Engineering microbial metabolic pathways allows the production of advanced biofuel and biochemical with desired structures and properties. Among them, the fatty acid biosynthetic pathway is particularly attractive because fatty acids are common precursors that can be readily converted to several types of chemicals including alkanes, alkenes, alcohols, and esters through either biological or chemical conversion. Moreover, introducing branches to the target molecule can improve the low-temperature operability of fatty acid-derived chemicals, which requires the synthesis of branched-chain fatty acids (BCFAs). However, previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production in Escherichia coli suffered from low titers and the significant co-production of straight-chain fatty acids (SCFAs), complicating efficient isolation of BCFA. In this work, we directed the carbon flux to the synthesis of BCFAs by replacing the acetyl-CoA-specific Escherichia coli FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase that catalyzes the initial fatty acid condensation reaction, with a branched-chain-acyl-CoA-specific FabH, enhancing BCFA titer by 81-fold compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further discovered that the overexpression of BKD, a member of the 2-oxoacid dehydrogenase family needed to synthesize branched-chain-acyl-CoA precursors, is toxic to E. coli due to depletion of protein lipoylation capacity. Engineering a complementary protein lipoylation pathway alleviated the toxicity and improved BCFA production to 276 mg/L and 85% of total free fatty acids, the highest BCFA titer and percentage to-date. Furthermore, we achieved fine-tuning of BCFA branch positions by engineering the upstream pathway to control the supply of branched-chain acyl-CoAs, eventually producing 181 mg/L BCFA at 72% of total FFA from glucose. Finally, we successfully converted BCFAs to high percentage of branched fatty alcohols, alkanes, and esters that can be used as lubricants and fuels with improved freezing point and cold flow properties. Overall, this work establishes a platform for the microbial synthesis of branched biofuels and chemical in high-percentage, paving the for bioproduction of structurally-defined molecules with desired properties.

Production of branched-chain fatty acid and derivatives in high percentage

253rd National Meeting (2017)

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