KR102653503B1 - An engineered yeast reducing ethyl carbamate in makgeolli and a preparation method of makgeolli using thereof - Google Patents

Abstract

The present invention is a recombinant Saccharomyces cerevisiae characterized by deleting CAR1 to reduce arginase production and deleting the GZF3 gene, which suppresses DUR 1, DUR 2, and DUR 3 gene expression. ), it is possible to produce fermented liquor with reduced ethyl carbamate content through the present invention.

Description

Improved yeast strain for manufacturing makgeolli with reduced ethyl carbamate and manufacturing method of makgeolli using the same {An engineered yeast reducing ethyl carbamate in makgeolli and a preparation method of makgeolli using the same}

The present invention relates to a recombinant strain in which ethyl carbamate production is reduced by genetically recombining Saccharomyces cerevisiae .

Ethyl carbamate is a naturally occurring substance in fermented beverages and foods and is known to be carcinogenic to humans. When consumed in small amounts, it causes dizziness and mouth pain, and when consumed in excessive amounts, it worsens intestinal health and causes cancer. Ethyl carbamate is continuously produced during fermentation and storage of alcoholic beverages, so efforts are needed to reduce it.

In the United States, the ethyl carbamate content is recommended to be less than 15ppb for table wine and less than 60ppb for fortified wine, and in Korea, the Ministry of Food and Drug Safety published the 'Manual for Reduction of Ethyl Carbamate in Alcoholic Beverages' in 2011. Efforts are being made to reduce ethyl carbamate.

In order to reduce ethyl carbamate, methods such as using yeast with a defective CAR1 gene, controlling fermentation temperature, and limiting arginine or urea in mainstream fermentation materials were used. However, this method has a small reduction effect, so when identifying and using a new brewing yeast with excellent fermentation ability, there is a problem that if the strain is a strain with a high production of ethyl carbamate, it cannot be used for actual mainstream fermentation.

Republic of Korea Patent Publication No. 10-2015-0045687 (publication date: 2015.04.29) relates to a method of reducing ethyl carbamate in fermented liquor, which minimizes the content of ethyl carbamate in fermented liquor by adding urease and adjusting reaction conditions. A possible method is described. Republic of Korea Patent No. 10-1813050 (publication date: December 21, 2017) relates to a method for producing plum wine with a reduced concentration of ethyl carbamate, in which plum leachate is distilled under reduced pressure under certain conditions to produce plum distillate. , it is described that plum wine with a reduced concentration of ethyl carbamate can be produced by using a method of producing plum wine by dilution.

The present invention provides a recombinant Saccharomyces cerevisiae that can reduce the amount of ethyl carbamate produced by genetically recombining the yeast Saccharomyces cerevisiae , and the strain is The aim is to provide fermented liquor with reduced ethyl carbamate content.

The present invention is a recombinant Saccharomyces cerevisiae characterized by deleting the CAR1 gene to reduce arginase activity and deleting the GZF3 gene to relieve inhibition of DUR 1, DUR 2, and DUR 3 gene expression. ( Saccharomyces cerevisiae ) is provided. On the other hand, due to the deletion of the GZF3 gene, the expression of DUR 1, DUR 2, and DUR 3 genes may preferably be increased. In addition, due to the increase in DUR 1 and DUR 2 gene expression, the elements accumulated in the bacterium are decomposed, and due to the increase in DUR 3 gene expression, elements other than the bacterium are introduced into the bacterium, increasing the amount of elements present outside the bacterium. It may have this decreasing characteristic.

The present invention provides a method for producing fermented liquor, characterized by using the recombinant Saccharomyces cerevisiae as a fermentation strain. Meanwhile, the fermented liquor may be makgeolli, preferably manufactured by using rice as a substrate and culturing it statically.

The present invention provides makgeolli produced by the above fermented liquor production method. Meanwhile, the makgeolli may preferably be characterized by reduced ethyl carbamate content.

According to the present invention, a fermented liquor with a reduced ethyl carbamate content is produced through genetic recombination of the yeast Saccharomyces cerevisiae strain and a recombinant strain with reduced ethyl carbamate production. It can be manufactured.

