18]) was used as the parental yeast strain to create a DON sensitive yeast strain as a tool to screen for microbial DNA fragments containing functional DON detoxifying/modifying enzymes or proteins. *S. cerevisiae* strain R2802 was kindly provided by J. Golin, The Catholic University, Washington, DC. A DON sensitive yeast strain (DSY) was created as described in [@bib1]. Briefly, *PDR5* and *PDR10* were deleted from wild-type RW2802 (these genes encode for ATP-binding cassette (ABC) multidrug resistance transporter proteins). This strain was then tested for sensitivity to 100 μg/mL DON. DSY5 as well as the parental yeast strain RW2802 were cultured in 2 mL of yeast peptone dextrose (YPD) media for 24 h at 28 °C. 10 μl samples of DSY5 and RW2802 were assayed in triplicate in a 96 well plate in 200 μl of YPD media supplemented with 100 μg/mL DON, 100 μg/mL de-epoxy-DON (DED), 50 μg/mL DON and 50 μg/mL DED, and YPD media without any toxins. The yeast was incubated for 24 h at 28 °C with shaking. Then, optical density (OD~600~) measurements were taken with a plate reader to measure growth.

2.3. Cloning into DON sensitive yeast plasmid {#sec2.3}
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Vector pCR8/GW/TOPO TA (Life Technologies, Carlsbad, CA) was combined with 5 μl of the microbial library fragments (described previously) and incubated for 30 min at room temperature before transforming into 50 μl of Clonetech® stellar competent cells (Clonetech Laboratories, Mountain View, CA). Cells were pooled and grown in 2 mL of lysogeny broth (LB) media with 50 μg/L spectinomycin. Plasmid DNA in pCR8 was recombined with pYesDEST-52 by combining 150 ng of plasmid with 50 ng of the pooled pCR8 library DNA using the Invitrogen Gateway LR Clonase II kit (Thermo Fisher Scientific, Waltham, MA) and incubated overnight at 25 °C. Aliquots of 5 μl of the transformation mixture were used to transform *E. coli* ccdB competent cells. Transformed cells were grown in 2 mL of LB media with ampicillin 