Rational design and screening system development of decarboxylases for production of amine compounds

Abstract

Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes. Combining directed evolution with rational design has proven to be synergistic in optimization and enhancement of enzyme activity. Accuracy and efficiency of screening system is of great demand in directed evolution. In this aspect, genetic circuit system is suitable because it provides high accuracy and efficiency by reduced false positive through tight regulation and simple detection method. For this reason, we devised the genetic circuit system for ODC library screening. Analyzing putrescine-inducible genes, we designed vectors containing putrescine-inducible promoters fused with a reporter gene (EGFP). The constructs were transformed in putrescine-de...