Figure 1 shows how CAR1 and GZF3 genes function in forming ethyl carbamate in the strain.
Figure 2 is a schematic diagram explaining the method for producing the recombinant strain of the present invention.
Figure 3 shows the chromosome number of the Saccharomyces cerevisiae BY4742 strain, known as a 1N strain with one chromosome, and the Saccharomyces cerevisiae GRL6 strain used in the present invention using a flow cytometer. This is the result of comparison.
Figure 4 shows the results of colony PCR to confirm whether the defect occurred in the recombinant strain of the present invention.
Figure 5 is a comparison result of the content of fermentation broth components of the recombinant strain of the present invention.
Figure 6 shows the results of comparison of RNA expression levels of the recombinant strains of the present invention.
Figure 7 shows the results of comparing the ethanol content of makgeolli using the recombinant strain of the present invention.
Figure 8 shows the results of comparing the ethyl carbamate content of makgeolli using the recombinant strain of the present invention.
Figure 9 is a comparative photograph of makgeolli using the recombinant strain of the present invention.

It is known that ethyl carbamate is naturally produced when urea excreted by yeast and urea present outside combine with ethanol produced during yeast fermentation.

Meanwhile, arginase is an enzyme that decomposes arginine, and when arginine is decomposed, urea is produced. Representative genes related to the decomposition and excretion of elements produced within the bacterial cell include the DUR 1, DUR 2 genes, and DUR 3 genes (Figure 1). Among them, the DUR 1 and DUR 2 genes are known to be genes that decompose urea (Urea) present within cells, and the DUR 3 gene is known to be a gene that absorbs extracellular urea (Urea). However, the expression of the DUR 1, DUR 2, and DUR 3 genes is regulated by the GZF3 gene, and it is known that the expression of the DUR 1, DUR 2, and DUR 3 genes is suppressed under normal circumstances.

In the present invention, an attempt was made to reduce the amount of elements produced within the bacterial cells and to reduce the amount of elements existing outside the bacterial cells by decomposing the produced elements. For this purpose, a three-step strategy was used. First, we wanted to reduce the amount of elements produced within the bacterial cell. We tried to reduce the activity of arginase by deleting the CAR1 gene (encoding the arginase enzyme), which is known to encode arginase. Second, we attempted to increase the expression of DUR 1 and DUR 2 genes to decompose urea present in the bacterial cells into ammonia and carbon dioxide. Third, we attempted to increase the expression of the DUR 3 gene in order to introduce elements present in the medium into the bacterial cells (after introduction, they are decomposed into ammonia and carbon dioxide). At this time, the expression of the DUR 1, DUR 2, and DUR 3 genes is regulated to be suppressed by a protein (transcriptional inhibitor) expressed by the GZF3 gene. In the present invention, by deleting the GZF3 gene, DUR 1, DUR 2, and DUR compared to the wild type 3 Gene expression levels were able to be increased. Through this, it was possible to reduce the production of urea present in the bacterial cells and also promote the decomposition of the urea present in the bacterial cells. In addition, urea present in the medium could be introduced into the bacterial cells and decomposed. Through this, it was ultimately possible to reduce the production of ethyl carbamate present in the extracellular medium (e.g., makgeolli).

Therefore, the present invention is a recombinant Saccharomyces seri characterized by deleting the CAR1 gene to reduce arginase activity and deleting the GZF3 gene to relieve the inhibition of DUR 1, DUR 2, and DUR 3 gene expression. Provides Saccharomyces cerevisiae . According to the following experimental example, it can be seen that the CAR1 gene of the recombinant strain of the present invention is hardly expressed, and the expression levels of DUR 1, DUR 2, and DUR 3 genes increase. In addition, it can be seen that the amount of ethyl carbamate produced has decreased, and accordingly, it can be determined that the amount of urea secreted has decreased.

Meanwhile, the Saccharomyces cerevisiae is not limited to a specific one, and existing known or newly isolated ones can be used. In the present invention, those isolated from yeast were used.

The present invention provides a method for producing fermented liquor, characterized by using the recombinant Saccharomyces cerevisiae as a fermentation strain. Meanwhile, the fermented liquor may correspond to any alcoholic beverage fermented using Saccharomyces cerevisiae . For example, it can be produced from wine, beer, Cheongju, Takju, and Yakju.

