Discovery, development and implementation of biomarker-specific peptide BRE for biosensing hydrocarbon-degrading fungi

Abstract

Fuel biocontamination is a significant problem that demands new tools for detection and mitigation. Due to the complexity of fuel microbiome, it is critical to differentiate the type of microorganisms present to apply appropriate countermeasures, and fuel-degrading fungi are among the main culprits of biodeterioration. To establish robust biosensors for detection of fuel biocontaminants, a conserved cell surface biomarker must be identified before selection of high-affinity peptide biorecognition elements (BRE) can be screened. To address this need, we applied functional genomics and transcriptomics to characterize a conserved cell surface protein of fuel-degrading fungi, Cytochrome P450 Alkane Hydroxylase (Alk), which is highly expressed during growth in fuel and is critical for alkane degradation. Multiple conserved extracellular ALK epitopes were used to screen a phage-displayed peptide library and a single BRE (5ALK-23) was identified. Molecular and fluorescence assays demonstrated high affinity and specificity of 5ALK-23 for fungi and a low limit of detection that ranged from 1 × 101 cells/mL to 9 × 105 cells/mL. 5ALK-23 remained stable in fuel and detected both planktonic cells and biofilms. The validated 5ALK-23 BRE can be used for functionalization of biosensors for detection of fungi in fuel.