Apples and apple-based products are among the most popular foods around the world for their delightful flavors and health benefits. However, the commonly found mold,
invades wounded apples, causing ...the blue mold decay and ensuing the production of patulin, a mycotoxin that negatively affects human health. Patulin contamination in apple products has been a worldwide problem without a satisfactory solution yet. A comprehensive understanding of the factors and challenges associated with patulin accumulation in apples is essential for finding such a solution. This review will discuss the effects of the pathogenicity of
species, quality traits of apple cultivars, and environmental conditions on the severity of apple blue mold and patulin contamination. Moreover, beyond the complicated interactions of the three aforementioned factors, patulin control is also challenged by the lack of reliable detection methods in food matrices, as well as unclear degradation mechanisms and limited knowledge about the toxicities of the metabolites resulting from the degradations. As apple-based products are mainly produced with stored apples, pre- and post-harvest strategies are equally important for patulin mitigation. Before storage, disease-resistance breeding, orchard-management, and elicitor(s) application help control the patulin level by improving the storage qualities of apples and lowering fruit rot severity. From storage to processing, patulin mitigation strategies could benefit from the optimization of apple storage conditions, the elimination of rotten apples, and the safe and effective detoxification or biodegradation of patulin.
Summary
The biological detoxification of mycotoxins, including deoxynivalenol (DON), represents a very promising approach to address the challenging problem of cereal grain contamination. The recent ...discovery of Devosia mutans 17‐2‐E‐8 (Devosia spp. 17‐2‐E‐8), a bacterial isolate capable of transforming DON to the non‐toxic stereoisomer 3‐epi‐deoxynivalenol, along with earlier reports of bacterial species capable of oxidizing DON to 3‐keto‐DON, has generated interest in the possible mechanism and enzyme(s) involved. An understanding of these details could pave the way for novel strategies to manage this widely present toxin. It was previously shown that DON epimerization proceeds through a two‐step biocatalysis. Significantly, this report describes the identification of the first enzymatic step in this pathway. The enzyme, a dehydrogenase responsible for the selective oxidation of DON at the C3 position, was shown to readily convert DON to 3‐keto‐DON, a less toxic intermediate in the DON epimerization pathway. Furthermore, this study provides insights into the PQQ dependence of the enzyme. This enzyme may be part of a feasible strategy for DON mitigation within the near future.
This report describes the discovery of a dehydrogenase responsible for the selective oxidation of mycotoxin deoxynivalenol (DON) at the C3 position. The enzyme was shown to readily convert DON to 3‐keto‐DON, a less toxic intermediate in the DON epimerization pathway. This enzyme may be part of a feasible strategy for DON mitigation within the near future.
Deoxynivalenol (DON) is one of the most common mycotoxins found in cereal grains and grains contaminated with DON can cause health issues for both humans and animals and result in severe economic ...losses. Currently there is no feasible method to remediate affected grains. The development of a biological method for detoxification is becoming increasingly more plausible with the discovery of microbes which can transform DON to a relatively non-toxic stereoisomer, 3-
-DON. Although bacteria capable of detoxifying DON have been known for some time, it is only recently an enzyme responsible was identified. In
17-2-E-8 (
sp. 17-2-E-8) a two-step DON epimerization (Dep) pathway, designated as the Dep system, completes this reaction. DepA was recently identified as the enzyme responsible for the conversion of DON to 3-keto-DON, and in this report, DepB, a NADPH dependent dehydrogenase, is identified as the second and final step in the pathway. DepB readily catalyzes the reduction of 3-keto-DON to 3-
-DON. DepB is shown to be moderately thermostable as it did not lose significant activity after a heat treatment at 55°C and it is amenable to lyophilization. DepB functions at a range of pH-values (5-9) and functions equally well in multiple common buffers. DepB is clearly a NADPH dependent enzyme as it utilizes it much more efficiently than NADH. The discovery of the final step in the Dep pathway may provide a means to finally mitigate the losses from DON contamination in cereal grains through an enzymatic detoxification system. The further development of this system will need to focus on the activity of the Dep enzymes under conditions mimicking industrially relevant conditions to test their functionality for use in areas such as corn milling, fuel ethanol fermentation or directly in animal feed.
