469011 Production of Branched-Chain Fatty Acid in High Percentage

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Gayle J. Bentley1, Wen Jiang2, Yanfang Jiang2,3, Di Liu2, Yi Xiao2 and Fuzhong Zhang2, (1)Division of Biological & Biomedical Sciences, Washington University in St. Louis, St. Louis, MO, (2)Department of Energy, Environmental, and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, (3)Sigma-Aldrich, St. Louis, MO

Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and the co-production of a large amount of straight-chain fatty acids (SCFAs), complicating efficient isolation of BCFA. Synthesis of both SCFAs and BCFAs requires FabH, the only β-ketoacyl-(acyl-carrier-protein) synthase that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. We demonstrated that replacement of the acetyl-CoA-specific Escherichia coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in 81-fold enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further identified the incomplete lipoylation of 2-oxoacid dehydrogenases as a key bottleneck in BCFA production, and thus improved the BCFA percentage to 85% of the total free fatty acids (FFAs) by engineering of protein lipoylation pathways. Furthermore, we achieved fine-tuning of BCFA branch positions by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, and eventually BCFA can be produced from glucose at 181 mg/L and 72% of total FFA.

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