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Acetogenic bacteria are already established as biocatalysts for production of high-value compounds from C1 substrates such as H2 + CO2 or CO. However, little is known about the physiology, biochemistry and bioenergetics of acetogenesis from formate, an interesting feedstock for biorefineries. Here, we analysed formate metabolism in the model acetogen Acetobacterium woodii. Cells grew optimally on 200 mM formate to an optical density of 0.6. Formate was exclusively converted to acetate (and CO2) with a ratio of 4.4:1. Transcriptome analyses revealed genes/enzymes involved in formate metabolism. Strikingly, A. woodii has two genes potentially encoding a formyl-THF synthetase, fhs1 and fhs2. fhs2 forms an operon with a gene encoding a potential formate transporter, fdhC. Deletion of fhs2/fdhC led to a reduced growth rate, formate consumption and optical densities. Acetogenesis from H2 + CO2 was accompanied by transient formate production; strikingly, formate reutilization was completely abolished in the Δfhs2/fdhC mutant. Take together, our studies gave the first detailed insights into the formatotrophic lifestyle of A. woodii.
The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2 + CO2, formate, pyruvate, lactate or alanine. In the absence of Na+, the Wood–Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.
The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2 + CO2, formate, pyruvate, lactate or alanine. In the absence of Na+, the Wood–Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.
More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2–3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end-product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood–Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.
Hydrogen is a promising fuel in a carbon-neutral economy, and many efforts are currently undertaken to produce hydrogen. One of the challenges is to store and transport the highly explosive gas in a safe and easy way. One option that is intensively analyzed by chemists and biologists is the conversion of hydrogen and CO2 to formic acid, the liquid organic hydrogen carrier. Here, we demonstrate for the first time that a bio-based system, using Acetobacterium woodii as the biocatalyst, allows multiple cycles of bi-directional hydrogenation of CO2 to formic acid in one bioreactor. The process was kept running over 2 weeks producing and oxidizing 330 mM formic acid in total. Unwanted side-product formation of acetic acid was prevented through metabolic engineering of the organism. The demonstrated process design can be considered as a future “bio-battery” for the reversible storage of electrons in the form of H2 in formic acid, a versatile compound.
Acetobacterium woodii utilizes the Wood‐Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non‐acetogenic growth on 1,2‐propanediol (1,2‐PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2‐PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC‐encapsulated propanol‐generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH‐dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2‐PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.
Das Modell-Acetogen Acetobacterium woodii ist bisher das Acetogen, das physiologisch, biochemisch und bioenergetisch am besten studiert ist. Während A. woodii als Homoacetogen bekannt ist, das Acetat als einziges Endprodukt produziert, hat seine genetische Information das Potenzial für die natürliche Produktion anderer reduzierter Produkte wie Ethanol oder Laktat angedeutet. Darüber hinaus wurde die Umsetzung bestimmter Substrate in A. woodii nur begrenzt untersucht, und ihre Physiologie, Biochemie und Bioenergetik sind noch nicht ausreichend erforscht. Das Ziel dieser Arbeit war es, unentdeckte Stoffwechselwege bestimmter Substrate in A. woodii zu untersuchen und neue metabolische Eigenschaften durch gentechnisches Engineering zu entdecken.
Das erste Kapitel dieser Arbeit konzentrierte sich auf die Untersuchung der formatotrophen Acetogenese von A. woodii. Formiat, eine der vielversprechenden C1-Verbindungen, kann in A. woodii verstoffwechselt werden. Es wurde festgestellt, dass ein Gencluster von A. woodii für eine zweite Formyl-Tetrahydrofolat-Synthetase zusammen mit einem potenziellen Formiat-Transporter kodiert. Um die Rolle dieser Gene zu verstehen, wurden sie in A. woodii deletiert. Die Charakterisierung der daraus resultierenden Mutanten zeigte, dass dieses Gencluster eine wichtige Rolle beim Formiat-Stoffwechsel spielt.
Im zweiten Kapitel wurde die Ethanologenese von A. woodii untersucht. Die Wachstums- und Zellsuspensionsversuche zeigten, dass höhere Fruktosekonzentrationen, Na+-Limitierung und niedriger Bikarbonatgehalt die Ethanolbildung in A. woodii stimulierten. Anschließend wurden Expressionsanalysen und Mutationsstudien durchgeführt, um die Ethanol-bildende Alkohol-Dehydrogenase zu identifizieren.
Das dritte Kapitel konzentrierte sich auf die Charakterisierung der formatogenen Lebensweise von A. woodii während des methylotrophen Stoffwechsels mittels der hdcr-Deletionsmutante. In der ∆hdcr-Mutante wurden die Methylgruppen in Formiat und Acetat umgewandelt, und die Zugabe eines alternativen Elektronenakzeptors, Kaffeat, ermöglichte die Umrichtung des Stoffwechsels auf Homoformatogenese.
Im vierten Kapitel wurde eine Mutante hergestellt und charakterisiert, die Methylene-Tetrahydrofolat-Reduktase (MTHFR) nicht mehr vorhanden ist. In der ∆metVF-Mutante, bei der zwei Untereinheiten von MTHFR genetisch deletiert wurden, wurde ein bisher unbekannter fermentativer Stoffwechsel von A. woodii, gemischte Säuregärung, während des Wachstums auf Fruktose beobachtet. Darüber hinaus wurden durch Transkriptomanalysen die mit diesem neuen fermentativen Stoffwechsel assoziierten Enzyme aufgedeckt.
Im letzten Kapitel wurde die Laktogenese in A. woodii unter Verwendung eines genetisch veränderten Stammes untersucht. A. woodii hat das Potenzial, Laktat durch die bifurkierende Laktatdehydrogenase, LDH/ETF-Komplex, zu produzieren. Die ∆metVF-Mutante war zwar in der Lage, Laktat zu produzieren, aber es wurde eine erhebliche Menge an Elektronen für die H2-Produktion verbraucht. Um die Laktatproduktion zu verbessern, wurde eine Doppeldeletionsmutante, ∆hydBA/hdcr, ausgewählt und die Laktogenese aus C1-Verbindungen in ruhenden Zellen der ∆hydBA/hdcr-Mutante untersucht.
