Improving biobutanol production in engineered Saccharomyces cerevisiae by manipulation of acetyl-CoA metabolism

Recently, butanols (1-butanol, 2-butanol and iso-butanol) have generated attention as alternative gasoline additives. Butanols have several properties favorable in comparison to ethanol, and strong interest therefore exists in the reconstruction of the 1-butanol pathway in commonly used industrial microorganisms. In the present study, the biosynthetic pathway for 1-butanol production was reconstructed in the yeast Saccharomyces cerevisiae. In addition to introducing heterologous enzymes for butanol production, we engineered yeast to have increased flux toward cytosolic acetyl-CoA, the precursor metabolite for 1-butanol biosynthesis. This was done through introduction of a plasmid-containing genes for alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA synthetase (ACS), and acetyl-CoA acetyltransferase (ERG10), as well as the use of strains containing deletions in the malate synthase (MLS1) or citrate synthase (CIT2) genes. Our results show a trend to increased butanol production in strains engineered for increased cytosolic acetyl-CoA levels, with the best-producing strains having maximal butanol titers of 16.3 mg/l. This represents a 6.5-fold improvement in butanol titers compared to previous values reported for yeast and demonstrates the importance of an improved cytosolic acetyl-CoA supply for heterologous butanol production by this organism. © 2013 Society for Industrial Microbiology and Biotechnology.
This work has been funded in part by the Chalmers Foundation, the Knut and Alice Wallenberg Foundation, and the European Research Council. C.S.A. is the recipient of an FPU predoctoral fellowship from the Spanish Ministerio de Educación. A. K. is a recipient of an Angpanneföreningens Forskningsstiftelse project grant.