KR100377380B1 - Recombinant Yeast producing phytase - Google Patents

Abstract

The present invention relates to a phytase-producing recombinant yeast using a phytase gene derived from Aspergillus ficuum , which controls the expression of phytase genes and phytase genes of Aspergillus picum amplified by PCR. GPD promoter (pGPD), 5 'UTR of GPD gene that improves translation efficiency of phytase gene mRNA, RAmy1A, which is a phytate secretion signal, and GAL7, which is a transcription terminator, were inserted into yEP352 vector and cloned. Pi produced by the carrier pYEGRPh vector and introduced into the yeast strain ( S. cerevisiae 2805) to produce a transformed recombinant yeast strain, and then mutated by ultraviolet treatment of the yeast strain again and secreted from the recombinant yeast mutant By measuring the titer of the taze by plate assay method, it is possible to produce phosphatase enzyme very efficiently. And transformed recombinant yeast strain, an excellent effect of providing a plate assay method of quickly and easily retrieve the activity of the pi Te-rise production from the strain.

Description

Recombinant Yeast producing phytase

The present invention relates to novel phytase producing recombinant yeast strains. More specifically, the present invention provides a translation efficiency of the phosphatase Aspergillus ficuum phosphate gene and GPD promoter (pGPD), a regulator that regulates phytase gene expression, phytate gene mRNA After constructing recombinant vector pYEGRPh containing 5 'UTR of GPD gene, RAmy1A, which is a phytase secretion, and GAL7, which is transcription terminator, the vector was introduced into S. cerevisiae 2805. The present invention relates to a plate assay method for comparing and retrieving transformed yeast strain recombinant yeast strain and the titer of the yeast strain produced by mutating the transformed recombinant yeast by irradiation with ultraviolet rays.

Yeast has traditionally contributed to human life with the history of alcohol and bread, and has played the most important role in the fermentation of liquor and bread. Recent advances in the natural sciences have revealed the mystery components of yeast cells and their metabolism, so that yeast cells are not only an excellent source of nutrients for higher animals, but also an important biochemical material, and also by metabolism of yeast, It is found that the production is possible, so that the usefulness of yeast is gradually increasing.

The usefulness of yeast is as follows: First, yeast can perform glycosylation, which plays an important role in structural integrity, solubility, biological activity of protein, and second, yeast is non-pathogenic and harmless GRAS ( Generally recognized as safe) microorganisms have proven stability in food and drug production, and third, yeast can contribute to the improvement of the purification process by secreting and producing proteins extracellularly.

Among the many factors that cause environmental and water pollution in livestock farming, phosphorus is one of the important constituents in vivo, but it is an essential nutrient for animals, but the utilization rate of phosphorus in feed is very low and it is the main culprit of environmental pollution. Phosphorus utilization in animal feed shows that only 20-30% of organic nutrients are used and the rest are excreted, resulting in severe water pollution, and the addition of inorganic phosphates to the feed due to the low phosphorus utilization leads to lower feed costs. In addition, the organic properties of grains have problems such as acting as anti-nutritional factors in combination with other nutrients or essential minerals. Therefore, in order to solve this problem, it is urgent to develop a low pollution feed that can make the best use of phosphorus acting as a potential pollutant and anti-nutritional factor in livestock feed.

Phytic acid, myo-inositol 1,2,3,4,5,6-hexakisphos-

Phate is a major component of cereals, the most common source of poultry and pig feed, and an important organophosphate storage compound. Phytinic acid is a substance in which six molecules of phosphorus of anion are combined with one molecule of inositol. It contains 0.14-7.50% by weight depending on the kinds of grains, seed oil, legumes, and protein by-products of livestock feed. According to the anionic nature of phosphorus, it is present in the state of being combined with cationic groups such as proteins, fats, and starches as well as minerals of cations important for livestock such as zinc, iron and magnesium. In addition, in the case of phosphorus contained in such feedstock, about 30-70% by weight is present in the form of phytic acid. The organic constituents present in the form of phytic acid are hydrolyzed into myo-inositol and inorganic phosphorus by the action of phytase (phytase: myo-inositol hexaphosphate phosphohydrolase EC 3.1.3.8) Not only is it transformed into an inorganic form, but it is also bound to phytic acid, which increases the availability of major minerals and proteins, such as iron, zinc, and magnesium, which decrease their availability. (Harper et al., 1997). However, the organic constituents present in more than two-thirds of these feeds have a very weak secretion of phytase, a degrading enzyme in unit animals such as chickens and pigs, and thus contain a large amount of phosphorus excreted in feces. In addition, the availability of major minerals and proteins is low.

