Multiple Global Transcription Factor Engineering Enables Rewiring of Regulatory Networks for Enhanced Biochemical Production in Escherichia coli

Cellular metabolism is governed by complex, multilayered regulatory networks coordinated by multiple global transcription factors (gTFs), thereby hindering rational metabolic rewiring. Single global transcription factor engineering (SgTFE), which involves introducing mutations into a single gTF or modulating its expression level, has been employed to reprogram the cellular metabolism. However, SgTFE perturbs one gTF at a time and therefore does not directly sample combinatorial interactions among multiple gTFs. We developed multiple global transcription factor engineering (MgTFE), a combinatorial approach that diversifies the ribosome-binding site (RBS) regions of seven gTF genes encoding Crp, Fis, Fnr, ArcA, H-NS, Lrp, and FadR to generate multi-locus regulatory variants. In Escherichia coli, MgTFE yielded a free fatty acid (FFA)-producing strain carrying RBS mutations in crp and fis, with a 66.5% increase in the FFA titer and differential expression of 1420 genes spanning key metabolic pathways. MgTFE was further applied to polyhydroxybutyrate (PHB) production, generating a strain with RBS mutations in crp, hns, and lrp and a 28.5% increase in the PHB content. These results demonstrate that MgTFE is an effective combinatorial strategy for metabolic network rewiring in E. coli to generate the desired phenotypes.