Global energy and climate problems have stimulated increased efforts in synthesizing fuels and chemicals from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. However, these alcohols cannot be synthesized economically using native organisms. Here we present a metabolic engineering approach using various microorganisms to produce higher alcohols (C3-C8) from renewable carbon source. This strategy leverages the host’s highly active amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis. In particular, we have achieved high yield, high specificity production of isobutanol from glucose. We further developed a non-natural chain-elongation pathway to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into E. coli, this non-natural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. This strategy has also been implemented in photosynthetic cyanobacteria, Synechococcus elongates to produce isobutyraldehyde and isobutanol directly from CO2, bypassing the need for plant or algal biomass processing. In addition, other non-natural metabolites which can be used as pharmaceutical intermediates can be produced using this strategy.
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