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 RoCYP01 by modulating substrate/product channels, enzyme–substrate interactions, the electron transfer chain, and O2/H2O exchange tunnels, effectively rebalancing the consecutive two-step oxidations from R–CH3 to R–CH2OH 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.