Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method

Abstract

The invention relates to a polypeptide for the enzymatic detoxification of zearalenone, said polypeptide being a monooxygenase which converts the keto group in position 7 of zearalenone into an ester group, the monooxygenase in particular being an amino acid sequence selected from the group comprising sequence ID No. 1, 2 and 3 or a functional variant thereof. The functional variant and at least one of the amino acid sequences has a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80% and most preferably 90%.

Claims

1. A method for enzymatic detoxification of zearalenone by a recombinant isolated polypeptide comprising the step of reacting the polypeptide with zearalenone, wherein the polypeptide is a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, and wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; wherein the monooxygenase is a Baeyer-Villiger monooxygenase and a cyclohexanone monooxygenase.

2. A method for enzymatic detoxification of zearalenone comprising the following steps: preparing a recombinant isolated polypeptide by utilizing a yeast strain, which is transformed with a polynucleotide for expressing the polypeptide; and reacting the polypeptide with zearalenone, wherein the polypeptide is a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, and wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; wherein the monooxygenase is a Baeyer-Villiger monooxygenase and a cyclohexanone monooxygenase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the following, the invention will be explained in more detail by way of exemplary embodiments and drawings. Therein,

(2) FIG. 1 depicts the partial transformation of ZEN in the negative control by S. cerevisiae;

(3) FIG. 2 depicts the transformation of ZEN in the test setup by S. cerevisiae, which expresses the polypeptide of SEQ ID No. 1;

(4) FIG. 3 illustrates the chemical structure of iZOM; and

(5) FIG. 4 shows the estrogenic activity of ZEN (FIG. 4A) and of iZOM (FIG. 4B).

DETAILED DESCRIPTION OF THE INVENTION

(6) Unless indicated in more detail, all molecular-biological and microbiological operations were performed using standard techniques (Methods in Enzymology, Volume 194, Pages 3-933 (1991); Guide to Yeast Genetics and Molecular Biology. Edited by Christine Guthrie, Gerald R. Fink. ISBN: 978-0-12-182095-4; J. Sambrook et al. 2012, Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor).

Example 1

Formation of iZOM from ZEN in a One-Step Reaction

(7) The yeast strain YZGA515 (Saccharomyces cerevisiae modified by inactivation of the ABC transporter gene PDR5 (Poppenberger et al. 2003, J. Biol. Chem., 278 (48) 47905-14) for the enhanced absorption of zearalenone (ZEN)) was transformed by the plasmid pCS57 using standard techniques. pCS57 is a plasmid derived from the yeast expression plasmid pADH-FW (Mitterbauer et. al 2002, Appl. Environ. Microbiol.; 68 (3), 1336-1346) with LEU2 as selection marker and ADH1 promoter.

(8) In the test setup, the yeast strain YZGA515 was transformed with the pCS57 vector additionally containing the polynucleotide of SEQ ID No. 4 as BamHI-Xhol fragment after the strongly constitutive ADH1 promoter (alcohol dehydrogenase 1). This enabled the formation of the polypeptide of SEQ ID No. 1.

(9) The yeast strain YZGA515 with the empty vector (pADH-FW without the polynucleotide of SEQ ID No.4) was transformed as negative control.

(10) The transgenic yeasts (test setup and negative control) were cultivated in SC-LEU medium (Sherman 1991, Methods Enzymol., 194, 3-21), centrifuged and resuspended in fresh SC-LEU medium at a concentration of 2 mg/l ZEN, wherein a cell density of OD600=4 was adjusted in both cases. After various incubation times, samples were taken, supplemented with 1 volume methanol, and the intracellular processes were thus stopped. The cell suspension was cleared by centrifugation, and the resulting supernatants were used for HPLC-MS measurements. The measurements, as described above, comprised the starting substance zearalenone (ZEN) and the transformation product ZOM-1 formed by Trichosporon mycotoxinivorans (Vekuri et al. 2010, Appl. Environ. Microbiol. 76(7) 2353-9).

(11) In the negative control transformed with the empty vector, no ZOM-1 and, in particular, no iZOM was formed. The respective HPLC analysis data are illustrated in FIG. 1, the y-axis indicating nanogram per milliliter (ng/ml) and the x-axis indicating the time in hours (h).

