Two different types of hydrolases co-degrade ochratoxin A in a highly efficient degradation strain Lysobacter sp. CW239.

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 µg/L OTA was completely degraded by 1.0 µg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-ß-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.

Glucose and HODEs regulate Aspergillus ochraceus quorum sensing through the GprC-AcyA pathway.

Aspergillus ochraceus is the traditional ochratoxin A (OTA)-producing fungus with density-dependent behaviors, which is known as quorum sensing (QS) that is mediated by signaling molecules. Individual cells trend to adapt environmental changes in a "whole" flora through communications, allowing fungus to occupy an important ecological niche. Signals perception, transmission, and feedback are all rely on a signal network that constituted by membrane receptors and intracellular effectors. However, the interference of density information in signal transduction, which regulates most life activities of Aspergillus, have yet to be elucidated. Here we show that the G protein-coupled receptor (GPCR) to cAMP pathway is responsible for transmitting density information, and regulates the key point in life cycle of A. ochraceus. Firstly, the quorum sensing phenomenon of A. ochraceus is confirmed, and identified the density threshold is 103 spores/mL, which represents the low density that produces the most OTA in a series quorum density. Moreover, the GprC that classified as sugar sensor, and intracellular adenylate cyclase (AcyA)-cAMP-PKA pathway that in response to ligands glucose and HODEs are verified. Furthermore, GprC and AcyA regulate the primary metabolism as well as secondary metabolism, and further affects the growth of A. ochraceus during the entire life cycle. These studies highlight a crucial G protein signaling pathway for cell communication that is mediated by carbohydrate and oxylipins, and clarified a comprehensive effect of fungal development, which include the direct gene regulation and indirect substrate or energy supply. Our work revealed more signal molecules that mediated density information and connected effects on important adaptive behaviors of Aspergillus ochraceus, hoping to achieve comprehensive prevention and control of mycotoxin pollution from interrupting cell communication.

Biodegradation of ochratoxin A by Brevundimonas diminuta HAU429: Characterized performance, toxicity evaluation and functional enzymes.

Ochratoxin A (OTA) is a notorious mycotoxin commonly contaminating food products worldwide. In this study, an OTA-degrading strain Brevundimonas diminuta HAU429 was isolated by using hippuryl-L-phenylalanine as the sole carbon source. The biodegradation of OTA by strain HAU429 was a synergistic effect of intracellular and extracellular enzymes, which transformed OTA into ochratoxin α (OTα) through peptide bond cleavage. Cytotoxicity tests and cell metabolomics confirmed that the transformation of OTA into OTα resulted in the detoxification of its hepatotoxicity since OTA but not OTα disturbed redox homeostasis and induced oxidative damage to hepatocytes. Genome mining identified nine OTA hydrolase candidates in strain HAU429. They were heterologously expressed in Escherichia coli, and three novel amidohydrolase BT6, BT7 and BT9 were found to display OTA-hydrolyzing activity. BT6, BT7 and BT9 showed less than 45 % sequence identity with previously identified OTA-degrading amidohydrolases. BT6 and BT7 shared 60.9 % amino acid sequence identity, and exhibited much higher activity towards OTA than BT9. BT6 and BT7 could completely degrade 1 µg mL-1 of OTA within 1 h and 50 min, while BT9 hydrolyzed 100 % of OTA in the reaction mixture by 12 h. BT6 was the most thermostable retaining 38 % of activity after incubation at 70 °C for 10 min, while BT7 displayed the highest tolerance to ethanal remaining 76 % of activity in the presence of 6 % ethanol. This study could provide new insights towards microbial OTA degradation and promote the development of enzyme-catalyzed OTA detoxification during food processing.

Structural and functional analysis of the key enzyme responsible for the degradation of ochratoxin A in the Alcaligenes genus.

