KR100777232B1 - [ 10796] Zeralenone Overproducing Microorganism and Methods of Zeralenone Overproduction By Using It - Google Patents

Abstract

A strain for mass-producing zearalenone is provided to increase the productivity of the zearalenone compared to conventional strains significantly. And a method for mass-producing the zearalenone through liquid or solid culturing the strain is provided to produce the zearalenone at low cost with high efficiency. A Gibberella zeae for mass-producing zearalenone is deposited as a deposition no. KCCM 10796P. A method for increasing the zearalenone production comprises a step of overexpressing ZEB2 in a strain in which ZEB1 is eliminated in zearalenone production pathways of the Gibberella zeae. A method for mass-producing the zearalenone comprises a step of culturing the strain in a solid medium.

Description

Zeralenone Overproducing Microorganism and Methods of Zeralenone Overproduction By Using It}

1 shows a method for overexpressing ZEB2 from the genome of ZEB2 removal strain GZ3639 in barley red fungus ( Gibberella zeae ). (A) E: Eco RI; β-tubulin, β-tubulin gene of barley red fungus; ZEB2, ZEB2 ORF; gen, geneticin resistance gene; amp, ampicillin resistance gene. (B) Gel blot made by treating genomic DNA of overexpressed strain with Eco RI. A portion of the ZEB2 ORF and a portion of the β-tubulin promoter region were used as probes for DNA gel blot hybridization. WT, wild type strain (GZ3639); Strains overexpressing ZEB2 in strains from which ODZ2-Z2 and ZEB2 were removed; ODZ2-ect, a transformant having a transformation vector at another location. The size of the lambda DNA standard is shown on the left side of the blot. (C) Pictures of strains grown in PDA medium for 6 days.

Figure 2 shows the mycelial amounts of GZ3639 and ODZ2-Z2 strains cultured in the improved CM medium. Results are expressed as mean ± standard deviation of three replicates. WT, wild type strain (GZ3639); Strain overexpressed ZEB2 in strains from which ODZ2-Z2 and ZEB2 were removed.

FIG. 3 shows the time course for zearalenone production of wild type strains and ZEB2 overexpressing strains in improved CM medium. Results are expressed as mean ± standard deviation of three replicates. WT, wild type strain (GZ3639); Strain overexpressed ZEB2 in strains from which ODZ2-Z2 and ZEB2 were removed.

Figure 4 shows RNA gel blots on days 3, 5 and 7 in GZ3639 and ODZ2-Z2 strains cultured in improved CM medium. Each probe was PKS13, PKS4, ZEB2 and shown on each blot. Agarose gels stained with ethidium bromide prior to blotting are shown in the left panel. WT, wild type strain (GZ3639); Strain overexpressed ZEB2 in strains from which ODZ2-Z2 and ZEB2 were removed.

Barley red fungus ( Gibberella zeae ) (incomplete generation: Fusarium graminearum ) is a geographically widely distributed fungal fungus that causes serious diseases in cereals such as corn, wheat and rice (McMullen). , M. et al, Plant Dis., 81 (1997) 1340-1348). These fungi produce a wide range of secondary metabolites, including mycotoxins, antibiotics, and pigments (Marasas, WFO et al, Toxigenic Fusarium Species; Identity and Mycotoxicology, The Pennsylvania State University Press (1984), Medentsev, AG et al, Phytochemistry, 47 (1998) 935-959, Nelson, PE et al, Fusarium species-An Illustrated Mannual for Identification, The Pennsylvania State University Press (1983)).

Among these metabolites, zearalenone ( ZEA ) causes estrogenic disorders in pigs fed corn, wheat, barley and sugar cane infected with Fusarium (Mirocha, CJ et al , Mycotoxins, (1974). 129-148). In many countries, along with deoxynivalenol, nivalenol, and trichothecene mycotoxins, the occurrence of girallenone is common in grains infected with G. zeae (Tanaka, T. et. al , J Agric Food Chem, 36 (1988) 979-983). Ginalenone is also known to occur naturally in foods made for human consumption, including maize flour, corn flakes, corn grits, and beer (Marasas, WFO et al , Toxigenic Fusarium Species; Identity and Mycotoxicology, The Pennsylvania State University). Press (1984).

