Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4-butanediol pathway in Escherichia coli.
1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.
Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon, 443-749, South Korea.
Hee Jin
HJ
Hwang
Jin Hwan
JH
Park
Jin Ho
JH
Kim
Min Kyung
MK
Kong
Jin Won
JW
Kim
Jin Woo
JW
Park
Kwang Myung
KM
Cho
Pyung Cheon
PC
Lee
D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D002072:Butylene Glycols / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism
D002072:Butylene Glycols; D050505:Mutant Proteins; C039681:1,4-butanediol; D000445:Aldehyde Oxidoreductases; C056375:butyraldehyde dehydrogenase (Coenzyme A-linked)
1,4-butanediol; butyraldehyde dehydrogenase; metabolic engineering