Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

Valencia, L. E.; Incha, M. R.; Schmidt, M.; Pearson, A. N.; Thompson, M. G.; Roberts, J.; Mehling, M.; Yin, K.; Sun, N.; Oka, A.; Shih, P. M.; Blank, L. M.; Gladden, J.; Keasling, J. D.

doi:10.1101/2022.08.20.504671

bioRxiv · 10.1101/2022.08.20.504671

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

Valencia, L. E.·Incha, M. R.·Schmidt, M.·Pearson, A. N.·Thompson, M. G.·Roberts, J.·Mehling, M.·Yin, K.·Sun, N.·Oka, A.·Shih, P. M.·Blank, L. M.·Gladden, J.·Keasling, J. D.

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Abstract

Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L, paving the road for the production of high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L.

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Valencia, L. E., Incha, M. R., Schmidt, M., Pearson, A. N., Thompson, M. G., Roberts, J., Mehling, M., Yin, K., Sun, N., Oka, A., Shih, P. M., Blank, L. M., Gladden, J., Keasling, J. D.. 2022-08-21. Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production. https://doi.org/10.1101/2022.08.20.504671

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