Girallenone and its derivatives, including zearalenol ( ZOL ), are all trans-configurations in 1'-2'double bonds. The activity of the giralenone is affected by the 6′-ketone modification, and the 1′-2 ′ double bond enhances the activity. α-Gyrallenol (α-ZOL) promotes estrus by 3-4 times as much as giralenone. However, the estrogenic effect of β-ziralenol (β-ZOL) is the same as or slightly less than that of giralenone (Hagler, WM et al , Appl Environ Microbiol, 37 (1979) 849-853). Cis-isomers are produced in the laboratory by the irradiation of trans-isomers using intense UV light (Mirocha, CJ et al , Appl Environ Microbiol, 35 (1978) 986 -987). A derivative called Zeranol produced as a result of this modification is used as a growth promoter for cattle (Ralgro; Pitman-Moore, Terre Haute, IN, USA). Moreover, giralenone , which has hormone-like activity in animals, is also involved in the regulation of sexual reproduction in G. zeae (Wolf, JC et al , Can J Microbiol, 19 (1973) 725-734).

Gyralenone is synthesized by PKS encoded from polyketide synthases ( PKS ) genes in barley red fungus. A total of 16 putative PKS genes have been identified in the barley red fungus genome (Kroken, S. et al, Proc. Natl. Acad. Sci. USA, 100 (2003) 15670-15675). By using methods to remove target PKS genes from barley red fungi, researchers recently found that PKS4 and PKS13 are required for giralenone biosynthesis (Gaffoor, I. et al , Eukaryotic Cell, 4 (2005) 1926-). 1933, Kim, YT et al , Mol. Microbiol., 58 (2005) 1102-1113).

Also, isoamyl alcohol oxidase (isoamyl alcohol oxidase) gene and a biosynthetic gene Fluorenone Fuck with high affinity biosynthesis of fluorenone Fuck (ze aralenone b iosynthesis gene 1; ZEB1) and a conserved basic region (basic region) leucine zipper (leucine zipper ) Fuck fluorenone biosynthesis gene having a domain 2 (ze aralenone b iosynthesis gene 2 ; need ZEB2). ZEB1 is required for chemical conversion of β-zearalenol ( β-ZOL ) to giralenone (ZEA) in the biosynthetic pathway and ZEB2 regulates the transcription of cluster members. Transcription of these genes is strongly influenced by other culture conditions such as malnutrition and ambient pH. Moreover, the gene sets regulated by ZEB2 in transgenic G. zeae strains with PKS13 or PKS4 removed are dramatically reduced. However, this is not the case with ZEB1 deficient strains. This indicates that giralenone (ZEA) or β-ziralenol (β-ZOL) is involved in the transcriptional activity of gene clusters required for giralenone biosynthesis in G. zeae strains.

Korean Patent Registration No. 10-0444124-0000 (Application Date: 2001.8.16) relates to a composition for inhibiting fungal toxin production, and discloses a composition for inhibiting fungal toxin production comprising a deficiency extract. In addition, for the composition for inhibiting fungal toxin production comprising at least one compound selected from the group consisting of Alizarin, Chrysophanic acid, Danson, Purpurin, and Quinizarin It is starting.

In Korean Patent Registration No. 10-0287382-0000 (filed date: Nov. 30, 1998), a heterologous protein is combined with a girallenone to obtain a giralenone protein conjugate, and the giralenone protein conjugate is administered to an animal, and the antibody produced in the animal. Disclosed is a method for producing a giralenone antibody for recovering a. However, there is no mention of how to produce giralenone in large quantities using barley red fungi ( Gibberella zeae ).

Giralenone is used for the production of giralenone-specific antibodies and is sold for 13,200,000 won per 1g (Http://www.sial.com/function/order/order.asp; September 19, 2006 Connection). Therefore, there is a demand for a method of mass production of giralenone in an economic manner. The present inventors overexpressed ZEB2 in a strain in which ZEB1 was removed from the production path of girallenone, and found that β-girallenol was mass-produced and completed the present invention.

It is an object of the present invention to provide a barley red fungus ( Gibberella zeae ) [KCCM 10796P] which mass-produces giralenone.