Preferably, rice can be used as a substrate and cultured statically to produce makgeolli, more preferably cultured at 24 to 26°C for the first 22 to 26 hours, and then cultured at 19 to 21°C for 150 to 180 hours. Makgeolli can be produced by culturing. According to the following experimental example, when makgeolli was produced by using the recombinant strain and rice of the present invention as a substrate and culturing it statically, there was no significant difference in ethanol productivity compared to the existing wild-type strain, and the color, aroma, and taste were all similar. could be confirmed. In addition, it was confirmed that the ethyl carbamate content was significantly reduced.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but also includes modifications of the technical idea equivalent thereto.

The strains used in the examples and experimental examples of the present invention are shown in Table 1, the plasmids are shown in Table 2, and the primers are shown in Tables 3 and 4.

Strains used in the examples and experimental examples of the present invention strain name Related features source S. cerevisiae GRL6 Industrial yeast strain (unknown genotype) this invention ΔCAR1 S. cerevisiae GRL6ΔCAR1 this invention ΔGZF3 S. cerevisiae GRL6ΔGZF3 this invention ΔCAR1ΔGZF3 S. cerevisiae GRL6 ΔCAR1 ΔGZF3 this invention E. coli TOP10 F-mcrA (mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG Invitrogen
(CA, USA)

Plasmids used in the examples and experimental examples of the present invention plasmid name Related features source pRS42K-gDNA-HXK2 HXK2 guide RNA expression plasmid, Geneticin marker Production of 1-butanol and butanol isomers by metabolically engineered Clostridium beijerinckii and Saccharomyces cerevisiae
Seung-Oh Seo, 2017, Ph.D. Thesis, University of Illinois at Urbana-Champaign pRS42K-gRNA-CAR1 Replacement of gRNA cassette for deletion of HXK2 with gRNA cassette for deletion of CAR1 this invention pRS42K-gRNA-GZF3 Replacement of gRNA cassette for deletion of HXK2 with gRNA cassette for deletion of GZF3 this invention pCas9-NAT Cas9 expression plasmid, Nourseothricin marker Addgene (#43802) pRS42H Yeast episomal plasmid, Hygromycin B marker System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae
Christof Taxis and Michael Knop
BioTechniques 2006 40:1, 73-78 pCas9-Hyg Replacement of Nourseothricin marker with Hygromycin B marker this invention

Primers used in Example 1 of the present invention Primer name sequence number ITS1 SEQ ID NO: 1 ITS4 SEQ ID NO: 2 SDM-gRNA-CAR1-F SEQ ID NO: 3 SDM-gRNA-CAR1-R SEQ ID NO: 4 SDM-gRNA-GZF3-F SEQ ID NO: 5 SDM-gRNA-GZF3-R SEQ ID NO: 6 Hyg-insert-F SEQ ID NO: 7 Hyg-insert-R SEQ ID NO: 8 Cas9-NAT-backbone-F SEQ ID NO: 9 Cas9-NAT-backbone-R SEQ ID NO: 10 Donor-CAR1-F SEQ ID NO: 11 Donor-CAR1-R SEQ ID NO: 12 Donor-GZF3-F SEQ ID NO: 13 Donor-GZF3-R SEQ ID NO: 14 Check-CAR1-F SEQ ID NO: 15 Check-CAR1-R SEQ ID NO: 16 Check-GZF3-F SEQ ID NO: 17 Check-GZF3-R SEQ ID NO: 18

Primers used in Experimental Example 2 of the present invention Primer name sequence number DUR1, 2-F SEQ ID NO: 19 DUR1, 2-R SEQ ID NO: 20 DUR3-F SEQ ID NO: 21 DUR3-R SEQ ID NO: 22 CAR1-F SEQ ID NO: 23 CAR1-R SEQ ID NO: 24 ACT1-F SEQ ID NO: 25 ACT1-R SEQ ID NO: 26

[Example 1: Recombinant Saccharomyces cerevisiae of the present invention ( Saccharomyces cerevisiae ) produce]

1) Overview

In this example, we attempted to produce recombinant Saccharomyces cerevisiae with deletion of CAR1 and GZF3 genes. Figure 1 shows how the genes function to produce ethyl carbamate, and Figure 2 shows a schematic diagram of producing a recombinant strain.

