title: A synthetic O2 -tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli.
abstract: Several synthetic metabolic pathways for butanol synthesis have been reported in Escherichia coli by modification of the native CoA-dependent pathway from selected Clostridium species. These pathways are all dependent on the O2 -sensitive AdhE2 enzyme from Clostridium acetobutylicum that catalyzes the sequential reduction of both butyryl-CoA and butyraldehyde. We constructed an O2 -tolerant butanol pathway based on the activities of an ACP-thioesterase, acting on butyryl-ACP in the native fatty acid biosynthesis pathway, and a promiscuous carboxylic acid reductase. The pathway was genetically optimized by screening a series of bacterial acyl-ACP thioesterases and also by modification of the physical growth parameters. In order to evaluate the potential of the pathway for butanol production, the ACP-dependent butanol pathway was compared with a previously established CoA-dependent pathway. The effect of (1) O2 -availability, (2) media, and (3) co-expression of aldehyde reductases was evaluated systematically demonstrating varying and contrasting functionality between the ACP- and CoA-dependent pathways. The yield of butanol from the ACP-dependent pathway was stimulated by enhanced O2 -availability, in contrast to the CoA-dependent pathway, which did not function well under aerobic conditions. Similarly, whilst the CoA-dependent pathway only performed well in complex media, the ACP-dependent pathway was not influenced by the choice of media except in the absence of O2 . A combination of a thioesterase from Bacteroides fragilis and the aldehyde reductase, ahr, from E. coli resulted in the greatest yield of butanol. A product titer of ~300 mg/L was obtained in 24 h under optimal batch growth conditions, in most cases exceeding the performance of the reference CoA-pathway when evaluated under equivalent conditions.
authors: Department of Biochemistry, University of Turku, Tykistökatu 6B 4krs, 20520, Turku, Finland.
authors: András
authors: A
authors: Pásztor
authors: Pauli
authors: P
authors: Kallio
authors: Dávid
authors: D
authors: Malatinszky
authors: M Kalim
authors: MK
authors: Akhtar
authors: Patrik R
authors: PR
authors: Jones
mesh_terms: D056804:Biofuels; D000440:Butanols / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*
chemical_list: D056804:Biofuels; D000440:Butanols; D005227:Fatty Acids
keywords: Escherichia coli; biofuel; butanol; fatty acid biosynthesis; synthetic pathway