A Single Mutation Enhancing the DNA-Binding Affinity of Cra Redirects Metabolism for High-Yield Succinate Production in Escherichia coli

Rational engineering of transcription factors is an attractive strategy to modulate microbial metabolism. This study demonstrated its effectiveness through a key mutation (R60A) in the understudied linker region of the global transcription factor Cra. Transcriptional and enzymatic analyses revealed that R60A upregulated multiple genes and enhanced several key enzyme activities in central carbon metabolism, including the direct Cra targets pck and aceB, which were selected for further mechanistic investigation. In vitro binding assays confirmed that R60A bound to the promoter regions of these genes with enhanced affinity. Molecular docking analysis predicted that the top-ranked binding poses of the WT and R60A complexes differ in DNA placement, with an altered set of predicted hydrogen-bond contacts. This mutation altered the central carbon metabolism, leading to improved CO2 fixation and succinate biosynthesis. Coupled with an optimized exponential feeding strategy that precisely controlled the specific growth rate, this approach achieved a high succinate titer of 100.8 g/L. Our work suggests a possible role for linker-mediated geometric modulation of DNA binding by Cra and presents an integrated metabolic and process engineering strategy for efficient succinate production.