Disentangling the regulatory response of Agrobacterium tumefaciens CHLDO to glyphosate for engineering whole-cell phosphonate biosensors

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 the source of genetic parts for constructing PHT biosensors. In this species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor and is part of the Pho regulon. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise optimization of the transcriptional cascade, we created a biosensor capable of detecting GP in the 0.25-50 uM range in various samples, including soil and water.