Engineering 2,4-Diacetylphloroglucinol-Hyper-Tolerant Escherichia coli via Adaptive Evolution for Efficient Biosynthesis and an Organic Solvent-Free Downstream Process

2,4-Diacetylphloroglucinol (DAPG) is a valuable antimicrobial compound with significant agricultural potential, suffers bioproduction limitations from host toxicity and inefficient downstream processing. This study engineered DAPG-hyper-tolerant Escherichia coli via adaptive laboratory evolution (ALE) starting from a phloroglucinol-tolerant strain. Optimized shake-flask fermentation of evolved Bdt03 yielded 330.52 mg/L DAPG (17.84-fold of the wild type), and the yield from the whole fermentation broth could be further increased to 391.36 mg/L. A novel organic solvent-free extraction method recovered DAPG from fermentation broth with over 98% yield via acidification, cold incubation and centrifugation, simplifying downstream processing. Genomic resequencing identified several key mutations underlying DAPG tolerance, which were validated and stacked to precisely construct strain Bb03 with enhanced production and tolerance. This work addresses the critical bottlenecks in DAPG biosynthesis by enhancing host tolerance and developing a sustainable downstream processing strategy, and also offers valuable genetic insights for constructing high-yield DAPG-producing strains and advancing the application of DAPG-responsive genetic circuits in synthetic biology.