Construction and transfer of a butanol pathway module from E. coli to cyanobacteria

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Andras Pasztor, Kalim M. Akhtar, Pauli Kallio, Fernando Guerrero, Patrik R. Jones

University of Turku, Finland

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. Although the AdhE2 pathways are capable of producing high titers of butanol they are limited to one carbon source only, glucose. The aim of our research was to develop a modular, oxygen-independent, synthetic pathway which would be active in both Escherichia coli and in photosynthetic organisms, like cyanobacteria. Engineering cyanobacteria to fix CO2 as a carbon source for butanol production instead of glucose, would deminish the needs of using a valuable food source for fuel production. To achieve this, we have combined a selection of bacterial acyl-ACP-thioesterases with carboxylic acid reductase in order to construct a novel oxygen-tolerant butanol-pathway. The thioesterase that resulted in the greatest yield of butanol was studied further in comparison with a previously established reference pathway that is CoA-dependent. The yield of butanol from the ACP-dependent pathway module was stimulated by enhanced O2-availability and co-expression with aldehyde reductase. A product titer of 300 mg/L was obtained under optimal batch growth conditions in E. coli, exceeding the performance of the reference CoA-pathway when evaluated under equivalent conditions. Preliminary experiments using cyanobacteria indicate that the transfer of the pathway module has influenced the metabolism of alkanes rather than alcohols. This illustrates the complexity of pathway transfer from one host to another.