Another object of the present invention is to provide a method for mass production of giralenone.

For the above purpose, the present invention provides a mass production of giralenone [KCCM 10796P, Korea Microorganism Conservation Center (KCCM), November 22, 2006]. Strains that produce large amounts of giralenone are GZ3639 barley red mold wild type strains (O'Donnell, KH et al, Proc. Natl. Acad.) That produce deoxynivalenol ( DON ) and zearalenone ( ZEA ). Sci. USA, 97 (2000) 7905-7910). For giralenone production, the aerial mycelia of the strains were incubated in 25 ml of improved CM liquid medium (Kim, YT et al, Mol. Microbiol. 58 (2005) 1102-1113). The ODZ2-Z2 strain according to the present invention increased the productivity of girallenone than GZ3639 in an improved CM liquid medium.

The ODZ2-Z2 strain is a filamentous filamentous fungus with a diaphragm similar to GZ3639, and has a slightly apricot-like morphological characteristic, and generally grows well at weakly acidic conditions of 25 ° C and pH 5.6. ODZ2-Z2 was inoculated with 100 ml of improved CM medium (NaNO3 2g, Sucrose 30g, KH2PO4 1g, MgSO4˙7H20 0.5g, KCl 0.5g, DW) incubated with PDA in mycelia and cultured at 25 ° C, 150rpm for 7 days. When cultured, about 800 ppb of giralenone could be detected. In addition, the strain ODZ2-Z2 was deposited on November 22, 2006 with the accession number KCCM 10796P to the Korea Microorganism Conservation Center (KCCM).

The second aspect of the present invention provides a method for mass-producing girallenone, which overexpresses ZEB2 in a strain from which ZEB2 has been removed from the giralenone production pathway. Methods for removing ZEB2 include deletion of biosynthetic genes, inhibition of transcription using antisense RNA of biosynthetic genes, knock-out of biosynthetic genes with transposons, inhibitors that inhibit the activity of proteins. A method of processing can be used. As a method of overexpressing ZEB2, a gene may be present in a multicopy using an expression vector and overexpressed, and an overexpression method through chromosomal integration may be used. More specifically, it provides a method for increasing girallenone production, which comprises deleting the ZEB2 gene of barley red fungus ( Gibberella zeae ) and overexpressing the ZEB2 gene. Gene deletion was carried out using gene recombination method and gene expression was overexpressed through chromosomal integration.

Giralenone is produced in several species of Fuzziaum in addition to the barley red fungi ( Gibberella zeae ) (incomplete generation: Fusarium graminearum ). Kenji et al. Reported the production in F. roseum, F. tricinctum, F. lateritium (Kenji Shii et al, Applied Microbiology, Vol. 27, pp. 625-628). Therefore, the method of over-expressing ZEB2 in the strain from which ZEB2 has been removed from the giralenone production pathway, thus producing girallenone, may be applicable to all the Furizium species.

The giralenone production culture can be used in both liquid culture and solid culture. The liquid culture medium may be improved liquid culture medium (CM modified media), solid culture medium wheat medium (wheat media).

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Mass Production of Giralenone in Transformed Barley Red Mold Strains

The present invention shows a method of overexpressing ZEB2 in a strain from which ZEB2 has been removed from the production path of giralenone, and mass-producing giralenone by culturing this strain.

Strains and Media

The GZ3639 strain was used as a barley red mold wild type strain producing deoxynivalenol ( DON ) and giralenone (zearalenone ( ZEA ) (O'Donnell, KH et al, Proc. Natl. Acad. Sci. USA). , 97 (2000) 7905-7910). Barley red mold strains ODZ2-Z2 and ODZ2-ect were derived from GZ3639. The former is a strain in which ZEB2 is overexpressed in a strain from which ZEB2 has been removed, and the latter is a transformant having a transformation vector at another position. For DNA isolation, fungal strains were incubated in a 250 ml Erlenmeyer flask containing 30 ml of complete medium ( CM ) in a rotary shake incubator at 25 ° C. and 150 rpm for 3 days (Correll, JC et al., Phytopathology 77 (1987) 1640-1646). For total RNA extraction and giralenone production, the aerial mycelia of the strains were cultured in 25 ml of improved CM liquid medium (Kim, YT et al, Mol. Microbiol. 58 (2005) 1102-1113).