2) Strain isolation

In the present invention, yeast was separated from nuruk and used. After performing colony PCR on the isolated yeast using ITS1 and ITS4 primers, it was identified through sequence determination that the isolated strain was a Saccharomyces cerevisiae strain commonly used in the manufacture of alcoholic beverages. The strain isolated in this experiment was named Saccharomyces cerevisiae GRL6. Afterwards, we attempted to confirm the chromosome number of the identified strain using flow cytometry. S. cerevisiae BY4742 strain, known as a 1N strain with one chromosome, and Saccharomyces cerevisiae GRL6 strain identified from yeast were simultaneously stained with propidium iodide dye and then analyzed using flow cytometry. The intensity of fluorescence was measured. The results are shown in Figure 3. As a result of comparing the fluorescence of the two strains using a flow cytometer, it was confirmed that the fluorescence of Saccharomyces cerevisiae GRL6 was twice that of Saccharomyces cerevisiae BY4742. As a result, it was found that Saccharomyces cerevisiae GRL6 is a 2N strain with two chromosomes.

3) Plasmid production

We attempted to construct pCas-Hyg plasmid to express Cas9 protein in yeast. In the case of the existing pCas-NAT plasmid, Nourseothricin was used as a marker. Since the Nourseothricin antibiotic is expensive, we attempted to create a pCas-Hyg plasmid using Hygromycin B antibiotic as a marker for more efficient and economical experiments. The Hygromycin B gene was PCR performed using the Hyg-insert primer in the pRS42H plasmid, and all parts except the Nourseothricin-related gene were PCR performed using the Cas9-NAT-backbone primer in the pCas-NAT plasmid. The detailed experimental method was produced according to the Gibson Assembly Kit protocol.

Meanwhile, we attempted to create pRS42K-gRNA-CAR1 and pRS42K-gRNA-GZF3 plasmids to express gRNA targeting the CAR1 and GZF3 genes to be deleted. NEB (MA, USE)'s Q5 Site-Directed Mutagenesis Kit was used to change the 20bp gRNA sequence targeting the HXK2 gene in the pRS42K-gRNA-HXK2 plasmid to match the CAR1 or GZF3 gene. The pRS42K-gRNA-CAR1 plasmid was created through PCR using the pRS42K-gRNA-HXK2 plasmid and SDM-gRNA-CAR1-R and SDM-gRNA-CAR1-B primers, and the pRS42K-gRNA-HXK2 plasmid and SDM-gRNA- pRS42K-gRNA-GZF3 plasmid was created through PCR using primers GZF3-F and SDM-gRNA-GZF3-R. The detailed experimental method was prepared according to the Q5 Site-Directed Mutagenesis Kit protocol.

Meanwhile, E. coli TOP10 strain was used for cloning and replication of all the above plasmids.

4) Donor DNA production

After cutting the target gene using Cas9 and gRNA, we attempted to create donor DNA to cause homologous recombination. Donor DNA of 90 bp per gene was prepared, and the primers used were Donor-CAR1-F, Donor-CAR1-R, Donor-GZF3-F, and Donor-GZF3-R. Each 60 bp forward and reverse primer has a corresponding 30 bp complementary region, and double-stranded donor DNA was produced through two PCRs.

5) Recombinant strain production

CRISPR/Cas9 was performed for gene deletion in yeast. pCas-Hyg plasmid and CAR1 expressing Cas9 protein for precise gene cutting, pRS42K-gRNA-CAR1 and pRS42K-gRNA-GZF3 plasmid expressing gRNA for targeting GZF3 gene, CAR1 to cause homologous recombination, After preparing GZF3 gene donor DNA, transformation was attempted. The transformation method followed 'High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method' (R Daniel Gietz & Robert H Schiestl, 2007, NATURE PROTOCOLS).

After transformation, gene deletion was confirmed through colony PCR. Primers Check-CAR1-R, Check-CAR1-B, Check-GZF3-R, and Check-GZF3-B were used in colony PCR, and the results are shown in Figure 4. Lines 1 to 4 in Figure 4 are the colony PCR results to confirm the CAR1 gene, and lanes 5 to 8 are the colony PCR results to confirm the GZF3 gene. As a result of colony PCR using the same primers, it can be seen that the band size is reduced compared to the wild type strain. Through this, it was confirmed that the CAR1 and GZF3 genes of the recombinant strain of the present invention were successfully deleted.

