Model‐Guided Rational Construction of Escherichia coli Synthetic Consortia for Enhanced 2‐Methylbutyric Acid Production

Abstract Synthetic consortia represent an innovative and effective platform that can significantly alleviate the metabolic burden on host organisms and enable flexible regulation of biosynthetic pathways. However, designing a stable synthetic consortium remains a significant challenge. In this study, a novel citramalate ‐derived pathway is first developed for 2‐methylbutyric acid (2MBA) biosynthesis in an E. coli mono‐culture system, achieving a titer of 678.78 ± 49.04 mg L −1 . Furthermore, it employs a CulECpy model‐guided strategy to design and optimize the division of labor within E. coli synthetic consortia, predicting the optimal pathway allocation for improved 2MBA production. The best‐performing consortium, using 2‐keto‐3‐methylvalerate (KMV) as a single node, achieved 1817.03 ± 103.73 mg L −1 of 2MBA, a 28‐fold increase over the initial mono‐culture strain, with the highest reported yield of 0.091 g/g glucose. This work demonstrates the effectiveness of synthetic consortia and model‐guided pathway optimization for improving high‐value products, a versatile strategy that can be applied to the production of other valuable metabolites.