In order to observe the giralenone production, the fungal strains were cultured in potato at 25 ° C. in cows in Potato-dextrose agar ( PDA ) (Difco Laboratories, Detroit, MI, USA), improved CM medium.

Nucleic acid manipulation

Fungal genomic DNA and plasmid DNAs were extracted as previously reported (Kereyi, Z. et al, Appl. Environ. Microbiol. 65 (1999) 4071-4076). Total RNA was extracted from mycelium (0.1 g) ground by liquid nitrogen using 1 ml of Trizol solution (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions. Standard procedures were performed by restriction enzyme treatment, ligation, agarose gel electrophoresis, and gel blotting (Sambrook, J. et al, Molecular Cloning: A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press (1989). P ³² labeled probes were used for DNA, RNA gel blot hybridization. PCR primers were obtained from Bioneer oligonucleotide synthesis team (Bioneer Co., Chungwon, Korea).

Double-Joint PCR and Transformation of Fungi

Transgenic DNA fragments with hygromycin resistance gene (hygB) based on DNA sequence homology located at the flanking 5 'and 3' genes of the barley red fungal strain ZEB2 were complemented by double-joint PCR (Double-Joint). PCR; DJ PCR ) (Yu, J.-H. et al, Fungal Genet. Biol. 41 (2004) 973-981).

bpro-5 (5′-CGAAGC CGACGACGAACA-3 ′) (SEQ ID NO: 1) / bpro3 (5′-ATTGACGGCTGTAG ATGTAAT-3 ′) (SEQ ID NO: 2) to amplify the β-tubulin promoter portion ZEB2 OEX-2 5R (5′-CATTACATCTAC AGCCGTCAATAACGGCCGTGACCTTAATTCA-3 ′) (SEQ ID NO: 3) / IAO-N-5'F (5′-CGTTTGATTC CCCGCAGTGT-3 ′) to amplify the ZEB2 ORF Number 4) was used and nested PCR primer pairs (IAO 3'F (5'-GGAAGAATGTGCAAGTCAGC-3 ') (SEQ ID NO: 5) / bpro / 5' (5'-CGAAGCCGACGACGAACA-) for fusion products 3 ') (SEQ ID NO: 6)). As a result, the β-tubulin promoter and ZEB2 ORF of 2.1 kb and 1.2 kb, respectively, were obtained and fused to obtain a 3.3 kb PCR product. This PCR product was cloned back into the TOPO vector to finally produce a vector of 6.8 kb size.

For transformation of the fungus, DJ-PCR with polyethyleneglycol was introduced into the protoplasts of the fungus prepared by the mycelium treated with a dryase (Driselase, InterSpex Products, Inc., San Mateo, Calif., USA). About 5 ug of fusion PCR product obtained by direct addition was added (Lee, T. et al, Appl. Environ. Microbiol. 68 (2002) 2148-2154). Stable transformants with hygB gene were selected from regeneration medium containing 75ug / ml of hygromycin B and stored at -80 ° C containing 25% glycerol.

Chemical analysis

Standard samples of giralenone, α-ZOL and β-ZOL were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). In order to detect Fuck Lennon from fungal strains, extracting a liquid culture solution was dried and the culture filtrate, and high performance liquid chromatography...; And analyzed by (High performance liquid chromatography HPLC) (Kim, JE et al, Appl Environ Microbiol 71 (2005) 1701-1708, Kim, YT et al, Mol. Microbiol. 58 (2005) 1102-1113).

result

1. Barley red fungus ( Gibberella zeae Gene expression and expression in

A target gene replacement strategy was used to create transformed barley red mold strains that overexpressed ZEB2 in strains that had ZEB2 removed.

