chinese Academy of Sciences), using primer pairs CPEC‐CA‐F/CPEC‐CA‐R and CPEC‐BT‐F/CPEC‐BT‐R. These two amplified DNA fragments were individually ligated with pTrc99A‐M‐M1‐46 backbone using CPEC, and correct clones were designated as pCA and pBT.…”

Synthetic biology for CO2 fixation

“…However, these studies were unable to determine the amount of CO2 that had been fixed by the central metabolic pathways. Recently, we have developed a relative quantification method to calculate the ratio of carbon flux from the CO2-fixation pathway and the central metabolic pathway by LC/MS/MS detection of 13 C and unlabeled metabolites (Gong et al, 2015).…”

“…We recently introduced cyanobacterial carbonic anhydrase, a key enzyme in the cyanobacterial CCM, into E. coli. Improved CO2-fixation efficiency was found in the engineered CO2-fixing E. coli, demonstrating that the CCM can also be transplanted into heterotrophic microbes (Gong et al, 2015). Ideas on introducing the CCM from cyanobacteria or C4 plants into C3 crops to improve the photosynthetic efficiency of the latter (Covshoff and Hibberd, 2012;Price et al, 2011;Price et al, 2013) have been reported, but much research is still needed on the topic.…”

Assimilation of formic acid and CO 2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways

“…However, a major limitation of biological C1 conversion is the inefficiency of the natural C1 assimilation pathway such as the Calvin-Benson-Bassham (CBB) cycle (4). To address this problem, extensive studies have been performed to enhance biological C1 assimilation by developing de novo pathways using efficient carboxylases (5)(6)(7)(8)(9), introducing the CBB cycle into Escherichia coli (10)(11)(12)(13), or by integration with an electrochemical method of reducing CO 2 to formic acid (FA) (14,15). FA can be easily and efficiently synthesized from CO 2 by various chemical processes employing metal catalysts (16) or electricity (17).…”