Genetic and biochemical characterization of a short‐chain alcohol dehydrogenase from the hyperthermophilic archaeon <i>Pyrococcus furiosu</i><i>s</i>

Oost, John van der; Voorhorst, W.G.B.; Kengen, Servé W. M.; Geerling, A.C.M.; Wittenhorst, V.; Gueguen, Yannick; Vos, Willem M. de

doi:10.1046/j.1432-1327.2001.02201.x

Cited by 52 publications

(26 citation statements)

References 27 publications

(46 reference statements)

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“…(19). "AdhA" was also used to describe the Pyrococcus furiosus short-chain dehydrogenase (adh_short) (20,21) (Fig. 3) and the PQQdependent alcohol dehydrogenases from Frateuria aurantia (22) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

et al. 2017

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Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at about 90% of the theoretical maximum yield (2 ethanol molecules per glucose equivalent) and a titer of 70 g/liter. Its ethanol-producing ability has drawn attention to its metabolic pathways, which could potentially be transferred to other organisms of interest. Here, we report that the iron-containing AdhA is important for ethanol production in the high-ethanol strain of T. saccharolyticum (LL1049). A single-gene deletion of adhA in LL1049 reduced ethanol production by ϳ50%, whereas multiple gene deletions of all annotated alcohol dehydrogenase genes except adhA and adhE did not affect ethanol production. Deletion of adhA in wild-type T. saccharolyticum reduced NADPH-linked alcohol dehydrogenase (ADH) activity (acetaldehyde-reducing direction) by 93%.IMPORTANCE In this study, we set out to identify the alcohol dehydrogenases necessary for high ethanol production in T. saccharolyticum. Based on previous work, we had assumed that adhE was the primary alcohol dehydrogenase gene. Here, we show that both adhA and adhE are needed for high ethanol yield in the engineered strain LL1049. This is the first report showing adhA is important for ethanol production in a native adhA host, which has important implications for achieving higher ethanol yields in other microorganisms.

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Section: Discussionmentioning

confidence: 99%

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

et al. 2017

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“…Pyrococcus furiosus DSM 3638 (half-life at 80°C, 150 h) (25) show somewhat greater thermostability than ST0053-ADH. The substrate specificity of ST0053-ADH was examined by using various alcohols, ketones, and aldehydes in the presence of NAD or NADH for oxidation or reduction, respectively.…”

Section: Vol 75 2009 Characterization Of St0053 Product 1761mentioning

confidence: 99%

Sulfolobus tokodaiiST0053 Produces a Novel Thermostable, NAD-Dependent Medium-Chain Alcohol Dehydrogenase

Yanai

Doi

Ohshima

2009

Appl Environ Microbiol

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An alcohol dehydrogenase (ADH) gene, ST0053, from Sulfolobus tokodaii was expressed in Escherichia coli. The purified recombinant enzyme was an NAD-dependent medium-chain ADH with high thermostability and tolerance of a wide range of pHs. This is the first step in creating an experimental functionality library of 10 genes annotated as ADHs in the S. tokodaii genome.NAD(P)-dependent alcohol dehydrogenases (ADHs; EC 1.1.1.1 and EC 1.1.1.2) catalyze the reversible oxidation of alcohols to their corresponding aldehydes or ketones. Many kinds of ADHs have been identified in a variety of microorganisms and are utilized for the production and detection of alcohols, aldehydes, and ketones (1, 11). Particularly useful are ADHs from thermophiles and hyperthermophiles, which are often more stable than their counterparts from mesophiles and psychrophiles (6, 7). These thermostable enzymes provide us with long-term functionality and novel reaction systems, such as the enzymatic vapor reaction under high temperature. Ten different genes have been annotated as NAD(P)-dependent ADHs in the genome of the hyperthermophilic and acidophilic archaeon Sulfolobus tokodaii strain 7 (13). We have been interested in compiling the structural and functional characteristics of their products into a library. Such a library would provide much useful information that could facilitate their application. We here describe the expression of ST0053, one of the 10 ADH genes in the S. tokodaii genome, and the subsequent purification and characteristics of its product.Cloning and expression of ST0053. A hybrid DNA plasmid, pET11a/ST0053, whose ST0053 open reading frame was amplified by PCR and inserted into the NdeI-BamHI site of pET11a (Novagen), was provided by Seiki Kuramitsu (Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan). The plasmid was transformed into E. coli strain Rosetta(DE3), after which the transformants were cultured for 16 h in 10 ml of Luria-Bertani medium containing ampicillin (50 g ml Ϫ1 ) at 37°C with shaking at 120 rpm. This culture medium was then added to 1 liter of Luria-Bertani medium containing the same amount of antibiotic in a 2-liter Erlenmeyer flask and incubated at 37°C with shaking at 220 rpm. Once the optical density at 660 nm reached 0.5, expression was induced by the addition of isopropyl-␤-D-thiogalactopyranoside (IPTG; 1 mM final concentration) and cultivation was continued at 37°C for an additional 4 h. The culture was harvested by centrifugation at 10,000 ϫ g for 10 min at 4°C, the cells were washed with 0.85% NaCl and pelleted, and then the cell pellet was stored at Ϫ80°C.Purification. To purify ADH, the stored cells (wet weight, about 2.55 g) were suspended in 10.2 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 0.01% 2-mercaptoethanol (2-ME) (buffer A) and then disrupted by ultrasonication on ice (Tomy Ultrasonic Disruptor UD-201 set at an output level of 2 and a 50% duty cycle; three sonications for 3 min each with 5-min intervals). The resultant homogenate was centrifuged a...

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“…The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (11,12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (3,4,14,16,19) or long-chain iron-activated ADHs (1,8,9). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (10,18) and only one archaeal aldo-keto reductase also from P. furiosus (11) have been characterized.…”

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confidence: 99%

“…TsAdh319 has an 85% degree of sequence identity with shortchain ADH from P. furiosus (AdhA; PF_0074) (18). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs.…”

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confidence: 99%

Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus

Stekhanova

Mardanov

Bezsudnova

et al. 2010

Appl Environ Microbiol

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Genetic and biochemical characterization of a short‐chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

Cited by 52 publications

References 27 publications

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Both adhE and a Separate NADPH-Dependent Alcohol Dehydrogenase Gene, adhA , Are Necessary for High Ethanol Production in Thermoanaerobacterium saccharolyticum

Sulfolobus tokodaiiST0053 Produces a Novel Thermostable, NAD-Dependent Medium-Chain Alcohol Dehydrogenase

Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus

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