KR20030046570A - β-glucosidase gene derived from Trichoderma sp. and yeast transformant introduced with the gene - Google Patents

Abstract

PURPOSE: A beta-glucosidase gene derived from Trichoderma sp. and a yeast transformant introduced with the same are provided, therefore a fibrous substrate is decomposed by the yeast transformant. CONSTITUTION: A beta-glucosidase has the amino acid sequence of SEQ ID NO: 2. A beta-glucosidase gene C4 beta glu has the nucleotide sequence of SEQ ID NO: 1, wherein the beta-glucosidase gene C4 beta glu is isolated from Trichoderma sp. C-4. A recombinant vector pVT-betaG contains the beta-glucosidase gene C4 beta glu. A yeast transformant, Saccharomyces cerevisiae SEY2102/pVT-betaG, is produced by transforming a yeast with the recombinant vector pVT-betaG. A feed additive contains the yeast transformant Saccharomyces cerevisiae SEY2102/pVT-betaG.

Description

Β-glucosidase gene derived from Trichoderma sp. Microorganisms and the yeast transformants into which the gene is introduced. and yeast transformant introduced with the gene}

The present invention relates to a beta-glucosidase gene derived from a Trichoderma genus microorganism and a yeast transformant to which the gene is introduced, and more particularly, SEQ ID NO: 1 derived from Trichoderma genus C-4 strain. It relates to a beta-glucosidase gene having a nucleotide sequence represented by, a recombinant vector into which the gene is inserted, and a yeast transformant transformed with the vector.

Yeast Saccharomyces cerevisiae is used in various bioprocessing fields utilizing starch or fiber, which is a vegetable raw material. However, yeast has a disadvantage in that it can not degrade the fibrin substrate at all by producing and secreting small amounts of starch degrading enzymes. New breeding of fibrinolytic yeast using genetic recombination technology can be used in a variety of applications, such as brewing, baking, alcohol production, probiotic development, the utilization of these recombinant yeast is expected. Currently, research for producing a recombinant yeast secreting fibrinolytic enzymes, but the situation is insufficient. In addition, for this purpose, it is necessary to secure a fibrinolytic gene having high thermal stability or excellent activity.

Meanwhile, the fungus Trichoderma reesei is the most studied fibrinolytic microorganism, and is known to produce various cellulase. The fibrinolytic enzymes produced by Trichoderma reesei show significant differences in substrate specificity and reaction mechanisms, but they all break down beta-1,4-glucoside (β-1,4-glycoside) bonds. Has been reported to be secreted into extracellular media (Nevalainen, H. et al ., Genetics and biotechnology , Pp 303-319, 1995). In addition, effective degradation of crystalline fibrin includes three cellulose degrading enzymes, endoglucanase, cellobiohydrolase (or exoglucanase) and beta-glucosidase ( β-glucosidase) is required simultaneously, and their synergy has been reported to significantly increase fibrin resolution (Henrissat, B., et al ., Bio / Technol . 3: 722-726, 1985; Tomme, P., et. al ., Adv. Microbiol. Physiol . 37: 1-81, 1995; Woodward, J., et al ., Bio / Technol . 6: 301-304, 1988).

To date, several kinds of trichoderma fibrinolytic genes have been cloned to reveal their nucleotide sequences. For example, cbh1 and cbh2 have been reported as cellobiohydrolase genes (Shoemaker, S., et al ., Bio / Technol . 1: 691-695, 1983; Teeri, TT, et al ., Gene 51 : 43-52, 1983), egl1 , egl2 , egl3 , egl4 and egl5 have been reported as endoglucanas genes (Penttila, M., et al ., Gene 45: 253-263, 1986; Saloheimo, M., et al , Eur. J. Biochem . 249: 584-591, 1997; Saloheimo, A., et al, Mol. Microbiol . 13: 219-228, 1994), and bgl1 as a beta-glucosidase gene has been reported. (Barnett, CC, et al ., Bio / Technol . 9: 562-567, 1991). However, since the cellulose degrading enzyme-producing strains developed to date, including the Trichoderma reesei, are mostly slow to decompose cellulose, research to search for new strains and enzymes is necessary. Furthermore, research on the production of transgenic microorganisms in which the fibrinolytic gene has been transferred to other microorganisms is very limited.

