Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

Abstract

c Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (k cat /K m ) of 6.4 mM ؊1 s ؊1 . The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

Deoxynivalenol: Masked forms, fate during food processing, and potential biological remedies

“…However, purifying detoxifying enzymes presents a challenge and is a time-consuming process. Ito et al (2013) reconstructed a cytochrome P450 system from the Sphingomonas sp. strain KSM1 in vitro and demon-strated its ability to hydroxylate DON and 3-acetyl DON.…”

Dual Function of a Novel Bacterium, Slackia sp. D-G6: Detoxifying Deoxynivalenol and Producing the Natural Estrogen Analogue, Equol

“…To isolate the bacterium that converts DON to DOM-1, a modified screening procedure ( Figure 1a) was set up based on a previous method [28]. After screening and four passages of the subculture, one single colony, Slackia sp.…”

A Novel Angular Dioxygenase Gene Cluster Encoding 3-Phenoxybenzoate 1′,2′-Dioxygenase in Sphingobium wenxiniae JZ-1

“…The HindIII-SacI-digested fusion PCR product was cloned into the corresponding sites of pBBR1MCS-5 to produce pBBRA1A2BT7. E. coli BL21(DE3) harboring pBBRA1A2BT7 was grown in 100 ml LB broth at 37°C to an optical density at 600 nm of 0.6, and 0.5 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) and 0.5 mM FeCl 3 were then added (27). After 12 h of incubation at 16°C, the cells were harvested by centrifugation and subjected to whole-cell transformation according to the method described above.…”