A quinone-dependent dehydrogenase and two NADPH-dependent aldo/keto reductases detoxify deoxynivalenol in wheat via epimerization in a Devosia strain
Introduction
Deoxynivalenol (DON), or vomitoxin, is the most widely distributed mycotoxin worldwide. It is associated primarily with the Fusarium head blight (FHB) fungal plant pathogen, which produces it during infection and disease development in wheat and other cereals including barley, maize, oats, and rye (Bai & Shaner, 2004). FHB epidemics in wheat and barley can cause huge losses in the field and during storage; historically, these epidemics have occurred frequently in central China and in recent decades they have caused huge yield reductions in North America and Europe (Bai and Shaner, 2004, Chen et al., 2000). Cereals contaminated with DON can enter human and animal food chains (Lancova et al., 2008), posing potentially serious negative consequences for human and animal health because of its various toxic effects (Pestka, 2010). DON is also phytotoxic: it damages tissues, ruins grains, and acts as a virulence factor that stimulates fungal colonization on plants (Bai & Shaner, 2004). Despite these ongoing impacts, plant genetic resources against DON and DON-producing pathogens are inadequate. Current control measures rely on applying fungicides in the field, resulting in fungicide-tolerant strains and an increase in DON biosynthesis (Audenaert et al., 2010, Chen et al., 2007). Thus, alternatives are needed to control mycotoxins in agricultural products.
Microbial and enzymatic detoxification is a promising approach for managing DON contamination. The DON C3 hydroxyl moiety was discovered to be a major determinant of toxicity (Ikunaga et al., 2011, Shima et al., 1997). This led to the proposal of a novel detoxification pathway: DON epimerization, in which DON is catabolized into 3-keto-deoxynivalenol (3-keto-DON) and 3-epi-deoxynivalenol (3-epi-DON) (Karlovsky, 2011). Recent studies revealed these resulting compounds exhibit little to no toxicity: 3-keto-DON has 10-fold lower toxicity than DON, while the toxicity of 3-epi-DON is 1181-fold lower, compared with that of DON (He et al., 2015, He et al., 2015, He et al., 2017, Pierron et al., 2016, Shima et al., 1997). Multiple bacterial species have been isolated that can convert DON to 3-keto-DON and 3-epi-DON (He et al., 2016, He et al., 2017, Ikunaga et al., 2011, Sato et al., 2012, Wang et al., 2017). DON epimerization is completed via two sequential reactions: DON oxidation forms the intermediate 3-keto-DON, and reduction of 3-keto-DON forms 3-epi-DON (He et al., 2016, He et al., 2017). Two different enzymes have been previously identified that catabolize DON into 3-keto-DON: an aldo/keto reductase (AKR) encoded by AKR18A1 that was cloned from the bacterium Sphingomonas sp. strain S3-4 which oxidizes DON to form 3-keto-DON (He et al., 2017), and a quinoprotein, DepA, encoded by the depA gene from Devosia mutans 17-2-E-8, which also converts DON into 3-keto-DON (Carere, Hassan, Lepp, & Zhou, 2017). An AKR protein, DepB, was also recently identified in D. mutans 17-2-E-8 that reduces 3-keto-DON into 3-epi-DON (Carere, Hassan, Lepp, & Zhou, 2018). However, recombinant enzymes from these two sequential reactions have not been shown to work together to complete the DON epimerization pathway.
AKRs are widely distributed across all phyla (Penning, 2015). This protein superfamily is subdivided into 18 distinct families (He et al., 2017, Penning, 2015). Most AKRs have a wide substrate spectrum, typically catalyzing the reversible reduction of multiple aldehyde- and/or ketone-containing compounds (Ellis, 2002, Penning, 2015, Simpson et al., 2009). AKR18A1, recently identified in Sphingomonas sp. strain S3-4, is the first AKR demonstrated to be capable of catabolizing DON to form 3-keto-DON (He et al., 2017). A nicotinamide adenine dinucleotide phosphate (NADPH)-dependent AKR, DepB, was recently reported to biotransform 3-keto-DON into 3-epi-DON in D. mutans 17-2-E-8 (Carere et al., 2018). However, the application of recombinant enzymes to degrade DON present in agricultural products has not yet been demonstrated.
