Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes

Abstract

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde.

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

“…Long-chain alcohols, such as butanol, have many desirable properties relative to existing biofuels such as ethanol (Atsumi et al 2010). In an effort to produce isobutanol through a 2-keto-acid based pathway in E. coli, Atsumi et al needed an alcohol dehydrogenase to convert isobutyraldehyde to isobutanol (Atsumi et al 2010).…”

“…In an effort to produce isobutanol through a 2-keto-acid based pathway in E. coli, Atsumi et al needed an alcohol dehydrogenase to convert isobutyraldehyde to isobutanol (Atsumi et al 2010). YqhD, due to its known activity as an aldehyde reductase, was a strong candidate for this desired activity.…”

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…The resulting aldehyde could then be reduced by an alcohol dehydrogenase to generate the primary alcohol product. The wide array of identified alcohol dehydrogenases created a high probability that an alcohol dehydrogenase could be found for conversion to the final alcohol 13,58,59 . S. cerevisiae Adh6p Sc was initially selected because it was previously found to be a broad specificity alcohol dehydrogenase with high activity on medium-and branchedchain aliphatic aldehydes 53 .…”

“…Early examples of this new paradigm have focused on introducing or combining portions of natural pathways in alternative host organisms or creating new products by altering the termination of natural pathways of a host organism with promiscuous enzymes 1,[3][4][5][6][7][8][9][10][11] . Recent work has been focused on improving modified natural pathways by substituting new enzymes to improve kinetics, improve expression or utilize alternate cofactors [12][13][14] . In some cases, pathways have been repurposed to synthesize new products by capitalizing on the natural capacity of enzymes to accept closely related substrates or by engineering protein specificity [15][16][17] .…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…In one study, a previously engineered biosynthetic pathway for isobutanol was pushed to its theoretical yield under anaerobic conditions by engineering the cofactor preference for NADH over NADPH in ilvC and adhA. [11,12] In another study, NADH accumulation was leveraged as a driving force to boost n-butanol production to 30 g L À1 . [13] An NADH-dependent CoA-reductase, ter, was heterologously introduced to an E. coli strain in which all fermentative NADH-consuming pathways were blocked.…”