In vitro production of n-butanol from glucose.
The heat treatment of recombinant mesophiles having heterologous thermotolerant enzymes results in the one-step preparation of highly selective biocatalytic modules. The assembly of these modules enables us to readily construct an artificial metabolic pathway in vitro. In this work, we constructed a non-natural, cofactor-balanced, and oxygen-insensitive pathway for n-butanol production using 16 thermotolerant enzymes. The whole pathway was divided into 7 parts, in each of which NAD(H)-dependent enzymes were assigned to be the last step, and the fluxes through each part were spectrophotometrically determined. This real-time monitoring technique enabled the experimental optimization of enzyme level to achieve a desired production rate. Through the optimized pathway, n-butanol could be produced from glucose with a molar yield of 82% at a rate of 8.2 µmol l(-1) min(-1). Our approach would be widely applicable to the rational optimization of artificial metabolic pathways as well as to the in vitro production of value-added biomolecules.
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Borimas
B
Krutsakorn
Kohsuke
K
Honda
Xiaoting
X
Ye
Takashi
T
Imagawa
Xiaoyu
X
Bei
Kenji
K
Okano
Hisao
H
Ohtake
D020001:1-Butanol / Q000737:chemistry / Q000378:metabolism*; D000091:Acetobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002474:Cell-Free System / Q000737:chemistry / Q000201:enzymology; D005947:Glucose / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*
D001426:Bacterial Proteins; D020001:1-Butanol; D005947:Glucose
Themophilic enzyme; in vitro metabolic pathway; n-Butanol