An aldo-keto reductase is responsible for Fusarium toxin-degrading activity in a soil Sphingomonas strain

Abstract

Degradation of toxins by microorganisms is a promising approach for detoxification of agricultural products. Here, a bacterial strain, Sphingomonas S3-4, that has the ability to degrade the mycotoxin deoxynivalenol (DON) was isolated from wheat fields. Incubation of Fusarium-infected wheat grains with S3-4 completely eliminated DON. In S3-4 DON is catabolized into compounds with no detectable phytotoxicity, 3-oxo-DON and 3-epi-DON, via two sequential reactions. Comparative analysis of genome sequences from two DON-degrading strains, S3-4 and Devosia D17, and one non-DON-degrading strain, Sphingobium S26, combined with functional screening of a S3-4 genomic BAC library led to the discovery that a novel aldo/keto reductase superfamily member, AKR18A1, is responsible for oxidation of DON into 3-oxo-DON. DON-degrading activity is completely abolished in a mutant S3-4 strain where the AKR18A1 gene is disrupted. Recombinant AKR18A1 protein expressed in Escherichia coli catalyzed the reversible oxidation/reduction of DON at a wide range of pH values (7.5 to 11) and temperatures (10 to 50 °C). The S3-4 strain and recombinant AKR18A1 also catabolized zearalenone and the aldehydes glyoxal and methyglyoxal. The S3-4 strain and the AKR18A1 gene are promising agents for the control of Fusarium pathogens and detoxification of mycotoxins in plants and in food/feed products.

Figure 1

The structures of DON and its metabolites. (A) The structure of DON; (B) The structure of 3-oxo-DON; (C) The structure of 3-epi-DON.

Figure 2

Growth and DON-degrading efficiency of strain S3-4. (A) Growth and degradation activity of strain S3-4 in mineral salts medium (MM) containing DON or 3A-DON (100 μg/mL). Levels of DON in the media and the amount of bacterial growth were determined every 12 h for 144 h. (B) Degradation activity of strain S3-4 in wheat grains contaminated with DON. Levels of DON were measured by HPLC at 0 h (gray bars) and after 72 h incubation (white bars) with or without S3-4. The values given are the means of three biological replicates. The error bars represent the standard deviation.

Figure 3

Chemical determination of DON and the products of DON catabolism in strain S3-4. (A) S3-4 was grown in mineral salts medium containing DON (100 μg/mL) and HPLC profiles were taken at 0 h (top panel) and 120 h (bottom panel). A representative result from three independent experiments is shown. (B) Depletion of DON and accumulation of its catabolites under the same conditions as in Fig. 2A during a period of 120 h. The values given are the means of three biological replicates. The error bars represent the standard deviation.

Figure 4

Gas chromatography (GC) and mass spectrometry (MS) of DON and its metabolites. (A) Total ion chromatogram of DON. Mass spectra for peaks representing DON are shown in the right insert. (B) Total ion chromatogram of compound A. Mass spectra for peaks representing compound A are shown in the right insert. (C) Total ion chromatogram of compound B. Mass spectra for peaks representing compound B are shown in the right insert. The structures of DON and its metabolites are shown in the left inserts.

Figure 5

Toxicity of DON, 3-oxo-DON and 3-epi-DON to wheat seedlings. (A) The lengths of wheat leaves (white bars) and coleoptiles (gray bars) 24 hours after inoculation with DON, 3-oxo-DON, 3-epi-DON and H₂O (control). (B) qRT-PCR determination of the expression levels of DON-responsive genes in the wheat samples from A. The levels of gene transcripts are calculated relative to the levels in the water inoculation sample.

Figure 6

Candidate genes for oxidation revealed by comparative genome sequence analysis. (A) The Venn diagram shows comparisons of gene numbers in the Sphingomonas sp. strain S3-4 (S3-4), Devosia sp. strain 17-2-E-8 (D17) and Sphingobium japonicum UT26 (S26). Overlapping regions represent genes common to different genomes. (B) Functional classification of the genes unique to strain S3-4 according to the COG database.

Figure 7

Functional classification of genes in one positive BAC clone isolated by functional screening. Genes functions were classified according to the COG database.

Figure 8

Expression and functional characterization of AKR18A1. (A) SDS-PAGE analysis of purified AKR18A1. Lane 1, crude extract of recombinant E. coli BL21 cells with IPTG induction; Lane 2, soluble fraction of crude extract of recombinant E. coli BL21 cells; Lane 3, purified recombinant AKR18A1. (B) HPLC profiles of 3-oxo-DON, the product of in vitro DON oxidation by recombinant AKR18A1, in the presence of cofactor NADP⁺. Insert: structure of DON and 3-oxo-DON. (C) Effect of pH (triangles), and temperature (circles) on recombinant AKR18A1 activity in the presence of NADP⁺. (D) Kinetic parameters of recombinant AKR18A1 activity in the presence of NADP⁺. The values given are the mean of three biological replicates. The error bars represent the standard deviation.

Figure 9

Proposed pathway for the transformation of DON into 3-oxo-DON and 3-epi-DON in strain S3-4.