Targeted Genome Editing of the ACC Deaminase Gene in <i>Bradyrhizobium</i> : Toward Enhanced Plant Growth and Stress Tolerance

Ensuring sufficient crop yields in an era of rapid population growth and limited arable land requires innovative strategies to enhance plant resilience and sustain, or even improve, growth and productivity despite environmental stress. Besides symbiotic nitrogen fixation, rhizobia may play a central role in sustainable agriculture by alleviating the detrimental effects of ethylene-a key stress hormone in plants-especially under conditions like drought through the deamination of 1-aminocyclopropane-1-carboxylic acid (ACC). In this study, we focused on genetically engineering a new Bradyrhizobium sp. isolate (Strain 9) from peanut root nodules to enhance its ACC deaminase activity. First, we developed a sacB-based genome-engineering tool and used it to knock out the ACC deaminase gene (acdS), confirming that its disruption severely diminished the strain's capacity to degrade ACC. Subsequently, we constructed an acdS-overexpressing strain by integrating a strong promoter and an optimized ribosome binding site upstream of acdS, achieving a five-fold increase in ACC deaminase activity relative to the wild-type. Peanut inoculation experiments demonstrated that both the acdS knockout and overexpression mutants effectively nodulated roots without impairing plant growth and nitrogen fixation, indicating that these modifications did not compromise symbiosis. Overall, this study highlights the utility of sacB-mediated counter-selection for precise genome editing in Bradyrhizobium and underscores the potential of enhanced ACC deaminase activity to improve plant growth under stress conditions. These findings pave the way for developing next-generation bioinoculants with superior ethylene mitigation capabilities, contributing to more productive and sustainable crop systems.