[Experimental Example 1: Comparison of fermentation products of wild-type strain and recombinant strain]

For comparison of fermentation products of recombinant strains, wild-type strain of Saccharomyces cerevisiae GRL6, ΔCAR1 strain with a deletion of the CAR1 gene, ΔGZF3 strain with a deletion of the GZF3 gene, ΔCAR1ΔGZF3 strain with both the CAR1 gene and GZF3 gene deletion, A total of four strains were fermented simultaneously under the same conditions. Fermentation was performed using YP medium containing 8% glucose, 50 mg/L urea, and 50 mM arginine, and the initial inoculation OD ₆₀₀ was set to 0.2, at 30°C and 200 rpm. proceeded. Fermentation broth was collected at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 168 h and stored at -80°C. Afterwards, the collected fermentation broth was analyzed through HPLC and GC/MS measurements. The measurement was repeated three times, and the average values are shown in Figure 5, Table 5, and Table 6. Figure 5 is a graph showing the glucose, glycerol, ethanol, and acetate concentrations and OD ₆₀₀ values of the fermentation broth of each strain. Among these, the values of the fermentation broth collected at 12 h are shown in Table 5 below. Table 6 shows the ethyl carbamate concentration of fermentation broth collected at 0 h, 24 h and 168 h.

strain name OD ₆₀₀ Ethanol (g/L) Ethanol yield
(g ethanol/g glucose) ethanol productivity
(g/L/h) S. cerevisiae GRL6 39.9±1.0 34.19 ± 0.24 0.45 ± 0.001 2.85 ± 0.02 ΔCAR1 41.3 ± 0.7 34.42 ± 0.07 0.44 ± 0.007 2.87 ± 0.005 ΔGZF3 40.7 ± 0.6 34.47 ± 0.63 0.47 ± 0.02 2.87 ± 0.05 ΔCAR1ΔGZF3 41.4 ± 0.3 34.34 ± 0.14 0.44 ± 0.02 2.86 ± 0.01

Table 5 shows the wild-type strain of Saccharomyces cerevisiae GRL6, the ΔCAR1 strain lacking the CAR1 gene, the ΔGZF3 strain lacking the GZF3 gene, and the ΔCAR1ΔGZF3 strain lacking both the CAR1 gene and GZF3 gene after 12 h of fermentation. It shows OD ₆₀₀ value, ethanol concentration, ethanol yield, and ethanol productivity. In the above items, it was confirmed that there was no significant difference among all four strains. Through this, it was confirmed that there was no difference in the growth and ethanol production of the strain even if the CAR1 and GZF3 genes were deleted in the wild type strain.

Ethyl carbamate concentration (μg/L) relative concentration value strain name 0 h 24h 168h 0h 24h 168h S. cerevisiae GRL6 13.2 ± 1.2 41.2 ± 5.2 56.9 ± 1.8 100 100 100 ΔCAR1 13.1 ± 1.5 28.0 ± 2.7 42.1 ± 0.9 99.3 67.9 73.9 ΔGZF3 13.0 ± 0.8 34.2 ± 2.9 48.2 ± 3.8 98.3 83.1 84.7 ΔCAR1ΔGZF3 12.9 ± 1.2 19.7 ± 5.1 37.6 ± 2.1 98.0 47.9 66.1

Table 6 shows the wild-type strain of Saccharomyces cerevisiae GRL6, the ΔCAR1 strain lacking the CAR1 gene, the ΔGZF3 strain lacking the GZF3 gene, and the ΔCAR1ΔGZF3 strain lacking both the CAR1 gene and the GZF3 gene at 0 h, 24 It shows the concentration and comparative amount of ethyl carbamate in the fermentation broth for 168 h and 168 h. From 24 h, all four strains showed significant differences from each other. The ΔCAR1 strain decreased to 67.9% compared to the wild type strain, the ΔGZF3 strain decreased to 83.1% compared to the wild type strain, and the ΔCAR1ΔGZF3 strain decreased to 47.9% compared to the wild type strain. In addition, even at 168 h, all four strains showed significant differences from each other. The ΔCAR1 strain decreased to 73.9% compared to the wild type strain, the ΔGZF3 strain decreased to 84.7% compared to the wild type strain, and the ΔCAR1ΔGZF3 strain decreased to 66.1% compared to the wild type strain. ΔCAR1ΔGZF3, which is defective in both CAR1 and GZF3 genes, shows the lowest ethyl carbamate concentration, confirming the excellent ethyl carbamate reduction effect of the recombinant strain of the present invention.

