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

Abstract

Droughts are the main reason for global annual crop loss [1]. Engineering microorganisms that positively influence plant growth and development is of interest for improving plant health and drought tolerance. Therefore, in order to combat the negative effects of droughts on crop production, it would be of paramount importance to develop methods of “microbiome engineering” [2]. The field of plant microbiome engineering focuses on genetically manipulating the plant microbiome with the goal of increasing plant fitness and health. Previous work from Finkel et al. 2020 demonstrated that the addition of the bacterial genus Variovorax in the bacterial synthetic community of Arabidopsis thaliana, henceforth Arabidopsis, significantly increased primary root elongation [3]. Specifically, the auxin degrading enzyme IadDE is required in Variovorax to maintain stereotypical primary root elongation [3, 4]. An increase in primary root elongation holds potential for increasing plant fitness during droughts. Here we express, purify and crystallize the Variovorax derived IadDE enzyme and attempt enzyme assays that demonstrate the integral role of a ferredoxin unit, IadC, in facilitating the enzymatic activity of IadDE. Furthermore, we express and purify IadC to varying levels of success. Finally, we express and purify an IadDE homologous enzyme, the Pseudomonas putida derived IacCD enzyme, in order to facilitate the eventual crystallization of IacCD for comparison to IadDE.