Rewiring the morphology and metabolism of <i>Escherichia coli</i> with an engineered CRISPRi system

AIMS: Microbial morphology is an increasingly leveraged target in metabolic engineering. To causally investigate the coupling between DNA replication, morphology, and metabolism, we constructed a CRISPR interference (CRISPRi) system in Escherichia coli BL21(DE3) to repress essential replication genes (dnaN, dnaG, polA, and ssb). METHODS AND RESULTS: Transcriptional repression disrupted cell division and yielded distinct, quantifiable shape changes: knockdown of dnaG (encoding primase) and ssb (encoding single-stranded DNA-binding protein), in particular, induced significant cellular elongation and widening. This morphological reprogramming concurrently reallocated metabolic flux, as evidenced by a substantial increase in lactic acid titer from 0.89 g L⁻¹ to 6.01 g L⁻¹. RNA-seq and subsequent analyses (differential expression, GO/KEGG enrichment, GSEA, and PPI) reveal that replication inhibition drives extensive metabolic reprogramming and cell envelope remodeling, with notable perturbations in peptidoglycan biosynthesis. CONCLUSION: Our results suggest that targeted replication stress can coordinately reshape bacterial morphology and alter metabolic output, providing a controllable strategy for engineering microbial cell factories.