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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.

ABSTRACT: We report the mixotrophic growth of Escherichia coli based on recombinant 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO₂ using malate as an auxiliary carbon source and hydrogen as an energy source. We employ a long-term (~184 days) two-stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli. In the first stage of evolution with serine, diauxic growth emerges as a prominent feature. At the end of the second stage of evolution with malate, the strain exhibits mixotrophy with CO₂ as an essential substrate for growth. We expect this work will open new possibilities in the utilization of OGOR for microbial CO₂ assimilation and future hydrogen-based electro-microbial conversion.

SUBMITTER: Cheng YC

PROVIDER: S-EPMC9967407 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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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.

Cheng Yu-Chen YC Huang Wei-Han WH Lo Shou-Chen SC Huang Eugene E Chiang En-Pei Isabel EI Huang Chieh-Chen CC Yang Ya-Tang YT

Microorganisms 20230119 2

We report the mixotrophic growth of Escherichia coli based on recombinant 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO2 using malate as an auxiliary carbon source and hydrogen as an energy source. We employ a long-term (~184 days) two-stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli. In the first stage of evolution with serine, diauxic growth emerges as a prominent feature. At the end of the second stage of evo ...[more]

PMID: 36838218

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Project description:Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.

Dataset Information

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.

Publications

Conversion of <i>Escherichia coli</i> into Mixotrophic CO<sub>2</sub> Assimilation with Malate and Hydrogen Based on Recombinant Expression of 2-Oxoglutarate:Ferredoxin Oxidoreductase Using Adaptive Laboratory Evolution.

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