Efficient production of medium-chain fatty acids in engineered Escherichia coli strains from ionic liquids pretreated bamboo enzymatic hydrolysate with carbon dioxide fixation.

The challenge of medium-chain fatty acids (MCFAs) biomanufacturing lies in controlling chain length and utilizing low-cost carbon sources. This work demonstrated MCFAs production from bamboo waste and CO2. The engineered E. coli strain LZK109/pLZ1Z, with deletions in fatty acyl-CoA synthetase (ΔfadD) and endogenous thioesterase (ΔtesA), and the glucose-specific PTS (ΔptsG) to enable the co-utilization of multiple carbon sources; and expression of Cuphea palustris FatB1 (CpFatB1) and (3R)-hydroxyacyl-ACP dehydratase (fabZ), produced 2.29 g/L of MCFAs. Protein engineering using AlphaFold3 created CpFatB1I228R, increasing MCFAs to 5.83 g/L. Glucose, xylose, arabinose, mixed sugars, and ionic liquids (ILs) pretreated bamboo enzymatic hydrolysate produced 6.46 g/L, 2.31 g/L, 2.04 g/L, 5.81 g/L, and 5.58 g/L of MCFAs, respectively. Overexpression of carbon fixation gene ecaA further improved MCFAs to 6.72 g/L, and fatty acid degradation regulator (fadR) enhanced production to 8.26 g/L (flask) and 9.05 g/L (5L bioreactor). This work offers pioneering strategies for MCFAs production with environmental and economic benefits.