Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Butanol may be made through chemical synthesis or by fermentation. The most popular fermentation process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE processes (Blaschek et al., U.S. Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations, and the pathways and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16:11-16 (1998)). Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (U.S. Patent Application Publication No. US20080182308A1), a 2-butanol biosynthetic pathway (U.S. Patent Application Publication Nos. US20070259410A1 and US 20070292927A1), and an isobutanol biosynthetic pathway (U.S. Patent Application Publication No. US 20070092957) have been described. However, biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in the fermentation.
Bacteria of the genus Clostridium naturally produce butanol. Strains of Clostridium with increased tolerance to 1-butanol have been isolated by chemical mutagenesis (U.S. Pat. No. 5,192,673; and U.S. Pat. No. 6,358,717), overexpression of certain classes of genes such as those that express stress response proteins (U.S. Pat. No. 6,960,465; and Tomas et al., Appl. Environ. Microbiol. 69(8):4951-4965 (2003)), and by serial enrichment (Quratulain et al., Folia Microbiologica (Prague) 40(5):467-471 (1995); and Soucaille et al., Current Microbiology 14(5):295-299 (1987)). Additionally, the isolation of 1-butanol tolerant strains from estuary sediment (Sardessai et al., Current Science 82(6):622-623 (2002)) and from activated sludge (Bieszkiewicz et al., Acta Microbiologica Polonica 36(3):259-265 (1987)) has been described.
It has been reported that in Pseudomonas putida, that cis unsaturated fatty acids are converted to the trans confirmation when cells are stressed with chemicals such as toluene. The increased trans fatty acid in the cell membrane plays a role in the toluene tolerance of P. putida (Junker and Ramos (1999) J. Bacteriol. 181:5693-5700).). In contrast, it has been reported that feeding a trans fatty acid to Clostridium acetobutylicum did not lead to improved butanol tolerance (Kuhn and Linden, Biotechnology and Bioengineering Symposium 17(Symp. Biotechnol. Fuels Chem., 8th, 1986), 197-207).
There is a need, therefore, for bacterial host strains which do not naturally produce butanol but can be engineered to express a butanol biosynthetic pathway to be more tolerant to these chemicals. In addition there is a need for methods of producing butanols using bacterial host strains engineered for butanol production that are more tolerant to these chemicals.
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