Fig. 1
The ABE butanol pathway does not lead to high levels of butanol production in S. cerevisiae. a Schematic diagram of a butanol production pathway utilised by a variety of clostridial species as part of ABE fermentation. The Hbd (3-hydroxybutyryl-CoA dehydrogenase), Crt (3-hydroxybutyryl-CoA dehydratase), Bcd (butyryl-CoA dehydrogenase) and Adhe2 (alcohol dehydrogenase) enzyme genes were derived from Clostridium beijerinckii, and the Erg10 (thiolase) sequence was taken from S. cerevisiae. b The strategy for expression of these genes via genomic integration into S. cerevisiae is depicted. Codon-optimised cassettes bearing C-terminal Flag epitope tags were expressed from the strong TDH3 gene promoter and CYC1 terminator sequences. Each cassette also carries a different marker downstream and was integrated at a precise location associated with high level expression (see Methods). c PCR analysis on genomic DNAs derived from either single integrant strains or a strain that has been back-crossed such that it harbours all five cassettes. The primers used are specific to the genomic integration loci and the cassettes labelled to the left of the gel pictures. d Western blotting using an anti-Flag antibody to detect the expressed proteins in either the single integrant strains or the strains bearing all five cassettes. Protein products are labelled to the right of the gel image. A blot probed with an anti-Pab1p antibody provides a loading control (lower panel). e and f Graphs depicting the level of ethanol or butanol produced from butanol sensitive (GCD1-S180) or butanol resistant (GCD1-P180) strains bearing the five butanol production genes (BS + 5 g or BR + 5 g) over a 21-day anaerobic fermentation. Error bars are ± SEM from five biological repeats