On the other hand, the present inventors confirmed that the Trichoderma sp. C-4 strain isolated from rice straw produces endoglucanase excellent in acid resistance and heat resistance, and the endoglucanase gene ( egl6). ) (Son, YJ, et al ., Kor. J. Appl. Microbiol. Biotechnol. 25: 346-353, 1997). The present inventors continue to study other fibrinolytic enzymes in addition to the endoglucanase produced by the Trichoderma genus C-4 strain, and the Trichoderma genus C-4 strain produces beta-glucosidase. The beta-glucosidase gene was isolated to develop a recombinant vector into which the gene was inserted and a yeast transformant transformed with the vector, as well as the yeast transformant to beta-glucosidase. This invention was completed by confirming that it secretes effectively.

It is therefore an object of the present invention to provide a beta-glucosidase gene isolated from Trichoderma genus C-4 strain.

Another object of the present invention is to provide a recombinant vector into which the gene is inserted.

Still another object of the present invention is to provide a yeast transformant transformed with the recombinant vector.

FIG. 1 shows the results of restriction mapping of plasmid DNA of six clones estimated to contain beta-glucosidase gene using restriction enzymes of Pvu II and Hae III. Is a molecular weight marker.

Figure 2 is a result of comparing the beta-glucosidase gene nucleotide sequence of the gene suspected of beta-glucosidase gene, isolated from the Trichoderma genus C-4 strains.

Figure 3 is a photograph showing the results of PCR to obtain a beta-glucosidase full-length gene.

Lane 1: molecular weight marker

Lanes 2-4: beta-glucosidase full-length gene obtained by PCR

In order to achieve the above object, the present invention provides a beta-glucosidase gene represented by SEQ ID NO: 1 isolated from Trichoderma genus C-4 strain.

In addition, to achieve another object of the present invention, the present invention provides a recombinant vector pVT-βG in which the gene is inserted.

In order to achieve another object of the present invention, the present invention provides a yeast transformant Saccharomyces cerevisiae SEY2102 / pVT-βG transformed with the recombinant vector pVT-βG.

Hereinafter, the present invention will be described in detail.

Cellulose (cellulose) is a major component of the cell wall of higher plants and occupies most of the wood, and is broken down by cellulase, a fibrinolytic enzyme. In the case of fiber intake, herbivores are decomposed by phytolytic enzymes secreted by the stomach parasites and used as nutrients, but in higher animals, they are excreted intact because they cannot break down fiber. Enzymes for degrading the cellulose include endoglucanase, xylanase, exoglucanase, beta-glucosidase, and the like. Among them, beta-glucosidase hydrolyzes β-D-glucoside (β-D-glucoside).

In the present invention, a recombinant yeast prepared by separating beta-glucosidase, another fibrinolytic enzyme, from the Trichoderma C-4 strain known to produce endoglucanase, which is excellent in acid resistance and heat resistance, and introducing the gene into yeast. It was confirmed that the strain expresses and secretes high levels of beta-glucosidase.

In an embodiment of the present invention, a beta-glucosidase gene-specific probe was prepared, and the beta-glucosidase clone was searched by screening the cDNA library using the beta-glucosidase gene-specific probe. As a result, six positive clones were obtained.

