Designing cell and protein-based DON biosensors for use in investigating DON-biotransforming microbes

Abstract

Fusarium head blight (FHB) poses a major threat to small-grain cereals, reducing yield, quality, and contaminating grains with the mycotoxin deoxynivalenol (DON). Once contaminated, it is difficult and costly to remove DON from grains. New rapid and low-cost assays for detecting the presence of DON are needed in order to screen for novel DON biotransforming microbial strains. This thesis develops and evaluates two novel biosensors for detecting DON: a cell-based Escherichia coli biosensor with a GFP reporter and a protein-based sensor engineered from Fusarium graminearum acetyltransferase TRI101. The Horizon Discovery E. coli Promoter Collection was screened using fluorescence-activated cell-sorting, followed by sequencing and fluorescence plate assays for DON-responsive strains. Strain rmf (ribosome modulation factor) exhibited a reproducible increase in fluorescence (~1.3 1.4× increase) when incubated in the presence of DON. Re-engineering this strain with the brighter GFP variant mClover3 improved peak ON/OFF fluorescence ratio to ~1.6-1.7. A medium-throughput fluorescence assay was developed and tested on 142 cultured soil microbial strains. Molecular dynamics (MD) simulations were performed to analyze the behaviour of TRI101 in its apo form as well as with a DON ligand. Computational analysis of TRI101 was used to determine the best residue fluorophore conjugation sites to produce a modified TRI101 that could report the presence of DON in a solution. Amino acid residues G421 and A218 were identified as the best candidates and a TRI101-G421C was designed and expressed recombinantly in E. coli. When conjugated to TRI101-G421C, 7-diethylamino-3-(4′ maleimidylphenyl)-4-methylcoumarin (CPM) produced a detectable response to DON in fluorescence assay with a decrease of 8-9% in emission intensity at the emission maximum wavelength (480 nm). Together, these results show that an E. coli rmf promoter-based biosensor can inexpensively detect DON in liquid samples with relatively high detection sensitivity. A TRI101-based biosensor demonstrated functionality in detecting DON however assay signal contrast and production of recombinant TRI101 remain as limiting factors. Improvements to TRI101 yield, testing A218C and other TRI101 mutants for improved performance, as well as further enhancing the sensitivity and assay signal contrast for both biosensor designs should be the focus of future research.Engineering Research Council of Canada (NSERC) Discovery Grants programFebruary 202