Rewiring the morphology and metabolism of Escherichia coli with an engineered CRISPRi system

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).

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.

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.