Dehydrogenases (DH) are a widespread subclass of oxidoreductase enzymes that oxidize substrates by reducing an electron acceptor, and are biologically important because they facilitate the conversion of alcohols or aldehydes. Pyrroloquinoline quinone (PQQ)-dependent dehydrogenases are quinoenzymes that localize in the bacterial periplasm and rapidly catalyze the oxidation of multiple alcohols, aldose sugars, and molecules that are harmful to cells (Matsushita, 2002, Toyama et al., 2004). However, reports on the detoxification of mycotoxins by quinoproteins are extremely rare. Only one quinoprotein from D. mutans 17-2-E-8 has been demonstrated to show DON oxidation activity (Carere et al., 2017). No studies have yet been reported that either describe molecular docking of DHs or identify which amino acids in DHs essential for interactions with DON.
In this study, genes encoding a quinone-dependent DON dehydrogenase (QDDH) and two AKRs were cloned from the highly active DON-degrading bacterium Devosia sp. strain D6-9. Affinity-purified, recombinant proteins efficiently degraded DON in wheat grains. We performed targeted mutagenesis on residues necessary for substrate binding, and found that in vitro QDDH detoxification activity was not detected for the QDDH protein triple mutant S497M/E499R/E535R. These results provide a foundation to advance the potential for enzymatic elimination of DON in agricultural samples. This work also reveals insights into the interactions between detoxifying enzymes and DON.
Section snippets
Soil, chemicals, and media
Soil was collected from a wheat field at Huazhong Agricultural University in Wuhan, China and these samples were used to isolate bacteria capable of DON biotransformation. Standard DON, 3-keto-DON, and 3-epi-DON were prepared as previously described (He et al., 2017). The capacity for DON degradation by microbial cultures was screened on minimum media (MM) supplemented with DON (Ikunaga et al., 2011). Nutrient agar (NA) was used to isolate single colonies.
Enrichment, isolation, and taxonomic characterization of bacteria with DON-transformation activity
Five grams of soil were suspended in
A highly active Devosia strain catabolizes DON into 3-keto-DON and 3-epi-DON
An aerobic bacterium was isolated from soil, shown to have high DON-degrading activity, and phylogenetically identified as a species of Devosia (Fig. S1), designated Devosia sp. D6-9. This strain completely degraded 500 μg/mL DON in media in 2 h when the cells had an OD595nm value of 0.05 (1 OD ≈ 5 × 109 cells) (Fig. 1A). Furthermore, DON was completely eliminated in 2 min when strain D6-9 cells reached 2 at OD595nm. Thus, strain D6-9 displays extremely high DON-degrading activity, with a rate
Discussion
The high DON-degrading activity of strain D6-9 shown in this study may be related to the environment where the strain was isolated and the method used for enrichment. Strain D6-9 had 50-fold higher activity than another well-characterized Devosia strain 17-2-E-8 (3 μg/h/108 cells) (He et al., 2016) and 180-fold higher activity than Sphingomonas sp. strain S3-4 (0.8 μg/h/108 cells) (He et al., 2017). D6-9 was isolated from wheat soil in Wuhan, located in the middle valley of the Yangtze river in
Conclusions
A new soil bacterium, Devosia sp. strain D6-9, that degrades DON at 2.5 μg/min/108 cells was isolated. Three genes responsible for DON degradation were cloned and heterologously expressed. We further demonstrated the ability of recombinant enzymes to eliminate DON from contaminated agricultural products and therefore established the basis for research into utilization of enzymes as an agent for control of DON. The key residues identified in the dehydrogenases may serve as a foundation to
Author contributions
Y.C.L. and J.B.Z. conceived, designed, and supervised the project. W.J.H. performed the biological and bioinformation studies. M.M.S. performed HPLC and GC–MS analysis. P.Y., T.H., Y.Z., A.B.W., W.B.D. and H.P.L. contributed reagents/materials. Y.C.L., J.B.Z. and W.J.H. wrote the paper. All authors reviewed the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This research was supported by the Ministry of Agriculture of China (2016ZX08002001-003), the National Key R&D Program of China (2016YFE0112900, 2018YFD02005), the National Natural Science Foundation of China (31901802), China Postdoctoral Science Foundation (2018M642805) and the Fundamental Research Funds for the Central Universities (2662016PY076).