(12) By contrast, zearalenone was significantly more rapidly transformed in the test setup, and a new metabolite with the expected mass of the intermediate postulated by Vekiru et al. (2010) and referred to as iZOM below was identified. In addition, ZOM-1 was also detectable, which clearly suggested that a hydrolysis of iZOM to ZOM had occurred to some extent. The respective HPLC analysis data are illustrated in FIG. 2. Compared to the negative control, a much smaller amount of ZEN was measured, the y-axis indicating nanogram per milliliter (ng/ml) and the x-axis indicating the time in hours (h).

(13) In a further test setup, the metabolite iZOM was additionally measured quantitatively. Table 1 shows the molar balance of the metabolization of ZEN. From this, it is clearly apparent that the major portion of the used zearalenone (ZEN) was already converted to iZOM after 6 hours. The small shortfall in the balance is explainable by the formation of further metabolites and the method-related analytical unsharpnesses. The results clearly show that the polypeptide of SEQ ID No. 1 is able to convert ZEN to its metabolite iZOM.

(14) TABLE-US-00001 TABLE 1 Molar balance of the metabolization of ZEN in test setup Time ZEN ZOM-1 iZOM [h] [nM] [nM] [nM] 0 3364 0 0 6 172.1 0 2163.1 24 4.8 214.8 2068.2

Example 2

Identification of the Chemical Structure of iZOM

(15) The chemical structure of the metabolite iZOM (FIG. 3) could be determined by NMR measurements of the isolated and purified metabolite. For measuring the NMR spectra, a sufficient amount of iZOM was prepared from several liters of the culture filtrate of a yeast culture (SC-LEU as described in Example 1). iZOM was purified by solid phase extraction and subsequent preparative HPLC.

(16) The use of the isolated reference substance for iZOM on the one hand allowed for the tracing of the conversion of ZEN to iZOM (cf. Example 1) and, on the other hand, enabled the clarification of the chemical structure by NMR.

(17) The NMR spectra for identifying iZOM were obtained in a CD3OD solution using a Bruker Avance DRX-400 FT NMR spectrometer at room temperature (20° C.) with a 5 mm inverse broadband z-gradient probe head. The chemical displacements were established based on the residual solvent resonance (3.31 ppm for .sup.1H NMR, 49.15 ppm for .sup.13C NMR). All pulse programs were taken from the Bruker software library. The NMR data were evaluated by means of TopSpin 1.3 (Bruker BioSpin GmbH). The determination of the complete structure and the assignment of signals were performed based on .sup.1H, .sup.13C-APT, .sup.1H.sup.1H correlation spectroscopy (COSY), .sup.1H.sup.13C heteronuclear single quantum correlation spectra (HSQC), and .sup.1H.sup.13C heteronuclear multiband correlation spectra (HMBC).

(18) The NMR spectroscopy data of iZOM show two essential differences as compared to ZEN. These confirm the conversion of the keto group to an ester group. The differences substantially comprise the shift of the carbonyl carbon atom from 212 ppm in ZEN (typical of a ketone) to 175 ppm (typical of a carboxylic acid derivative) and the shift of the adjacent CH.sub.2 group from 36 ppm and 2.90/2.30 ppm (position 8 in ZEN) to 65 ppm and 4.20/4.05 (position 9 in iZOM) for .sup.13C and .sup.1H, respectively, caused by the adjacent oxygen atom. Unlike the open ring of ZOM1, the macrocyclic ring in iZOM is still intact as evidenced by a long-range correlation between C7 and H9. The NMR data are indicated in Table 2. The chemical structure of iZOM is illustrated in FIG. 3.

(19) TABLE-US-00002 TABLE 2 .sup.1H and .sup.13C NMR date for identifying iZOM .sup.1H .sup.13C Position δ (ppm) Multiplicity, J (Hz) δ (ppm) 1 — 172.9 3 5.14 m 74.2 3-CH.sub.3 1.36 d, 6.2 20.2 4 1.65-1.80 m 36.5 5 1.90, 1.76 m 23.1 6 2.30-2.45 m 35.8 7 — 175.4 9 4.18, 4.05 m 64.6 10 1.75-1.90 m 28.9 11 2.30-2.50 m 30.8 12 5.93 ddd, 15.4, 8.3, 6.1 132.4 13 7.00 d, 15.4 133.2 14 6.40 d, 2.2 109.6 15 — 165.0 16 6.19 d, 2.2 103.3 17 — 165.6 18 — 104.9 19 — 143.9

Example 3

Strongly Reduced Estrogenic Activity of iZOM as Compared to ZEN

(20) The purified metabolite iZOM as described in Example 2 was used to determine its toxicity, expressed by its estrogenic activity, and compared to that of zearalenone.