The potential to degrade ochratoxin A (OTA), a highly poisonous mycotoxin, was investigated in cultures from Alcaligenes-type strains. Genome sequence analyses from different Alcaligenes species have permitted us to demonstrate a direct, causal link between the gene coding a known N-acyl-L-amino acid amidohydrolase from A. faecalis (AfOTH) and the OTA-degrading activity of this bacterium. In agreement with this finding, we found the gene coding AfOTH in two additional species included in the Alcaligenes genus, namely, A. pakistanensis, and A. aquatilis, which also degraded OTA. Notably, A. faecalis subsp. faecalis DSM 30030T was able to transform OTα, the product of OTA hydrolysis. AfOTH from A. faecalis subsp. phenolicus DSM 16503T was recombinantly over-produced and enzymatically characterized. AfOTH is a Zn2+-containing metalloenzyme that possesses structural features and conserved residues identified in the M20D family of enzymes. AfOTH is a tetramer in solution that shows both aminoacylase and carboxypeptidase activities. Using diverse potential substrates, namely, N-acetyl-L-amino acids and carbobenzyloxy-L-amino acids, a marked preference towards C-terminal Phe and Tyr residues could be deduced. The structural basis for this specificity has been determined by in silico molecular docking analyses. The amidase activity of AfOTH on C-terminal Phe residues structurally supports its OTA and OTB degradation activity.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Identification of genes showing altered DNA methylation and gene expression in the renal proximal tubular cells of rats treated with ochratoxin A for 13 weeks.

Ochratoxin A (OTA) is a mycotoxin that causes renal carcinogenicity following the induction of karyomegaly in proximal tubular cells after repeated administration to rats. Here, we performed gene profiling regarding altered DNA methylation and gene expression in the renal tubules focusing on the mechanism of OTA-induced carcinogenesis. For this purpose, OTA or 3-chloro-1,2-propanediol (3-MCPD), a renal carcinogen not inducing karyomegaly, was administered to rats for 13 weeks, and DNA methylation array and RNA sequencing analyses were performed on proximal tubular cells. Genes for which OTA altered the methylation status and gene expression level, after excluding genes showing similar expression changes by 3-MCPD, were subjected to confirmation analysis of the transcript level by real-time reverse-transcription PCR. Gene Ontology (GO)-based functional annotation analysis of validated genes revealed a cluster of hypermethylated and downregulated genes enriched under the GO term "mitochondrion," such as those associated with metabolic reprogramming in carcinogenic process (Clpx, Mrpl54, Mrps34, and Slc25a23). GO terms enriched for hypomethylated and upregulated genes included "response to arsenic-containing substance," represented by Cdkn1a involved in cell cycle arrest, and "positive regulation of IL-17 production," represented by Osm potentiating cell proliferation promotion. Other genes that did not cluster under any GO term included Lrrc14 involved in NF-κB-mediated inflammation, Gen1 linked to DNA repair, Has1 related to chromosomal aberration, and Anxa3 involved in tumor development and progression. In conclusion, a variety of genes engaged in carcinogenic processes were obtained by epigenetic gene profiling in rat renal tubular cells specific to OTA treatment for 13 weeks.

The Effect of Mushroom Culture Filtrates on the Inhibition of Mycotoxins Produced by Aspergillus flavus and Aspergillus carbonarius.

Two of the mycotoxins of greatest agroeconomic significance are aflatoxin B1 (AFB1), and ochratoxin A (OTA). It has been reported that extracts from some wood-decaying mushrooms, such as Lentinula edodes and Trametes versicolor showed the ability to inhibit AFB1 or OTA biosynthesis. Therefore, in our study, a wide screening of 42 isolates of different ligninolytic mushrooms was assayed for their ability to inhibit the synthesis of OTA in Aspergillus carbonarius and AFB1 in Aspergillus flavus, in order to find a metabolite that can simultaneously inhibit both mycotoxins. The results showed that four isolates produce metabolites able to inhibit the synthesis of OTA, and 11 isolates produced metabolites that inhibited AFB1 by >50%. Two strains, the Trametes versicolor strain TV117 and the Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. Preliminary results suggest that the mechanism of efficacy of the S. commune rough and semipurified polysaccharides could be analogous to that found previously for Tramesan®, by enhancing the antioxidant response in the target fungal cells. The overall results indicate that S. commune's polysaccharide(s) could be a potential agent(s) in biological control and/or a useful component of the integrated strategies able to control mycotoxin synthesis.

Interacted toxic mechanisms of ochratoxin A and tricyclazole on the zebrafish (Danio rerio).

