Ethanol Metabolism Dynamics in Clostridium ljungdahlii Grown on Carbon Monoxide

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p>Bioethanol production from syngas using acetogenic bacteria has attracted considerable attention in recent years. However, low ethanol yield is the biggest challenge that prevents the commercialization of syngas fermentation into biofuels using microbial catalysts. The present study demonstrated that ethanol metabolism plays an important role in recycling NADH/NAD<jats:sup>+</jats:sup> during autotrophic growth. Deletion of bifunctional aldehyde/alcohol dehydrogenase (<jats:italic>adhE</jats:italic>) genes leads to significant growth deficiencies in gas fermentation. Using specific fermentation technology in which the gas pressure and pH were constantly controlled at 0.1 MPa and 6.0, respectively, we revealed that ethanol was formed during the exponential phase, closely accompanied by biomass production. Then, ethanol was oxidized to acetate via the aldehyde ferredoxin oxidoreductase pathway in <jats:named-content content-type="genus-species">Clostridium ljungdahlii</jats:named-content>. A metabolic experiment using <jats:sup>13</jats:sup>C-labeled ethanol and acetate, redox balance analysis, and comparative transcriptomic analysis demonstrated that ethanol production and reuse shared the metabolic pathway but occurred at different growth phases.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> Ethanol production from carbon monoxide (CO) as a carbon and energy source by <jats:named-content content-type="genus-species">Clostridium ljungdahlii</jats:named-content> and “<jats:named-content content-type="genus-species">Clostridium autoethanogenum</jats:named-content>” is currently being commercialized. During gas fermentation, ethanol synthesis is NADH-dependent. However, ethanol oxidation and its regulatory mechanism remain incompletely understood. Energy metabolism analysis demonstrated that reduced ferredoxin is the sole source of NADH formation by the Rnf-ATPase system, which provides ATP for cell growth during CO fermentation. Therefore, ethanol production is tightly linked to biomass production (ATP production). Clarification of the mechanism of ethanol oxidation and biosynthesis can provide an important reference for generating high-ethanol-yield strains of <jats:italic>C. ljungdahlii</jats:italic> in the future.</jats:p>

The Metabolism of Clostridium ljungdahlii in Phosphotransacetylase Negative Strains and Development of an Ethanologenic Strain

“…The metabolic effect of these enzymes change depending on the state of cells. For instance, during growth on syngas, ethanol can be both produced and consumed, which is dependent on both AdhE and AOR (Liu et al, 2020). Additionally, there are many potential redundant enzymes and their regulation is unknown, and while many of these genes are annotated as alcohol or aldehyde dehydrogenases, they may not be specific for acetaldehyde/ethanol (Tan et al, 2015).…”

Identifying and Engineering Bottlenecks of Autotrophic Isobutanol Formation in Recombinant C. ljungdahlii by Systemic Analysis

“…Further strain optimization may aim to detour said reductive power into isobutanol formation. In case of ethanol this goal is very challenging as ethanol may be considered as a vital by-product of the acetaldehyde:ferredoxin oxidoreductase (AOR) which links its formation with ATP synthesis ( Mock et al, 2015 ; Liew et al, 2017 ; Hermann et al, 2020 ; Liu et al, 2020 ; Zhu et al, 2020 ). However, elimination of 2,3-butanediol production may increase isobutanol formation as both products originate from the precursor pyruvate requiring NADPH as electron donor.…”

A Heterodimeric Reduced-Ferredoxin-Dependent Methylenetetrahydrofolate Reductase from Syngas-Fermenting Clostridium ljungdahlii

“…MTHFR and the reactions it catalyzes in C. ljungdahlii have been poorly understood but are vital for understanding the maintenance of the redox balance and the energy metabolism of the WLP. The enzyme has been suggested to be an NADH-dependent or electron-bifurcating NADH- and Fd-dependent multimeric complex ( 16 , 17 , 19 , 27 , 42 ). However, the MTHFR purified from C. ljungdahlii was found in this study to be composed of only MetFV and could not catalyze the proposed reactions.…”

“…Trx red 2− could also serve as an electron donor for the reduction of methylene-THF, but its specific activity was much lower than that with Fd red 2− . Considering that the transcription level of Fd gene is much higher than that of Fld and Trx in C. ljungdahlii ( 27 , 48 ), Fd can be reduced in various reactions catalyzed by the enzymes such as CO dehydrogenase (grown on CO), HytA-E (grown on H 2 ), and pyruvate:Fd oxidoreductases (grown on fructose), and the enzyme presented a high specific activity with Fd red 2− as electron donor and low apparent K m value for Fd, we therefore suggest that Fd red 2− might be the physiological electron donor for MTHFR in C. ljungdahlii , and Fld red 2− may be used as the electron donor under the iron-deficient conditions.…”

“…In recent years, two hypotheses about the MTHFR from C. ljungdahlii / C. autoethanogenum have been proposed. One is that it is an NADH- and Fd-dependent electron-bifurcating enzyme, and the other is that it is NADH-dependent but non-electron-bifurcating ( 16 , 17 , 19 , 27 ). However, the binding site of NAD(P)H was not detected on MetFV in all the multimeric MTHFRs from acetogens, while it was found on the RnfC subunit of the enzyme from A. woodii and the HdrA subunit of M. thermoacetica ( 22 , 23 ).…”