(21) For measuring the estrogenic activity, a specially prepared reporter yeast strain, YZHB817 (Bachmann, H.: Phenotypic detection of zearalenone in Saccharomyces cerevisiae. Master thesis BOKU Vienna, 2003) was used. This strain is derived from yeast-two hybrid strain PJ69-4a (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4-(deleted) gal80(deleted) LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) (James et al. 1996, Genetics; 144(4), 1425-1436). PJ69-4a was further developed by the disruption of the ABC transporters PDR5 and SNQ2. Strains with such double mutations pdr5 and snq2 exhibit a particularly high ZEN absorption (Mitterbauer et al. 2003, Appl. Environ. Microbiol.; 69(2), 805-811). This strain was subsequently transformed with an expression vector (pTK103) that triggers the production of a fusion protein containing the hormone-dependent activation domain of the human estrogen receptor alpha and the DNA binding domain of the yeast Gal4 protein, in order to obtain the strain YZHB817.

(22) In the presence of estrogenic substances, the yeast strain YZHB817 induces the expression of the GAL7 lacZ reporter gene, whose product, the enzyme beta-galactosidase, is easy to measure by the hydrolyzation of the chromogenic substrate ONPG (ortho-nitrophenyl-beta-galactoside) at 420 nm (Current Protocols in Molecular Biology, Chapter 13, Yeast (Eds. Lundblad V, Struhl, K.)).

(23) 100 ml of the estrogenic test solution (ZEN or iZOM) were dissolved in 50% ethanol, mixed with 1.9 ml of the yeast culture of strain YZGA817 in SC-TRP medium at a cell density of OD.sub.600=0.1, and incubated for 18 hours at 30° C. and 180 rpm. After this, the OD.sub.600 of the culture was determined, and the cell pellet (10 min, 13 krpm) of 1 ml was resuspended in 500 ml Z buffer and permeabilized with 25 μl chloroform. The enzyme reaction was started by the addition of 100 μl ONPG solution (4 mg/ml) and stopped after yellow coloring upon addition of 250 μl of a 1M Na.sub.2CO.sub.3 solution. The supernatant was measured at 420 nm, and the relative units (based on the used cell amount) were calculated as:
RU=(OD.sub.420×1000)/(OD.sub.600×incubation time in min)

(24) As illustrated in FIG. 4, ZEN induces the expression of the GAL7-lacZ reporter gene in the low ppb range, thus approximately 45 ppb ZEN are necessary to express 7.5 relative units of beta-galactosidase (FIG. 4A). By contrast, iZOM exhibits a far lower estrogenicity. In comparison, about 500 ppb were required to achieve approximately the same effect (FIG. 4B). Hence results that iZOM has an estrogenic activity approximately reduced by a factor 100 as compared to ZEN.

Example 4

Determination of the Sequence Identity

(25) The determination of the percentual sequence identity of the polypeptides with the amino acid sequences SEQ ID Nos. 1, 2 and 3 relative to one another was performed by the alignment of two sequences using the program BLAST (Basic Local Alignment Search Tool), in particular BLASTP, which can be used on the homepage of the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov). It is thereby possible to compare two or several sequences with one another according to the algorithm of Altschul et al., 1397 (Nucleic Acids Res. (1997) 25: 3389-3402). The base adjustments were used as program adjustments, but in particular: “max target sequence”=100; “expected threshold”=10; “word size”=3; “matrix”=BLOSOM62; “gap costs”=“Existence: 11; Extension: 1”; “computational adjustment”=“Conditional compositional score matrix adjustment”. The sequence identity between the sequences of SEQ ID No. 1 and SEQ ID No. 3 is 73%.

(26) This application incorporates by reference the electronically submitted Sequence Listing filed on Jul. 9, 2020 in ASCII text file (CRF format) with the file name of P75529US0_SEQ_LIST_ST25.txt, created on May 1, 2017 and with the size of 11,026,432 bytes.