2,3-Butanediol synthesis from glucose supplies NADH for elimination of toxic acetate produced during overflow metabolism

Overflow metabolism-caused acetate accumulation is a major problem that restricts industrial applications of various bacteria. 2,3-Butanediol (2,3-BD) synthesis in microorganisms is an ancient metabolic process with unidentified functions. We demonstrate here that acetate increases and then decreases during the growth of a bacterium Enterobacter cloacae subsp. dissolvens SDM. Both bifunctional acetaldehyde/ethanol dehydrogenase AdhE-catalyzed ethanol production and acetate-induced 2,3-BD biosynthesis are indispensable for the elimination of acetate generated during overflow metabolism. 2,3-BD biosynthesis from glucose supplies NADH required for acetate elimination via AdhE-catalyzed ethanol production. The coupling strategy involving 2,3-BD biosynthesis and ethanol production is widely distributed in bacteria and is important for toxic acetate elimination. Finally, we realized the co-production of ethanol and acetoin from chitin, the second most abundant natural biopolymer whose catabolism involves inevitable acetate production through the coupling acetate elimination strategy. The synthesis of a non-toxic chemical such as 2,3-BD may be viewed as a unique overflow metabolism with desirable metabolic functions.

Lanthanide-Dependent Methanol and Formaldehyde Oxidation in Methylobacterium aquaticum Strain 22A

Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.

Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates

ABSTRACT Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain ( mxaF xoxF1 xoxF2 ; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF , was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La 3+ ). ExaF was purified from cells grown with both calcium (Ca 2+ ) and La 3+ and with Ca 2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La 3+ over Ca 2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca 2+ -plus-La 3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca 2+ -only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La 3+ is present, expanding the role of lanthanides to multicarbon metabolism. IMPORTANCE ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from ‘Aromatoleum aromaticum' EbN1

The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of <i>p</i>-ethylphenol in the denitrifying bacterium ‘<i>Aromatoleum aromaticum</i>' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked <i>in-frame</i> deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the ‘classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in <i>Escherichia coli</i>, purified and crystallized. The X-ray structures of the apo- and NAD⁺-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict <i>(R)</i>-specificity of Hped (Phe¹⁸⁷), contrasting the <i>(S)</i>-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr⁹³) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93].

Aerobic and anaerobic enzymatic activity and allometric scaling of the deep benthic polychaete Hyalinoecia artifex (Polychaeta: Onuphidae)

The enzymatic activity of aerobic and anaerobic metabolic pathways in Hyalinoecia artifex, a polychaete inhabiting the deep ocean, is reported. In addition, the allometry of its anaerobic and aerobic enzymatic activity is analysed. The aerobic metabolism was measured using the electron transport system activity technique (ETS), whereas the anaerobic metabolism was estimated using the activity of lactate dehydrogenase (LDH), octopine dehydrogenase (OPDH), alanopine dehydrogenase (ALPDH), strombine dehydrogenase (STRDH), and ethanol dehydrogenase (EtOHDH). The ETS activity was about 296.18 (µLO2 h−1 g−1), which is within the range described for polychaetes and other benthic metazoans. The anaerobic enzymatic activity expressed as µmol NADH min−1 g−1 was: LDH = 0.35, OPDH = 0.11, ALPDH = 12.66, STRDH = 10.78 and SDH = 0.48. The slope of the allometric relationship between specific aerobic metabolism and body size was −0.35. In the case of the allometric scaling of the anaerobic metabolism, only LDH presented a significant relationship, with a slope of b = 0.44. This positive scaling is consistent with the pattern emerging from the scarce literature on the allometry of anaerobic metabolism in marine biota.