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.

Synthesis, structure and properties of poly(ester-urethane)s obtained using bio-based and petrochemical 1,3-propanediol and 1,4-butanediol

“…This contamination can significantly influence on the chemical structure and properties of polyurethanes, and this is resulted from the lower mass of chain extenders in comparison with the mass of polyols or isocyanates used during the synthesis of PUs. According to the literature, the bio-based glycols can contain contaminations like 2,3-butanediol, lactic acid, acetic acid, ethanol, succinic acid 1,4-butanediol monoacetate, 4-hydroxybutyraldehyde and another ketones and aldehydes, enzymes and inorganic compounds [43][44][45].…”

Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker

“…We then integrated the kgd (NCgl1084) gene from Corynebacterium glutamicum to mediate the conversion of α-ketoglutarate into succinyl semialdehyde, further increasing the metabolic flux for synthesis of 4HB, a necessary intermediate in the 1,4-BDO pathway. Finally, the aldehyde dehydrogenation step, which is considered to be limiting for the conversion of 4HB to 1,4-BDO, was enhanced by replacing ald with the mutated bld ( bld M227L, L273I) from Clostridium saccharoperbutylacetonicum 24, as this has been shown to encode a protein with higher activity than that encoded by the C. beijerinckii ald gene (data not shown).…”

“…We utilized the previously reported W023 strain24, an E. coli W derivative with the modifications described above: deletions of mdh, ldhA , arcA , adhE , and pflB , as well as the gltA R164L mutation and a substitution of lpdA with the homologue from K. pneumoniae containing E354K.…”

“…We then substituted the wild-type lpdA gene with lpdA E354K mutant from K. pneumoniae , to generate YSB11183536. E. coli W023 (Δ ldhA , Δ pflB , Δ adhE , Δ lpdA :: K. lpd E354K, Δ mdh , Δ arcA , gltA R164L), a derivative of strain W, was described in a previous report1824. The development of strain W029, the precursor for insertion of the 1,4-BDO biosynthetic pathway is described in Supplementary Note 2.…”

“…Following the cat2 cloning, the mutated bld gene which constains M227L and L273I mutation was cloned to pTrc99A cat2 to generate pTrc99a bld M -cat2. The bld gene was amplified from genomic DNA of C. saccharoperbutylacetonicum , and M277L and L273I mutations were introduced by site-directed mutagenesis24. Sequences of oligos for construction of engineered 1,4-BDO biosynthetic pathway plasmids are listed in Supplementary Table 10.…”