UC Berkeley

Microbial fermentations are often the most efficient and environmentally friendly option for producing valuable chemicals. However, producing the correct molecule without losing material to side products and spontaneous degradation can prove challenging, especially working within the constraints of a living organism. To address this problem, we present three separate strategies for directing flux and controlling reactions, each operating on a different pathway. We apply enzyme-level engineering to enhance the benzylisoquinoline alkaloid (BIA) pathway, where a cytochrome P450 is responsible for both product formation and degradation. Next, we demonstrate compartment engineering by sequestering prodeoxyviolacein (PDV) pathway enzymes in the peroxisome to control the flow of intermediates and reduce side products. Finally, we rearrange pathways themselves by using biological protecting groups to protect reactive and unstable molecules, producing stable indican from unstable indoxyl in the indigo pathway. These three strategies – enzyme, compartment, and protecting group engineering – can be applied individually or together to enhance microbial yields of valuable products while minimizing byproducts.