Despite the current efforts to identify the mixtures of chemical pollutants, they are often "binned" into their corresponding pollutant groups. Limited studies have investigated complex mixtures of chemical pollutants co-occurring across different groups. The combined toxic impacts of several substances become a critical consideration in toxicology because chemical combinations can exert a greater deleterious effect than the single components in the mixture. In the current work, we assessed the joint impacts of ochratoxin A and tricyclazole on the zebrafish (Danio rerio) embryos and explored their underlying signaling pathways. Ochratoxin A displayed higher toxicity than tricyclazole, with a 10-day LC50 of 0.16 mg L-1, whereas that for tricyclazole was 1.94 mg L-1. The combination of ochratoxin A and tricyclazole exhibited a synergistic impact on D. rerio. The activities of detoxification enzymes GST and CYP450, as well as apoptosis-associated enzyme caspase 3, were distinctly changed in most individual and mixture exposures comparing to the untreated group. Upon both individual and mixture exposures, more dramatic variations were detected in the expressions of nine genes, such as the apoptosis genes cas3 and bax, antioxidant gene mn-sod, immunosuppression gene il-1ß, and the endocrine system genes trα, dio1, trß, ugtlab, and crh, compared with the untreated group. These findings suggested that the simultaneous exposure to low doses of mycotoxins and pesticides in food commodities was more toxic than predicted from the individual chemicals. Considering the frequent co-occurrence of mycotoxins and pesticides in the diet, this synergy should be considered in future assessments.

Toxicokinetics of a Single Oral Dose of OTA on Dezhou Male Donkeys.

Ochratoxin (OTA) is widely present in a wide range of foods and feeds, causing adverse effects on animals and humans. This study aims to explore the toxicokinetics of OTA-contaminated materials on the Dezhou male donkey. Donkeys received a single orally dose of 2500 µg OTA/kg BW, obtained from Aspergillus ochraceus culture material. The concentrations of OTA in plasma collected at 0, 5, 10, 15, 20, 30, 45 min, and at 1, 1.5, 2, 3, 6, 9, 12, 24, 48, 72, 96 and 120 h were detected by HPLC. OTA eliminated in urine and feces were quantified at 6-h intervals up to 24 h and then at 4-h intervals up to 120 h. The results suggested that the maximum concentration of OTA in plasma was observed at 12 h after administration, with a mean value of 10.34 µg/mL. The total excretion in both urine and feces was about 10% of the intake until 120 h.

AnAzf1 acts as a positive regulator of ochratoxin A biosynthesis in Aspergillus niger.

Aspergillus niger produces genotoxic and carcinogenic ochratoxin A (OTA) that severely threatens human and animal health. Transcription factor Azf1 is essential in regulating fungal cell development and primary metabolism. However, its effect and mechanism on secondary metabolism are unclear. Here, we characterized and deleted a Azf1 homolog gene, An15g00120 (AnAzf1), in A. niger, which completely blocked OTA production, and repressed the OTA cluster genes, p450, nrps, hal, and bzip at the transcriptional level. The results indicated that AnAzf1 was a positive regulator of OTA biosynthesis. Transcriptome sequencing results showed that the AnAzf1 deletion significantly upregulated antioxidant genes and downregulated oxidative phosphorylation genes. Enzymes involved in reactive oxygen species (ROS) scavenging, including catalase (CAT) and peroxidase (POD) were increased, and the corresponding ROS levels were decreased. Upregulation of genes (cat, catA, hog1, and gfd) in the MAPK pathway and downregulation of genes in iron homeostasis were associated with decreased ROS levels, linking the altered MAPK pathway and iron homeostasis to lower ROS levels caused by AnAzf1 deletion. Additionally, enzymes including complex I (NADH-ubiquinone oxidoreductase), and complex V (ATP synthase), as well as ATP levels, were significantly decreased, indicating impaired oxidative phosphorylation caused by the AnAzf1-deletion. During lower ROS levels and impaired oxidative phosphorylation, OTA was not produced in ∆AnAzf1. Together, these results strongly suggested that AnAzf1 deletion blocked OTA production in A. niger by a synergistic interference of ROS accumulation and oxidative phosphorylation. KEY POINTS: • AnAzf1 positively regulated OTA biosynthesis in A. niger. • Deletion of AnAzf1 decreased ROS levels and impaired oxidative phosphorylation. • An altered MAPK pathway and iron homeostasis were associated with lower ROS levels.

Isolation, identification and safety evaluation of OTA-detoxification strain Pediococcus acidilactici NJB421 and its effects on OTA-induced toxicity in mice.

