Engineering of Integral Membrane Metalloenzyme UndB and Designing of a Cell-Free Biocatalytic Platform Enabled Efficient 1‑Alkene Production.

The bioproduction of 1-alkenes is of significant global interest due to their potential as green commodity chemicals and next-generation 'drop-in' biofuels. Here, we report an engineering strategy to enhance the catalytic activity and substrate specificity of the membrane-bound metalloenzyme UndB, significantly improving its utility in biocatalytic 1-alkene production. We developed a highly efficient UndB-based cell-free biocatalytic platform for high-yield medium-chain 1-alkene production. This system achieved a 262-fold improvement in UndB activity toward 1-undecene production, with a total turnover of 3412. Through structural analysis of the UndB family of proteins, we engineered UndB by domain-swapping, enhancing its selectivity toward naturally abundant long-chain fatty acids, enabling efficient long-chain 1-alkene production. Our large-scale simulations unveiled a crucial ion-pair network that orchestrates substrate-protein interactions, providing a framework for substrate stabilization. We identified a highly dynamic and functionally pivotal Arg121 residue that governs substrate uptake and stabilization, providing mechanistic insights into UndB's substrate recognition. Furthermore, simulations revealed that precise modulation of the substrate-binding pocket volume serves as the key determinant of substrate specificity across UndB variants, offering insights into the evolutionary adaptability of the UndB family. Our system achieved 98% 1-alkene yield using only 0.04 mol % catalyst loading under mild conditions, presenting a promising bioproduction strategy.