In another embodiment of the present invention, the six clones are converted into plasmids by in vivo excision and then restricted to the plasmid DNA of the six clones using Pvu II and Hae III restriction enzymes. Restriction enzyme mapping was performed. As a result, as shown in Figure 1, it was confirmed that all six clones are clones made from the same gene. In addition, as a result of analyzing the nucleotide sequences, it was confirmed that all of the six clones did not perform full-size cDNA synthesis during the cloning process, so that a part of the 5 'side including the start codon was deleted for each clone. Therefore, again a cDNA library of Trichoderma genus C-4 strain was prepared and rescreened. As a result of analyzing the sequencing of the obtained positive clones, part of the 5 'side including the initiation codon was identically deleted. It could be confirmed. This is probably due to the fact that the mRNA transcript of the deleted site is highly fixed, and that the synthesis of cDNA by reverse transcriptase no longer proceeds at this site. In addition, the base sequence of the beta-glucosidase of Trichoderma reesei and a partial sequence of the positive clone was compared, and the results are shown in FIG. 2.

On the other hand, since a part of the gene obtained from the Trichoderma genus C-4 strain has been confirmed to have high homology with the beta-glucosidase sequence of Trichoderma lysase, the beta-glucosid of the tricoderma lysase A full length gene estimated to be a beta-glucosidase gene was obtained by preparing a primer containing an ATG codon with reference to the nucleotide sequence of the agent, and named the gene "C-4 β glu" (FIG. 3). Then, the nucleotide sequence of the C-4 β glu gene and the amino acid sequence inferred from the nucleotide sequence were analyzed and shown in SEQ ID NOs: 1 and 2, respectively.

In another embodiment of the invention the C-4 glu β gene yeast - E. coli and subcloned into the shuttle vector (yeast- E. coli shuttle vector) to prepare a recombinant vector. The yeast-E. Coli shuttle vector can be used without limitation as long as it is a yeast-E. Coli shuttle vector commonly used in the art, more preferably pVT-103U is used. Then, the yeast host cell was transformed using the recombinant vector into which the C-4 β glu was inserted. The yeast host cells that can be used at this time may be used without limitation, Saccharomyces cerevisiae strains, such as S. cerevisiae YNN27, S. cerevisiae 2805, S. cerevisiae SEY2102 and excellent stability and breeding ability, in particular Preference is given to using S. cerevisiae SEY2102. Then, to investigate that the protein expressed from the C-4 β glu gene is beta-glucosidase, beta-glucosidase activity was measured by culturing a recombinant yeast transformant with C-4 β glu introduced. As a result, it was confirmed that the recombinant yeast transformant introduced with C-4 β glu according to the present invention showed significantly higher beta-glucosidase activity than the wild type S. cerevisiae SEY2102 strain. As a result, the C-4 β glu gene isolated from the Trichoderma C-4 strain according to the present invention was confirmed to be a beta-glucosidase gene.

In addition, as a result of examining the in vitro NDF (acid detergent fiber) and ADF (acid detergent fiber) degradation rate of the recombinant yeast transformant, it was confirmed that the degradation rate of NDF and ADF increased as the incubation time increased. This indicates that the beta-glucosidase gene (C-4 β glu) derived from Trichoderma genus C-4 strain is expressed and secreted very efficiently in S. cerevisiae SEY2102 strain.

Beta-glucosidase gene derived from Trichoderma genus C-4 strain according to the present invention, beta-glucosidase expressed from the gene and recombinant yeast strains into which the gene is introduced are produced by brewing, baking, alcohol production and probiotic. It can be usefully used for development. In particular, it can be usefully used as a feed additive showing a superior digestive effect.

Hereinafter, the present invention will be described in detail by way of 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>

Exploring Beta-glucosidase Clones

cDNA library was prepared from Trichoderma genus C-4 strain (Accession No .: KFCC-10906) using a cDNA library kit (cDNA library kit, Stratagene). After amplifying the cDNA library, phage stock was prepared. The phage stock was then titered and then cultured in agar-LB medium with a diameter of 15 mm. Then about 5 × 10 per plate⁴Plates were inoculated to phage and blotted with nylon membranes. Trikoderma Reese (Trichoderma reeseiPCR was performed by constructing a specific primer set of the beta-glucosidase genes represented by SEQ ID NOs: 3 and 4 with reference to the nucleotide sequence of the gene. The PCR reaction was denatured by heating at 94 ° C. for 5 minutes, and then repeated 30 times under conditions of 1 minute at 94 ° C., 1 minute at 45 ° C., and 1 minute at 72 ° C., followed by final amplification at 72 ° C. for 10 minutes. The reaction was terminated. Subsequently, the PCR product Agarose gel After confirmation by electrophoresis, the PCR product was separated from the gel,³²Beta-glucosidase gene specific probes were prepared by labeling with P-dCTP.

