A Versatile Aldehyde: Ferredoxin Oxidoreductase from the Organic Acid Reducing Thermoanaerobacter sp. Strain X514.

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

Lactate formation from fructose or C1 compounds in the acetogen Acetobacterium woodii by metabolic engineering.

Anaerobic, acetogenic bacteria are promising biocatalysts for a sustainable bioeconomy since they capture and convert carbon dioxide to acetic acid. Hydrogen is an intermediate in acetate formation from organic as well as C1 substrates. Here, we analyzed mutants of the model acetogen Acetobacterium woodii in which either one of the two hydrogenases or both together were genetically deleted. In resting cells of the double mutant, hydrogen formation from fructose was completely abolished and carbon was redirected largely to lactate. The lactate/fructose and lactate/acetate ratios were 1.24 and 2.76, respectively. We then tested for lactate formation from methyl groups (derived from glycine betaine) and carbon monoxide. Indeed, also under these conditions lactate and acetate were formed in equimolar amounts with a lactate/acetate ratio of 1.13. When the electron-bifurcating lactate dehydrogenase/ETF complex was genetically deleted, lactate formation was completely abolished. These experiments demonstrate the capability of A. woodii to produce lactate from fructose but also from promising C1 substrates, methyl groups and carbon monoxide. This adds an important milestone towards generation of a value chain leading from CO2 to value-added compounds. KEY POINTS: • Resting cells of the ΔhydBA/hdcr mutant of Acetobacterium woodii produced lactate from fructose or methyl groups + CO • Lactate formation from methyl groups + CO was completely abolished after deletion of lctBCD • Metabolic engineering of a homoacetogen to lactate formation gives a potential for industrial applications.

A new metabolic trait in an acetogen: Mixed acid fermentation of fructose in a methylene-tetrahydrofolate reductase mutant of Acetobacterium woodii.

To inactivate the Wood-Ljungdahl pathway in the acetogenic model bacterium Acetobacterium woodii, the genes metVF encoding two of the subunits of the methylene-tetrahydrofolate reductase were deleted. As expected, the mutant did not grow on C1 compounds and also not on lactate, ethanol or butanediol. In contrast to a mutant in which the first enzyme of the pathway (hydrogen-dependent CO2 reductase) had been genetically deleted, cells were able to grow on fructose, albeit with lower rates and yields than the wild-type. Growth was restored by addition of an external electron sink, glycine betaine + CO2 or caffeate. Resting cells pre-grown on fructose plus an external electron acceptor fermented fructose to two acetate and four hydrogen, that is, performed hydrogenogenesis. Cells pre-grown under fermentative conditions on fructose alone redirected carbon and electrons to form lactate, formate, ethanol as well as hydrogen. Apparently, growth on fructose alone induced enzymes for mixed acid fermentation (MAF). Transcriptome analyses revealed enzymes potentially involved in MAF and a quantitative model for MAF from fructose in A. woodii is presented.

Reprogramming the metabolism of an acetogenic bacterium to homoformatogenesis.

Methyl groups are abundant in anoxic environments and their utilization as carbon and energy sources by microorganisms involves oxidation of the methyl groups to CO2, followed by transfer of the electrons to an acceptor. In acetogenic bacteria, the electron acceptor is CO2 that is reduced to enzyme bound carbon monoxide, the precursor of the carboxyl group in acetate. Here, we describe the generation of a mutant of the acetogen Acetobacterium woodii in which the last step in methyl group oxidation, formate oxidation to CO2 catalyzed by the HDCR enzyme, has been genetically deleted. The mutant grew on glycine betaine as methyl group donor, and in contrast to the wild type, formed formate alongside acetate, in a 1:2 ratio, demonstrating that methyl group oxidation stopped at the level of formate and reduced electron carriers were reoxidized by CO2 reduction to acetate. In the presence of the alternative electron acceptor caffeate, CO2 was no longer reduced to acetate, formate was the only product and all the carbon went to formate. Apparently, acetogenesis was not required to sustain formatogenic growth. This is the first demonstration of a genetic reprogramming of an acetogen into a formatogen that grows by homoformatogenesis from methyl groups. Formate production from methyl groups is not only of biotechnological interest but also for the mechanism of electron transfer in syntrophic interactions in anoxic environments.

Physiology and genetics of ethanologenesis in the acetogenic bacterium Acetobacterium 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.

Formate metabolism in the acetogenic bacterium Acetobacterium woodii.

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.

Growth of the acetogenic bacterium Acetobacterium woodii on glycerol and dihydroxyacetone.

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.

Adh4, an alcohol dehydrogenase controls alcohol formation within bacterial microcompartments in the acetogenic bacterium Acetobacterium woodii.

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.

Homoacetogenic Conversion of Mannitol by the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui Requires External CO2.

Acetogenic microorganisms utilize organic substrates such as sugars in addition to hydrogen (H2) + carbon dioxide (CO2). Recently, we reported that the thermophilic acetogenic microorganism Thermoanaerobacter kivui is among the few acetogens that utilize the sugar alcohol mannitol, dependent on a gene cluster encoding mannitol uptake, phosphorylation and oxidation of mannitol-1-phosphate to fructose-6-phosphate. Here, we studied mannitol metabolism with resting cells of T. kivui; and found that mannitol was "fermented" in a homoacetogenic manner, i.e., acetate was the sole product if HCO3 - was present. We found an acetate:mannitol ratio higher than 3, indicating the requirement of external CO2, and the involvement of the WLP as terminal electron accepting pathway. In the absence of CO2 (or bicarbonate, HCO3 -), however, the cells still converted mannitol to acetate, but slowly and with stoichiometric amounts of H2 formed in addition, resulting in a "mixed" fermentation. This showed that-in addition to the WLP-the cells used an additional electron sink-protons, making up for the "missing" CO2 as electron sink. Growth was 2.5-fold slower in the absence of external CO2, while the addition of formate completely restored the growth rate. A model for mannitol metabolism is presented, involving the major three hydrogenases, to explain how [H] make their way from glycolysis into the products acetate or acetate + H2.

A thermostable mannitol-1-phosphate dehydrogenase is required in mannitol metabolism of the thermophilic acetogenic bacterium Thermoanaerobacter kivui.

Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO2 ) with hydrogen (H2 ) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h-1 to a final optical density (OD600 ) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre-grown on glucose, not in those pre-grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol-1-phosphate dehydrogenase (MtlD) activity was observed in cell-free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830-TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose-6-phosphate with NADH, at a high VMAX of 1235 U mg-1 at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.