For this reason, many studies have reported that phytase enzymes, like other enzymes, are used as enzyme additives in unit livestock feed, increasing the availability of organic foods in feeds by more than 60% and reducing the content of organic foods excreted in animal manure. Denbow, 1995). The reduction of organic nutrients due to the addition of phytase is expected to play a role not only in solving environmental problems but also as a substitute for expensive minerals. However, the phytase sold on the market loses most of its activity due to the low pH of the animal even when administered to a unit animal because the pH is unstable. In addition, the production process is more difficult than other enzymes, and expensive production facilities, technology, and manpower are required, so that expensive culture medium is used for mass production in order to obtain phytate from microorganisms. There was a problem.

The present invention relates to a 5 'UTR of GPD gene, RAmy1A which is a secretion signal of phytate, and a transcription terminator, which enhance the translation efficiency of glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter (pGPD) and phytase gene mRNA of S. cerevisiae . By producing a recombinant vector using GAL7, etc. and introducing it into the yeast, it is possible to easily produce a large amount of phosphatase, a phosphatase by producing a recombinant yeast that efficiently transforms the phytase. By adding the yeast culture itself to the feed, there is an advantage of producing an enzyme probiotic that can continuously express phytase in the animal's stomach. In addition, the plate assay of the mutants generated by ultraviolet irradiation of yeast provides a method for quickly and easily comparing and searching the phytate production titres, which is very useful.

Accordingly, an object of the present invention is to provide a recombinant plasmid pYEGRPh into which the phytase gene is inserted in view of the above facts. Another object of the present invention is to provide a novel recombinant yeast strain transformed by introducing the recombinant plasmid pYEGRPh inserted with the phytate gene. Still another object of the present invention is to provide a method for quickly and easily searching the titer of phytase secreted from recombinant yeast by plate assay of the transformed mutant yeast strain generated by UV irradiation of the yeast strain.

The object of the present invention is to construct a recombinant plasmid pYEGRPh for high efficiency production of phytase using a phytase gene of Aspergillus ficuum and a yEP352 vector, a kind of yeast episomal vector. This vector was then introduced into co-yeast yeast ( S. cerevisiae 2805, Y2805) to prepare a recombinant recombinant yeast strain, and the strain was irradiated with UV light again to mutate the strain, and the mutants were used to produce yeast produced in yeast. This was accomplished by providing a way to quickly and easily retrieve potency.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a schematic diagram of a gene map of the recombinant vector pYEGRPh1 which is a gene carrier.

FIG. 2 is a photograph showing phytase separation using sodium phytate as a substrate for colorimetric detection.

3 is a plate assay photograph for searching the titer of phytase produced in recombinant yeast.

Figure 4 is a plate assay photograph of the phytase carried out in the medium except agar (agar) component.

The present invention is aspergillus picum (phosphate degrading enzyme) in the yEP352 vector, a kind of yeast episomal vector (Aspergillus ficuum), The GPD promoter (pGPD), a regulator that regulates phytase gene expression, the 5 'UTR of the GPD gene that enhances the translation efficiency of phytase gene mRNA, RAmy1A, a secretion signal of phytase, and transcription. Constructing a recombinant vector pYEGRPh by inserting a terminator, such as GAL7; Yeast Disclosing the Recombinant Vector pYEGRPhS. cerevisiae2805 (Y2805) to prepare a transformed recombinant yeast strain; Irradiating and transforming the transformed yeast strain with ultraviolet light (UV mutagenesis) to improve the performance of the recombinant yeast strain; Comprising the step of conducting a plate assay to search for phytase titer secreted from the transgenic recombinant yeast mutant.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Preparation of Recombinant Vector pYEGRPh, a Recombinant Phytase Gene Carrier