Ochratoxin A (OTA) is a potent mycotoxin found in foods and feeds, posing a health risk to animals and humans. Biological detoxification of OTA is considered a promising method, and some bacteria and fungi which can degrade OTA are isolated. However, research on safety and alleviating toxic effects are scarce. This study aims to isolate OTA-detoxification probiotics from natural samples and evaluate their safety and protective effects in mice. Here, a new OTA-detoxification strain named Pediococcus acidilactici NJB421 (P. acidilactici NJB421) was isolated from cow manure, which exhibited a removal rate of OTA at 48.53% for 48 h. P. acidilactici NJB421 exhibited high temperature resistance, acid tolerance, 0.3% bile salt and 1.4% trypsin resistance. The safety evaluation showed that P. acidilactici NJB421 at 2 × 108 CFU/per mouse had no abnormalities in body weight, organ indices, ALT, AST and ALP activities, BUN, CRE and TP contents. And P. acidilactici NJB421 alleviated the decreases in body weight, organ indices and small intestinal length, and alleviated intestinal injury, liver injury and kidney injury. These results suggest P. acidilactici NJB421 is safe and has protection against OTA poisoning, which provides a new OTA-detoxification strain for livestock and food industries.

Active packaging of poly(lactic acid) nanofibers and essential oils with antifungal action on table grapes.

The table grape is a non-climateric fruit that is very susceptible to fungal contamination, in addition to suffering an accelerated loss of quality during storage. The in vitro and in grape antifungal and antiocratoxigenic effects of the essential oils from Alpinia speciosa and Cymbopogon flexuosus against Aspergillus carbonarius and Aspergillus niger were studied. The oils were encapsulated in poly(lactic acid) (PLA) nanofibers as a potential active packaging to be applied to control the degradation of grapes stored during the post-harvest period. Fungal proliferation and ochratoxin A synthesis in A. carbonarius and A. niger decreased in the presence of the active packaging. However, the nanofiber containing the essential oil from C. flexuosus was more efficient in providing a fungicidal effect against A. carbonarius (10% and 20%) and A. niger (20%). In addition, weight loss and color changes were controlled and the parameters of acidity, °Brix, softening and the texture of the grape were maintained. A very small mass loss of the essential oils encapsulated in nanofibers was observed by thermogravimetric analysis, showing that the nanofiber was efficient in enabling the controlled release. The quality and safety of table grapes were maintained for longer periods of storage in the presence of active packaging, so the incorporation of these oils in nanofibers can be a promising way to increase the shelf life of grapes.

Biodegradation of ochratoxin A by endophytic Trichoderma koningii strains.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus spp. and Penicillium spp. that causes a threat to food safety and human health. Fungal biodegradation might be a promising strategy for reducing the OTA contamination in the future. In this study, the ability of Trichoderma koningii strains to degrade OTA produced by Aspergillus niger T2 (MW513392.1) isolated from tomato seeds was investigated. Among T. koningii strains tested, three strains; AUMC11519, AUMC11520 and AUMC11521 completely eliminated OTA from the culture medium, while AUMC11522 strain eliminated only 41.82% of OTA. OTα-amide, 3-phenylpropionic acid, OTα and phenylalanine were assayed as degradation products by FTIR analysis and LC-MS/MS spectra. Carboxypeptidase A (CPA) was found responsible for OTA degradation when a metal ion chelator, EDTA, was added to cell free supernatants of the three effective strains. OTA detoxification by T. koningii could present new prospective strategies for a possible application in food commodities intoxicated with ochratoxin.

Ochratoxin A Defective Aspergillus carbonarius Mutants as Potential Biocontrol Agents.

Aspergillus carbonarius is one of the main species responsible for wine, coffee and cocoa toxin contamination. The main mycotoxin produced by this fungus, ochratoxin A (OTA), is a secondary metabolite categorized as a possible carcinogen because of its significant nephrotoxicity and immunosuppressive effects. A polyketide synthase gene (otaA) encodes the first enzyme in the OTA biosynthetic pathway. It is known that the filamentous fungi, growth, development and production of secondary metabolites are interconnected processes governed by global regulatory factors whose encoding genes are generally located outside the gene clusters involved in the biosynthesis of each secondary metabolite, such as the veA gene, which forms part of the VELVET complex. Different fungal strains compete for nutrients and space when they infect their hosts, and safer non-mycotoxigenic strains may be able to outcompete mycotoxigenic strains during colonization. To determine the possible utility of biopesticides based on the competitive exclusion of mycotoxigenic strains by non-toxigenic ones, we used A. carbonarius ΔotaA and ΔveA knockout mutants. Our results showed that during both in vitro growth and infection of grapes, non-mycotoxigenic strains could outcompete the wild-type strain. Additionally, the introduction of the non-mycotoxigenic strain led to a drastic decrease in OTA during both in vitro growth and infection of grapes.

