Unveiling Metabolic Engineering Strategies by Quantitative Heterologous Pathway Design

Abstract Constructing efficient cell factories requires the rational design of metabolic pathways, yet quantitatively predicting the potential pathway for breaking stoichiometric yield limit in hosts remains challenging. This leaves it uncertain whether the pathway yield of various products can be enhanced to surpass the stoichiometric yield limit and whether common strategies exist. Here, a high‐quality cross‐species metabolic network model (CSMN) and a quantitative heterologous pathway design algorithm (QHEPath) are developed to address this challenge. Through systematic calculations using CSMN and QHEPath, 12,000 biosynthetic scenarios are evaluated across 300 products and 4 substrates in 5 industrial organisms, revealing that over 70% of product pathway yields can be improved by introducing appropriate heterologous reactions. Thirteen engineering strategies, categorized as carbon‐conserving and energy‐conserving, are identified, with 5 strategies effective for over 100 products. A user‐friendly web server is developed to quantitatively calculate and visualize the product yields and pathways, which successfully predicts biologically plausible strategies validated in literature for multiple products.