Aspergillus picum, the first stage disclosure strain ( Aspergillus ficuum Isolation of Phytase Gene and PCR Amplification of Phytase Gene

The phytease gene was isolated from the disclosed strain Aspergillus ficuum . After separating total RNA from Aspergillus picum, 5 ㎍ of the total RNA isolated from above was incubated at 65 ° C for 10 minutes to synthesize first-strand cDNA, and then cooled with ice water. 11 μl of a bulk first-strand cDNA reaction mixture, 1 μl of DTT (1 M), and 1 μl of Not Id (T) ₁₈ (5 μg / μl) were added and reacted at 37 ° C. for 1 hour. . Thus synthesized first-strand cDNA (first-strand cDNA) in 5 μl 10 μl 10X PCR buffer 8 μl, 2.5 mM dNTP 8 μl, 5 '-/ 3'-primer each 150 ng, dH2O 77 μl, rTaq (Takara) 2.5 U was added, and PCR was repeatedly performed at 94 ° C. for 5 minutes (94 ° C., 1 minute-45 ° C., 72 ° C.-1, 3 minutes) X30 cycles at 72 ° C. for 7 minutes. 1% agarose gel electrophoresis of the DNA amplified by PCR showed a DNA band of about 1.4 kbp, which was in agreement with the expected PCR product size. As a result of ligation to the pGEM-T Easy (Promega Co.) vector to determine the base sequences of the 5 'and 3' sides, aspergillus picum was compared. ( Aspergillus ficuum ) was found to be a gene encoding the phytate protein.

Step 2 Construction of Recombinant Vector pYEGRPh Inserted with the Amplified Pytease Gene

A recombinant vector was constructed to efficiently produce phytase using yEP352 vector, a kind of yeast episomal vector.

The α-amylase 1A gene (RAmy1A), a secretion signal for secreting the phytase gene amplified by PCR in the first step and the phytase enzyme produced by being expressed in the yeast strain into the medium, was overlapped. After the cleaved fusion gene with restriction enzyme BamH1, it is a regulator that regulates phytase gene expression.Saccharomyces cerevisiaeOf glyceraldehyde-3-phosphate dehydro-genase (GPD) Promoter (pGPD), 5 'UTR of GPD gene, 5' UTR to increase efficiency of plasmid gene mRNA translation, galactose-1-P uridyl transferase for transcription termination of phytase gene The terminator of GAL7 was inserted into the yeast expression vector yEP352 to construct a recombinant vector pYEGRPh. The sequence information of the junction region of the recombinant vector pYEGRPh (pGPD + RAmy1A + mPhyA + tGal7) thus constructed is as follows.

Sequence information of the intergenic linkage site of the recombinant vector pYEGRPh (pGPD + RAmy1A + mPhyA + tGal7)

GPD promoter + α-amylase 1A signal peptide + mphyA + tGAL7

* **

first junction: aa a cacca ag aacttagtttcga-ggggatcc-gcatgcaggtgctga

pGPD BamHI 1Asp

second junction: tgacagccggg-ctggcagtcccccg

1Asp mPhyA

[* Is the transcription initiation site of pGPD (promotor).]

A genetic map of the recombinant vector pYEGRPh is shown in FIG. 1 (FIG. 1).

