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The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.
The quarternary, trimethylated amine glycine betaine (GB) is widespread in nature but its fate under anoxic conditions remains elusive. It can be used by some acetogenic bacteria as carbon and energy source but the pathway of GB metabolism has not been elucidated. We have identified a gene cluster involved in GB metabolism and studied acetogenesis from GB in the model acetogen Acetobacterium woodii . GB is taken up by a secondary active, Na+ coupled transporter of the betaine‐choline‐carnitine (BCC) family. GB is demethylated to dimethylglycine, the end product of the reaction, by a methyltransferase system. Further conversion of the methyl group requires CO2 as well as Na+ indicating that GB metabolism involves the Wood‐Ljungdahl pathway. These studies culminate in a model for the path of carbon and electrons during acetogenensis from GB and a model for the bioenergetics of acetogenesis from GB.
Methanol is the simplest of all alcohols, is universally distributed in anoxic sediments as a result of plant material decomposition and is constantly attracting attention as an interesting substrate for anaerobes like acetogens that can convert bio-renewable methanol into value-added chemicals. A major drawback in the development of environmentally friendly but economically attractive biotechnological processes is the present lack of information on biochemistry and bioenergetics during methanol conversion in these bacteria. The mesophilic acetogen Eubacterium callanderi KIST612 is naturally able to consume methanol and produce acetate as well as butyrate. To grasp the full potential of methanol-based production of chemicals, we analysed the genes and enzymes involved in methanol conversion to acetate and identified the redox carriers involved. We will display a complete model for methanol-derived acetogenesis and butyrogenesis in Eubacterium callanderi KIST612, tracing the electron transfer routes and shed light on the bioenergetics during the process.
Methanol derived from plant tissue is ubiquitous in anaerobic sediments and a good substrate for anaerobes growing on C1 compounds such as methanogens and acetogens. In contrast to methanogens little is known about the physiology, biochemistry and bioenergetics of methanol utilization in acetogenic bacteria. To fill this gap, we have used the model acetogen Acetobacterium woodii to study methanol metabolism using physiological and biochemical experiments paired with molecular studies and transcriptome analysis. These studies identified the genes and enzymes involved in acetogenesis from methanol and the redox carriers involved. We will present the first comprehensive model for carbon and electron flow from methanol in an acetogen and the bioenergetics of acetogenesis from methanol.
The production of bulk chemicals mostly depends on exhausting petroleum sources and leads to emission of greenhouse gases. Within the last decades the urgent need for alternative sources has increased and the development of bio-based processes received new attention. To avoid the competition between the use of sugars as food or fuel, other feedstocks with high availability and low cost are needed, which brought acetogenic bacteria into focus. This group of anaerobic organisms uses mixtures of CO2, CO and H2 for the production of mostly acetate and ethanol. Also methanol, a cheap and abundant bulk chemical produced from methane, is a suitable substrate for acetogenic bacteria. In methylotrophic acetogens the methyl group is transferred to the Wood-Ljungdahl pathway, a pathway to reduce CO2 to acetate via a series of C1-intermediates bound to tetrahydrofolic acid. Here we describe the biochemistry and bioenergetics of methanol conversion in the biotechnologically interesting group of anaerobic, acetogenic bacteria. Further, the bioenergetics of biochemical production from methanol is discussed.
Acetogenic bacteria such as Acetobacterium woodii use the Wood–Ljungdahl pathway (WLP) for fixation of CO2 and energy conservation. This pathway enables conversion of diverse substrates to the main product of acetogenesis, acetate. Methyl group containing substrates such as methanol or methylated compounds, derived from pectin, are abundant in the environment and a source for CO2. Methyl groups enter the WLP at the level of methyltetrahydrofolic acid (methyl-THF). For methyl transfer from methanol to THF a substrate-specific methyltransferase system is required. In this study, we used genetic methods to identify mtaBC2A (Awo_c22760-Awo_c22740) as the methanol-specific methyltransferase system of A. woodii. After methyl transfer, methyl-THF serves as carbon and/or electron source and the respiratory Rnf complex is required for redox homeostasis if methanol + CO2 is the substrate. Resting cells fed with methanol + CO2, indeed converted methanol to acetate in a 4:3 stoichiometry. When methanol was fed in combination with other electron sources such as H2 + CO2 or CO, methanol was converted Rnf-independently and the methyl group was condensed with CO to build acetate. When fed in combination with alternative electron sinks such as caffeate methanol was oxidized only and resulting electrons were used for non-acetogenic growth. These different pathways for the conversion of methyl-group containing substrates enable acetogens to adapt to various ecological niches and to syntrophic communities.
Flavin-based electron bifurcation is a long hidden mechanism of energetic coupling present mainly in anaerobic bacteria and archaea that suffer from energy limitations in their environment. Electron bifurcation saves precious cellular ATP and enables lithotrophic life of acetate-forming (acetogenic) bacteria that grow on H2 + CO2 by the only pathway that combines CO2 fixation with ATP synthesis, the Wood–Ljungdahl pathway. The energy barrier for the endergonic reduction of NADP+, an electron carrier in the Wood–Ljungdahl pathway, with NADH as reductant is overcome by an electron-bifurcating, ferredoxin-dependent transhydrogenase (Nfn) but many acetogens lack nfn genes. We have purified a ferredoxin-dependent NADH:NADP+ oxidoreductase from Sporomusa ovata, characterized the enzyme biochemically and identified the encoding genes. These studies led to the identification of a novel, Sporomusa type Nfn (Stn), built from existing modules of enzymes such as the soluble [Fe–Fe] hydrogenase, that is widespread in acetogens and other anaerobic bacteria.