Development of a fumonisin-sensitive Saccharomyces cerevisiae indicator strain and utilization for activity testing of candidate detoxification genes

Abstract

Fumonisins can cause diseases in animals and humans consuming Fusarium-contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food.

Keywords: S. cerevisiae; bioassay; bioindicator; mycotoxin detoxification.

FIG 1

Growth inhibition of yeast by a crude fumonisin-containing extract (“crude FB1”) on solid and in liquid media. The wild-type yeast laboratory strain YPH500 and three derived multi-deletion strains YRU74 (triple ABC transporter mutant, yor1 pdr12 snq2), YRU94ML (yor1 pdr12 snq2 plus cka2 and lcb3), and YTKT33 (yor1 pdr12 snq2 cka2 lcb3 plus vps51) were grown in complex (YPD) and synthetic (SC) media overnight and rediluted to an OD₆₀₀ of 0.1 in the morning. After reaching (OD₆₀₀ >0.3), they were diluted again for spotting on (A) YPD and (B) SC plates containing crude FB1, as indicated. (C and D) The inoculum was pipetted into a 96-well microtiter plate with different concentrations of crude FB1 and put into an incubator, where the absorbance at OD₆₀₀ was measured after 24 h. At least three replicates were used for each strain. On the x-axis, the graph shows the final FB1 concentration that the strains were exposed to, while the y-axis shows the inhibition of growth in % compared to each strain growing without exposure to FB1. The standard deviation was calculated for the replicates and displayed in the error bars.

FIG 2

Sensitivity testing with pure FB1. For strain genotypes, refer to Fig. 1 or Table 1. Growth inhibition of yeast by a 98% pure FB1 stock on solid and liquid media. The wild-type yeast laboratory strain YPH500 and three derived multi-deletion strains YRU74 (triple ABC transporter mutant, yor1 pdr12 snq2), YRU94ML (yor1 pdr12 snq2 plus cka2 and lcb3), and YTKT33 (yor1 pdr12 snq2 cka2 lcb3 plus vps51) were grown in complex (YPD) and synthetic (SC) media overnight and rediluted to an OD₆₀₀ of 0.1 in the morning. After reaching OD₆₀₀ >0.3, they were diluted again for spotting on (A) YPD and (B) SC plates containing crude FB1, as indicated. (C and D) Inoculum was pipetted into a 96-well microtiter plate with different concentrations of pure FB1 and put into an incubator, where the absorbance at OD₆₀₀ was measured after 24 h. At least three replicates were used for each strain.

FIG 3

Growth inhibition of YTKT33 in microtiter plates in SC medium by B-type fumonisins FB1-FB4. The sensitive yeast strain YTKT33 was grown in SC medium and exposed to different concentrations of FB1, FB2, FB3, and FB4. Strain inoculum was pipetted into a microtiter well plate with 0.6^N dilutions of the respective fumonisins. After 24 h at 30°C, the optical density at 600 nm (OD₆₀₀) was measured to monitor growth. The blank (medium without yeast) was subtracted from the measured OD₆₀₀ values. Means and standard deviations were calculated for four replicates each, as depicted in the error bars of the graphs.

FIG 4

Growth of YTKT33-derived strains on URA-dropout SC agar media containing increasing concentrations of FB1 carrying either the empty expression vector pYes2-P_TEF1 (negative control) or expressing one of the following genes: FUM18 (ceramide synthase from the F. verticillioides fumonisin cluster, positive control), FumD (Sphingopyxis fumonisin carboxylesterase FumD), FumD_noL (FumD lacking the N-terminal secretion signal), and AspAmOx (Aspergillus amine oxidase). Three dilutions of the transformed YTKT33 strain were spotted for each culture (OD₆₀₀ = 0.1, 0.01, and 0.001).

FIG 5

One hundred microliters of YTKT33 (OD₆₀₀ = 0.3) was embedded in SC top agar after equilibration at 48°C and left at room temperature to solidify on SC media (bottom agar). Five microliters of crude FB1 (3,180 mg/L) was added to paper discs (left), while water or FUMzyme was added on the right.