Expression, Purification, and Analysis of Essential Indole-3-Acetic Acid Degradation Enzymes from ROOT Microbiome Isolates.

Michal Kozlowski1, Ting Jiang1, Jonathan M. Conway1

1Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, USA

Auxins are plant hormones that play a major role in regulating plant growth and development. Previous experiments demonstrate that certain microorganisms in the plant root microbiome degrade and/or produce auxins and that these plant-microbe interactions involving auxins greatly impact plant growth and development. These experiments also demonstrated that the bacterial genus Variovorax was responsible for the degradation of auxins, primarily Indole-3-Acetic Acid (IAA), through a novel IAA degradation locus. Here, we express and purify essential, multimeric IAA degradation enzymes: IadDE from Variovorax paradoxus and IacCD from Pseudomonas putida. The expression and purification of these essential enzymes enables subsequent biochemical characterization of the enzymes. Through the development of enzyme assays, we support previous experiments that demonstrated the IAA catabolic capabilities of Variovorax paradoxus and Pseudomonas putida derived enzymes. We have also undertaken protein crystallography efforts on these enzymes to enable structure-function analysis and rational engineering towards the optimization of IAA degradation in the plant root microbiome.