Example 2 Production of Transformed Recombinant Yeast Strain Transformed by Introducing Recombinant Vector pYEGRPh and Determination of Activity of Recombinant Pytease

Production of Transgenic Recombinant Yeast Strain Incorporating First Stage Recombinant Vector pYEGRPh

In order to produce the recombinant yeast of the present invention, a strain of Saccharomyces cerevisiae 2805 [MATa pep4 :: HIS3 prb1-d Can1 GAL2 his3 ura (3-52) 1-1] was received from Dr. Sang Ki Lee of the Korea Research Institute of Bioscience and Biotechnology. 1% yeast extract, 2% peptone, 2% dextrose) medium and incubated in 30 ^o C incubator. The recombinant vector pYEGRPh of Example 1 was transformed by inserting into yeast S. cerevisiae 2805 (Y2805) cultured according to Li-acetate method such as Ito (Ito et al., 1983). This transformed yeast was plated in ura-medium and the transformants were selected.

The present inventors named the transformed recombinant yeast strain thus obtained as S. cerevisiae TSA05 and deposited it with the Korean Bacillus Association on July 25, 2000 with accession number KFCC 11188.

Purification and Activity Measurement of Recombinant Phytase Produced in Stage 2 Transgenic Recombinant Yeast

After inoculating the transformed recombinant yeast S. cerevisiae TSA05 of the first step in a concentration of 1 × 10 ⁷ in 40 ml of expression medium YEPD, shaking the culture at 200 rpm, and filtering the supernatant, the titer titer in the culture filtrate every 24 hours. Was measured. The phytase titer was reacted for 30 minutes at 58 ° C by adding 50ul of culture filtrate to 354ul buffer containing sodium phytate and 800ul of AAM (10mM Ammonium molybdate, 5N H2SO4, Acetone = 1: 2: 2). 100ul of the reaction solution was mixed and 80ul of 1M citric acid was added thereto, and then molybdate produced was measured.

The phytase activity of the recombinant yeast strain measured by absorbance OD _{405 nm} is shown in Table 1.

Phytease Activity of Transgenic Recombinant Yeast Strain replication 1 day 2 days 3 days 4 days 5 days S. cerevisiae TSA05 One 0.279 0.432 0.341 0.249 0.118 2 0.304 0.515 0.264 0.278 0.121 3 0.302 0.351 0.344 0.319 0.131 Average 0.307 0.432 0.316 0.282 0.153 pYEGPD-phy A One 0.154 0.129 0.318 0.137 0.248 2 0.180 0.138 0.224 0.330 0.194 3 0.175 0.139 0.337 0.188 0.260 Average 0.169 0.135 0.294 0.162 0.234 S.cerevisiae 2805 <0.02 <0.02 <0.02 <0.02 <0.02 NOTE The absorbance was measured at OD _{405 nm} .

The phytase activity of the transformed recombinant yeast strain was greater than that of the control pYEGPD-p hy A or S. cerevisiae 2805 strains, especially after 2 days of culture. Recombinant yeast from which the yeast strain was recombined increased phytase production efficiency.

In addition, the recombinant yeast was inoculated at a concentration of 1 × 10 ⁷ in 40 ml of expression medium YEPD, followed by shaking culture at 200 rpm to measure the number of cells in the culture solution at an absorbance of OD _{600 nm} every 24 hours. The results are shown in Table 2 below.

Yeast growth rate Strains Replication 1 day 2 days 3 days 4 days 5 days S.cerevisiae TSA05 One 3.82 10.00 13.62 10.76 14.30 2 4.26 10.08 14.18 13.34 14.44 3 4.15 8.94 12.72 13.92 14.44 Average 4.07 9.91 13.50 12.67 14.39 pYEGPD-phyA One 3.62 8.80 11.74 13.14 15.32 2 3.67 9.36 13.98 12.38 15.06 3 3.85 8.58 13.02 15.44 15.02 Average 3.71 8.91 13.62 13.65 15.13 S.cerevisiae Y2805 3.95 10.01 12.05 13.75 13.51 NOTE Absorbance was measured at OD _{600 nm} .

Recombinant yeast and recipient strains reached a stationary phase after exponential growth on day 3 after culture, and no inhibition of yeast growth by phytase expression was found.