Abstract
Deoxynivalenol (DON) is a mycotoxin, produced by filamentous fungi such as
Fusarium graminearum
, that causes significant yield losses of cereal grain crops worldwide. One of the most ...promising methods to detoxify this mycotoxin involves its enzymatic epimerization to 3-
epi
-DON. DepB plays a critical role in this process by reducing 3-keto-DON, an intermediate in the epimerization process, to 3-
epi
-DON. DepB
Rleg
from
Rhizobium leguminosarum
is a member of the new aldo–keto reductase family, AKR18, and it has the unusual ability to utilize both NADH and NADPH as coenzymes, albeit with a 40-fold higher catalytic efficiency with NADPH compared to NADH. Structural analysis of DepB
Rleg
revealed the putative roles of Lys-217, Arg-290, and Gln-294 in NADPH specificity. Replacement of these residues by site-specific mutagenesis to negatively charged amino acids compromised NADPH binding with minimal effects on NADH binding. The substrate-binding site of DepB
Rleg
is larger than its closest structural homolog, AKR6A2, likely contributing to its ability to utilize a wide range of aldehydes and ketones, including the mycotoxin, patulin, as substrates. The structure of DepB
Rleg
also suggests that 3-keto-DON can adopt two binding modes to facilitate 4-
pro
-R hydride transfer to either the
re
- or
si
-face of the C3 ketone providing a possible explanation for the enzyme’s ability to convert 3-keto-DON to 3-
epi
-DON and DON in diastereomeric ratios of 67.2% and 32.8% respectively.
Summary
The fungal pathogen Fusarium pseudograminearum causes important diseases of wheat and barley. During a survey of secondary metabolites produced by this fungus, a novel class of cytokinins, ...herein termed Fusarium cytokinins, was discovered. Cytokinins are known for their growth‐promoting and anti‐senescence activities, and the production of a cytokinin mimic by what was once considered as a necrotrophic pathogen that promotes cell death and senescence challenges the simple view that this pathogen invades its hosts by employing a barrage of lytic enzymes and toxins. Through genome mining, a gene cluster in the F. pseudograminearum genome for the production of Fusarium cytokinins was identified and the biosynthetic pathway was established using gene knockouts. The Fusarium cytokinins could activate plant cytokinin signalling, demonstrating their genuine hormone mimicry. In planta analysis of the transcriptional response to one Fusarium cytokinin suggests extensive reprogramming of the host environment by these molecules, possibly through crosstalk with defence hormone signalling pathways.
DepA, a pyrroloquinoline quinone (PQQ)-dependent enzyme isolated from
mutans 17-2-E-8, exhibits versatility in oxidizing deoxynivalenol (DON) and its derivatives. This study explored DepA's substrate ...specificity and enzyme kinetics, focusing on DON and 15-acetyl-DON. Besides efficiently oxidizing DON, DepA also transforms 15-acetyl-DON into 15-acetyl-3-keto-DON, as identified via LC-MS/MS and NMR analysis. The kinetic parameters, including the maximum reaction rate, turnover number, and catalytic efficiency, were thoroughly evaluated. DepA-PQQ complex docking was deployed to rationalize the substrate specificity of DepA. This study further delves into the reduced toxicity of the transformation products, as demonstrated via enzyme homology modeling and in silico docking analysis with yeast 80S ribosomes, indicating a potential decrease in toxicity due to lower binding affinity. Utilizing the response surface methodology and central composite rotational design, mathematical models were developed to elucidate the relationship between the enzyme and cofactor concentrations, guiding the future development of detoxification systems for liquid feeds and grain processing. This comprehensive analysis underscores DepA's potential for use in mycotoxin detoxification, offering insights for future applications.
Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these ...defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes.
Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed.
Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted.
The method presented here will aid in our understanding of plant–pathogen interactions and may lead to the development of new plant protection strategies.
