Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4-butanediol pathway in Escherichia coli

ABSTRACT 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. In this study, the authors selected Butyraldehyde dehydrogenase (Bld) and engineered Bld to be fit into the non‐natural 1,4‐BDO pathway through random mutagenesis. Subsequent site‐directed mutagenesis of Bld generated the best Bld mutant L273T, which produced 1,4‐BDO titers 4‐fold greater than those of wild‐type Bld. The enhanced 1,4‐BDO titer obtained using L273T clearly correlated with its enhanced activity, which was 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)].