Î’-Keto Acids Platform for Ketones and Alcohols Production in Saccharomyces Cerevisiae

Depleting oil reserves and growing environmental concerns have aroused general interest for developing sustainable processes to fuels and chemicals. β-Keto acids, or 3-oxoacids, such as acetoacetate, have the ketone group at the second carbon from the carboxylic acid. They are easily decarboxylated to produce ketones and ketones could be reduced to secondary alcohols. Ketones and alcohols are important platform chemicals with widespread applications in the fragrance, flavor and pharmacological industries. In nature, β-keto acids could be formed by the hydrolysis of β-ketoacyl-CoA which is an intermediate of fatty acid β-oxidation. Saccharomyces cerevisiae is a very important cell factory and has been widely used for many biomolecules production. In S. cerevisiae. In yeast, fatty acid β-oxidation process was performed in peroxisome which is a natural workshop for our target chemicals production. Hereby, (a) POT1 gene was deleted to interrupt fatty acid β-oxidation process and increase the precursor β-ketoacyl-CoA, (b) native acyl-CoA oxidase, enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase were overexpressed to increase the accumulation of β-ketoacyl-CoA, (c) fatty acids transporters PXA1 and PXA2 were also expressed to improve the provision of fatty acids from cytosol to peroxisome, (d) a thioesterase FadM from E. coli was expressed with peroxisome signal peptide to convert β-ketoacyl-CoA to β-keto acid. To prove the concept, different chain length fatty acids were added into the culture medium to test the production of methyl ketones. Results showed methyl ketones (2-Undecanone, 2-Tridecanone, 2-Heptadecanone) could be detected when adding dodecanoic acid.