Nanomolar biosensor for detection of phenylacetic acid and L-phenylalanine

Abstract

Phenylacetic acid (PAA) plays an important role in the plant hormone biology, possess a substantial antimicrobial activity and widely used building block for several pharmaceutical drugs. Technological advancement for PAA and other small molecule investigation in vivo can help to yield interesting insights into their involvement in biological processes. Transcription factor-based whole-cell biosensors have recently become useful analytical tools for detection and monitoring of various molecules. Compared to conventional analytical methods, the whole-cell biosensor offers cost-effective, rapid and high-throughput solution. In this paper, we identify and characterize PAA-inducible gene expression system CnPaaX/PpaaA2 from Cupriavidus necator H16. Nucleotide sequence analysis and mutagenesis reveal a conservative region with palindromic sequence motifs upstream to paaA2, which are likely used for PaaX-mediated transcriptional regulation. Using inducible system CnPaaX/PpaaA2 we develop C. necator-based whole-cell biosensor that exhibits a dose-dependent response to the exogenous PAA. Moreover, the biosensor is highly specific to the PAA compared to naturally occurring hydroxylated forms of this compound. Importantly, it exhibits a limit of measurement as low as 9.54 nM demonstrating the highest level of sensitivity reported so far. By combining genetic elements of PAA-inducible system with phenylpyruvate decarboxylase gene ipdC, we develop biosensor suitable for determining of L-phenylalanine.