Disentangling the Regulatory Response of Agrobacterium tumefaciens CHLDO to Glyphosate for Engineering Whole-Cell Phosphonate Biosensors

Abstract

Phosphonates (PHTs), organic compounds with a stable C–P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as a host and the source of genetic parts for constructing PHT biosensors. In this bacterial species, the phn gene cluster, encoding the C–P lyase pathway, is regulated by the PhnF transcriptional repressor. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise genetic optimization of the transcriptional cascade, we created a whole-cell biosensor capable of detecting GP in the 0.25–50 μM range in various samples, including soil and water.