Fermentive production of four carbon alcohols

A method for producing a fuel or fuel additive comprising bioproduced isobutanol comprising:

• a) providing a recombinant yeast host cell expressing an engineered isobutanol biosynthetic pathway comprising the following substrate to product conversions:
• i) pyruvate to acetolactate;
• ii) the acetolactate from i) to 2,3-dihydroxyisovalerate;
• iii) the 2,3-dihydroxyisovalerate from ii) to α-ketoisovalerate;
• iv) the α-ketoisovalerate from iii) to isobutyraldehyde;
• and
• v) the isobutyraldehyde from iv) to isobutanol;
• and wherein the substrate to product conversion i) is catalyzed by art acetolactate synthase;
• the substrate to product conversion ii) is catalyzed by an acetohydroxy acid reductoisomerase;
• the substrate to product conversion iii) is catalyzed by an acetohydroxy acid dehydratase;
• the substrate to product conversion iv) is catalyzed by a branched-chain α-keto acid decarboxylase;
• and the substrate to product conversion v) is catalyzed by an alcohol dehydrogenase;
• and wherein the acetolactate synthase, acetohydroxy acid reductoisomerase, acetohydroxy acid dehydratase, branched-chain α-keto acid decarboxylase, and alcohol dehydrogenase are each encoded by a heterologous polynucleotide that lacks a mitochondrial targeting sequence,
• b) contacting the recombinant yeast host cell of a) with a fermentable sugar in fermentation medium under conditions whereby the recombinant yeast host cell produces more moles of isobutanol per mole of sugar through the substrate to product conversions of i) to v) as compared to naturally occurring amounts of isobutanol made by unmodified yeast;
• and
• c) recovering the bioproduced isobutanol to produce a fuel or fuel additive.