Recent advances in microbial 3-methyl-1-butanol production.

3-Methyl-1-butanol (3MB), also known as isoamyl alcohol, is an emerging bio-based solvent, platform chemical, and advanced biofuel candidate whose demand continues to grow across chemical, energy, and consumer product sectors. Microbial synthesis offers a sustainable alternative to petrochemical routes, yet achieving industrially viable titers remains challenging due to pathway complexity, byproduct formation, redox imbalance, and product toxicity. This review provides a comprehensive summary of current advances in microbial 3MB production, including host strain and pathway engineering, feedstock diversification, and fermentation design. We compare the three principal biosynthetic routes toward 3MB-the valine-leucine-Ehrlich pathway, the mevalonate pathway, and the isovaleryl-CoA pathway-and evaluate their implementation across bacterial and yeast chassis. Particular focus is placed on strategies that enhance flux through leucine biosynthesis, reduce byproduct formation such as isobutanol, and rebalance NAD(P)H cofactors. Mechanisms of 3MB toxicity and recent insights from adaptive laboratory evolution and omics analyses are discussed as emerging guides for improving product tolerance. Beyond genetic interventions, we highlight process-level opportunities such as in situ product extraction, oxygen-supply optimization, and fed-batch operation, which remain underexplored yet are critical for achieving high 3MB titers. Looking forward, leveraging isobutanol chassis strains, employing high-throughput technologies such as biosensor-guided evolution, adopting intensified fermentation strategies, and co-producing 3MB alongside bioethanol may accelerate the development of scalable and economically competitive microbial platforms for 3MB production.