Unveiling ochratoxin a controlling and biodetoxification molecular mechanisms: Opportunities to secure foodstuffs from OTA contamination.

Anarchic growth of ochratoxin A (OTA) producing fungi during crop production, prolonged storage, and processing results in OTA contamination in foodstuffs. OTA in food exacerbates the risk of health and economic problems for consumers and farmers worldwide. Although the toxic effects of OTA on human health have not been well established, comprehensive preventive and remedial measures will be essential to eliminate OTA from foodstuffs. Strict regulations, controlling OTA at pre- or post-harvest stage, and decontamination of OTA have been adopted to prevent human and animal OTA exposure. Biological control of OTA and bio-decontamination are the most promising strategies due to their safety, specificity and nutritional value. This review addresses the current understanding of OTA biodegradation mechanisms and recent developments in OTA control and bio-decontamination strategies. Additionally, this review analyses the strength and weaknesses of different OTA control methods and the contemporary approaches to enhance the efficiency of biocontrol agents. Overall, this review will support the implementation of new strategies to effectively control OTA in food sectors. Further studies on efficacy-related issues, production issues and cost-effectiveness of OTA biocontrol are to be carried out to improve the knowledge, develop improved delivery technologies and safeguard the durability of OTA biocontrol approaches.

The Neurotoxic Effect of Ochratoxin-A on the Hippocampal Neurogenic Niche of Adult Mouse Brain.

Ochratoxin A (OTA) is a common secondary metabolite of Aspergillus ochraceus, A. carbonarius, and Penicillium verrucosum. This mycotoxin is largely present as a contaminant in several cereal crops and human foodstuffs, including grapes, corn, nuts, and figs, among others. Preclinical studies have reported the involvement of OTA in metabolic, physiologic, and immunologic disturbances as well as in carcinogenesis. More recently, it has also been suggested that OTA may impair hippocampal neurogenesis in vivo and that this might be associated with learning and memory deficits. Furthermore, aside from its widely proven toxicity in tissues other than the brain, there is reason to believe that OTA contributes to neurodegenerative disorders. Thus, in this present in vivo study, we investigated this possibility by intraperitoneally (i.p.) administering 3.5 mg OTA/kg body weight to adult male mice to assess whether chronic exposure to this mycotoxin negatively affects cell viability in the dentate gyrus of the hippocampus. Immunohistochemistry assays showed that doses of 3.5 mg/kg caused a significant and dose-dependent reduction in repetitive cell division and branching (from 12% to 62%). Moreover, the number of countable astrocytes (p < 0.001), young neurons (p < 0.001), and mature neurons (p < 0.001) negatively correlated with the number of i.p. OTA injections administered (one, two, three, or six repeated doses). Our results show that OTA induced adverse effects in the hippocampus cells of adult mice brain tissue when administered in cumulative doses.

Protective Effect of SeMet on Liver Injury Induced by Ochratoxin A in Rabbits.

Ochratoxin A (OTA) is second only to aflatoxin in toxicity among mycotoxins. Recent studies have shown that selenomethionine (SeMet) has a protective effect on mycotoxin-induced toxicity. The purpose of this study was to investigate the protective effect and mechanism of SeMet on OTA-induced liver injury in rabbits. Sixty 35-day-old rabbits with similar body weight were randomly divided into five groups: control group, OTA group (0.2 mg/kg OTA), OTA + 0.2 mg/kg SeMet group, OTA + 0.4 mg/kg SeMet group and OTA + 0.6 mg/kg SeMet group. Rabbits were fed different doses of the SeMet diet for 21 d, and OTA was administered for one week from day 15 (the control group was provided the same dose of NaHCO3 solution). The results showed that 0.4 mg/kg SeMet could significantly improve the liver injury induced by OTA poisoning. SeMet supplementation can improve the changes in physiological blood indexes caused by OTA poisoning in rabbits and alleviate pathological damage to the rabbit liver. SeMet also increased the activities of SOD, GSH-Px and T-AOC and significantly decreased the contents of ROS, MDA, IL-1ß, IL-6 and TNF-α, effectively alleviating the oxidative stress and inflammatory response caused by OTA poisoning. In addition, OTA poisoning inhibits Nrf2 and HO-1 levels, ultimately leading to peroxide reaction, while SeMet activates the Nrf2 signaling pathway and enhances the expression of the HO-1 downstream Nrf2 gene. These results suggest that Se protects the liver from OTA-induced hepatotoxicity by regulating Nrf2/HO-1 expression.