Patulin (PAT) is a fungal secondary metabolite which is mainly detected in rotten apples, and possesses serious risks to human health. Saccharomyces cerevisiae has become a promising biological ...control candidate due to its ability to degrade PAT. However, the degradation mechanism remains elusive. In this study, the degradation system and potential other metabolisms of PAT by S. cerevisiae were investigated. After a 96 h fermentation, 5.0 mg/L of PAT was completely metabolized by S. cerevisiae. Among the mixture of metabolites, ascladiol was identified but only accounted for 3.3% of the initial PAT. Yeast cell lysates, attained from a 96 h culture, altered 10 mg/L of PAT within 8 h. As this activity was reduced by heat treatment, it indicated that soluble intracellular enzymes were key components in PAT transformation. In addition, these effective intracellular enzymes were obtained in PAT-treated yeast cells instead of the untreated cells, indicating the activity was induced by PAT. LC-MS/MS revealed a drastic decrease of glutathione (GSH) in cell lysate upon addition of PAT. Several glutathione-patulin (GSH-PAT) conjugates (mainly c-GSH-PAT and l-GSH-PAT) were later identified. The conjugation of GSH and PAT plays an important role in PAT reduction by yeast cell lysate, even after enzymes in the lysate have been heat inactivated. Therefore, the PAT reduction by S. cerevisiae was a consequence of PAT-induced intracellular enzymes and GSH-mediated conjugation.
•Saccharomyces cerevisiae S288C could degrade patulin into ascladiol and unknown metabolites.•The soluble intracellular enzymes were important in the degradation and may not relate to ascladiol production.•The glutathione was indispensable in the detoxification by forming glutathione-patulin conjugates in vivo.
Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE ...pilus), along with a two-component system (TCS) designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS in the regulation of pilin production. Isogenic
,
, and
null mutants were generated from the parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1
mutant, while production in its complement (CP1
+) was greater than wild-type levels. In contrast, pilus production in the
and
mutants was comparable or higher than the wild type but reduced in their respective complemented strains. When examined for collagen-binding activity, the
mutant showed significantly lower binding to most collagen types (types I to V) than parental CP1 (
0.05), whereas this activity was restored in the complemented strain (
0.05). In contrast, binding of
and
mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 (
0.05), whereas the complemented strains exhibited significantly reduced binding (
0.05). These data suggest the PilRS TCS positively regulates pilus production in C. perfringens, while the Agr-like QS system may serve as a negative regulator of this operon.
Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the postantibiotic era. Multiple factors in C. perfringens, including both virulent and nonvirulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development, along with a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens. In addition, the Agr-like quorum sensing signaling pathway was found to be involved in the regulation. These findings have identified additional targets for developing nonantibiotic strategies to control NE disease.
Fusarium crown rot caused by the fungal pathogen Fusarium pseudograminearum is a disease of wheat and barley, bearing significant economic cost. Efforts to develop effective resistance to this ...disease have been hampered by the quantitative nature of resistance and a lack of understanding of the factors associated with resistance and susceptibility. Here, we aimed to dissect transcriptional responses triggered in wheat by F. pseudograminearum infection.
We used an RNA-seq approach to analyse host responses during a compatible interaction and identified >2700 wheat genes differentially regulated after inoculation with F. pseudograminearum . The production of a few key metabolites and plant hormones in the host during the interaction was also analysed.
Analysis of gene ontology enrichment showed that a disproportionate number of genes involved in primary and secondary metabolism, signalling and transport were differentially expressed in infected seedlings. A number of genes encoding pathogen-responsive uridine-diphosphate glycosyltransferases (UGTs) potentially involved in detoxification of the Fusarium mycotoxin deoxynivalenol (DON) were differentially expressed. Using a F. pseudograminearum DON-non-producing mutant, DON was shown to play an important role in virulence during Fusarium crown rot. An over-representation of genes involved in the phenylalanine, tryptophan and tyrosine biosynthesis pathways was observed. This was confirmed through metabolite analyses that demonstrated tryptamine and serotonin levels are induced after F. pseudograminearum inoculation.
Overall, the observed host response in bread wheat to F. pseudograminearum during early infection exhibited enrichment of processes related to pathogen perception, defence signalling, transport and metabolism and deployment of chemical and enzymatic defences. Additional functional analyses of candidate genes should reveal their roles in disease resistance or susceptibility. Better understanding of host responses contributing to resistance and/or susceptibility will aid the development of future disease improvement strategies against this important plant pathogen.
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