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

Abstract

ABSTRACT Fumonisins, produced mainly by various Fusarium species, are sphinganine-analog mycotoxins and potent inhibitors of ceramide synthases in animals, plants, and other eukaryotes, leading to toxicity and programmed cell death. Wild-type baker’s yeast is naturally highly resistant to fumonisin B 1 (FB1); on rich medium, it tolerates more than 1,000 µM. We report the construction of a fumonisin-sensitive Saccharomyces cerevisiae strain with six disrupted genes: SNQ2 , PDR12 , and YOR1 , coding for ATP-binding cassette transporters involved in active efflux; CKA2 , encoding casein kinase II, regulating ceramide synthase activity by phosphorylation; and LCB3 and VPS51 , encoding proteins involved in the biosynthesis and recycling of sphingolipid precursors, respectively. The constructed FB1-sensitive strain YTKT33 was tested on complex yeast extract-peptone-dextrose (YPD) and synthetic complete medium alongside the wild-type strain and strains with only three (YRU74) or five (YRU94ML) disrupted genes. YTKT33, exhibiting full growth inhibition at a concentration below 5 µM FB1 on synthetic complete agar lacking uracil (SC-URA), was used as a host for heterologous expression of previously reported fumonisin detoxification genes. Transformants expressing either the Sphingopyxis fumD esterase, which removes removing tricarballylic acid side chains, or an Aspergillus amine oxidase, which converts the amino- to a keto-group, can grow at FB1 concentrations inhibitory for transformants containing the empty vector. YTKT33 was inhibited by other type B fumonisins (B 2 , B 3 , B 4 ) at concentrations very similar to FB1, allowing rapid evaluation of total fumonisin toxicity in crude fungal extracts. IMPORTANCE 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.