Then, the probe was hybridized to the prepared cDNA library membrane, and then exposed to an X-ray film to confirm a positive signal. The phage group obtained by primary screening as described above was second screened again in the same manner to obtain six monoclones.

<Example 2>

Mapping of Beta-Glucosidase Genes

The phage clone obtained in Example 1 was converted to a plasmid by performing in vivo o excision according to the experimental method of Stratagene, and then the recombinant plasmid of the clone was extracted. Then, restriction enzyme mapping was performed on plasmid DNA of each clone using restriction enzymes of Pvu II and Hae III. As a result, it was confirmed that all clones as shown in FIG.

<Example 3>

Sequence analysis of the beta-glucosidase gene

As a result of analyzing the base sequence from the 5 'direction of the six clones obtained in Example 1, it was confirmed that all clones were made from the same gene, but 5' was not synthesized due to the full size cDNA synthesis during the cloning process. It was found that the flanks (some containing the codon) were differently lost clones. Then, one of the six clones was selected to compare the nucleotide sequence with the beta-glucosidase gene of Trichoderma Risei species, and the results are shown in FIG. In order to repair the deleted portion of the 5 'side, primers were synthesized by referring to the nucleotide sequence of the beta-glucosidase gene of the Trichoderma lysase, which was confirmed to have high homology with the gene of the present invention. Thereafter, PCR was performed using the primers represented by SEQ ID NOs: 5 and 6. The PCR reaction was denatured by heating at 94 ° C. for 5 minutes, and then repeated 30 times under conditions of 1 minute at 94 ° C., 1 minute at 50 ° C., and 1 minute at 72 ° C., followed by final amplification at 72 ° C. for 10 minutes. The reaction was terminated. Subsequently, agarose gel electrophoresis was performed to identify PCR products presumed to be beta-glucosidase genes, and the results are shown in FIG. 3. The PCR product was separated from the gel and cloned into the pGEM T-easy vector, and the entire nucleotide sequence of the PCR product was analyzed. As a result, as described in SEQ ID NO: 1, it could be confirmed that the gene is about 2220 bp in size, and the gene was named "C4 βglu". In addition, it was confirmed that the protein expressed from the gene is composed of 733 amino acids, as described in SEQ ID NO: 2.

<Example 4>

Cloning of Beta-Glucosidase Gene and Introduction into Yeast

The embodiment was cut βglu C4 gene is inserted into a pGEM T-easy vector obtained in Example 3 by Xba Ⅰ, again with parts cut Xho Ⅰ (partial digestion) and yeast - E. coli shuttle vector (yeast- E.coli shuttle vector) Was subcloned to pVT-103U (available from Dr. Thomas David of Canada biotech. Research institute) and the recombinant vector was named pVT-βG. Thereafter, the recombinant vector pVT-βG was used to transform yeast host cells according to Ito et al . (Ito, H., et al ., J. Bacteriol . 153: 163-168, 1983). As the yeast host cell, S. cerevisiae SEY2102 ( MATα ura3-52 leu2-3, 112 his4-419 suc2-Δ9 ) (obtained from Dr. Nam Su-wan of Dong- Eui University) was used. Subsequently, transformants were selected using uracil deficient SD medium (0.67% Bacto-yeast, nitrogen base without amino acids, 2% glucose, 0.5% Casamino acid), which was named S. cerevisiae SEY2102 / pVT-βG. .

