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Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4‐butanediol pathway in Escherichia coli
Author(s) -
Hwang Hee Jin,
Park Jin Hwan,
Kim Jin Ho,
Kong Min Kyung,
Kim Jin Won,
Park Jin Woo,
Cho Kwang Myung,
Lee Pyung Cheon
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25196
Subject(s) - escherichia coli , strain (injury) , chemistry , mutagenesis , biochemistry , mutant , butyraldehyde , 2,3 butanediol , clostridium , biology , microbiology and biotechnology , bacteria , fermentation , gene , genetics , anatomy , catalysis
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. Biotechnol. Bioeng. 2014;111: 1374–1384. © 2014 Wiley Periodicals, Inc.