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A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate.

ABSTRACT: Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and ?-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65-70?°C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36?±?0.26?mM acetoin was produced from 10?mM pyruvate after reaction for 24?h at 65?°C. The productivity of acetoin was 0.14?mM?h-1, and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

SUBMITTER: Jia X

PROVIDER: S-EPMC5489476 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate.

Jia Xiaojing X Liu Ying Y Han Yejun Y

Scientific reports 20170628 1

Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-ace ...[more]

PMID: 28659601

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Project description:UNLABELLED: BACKGROUND:Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination. RESULTS:This work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7?g/L of acetoin and 14.5?g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography-mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. ?-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C. CONCLUSIONS:Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work.

Dataset Information

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate.

Publications

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate.

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