Isoenzyme N-Acyl-l-Amino Acid Amidohydrolase NA Increases Ochratoxin A Degradation Efficacy of Stenotrophomonas sp. CW117 by Enhancing Amidohydrolase ADH3 Stability.

Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. OTA detoxification strategies are significant to food safety. A superefficient enzyme ADH3 to OTA hydrolysis was isolated from the difunctional strain Stenotrophomonas sp. CW117 in our previous study. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA, which is an isoenzyme of ADH3. However, it is not as efficient a hydrolase as ADH3. The kinetic constant showed that the catalytic efficiency of ADH3 (Kcat/Km = 30,3938 s-1 · mM-1) against OTA was 29,113 times higher than that of NA (Kcat/Km = 10.4 s-1 · mM-1), indicating that ADH3 was the overwhelming superior detoxifying gene in CW117. Intriguingly, when gene na was knocked out from the CW117 genome, degradation activity of the Δna mutant was significantly reduced at the first 6 h, suggesting that the two enzymes might have an interactive effect on OTA transformation. Gene expressions and Western blotting assay showed that the Δna mutant and wild-type CW117 showed similar adh3 expression levels, but na deficiency decreased ADH3 protein level in CW117. Collectively, isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 but not as a dominant hydrolase for OTA transformation. IMPORTANCE Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. Previous OTA detoxification studies mainly focused on characterizations of degradation strains and detoxifying enzymes. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA from strain CW117, which is an isoenzyme of the efficient detoxifying enzyme ADH3. Isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 and, thus, enhanced the degradation activity of the strain. This is the first study on an isoenzyme improving the stability of another efficient detoxifying enzyme in vivo.

A Newly Isolated Alcaligenes faecalis ANSA176 with the Capability of Alleviating Immune Injury and Inflammation through Efficiently Degrading Ochratoxin A.

Ochratoxin A (OTA) is one of the most prevalent mycotoxins that threatens food and feed safety. Biodegradation of OTA has gained much attention. In this study, an Alcaligenes faecalis strain named ANSA176, with a strong OTA-detoxifying ability, was isolated from donkey intestinal chyme and characterized. The strain ANSA176 could degrade 97.43% of 1 mg/mL OTA into OTα within 12 h, at 37 °C. The optimal levels for bacterial growth were 22-37 °C and pH 6.0-9.0. The effects of ANSA176 on laying hens with an OTA-contaminated diet were further investigated. A total of 36 laying hens were assigned to three dietary treatments: control group, OTA (250 µg/kg) group, and OTA + ANSA176 (6.2 × 108 CFU/kg diet) group. The results showed that OTA decreased the average daily feed intake (ADFI) and egg weight (EW); meanwhile, it increased serum alanine aminopeptidase (AAP), leucine aminopeptidase (LAP), ß2-microglobulin (ß2-MG), immunoglobulin G (IgG), tumor necrosis factor-α (TNF-α), and glutathione reductase (GR). However, the ANSA176 supplementation inhibited or attenuated the OTA-induced damages. Taken together, OTA-degrading strain A. faecalis ANSA176 was able to alleviate the immune injury and inflammation induced by OTA.

LaeA regulates morphological development and ochratoxin A biosynthesis in Aspergillus niger.

The global regulator LaeA and its orthologs govern the morphogenetic development and secondary metabolism of several filamentous ascomycetes. In Aspergillus niger, it has been shown that an LaeA ortholog (AnLaeA) regulates the production of citric acid and secondary metabolites. In this work, we constructed AnlaeA disruption and overexpression strains to investigate the roles of AnLaeA in morphological development and ochratoxin A (OTA) biosynthesis in A. niger. Phenotypic observation, chemical analysis, and gene expression analysis indicated that AnLaeA acts as a negative regulator of conidial morphogenesis and positively regulates gene expression of the OTA cluster in A. niger grown in CYA medium. However, it was observed that the upregulation of gene expression of the OTA cluster does not necessarily increase OTA production. Our results contribute to a better understanding of the AnlaeA regulatory mechanism and suggest the AnlaeA gene as a potential target for developing control strategies for A. niger infection and OTA biosynthesis.