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

• Published in: Scientific reports Vol. 7; no. 1; pp. 4333 - 10
• Main Authors: Jia, Xiaojing, Liu, Ying, Han, Yejun
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28-06-2017; Nature Publishing Group; Nature Portfolio
• Subjects: 38/22; 38/23; 38/77; 631/45/603; 631/61/338/552; 82/29; 82/80; 82/83; Acetoin; Acetoin - metabolism; Acetolactate decarboxylase; Acetolactate synthase; Acetolactate Synthase - genetics; Acetolactate Synthase - metabolism; Biosynthetic Pathways; Catalysis; Enzyme Activation; Enzyme kinetics; Fermentation; Gene Expression Regulation, Enzymologic; Humanities and Social Sciences; Hydrogen-Ion Concentration; Kinetics; multidisciplinary; NADP; pH effects; Pyruvic acid; Pyruvic Acid - metabolism; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-04684-8

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-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 Mn
2+
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.