Systems metabolic engineering of Escherichia coli for high-level branched-chain fatty acid production via dual...

Branched-chain fatty acids (BCFAs), naturally synthesized by Gram-positive bacteria, are promising feedstocks for the production of advanced biofuels. However, efficient BCFA biosynthesis in Gram-negative bacteria such as Escherichia coli remains challenging because of insufficient supply of branched-chain acyl-CoA precursors, poor compatibility of the endogenous fatty acid synthesis pathway with branched-chain substrates, and limited cellular robustness toward non-native fatty acids. In this study, we first engineered an orthogonal isovaleryl-CoA biosynthetic pathway in E. coli and demonstrated its functionality in supporting BCFA production. Importantly, we identified a strong synergistic interaction between the isovaleryl-CoA pathway and the branched-chain α-keto acid dehydrogenase pathway, and thereby established a dual-route strategy for precursor supply. To further enhance BCFA production, we rewired central carbon metabolism by eliminating competing pathways and introducing a non-oxidative glycolysis pathway to increase acetyl-CoA availability while minimizing byproduct formation. We additionally optimized the fatty acid biosynthetic module through expression of a highly active 'TesA (R65C) variant and the transcriptional regulator FadR, and enhanced cellular robustness via introduction of N138H mutation into PcnB and overexpression of the stress resistance associated genes rfaY and yafL. The final engineered strain produced 2.96 g/L BCFAs (approximately 10-fold higher than the previously reported titers), representing 55% of total fatty acids, with a yield of 0.08 g/g glucose. Overall, this work established a dual-precursor supply strategy combined with systems metabolic engineering for BCFA production, providing a foundation for the development of sustainable bioprocesses for advanced branched-chain biofuels.