Acetate is a cost-effective and sustainable carbon source that, despite its potential, remains underutilized. This study employed biosensor-assisted adaptive laboratory evolution (ALE) to enhance itaconic acid production and acetate metabolism in Escherichia coli. The evolved E. coli W strains exhibited 65% increase in itaconic acid production and 71% increase in growth rate, and 45% increase in itaconic acid yield. A common 31-kb genomic deletion was identified in the evolved strains, with two genes, ecw_m2276 and ecw_m2277, driving the observed phenotypic changes. The evolved strains exhibited an intensified stringent response, which enhanced the acetate-utilizing pathway and resulted in over a 5,000% increase in the expression of the glyoxylate shunt, thereby boosting microbial growth. Overexpression of relA further replicated these enhanced phenotypes. Our findings highlight not only significant physiological improvements but also present a novel strategy for enhancing microbial growth and bioproduction from acetate, offering valuable insights for industrial biotechnology applications.