References (40)
Microbial aldo-keto reductases
FEMS Microbiology Letters
(2002)- et al.
Toxicology of 3-epi-deoxynivalenol, a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8
Food and Chemical Toxicology
(2015) - et al.
Protective effects of Devosia sp. ANSB714 on growth performance, immunity function, antioxidant capacity and tissue residues in growing-finishing pigs fed with deoxynivalenol contaminated diets
Food and Chemical Toxicology
(2018) The aldo-keto reductases (AKRs): Overview
Chemico-Biological Interactions
(2015)- et al.
Characterization of two novel aldo-keto reductases from Arabidopsis: Expression patterns, broad substrate specificity, and an open active-site structure suggest a role in toxicant metabolism following stress
Journal of Molecular Biology
(2009) - et al.
Quinohemoprotein alcohol dehydrogenases: Structure, function, and physiology
Archives of Biochemistry and Biophysics
(2004) - et al.
Protective effect of Devosia sp. ANSB714 on growth performance, serum chemistry, immunity function and residues in kidneys of mice exposed to deoxynivalenol
Food and Chemical Toxicology
(2016) - et al.
Strategies and methodologies for developing microbial detoxification systems to mitigate mycotoxins
Toxins
(2017) - et al.
Hydrogen peroxide induced by the fungicide prothioconazole triggers deoxynivalenol (DON) production by Fusarium graminearum
BMC Microbiology
(2010) - et al.
Management and resistance in wheat and barley to Fusarium head blight
Annual Review of Phytopathology
(2004)
The enzymatic detoxification of the mycotoxin deoxynivalenol: Identification of DepA from the DON epimerization pathway
Microbial Biotechnology
The identification of DepB: An enzyme responsible for the final detoxification step in the deoxynivalenol epimerization pathway in Devosia mutans17-2-E-8
Frontiers in Microbiology
Characterization and fitness of carbendazim-resistant strains of Fusarium graminearum (wheat scab)
Pest Management Science
Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data
Nature Methods
Improved microbial gene identification with GLIMMER
Nucleic Acids Research
The interaction pattern between a homology model of 40S ribosomal S9 protein of Rhizoctonia solani and 1-hydroxyphenaize by docking study
Biomed Research International
The enzymatic epimerization of deoxynivalenol by Devosia mutans proceeds through the formation of 3-keto-DON intermediate
Scientific Reports
Draft genome sequences of Devosia sp. strain 17-2-E-8 and Devosia riboflavina strain IFO13584
Genome Announcements
Bacterial epimerization as a route for deoxynivalenol detoxification: The influence of growth and environmental conditions
Frontiers in Microbiology
Cited by (51)
A comprehensive review of biodetoxification of trichothecenes: Mechanisms, limitations and novel strategies
2024, Food Research InternationalEngineering substrate specificity of quinone-dependent dehydrogenases for efficient oxidation of deoxynivalenol to 3-keto-deoxynivalenol
2024, International Journal of Biological MacromoleculesSelf-cascade deoxynivalenol detoxification by an artificial enzyme with bifunctions of dehydrogenase and aldo/keto reductase from genome mining
2024, International Journal of Biological MacromoleculesDeoxynivalenol: Occurrence, toxicity, and degradation
2024, Food Control