Balancing Consecutive Two-Step Oxidations Catalyzed by a Single Cytochrome P450 Enables Efficient Biosynthesis of Plant Natural Products

Consecutive two-step oxidations catalyzed by a single cytochrome P450 are often reported in the biosynthesis of plant natural products (PNPs). However, due to unbalanced catalytic activity, intermediates and byproducts are always substantially accumulated, creating a major bottleneck in the efficient heterologous synthesis of many important PNPs. Despite this, efforts to balance these consecutive two-step oxidations to enhance the production of final PNPs are often overlooked. Here, guided by rational design, we engineered Ro CYP01 by modulating substrate/product channels, enzyme–substrate interactions, the electron transfer chain, and O 2 /H 2 O exchange tunnels, effectively rebalancing the consecutive two-step oxidations from R–CH 3 to R–CH 2 OH to R–COOH. Consequently, the product, betulinic acid, was greatly improved in an engineered yeast strain, substantially reducing the accumulation of intermediates. Thus, this work establishes a mechanism-driven strategy to rebalance consecutive two-step oxidations, providing a potential strategy for the efficient biosynthesis of other PNPs that involve P450-catalyzed consecutive two-step oxidations.