Recent advances in pathway engineering for the biosynthesis of 1,4-butanediol.

1,4-Butanediol (1,4-BDO) is a short-chain diol with extensive applications in various industries. Traditional petroleum-based synthesis methods face challenges such as harsh reaction conditions and environmental pollution. Recent advances in bio-based 1,4-BDO synthesis harness synthetic biology tools to engineer the microbial pathways for sustainable production. Three principal biosynthetic routes, the CoA-dependent route, the lignocellulose-derived sugar-catabolizing route, and the amino acid-catabolizing route, have been rationally optimized using enzyme engineering, and dynamic metabolic regulation strategies. Besides these in vivo strategies, whole-cell transformation and enzyme cascades were engineered in vitro. Despite these advances, industrial scalability remains constrained by unresolved intracellular catabolism, host tolerance limitations, and suboptimal economic feasibility. Future priorities involve elucidating 1,4-BDO degradation mechanisms via multi-omics, designing high-specificity dehydrogenases, and developing precision genetic tools for dynamic flux control. This review critically assesses the pathway engineering breakthroughs and the translational challenges for sustainable 1,4-BDO production. KEY POINTS: • Three sets of novel pathway designs and engineering strategies were developed for de novo 1,4-BDO production in vivo. • Two biotransformation systems were established for 1,4-BDO production via in vitro enzyme cascades and in vivo whole-cell biocatalysis. • High-efficiency bioproduction of 1,4-BDO still faces multiple core challenges and technical bottlenecks.