title: Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.
abstract: 2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Sabine
authors.initials: S
authors.lastname: Bastian
authors.forename: Xiang
authors.initials: X
authors.lastname: Liu
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Meyerowitz
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Snow
authors.forename: Mike M Y
authors.initials: MM
authors.lastname: Chen
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000426:Alcohol Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D056804:Biofuels; D000440:Butanols / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D006019:Glycolysis / Q000235:genetics; D050540:Ketol-Acid Reductoisomerase / Q000096:biosynthesis* / Q000235:genetics; D009249:NADP / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D056804:Biofuels; D000440:Butanols; D029968:Escherichia coli Proteins; D009249:NADP; C040507:isobutyl alcohol; D000426:Alcohol Dehydrogenase; D050540:Ketol-Acid Reductoisomerase