A Synthetic O-2-Tolerant Butanol Pathway Exploiting Native Fatty Acid Biosynthesis in Escherichia coli

摘要

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 O-2-sensitive AdhE2 enzyme from Clostridium acetobutylicum that catalyzes the sequential reduction of both butyryl-CoA and butyraldehyde. We constructed an O-2-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) O-2-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 O-2-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 O-2. 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 similar to 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. (C) 2014 Wiley Periodicals, Inc.