Example 3 Mutation of Recombinant Yeast (UV mutagenesis) and Plate Assay to Improve the Performance of Transformed Recombinant Yeast Strains

Production of the first stage recombinant yeast mutant

The transformed recombinant yeast strain (parent strain) was grown in ura- medium until the absorbance reached OD _600nm 0.5, and then the cells were cultured, washed twice with distilled water and dissolved in 10mM MgSO ₄ , and then the cells. After counting and diluting to 1.0 × 10 ⁷ / ml, 5 ml of cell suspension is mutated by irradiating with ultraviolet rays to plate in a petri dish. Cell survival ratio according to ultraviolet dose for mutations is shown in Table 3 below.

UV Dosage for Mutation of Recombinant Yeast Condition Cell count percent Control 304 100% 600x100uJ / cm ² 0.1 min 58 19% 600x100uJ / cm ² 0.2min 24 7% 600x100uJ / cm ² 0.3min 5 One% 600x100uJ / cm ² 0.4min 0 0% 600x100uJ / cm ² 0.5min 0 0% Note: Ultraviolet radiation of 600x100 micro joule energy was investigated at 600x100uJ / cm ² = 1cm ² (1J = 0.239 cal = 1 watt).

The mutated cells were diluted 10-fold to about 300 / plate.

The mutant cell line was prepared by placing the cells in CYEPD medium and growing them in a cancerous state for 2-3 days.

Step 2 plate assay for detecting the titer of phytase secreted from the recombinant mutant yeast

To improve the performance of the phytase producing strains, titers of phytase produced by the mutated recombinant yeast mutants were compared and searched on plates. The putative mutant surviving in Example 3 was transferred to plateassay medium to measure the titer of phytase.

Sterilized 2 X nutrient solution (Nutrient solution) and 2 X vitamin solution (vitamine solution) composed of the composition of Table 4 and Table 5 were added, and then sterilized agar (final concentration = 1.5%) was added and 1M HCl was added. The medium was prepared by adjusting the pH of the medium to 5.5 while mixing little by little. The recombinant mutant yeast strain was streaked in this medium, put in a 30 ° C. incubator, and cultured for 2-3 days, and then white precipitate was observed.

Ingredients of Nutrient solution Configuration (g / L) Na-phytate 5 NH ₄ NO ₃ 3 MgSO ₄ 0.5 CaCl ₂ -2H ₂ O 0.13 MnSO ₄ -5H ₂ O 0.05 FeSO ₄ -7H ₂ O 0.18 Glucose 10

Ingredients of Vitamin Solution Configuration (mg / L) Biotin 10.0 Ca-pantothenic acid 120 Inositol 600 Pyridoxine 120 Thiamine HCl 120

As a result of plate assay, white precipitate was formed around colony of phytase-producing recombinant yeast strain. As a reference, the difference in yeast phytate production capacity was also searched (Fig. 2, 3, 4).

As is apparent from the above examples, the present invention provides a recombinant yeast strain S. which effectively produces a phytase enzyme by introducing and transforming the recombinant vector pYEGRPh into which the phytate gene is inserted into the yeast strain Saccharomyces cerevisiae 2805 (Y2805) . It is effective in providing cerevisiae TSA05. In addition, by mutating the recombinant yeast strain by ultraviolet irradiation and plate assay, there is an excellent effect of providing a method for quickly and easily searching the titer of phytase secreted from recombinant yeast, which is a very useful inventor in the animal feed industry and environmental conservation industry. will be.

Claims (5)

Recombinant vector pYEGRPh comprising fragments of the phytase gene and portions thereof A high yielding recombinant yeast S. cerevisiae TSA05 (KFCC 11188), characterized in that the recombinant vector pYEGRPh according to claim 1 containing the phytate gene was introduced. Recombinant phytase enzyme expressed from recombinant yeast according to claim 2 Unit animal feed containing a recombinant phytase enzyme produced from the recombinant yeast according to claim 2 Plate assay method for detecting phytase titer secreted from mutated by ultraviolet irradiation of phytase high efficiency production recombinant yeast according to claim 2, wherein the recombinant vector pYEGRPh containing phytase gene is introduced