Expression of the Clonostachys rosea lactonohydrolase gene by Lactobacillus reuteri to increase its zearalenone-removing ability

Abstract

Background: Mycotoxins are secondary metabolites produced by filamentous fungi that can contaminate agricultural crops in the field as well as during harvest, transportation, processing, or storage. Zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, produced by Fusarium species, has been shown to be associated with reproductive disorders in farm animals and to a lesser extent in hyperoestrogenic syndromes in humans. Thus, the decontamination of ZEN in foods and feeds is an important issue.

Results: In this study, the gene encoding ZHD101, a ZEN-degrading enzyme produced by Clonostachys rosea IFO 7063, was cloned into an Escherichia coli-Lactobacillus shuttle vector, pNZ3004, and the resultant plasmid pNZ-zhd101 was then introduced via electroporation into Lactobacillus reuteri Pg4, a probiotic strain isolated from the gastrointestinal tract of broilers. The transformed strain L. reuteri pNZ-zhd101 acquired the capacity to degrade ZEN. In addition, the production of recombinant ZHD101 did not affect cell growth, acid and bile salt tolerance, and had only a minor effect on the adhesion ability of L. reuteri pNZ-zhd101.

Conclusions: To the best of our knowledge, this is the first report of successful expression of a ZEN-degrading enzyme by intestinal lactobacilli.

Keywords: Lactobacillus reuteri; Lactonohydrolase; Probiotics; Zearalenone.

Fig. 1

Lactobacillus expression plasmid harboring the Clonostachys rosea zearalenone hydrolase gene zhd101

Fig. 2

Expression of Zhd101 in Lactobacillus reuteri cells. a RT-PCR for Zhd101 in Lb. reuteri Pg4, Lb. reuteri pNZ-zhd101, and Lb. reuteri pNZ3004, along with the housekeeping gene 16S rRNA. b Western blot for purified recombinant Zhd101 and Zhd101 in the intracellular extracts of Lb. reuteri Pg4, Lb. reuteri pNZ3004, and Lb. reuteri pNZ-zhd101. The samples (2 μg of protein in each lane) were separated by SDS-PAGE with a 12.5% gel and probed with mouse polyclonal anti-zhd101 antibody and horseradish-peroxidase-linked anti-mouse IgG antibody as the primary and secondary antibodies, respectively

Fig. 3

Changes in ZEN concentrations of MRS media containing ZEN (4.5 mg/L) incubated with L. reuteri Pg4 or L. reuteri pNZ-zhd101. The pH values of culture media were adjusted to pH 7.0 by adding 0.1 N NaOH every hour

Fig. 4

Survival of L. reuteri Pg4, L. reuteri pNZ3004 and L. reuteri pNZ-zhd101 after incubation at pH 3.0 (a) or in the presence of 0.5% ox gall (b). The bars represent standard errors of the means calculated from three independent experiments performed in triplicate

Fig. 5

Flow cytometric analysis of Lactobacillus adherence to Caco-2 cells. a Autofluorescence of Caco-2 cells. b Caco-2 cells exposed to fluorescently labeled L. reuteri Pg4. c Caco-2 cells exposed to fluorescently labeled L. reuteri pNZ3004. d Caco-2 cells exposed to fluorescently labeled L. reuteri pNZ-zhd101. Bacterial cells were labeled with hexidium iodide (HI) and incubated with Caco-2 cells for 2 h. For each experiment, 10,000 Caco-2 cells were analyzed. The red fluorescence intensity of the Caco-2 cells with adherent lactobacilli was measured by flow cytometry at 620 nm emission wavelengths. Mean red fluorescence intensity (MFI) was calculated from two independent experiments performed in triplicate