Reverse beta-oxidation for biochemical production: insights into the functional properties of key enzymes

The reverse β-oxidation (rBOX) pathway is emerging as a promising alternative to fossil fuel-based chemical production, providing a versatile platform for the synthesis of various valueadded biochemicals. Efficient application of rBOX depends on the selection of enzymes with high catalytic activity, suitable substrate specificity, and strong functional compatibility within the pathway. In this review, we focus on the structural and biochemical characteristics of four key enzymes-thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase-and explore how their individual features and combinations influence pathway performance. We then summarize previous studies that highlight the importance of enzyme cooperation in achieving optimal production outcomes. These insights provide valuable guidance for the rational design of rBOX-based biosynthetic pathways tailored to specific chemical targets. [BMB Reports 2026; 59(4): 209-218].