Example 5

Measurement of Beta-glucosidase Activity of Yeast Transformants

Transformant obtained in Example 4S. cerevisiaeTo measure the beta-glucosidase activity of SEY2102 / pVT-βG, transformants on SD medium were placed in 5 ml YNBCAD medium (0.67% yeast nitrogen without amino acid, 0.5% casamino acid, 2% glucose) for 3 days. After incubating the culture solution for each hour while incubating, 0.1 mL of the culture solution was mixed with 0.4 mL of 1 mM PNPG (p-nitrophenyl-β-D glucopyranoside) dissolved in acetic acid buffer (pH5.0) and reacted at 40 ° C for 30 minutes. . 1 ml 1 M Na₂CO₃Was added and diluted with 5 ml of distilled water, and the absorbance was measured at 420 nm. Beta-glucosidase activity was measured by examining the amount of free PNP by the above method. The results are shown in Table 1 below. In this case, the control was not transformed wild type (wild type)S. cerevisiaeSEY1202.

Beta-glucosidase activity (Unit / L) Incubation time 24 hours 48 hours 72 hours Control 10 15 17 Yeast transformants 24 70 85

As shown in Table 1, as the incubation time increased, it was confirmed that the beta-glucosidase was expressed in the yeast transformant S. cerevisiae SEY1202 / pVT-βG strain to which the C4 βglu gene was introduced according to the present invention. . From this, it can be seen that the C4 βglu gene according to the present invention is a beta-glucosidase gene.

<Example 6>

Yeast transformants S. cerevisiae Of SEY2102 / pVT-βG in vitro Efficacy test

The degradation rate of in vitro natural detergent fiber (NDF) and acid detergent fiber (ADF) of S. cerevisiae SEY2102 / pVT-βG strains obtained in Example 4 were investigated. The yeast transformants were added to 0.2 mg / ml in 0.5 g of orchard grass ground finely ground to a size of 1 mm and incubated at 39 ° C. for 24 and 48 hours using a DAISY incubator. In addition, the in vitro NDF and ADF degradation rates (%) were analyzed using a Dairy crude analyzer. The results are shown in Table 2 below. All experiments were repeated four times, using the covariance analysis of the SAS statistical package, and the significant tests between treatments were analyzed by Duncan's multiple comparison method by full randomization.

Hour (hr) NDF decomposition rate (%) ADF decomposition rate (%) Yeast transformants 24 3.08 0.11 48 10.62 4.78

As shown in Table 2, as the incubation time was increased it was confirmed that the NDF and ADF degradation rate increased. This shows that the beta-glucosidase protein is expressed and secreted in yeast S. cerevisiae SEY1202 very efficiently, and as a feed additive showing the excellent digestive effect of the S. cerevisiae SEY2102 / pVT-βG strain, the recombinant strain of the present invention. The result shows that it can be useful.

As described above, in the present invention, beta-glucosidase gene was isolated from Trichoderma genus C-4 strain, and the gene was introduced into yeast strain. Then, it was confirmed that the beta-glucosidase gene is expressed and secreted at a high level in the yeast strain. According to the present invention, by transforming the yeast using a beta-glucosidase gene derived from the Trichoderma genus C-4 strain, the disadvantage of the yeast that can not degrade the fibrin substrate at all can be overcome. In addition, according to the present invention, the yeast transformants into which the beta-glucosidase gene derived from the Trichoderma genus C-4 strain is introduced and the beta-glucosidase expressed from the gene are used for brewing, baking, alcohol production, feed It can be usefully used for production and probiotic development.

Claims (9)

Β-glucosidase having the amino acid sequence represented by SEQ ID NO: 2. Β-glucosidase gene encoding the β-glucosidase of claim 1. The β-glucosidase gene C4 βglu according to claim 2, which has a nucleotide sequence represented by SEQ ID NO: 1. 4. The β-glucosidase gene C4 βglu according to claim 3, which is isolated from Trichoderma sp . C-4. Recombinant vector having the gene of claim 2 inserted. The recombinant vector pVT-βG according to claim 5. A transformant transformed with the vector of claim 6. 8. The Saccharomyces cerevisiae SEY2102 / pVT-βG of claim 7. A feed additive comprising the Saccharomyces cerevisiae SEY2102 / pVT-βG of claim 8.