The ΔZEB2 strain was a previously prepared strain (Kim, YT et al, Mol. Microbiol. 58 (2005) 1102-1113), and overexpression of ZEB2 resulted in the β-tubulin promoter portion and the ZEB2 ORF. A ZEB2 / β-tubulin vector prepared by fusing the fused PCR product with the TOPO vector was performed by simultaneously transforming the ΔZEB2 strain with a pII99 plasmid having a geneticin marker. The transgenic rims were selected primarily through the geneticin antibiotic markers and secondly the genomic DNA of the transgenic rims was beta-tubulin in the β-tubulin promoter. PCR was confirmed by probe 3 REV (5′-CGTCACTGCAGGTACCACATCTTG-3 ′) (SEQ ID NO 7) and zeb2 probe 5FOR (5′-GATAGAAAGGCCTCGCTGTG-3 ′) (SEQ ID NO 8), and the insertion position was determined by Southern blot. It was confirmed. As a result, it was confirmed that the β-tubulin promoter portion and the vector of the ΔZEB2 strain were inserted through the cross. In the case of Fig. 1A , the ZEB2 / β-tubulin vector is inserted into the β-tubulin promoter portion of the genomic DNA through a simple schematic diagram. . The vector is expected to be inserted into the 5 'of the genomic β-tubulin through the intersection. FIG. 1 (B) is cultured in a 50 mL liquid medium at 25 ° C. and 150 rpm for 3 days after transformants expected to have been inserted by the method of FIG. 1 (A) , followed by extraction of DNA and treatment with EcoRI. Is the result of Probes were used as part of the ZEB2 ORF and part of the β-tubulin promoter region. As a result, in case of wild type strain GZ3639, the EcoRI treatment resulted in the ZEB2 band at about 11.5 kb and the β-tubulin promoter band at 3.8 kb. In the case of ODZ2-Z2, EcoRI is present in the vector when the vector is inserted and the EcoRI is processed. Thus, two bands of β-tubulin appear to be 3kb and 4.4kb.

2. Phenotypes of Transformed PKS Removal Strains

Mycelia of wild type strains GZ3639 and ODZ2-Z2 were incubated for 6 days in PDA medium ( FIG. 1C ). GZ3639 is a wild type strain and ODZ2-Z2 overexpresses ZEB2 and shows about 20% less growth than wild type strain. This seems to be due to the overproduction of giralenone. The ODZ2-Ect strains were inserted into different positions, so the strains could not overproduce giralenone, so the growth rate was not different from that of the wild type strains ( FIG. 1C ). The ODZ2-Z2 strain in the improved CM medium showed an increase of about 2-3 times from the 5th day when compared to the GZ3639 strain ( FIG. 2 ).

3. Production of Giralenone in Transgenic Barley Red Mold Strains

In improved CM medium, wild type strain GZ3639 produced little giralenone until 7 days after inoculation. However, ODZ2-Z2 strain was detected after 3 days of inoculation of girallenone and gradually increased until day 7 ( FIG. 3 ).

4. Expression of PKS Genes

In the improved CM medium, the wild type strain GZ3639 showed no expression of the two PKS genes and ZEB2 genes required for giralenone biosynthesis. On the other hand, ODZ2-Z2 strains were detected on RNA blot for the first time on the 5th day and maintained until 7 days, and transcripts of ZEB2 were also detected on the 5th day and increased slightly on day 7. From day 5 when the expression levels of PKS13, PKS4 and ZEB2 were detected, the detection amount of giralenone also increased. In comparison, no Gyralenone was detected in the GZ3639 strain in which no expression of the girallenone biosynthesis gene was induced. ( FIG. 3 ) This means that the expression of the girallenone biosynthesis gene is associated with the regulation of giralenone production. . In addition, ZEB2 overexpressed strain means that unlike the wild-type strain is less affected by the external environment.

The giralenone mass production strain [KCCM 10796P] according to the present invention greatly increased the productivity of the giralenone than the conventional strain. In addition, the method for mass production of girallenone through liquid or solid culture using the strain may produce girallenone at low cost and high efficiency.

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Claims (3)

Barley red fungus ( Gibberella zeae ), which mass-produces giralenone [KCCM 10796P]. A method for increasing girallenone production, characterized by overexpressing ZEB2 in a strain in which ZEB2 has been removed from the giralenone production pathway of barley red fungus ( Gibberella zeae ). A method for mass production of giralenone, characterized in that to produce a giralenone by solid culture of the strain of claim 1.

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