Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures.
There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30 degrees C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30 degrees C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of approximately 1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30 degrees C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183.
Machielsen
Ronnie
R
Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. rmachielsen@hotmail.com
Leferink
Nicole G H
NG
Hendriks
Annemarie
A
Brouns
Stan J J
SJ
Hennemann
Hans-Georg
HG
Daussmann
Thomas
T
van der Oost
John
J
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006588:Hexanones / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D008956:Models, Chemical; D008968:Molecular Conformation; D009154:Mutation; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013696:Temperature; D013912:Threonine / Q000737:chemistry
D006588:Hexanones; D013912:Threonine; C011269:2,5-hexanedione; D000426:Alcohol Dehydrogenase