[Experimental Example 2: Confirmation of RNA expression level of wild-type strain and recombinant strain]

In this experimental example, the purpose was to confirm the RNA expression level of genes related to ethyl carbamate production in the wild-type strain, the ΔCAR1 strain with a deletion of the CAR1 gene, the ΔGZF3 strain with a deletion of the GZF3 gene, and the ΔCAR1ΔGZF3 strain with both the CAR1 gene and the GZF3 gene. did. To confirm the amount of RNA expression, qRT-PCR was performed on 6 h of yeast from the fermentation broth collected in Experimental Example 1, and the relative expression of CAR1 , DUR1 , DUR2 , and DUR3 genes was determined based on the ACT1 gene, which is known to be always expressed consistently. The expression level was calculated, and the results are shown in Figure 6.

Looking at the results in Figure 6, the expression level of the CAR1 gene was not expressed in the ΔCAR1 and ΔCAR1ΔGZF3 strains, and the expression level of the ΔGZF3 strain was not significantly different from that of the wild type strain. In the case of DUR1 and DUR2 genes, ΔCAR1 was not significantly different from the wild type strain, and the expression level of ΔGZF3 and ΔCAR1ΔGZF3 strains was significantly increased compared to the wild type strain. The expression levels of DUR1 and DUR2 genes increased 2.12-fold in the ΔGZF3 strain and 1.92-fold in the ΔCAR1ΔGZF3 strain compared to the wild type strain. In the case of the DUR3 gene, the ΔCAR1 strain was not significantly different from the wild type strain, and the expression level of the ΔGZF3 and ΔGZF3 strains was significantly increased compared to the wild type strain. The expression level of the DUR3 gene increased 1.41-fold in the ΔGZF3 strain and 1.31-fold in the ΔCAR1ΔGZF3 strain compared to the wild type strain. Through this, it was confirmed that the recombinant strain of the present invention was well recombined to reduce the amount of ethyl carbamate produced as intended.

[Experimental Example 3: Comparison of makgeolli using wild-type strain and recombinant strain]

In this experimental example, to determine whether there was a difference in the amount of ethyl carbamate production between the recombinant strain and the wild-type strain in actual liquor, makgeolli was mixed with 100 g of rice, 10 g of Chrysanthemum fungus powder, and 300 ml of water, and the cells of each strain were recovered at an OD of ₆₀₀ = 50. It was prepared by inoculating. Makgeolli was prepared using the wild-type strain, the ΔCAR1 strain with a deletion of the CAR1 gene, the ΔGZF3 strain with a deletion of the GZF3 gene, and the ΔCAR1ΔGZF3 strain with both the CAR1 and GZF3 genes, and were incubated at 25°C for the first 24 hours, and then cultured at 25°C for the first 24 hours. The culture was incubated at 20°C until the end of time. Culture fluid was collected for each strain at 0 h, 24 h, and 168 h, and ethyl carbamate and ethanol production were analyzed through HPLC and GC/MS measurements. The measured amount of ethanol is shown in Figure 7, and the measured amount of ethyl carbamate is shown in Figure 8. Looking at the results in Figure 7, it can be seen that there is no significant difference in ethanol content. Looking at the results in Figure 8, it can be seen that the recombinant strain according to the present invention excellently reduced the ethyl carbamate content of makgeolli.

Afterwards, the color, taste and aroma of makgeolli from the culture solution collected at 168 h were compared. Taste and aroma were compared through tasting and tasting, and both taste and aroma showed no significant differences. Additionally, looking at Figure 9, it can be seen that there was no significant difference in the color of the makgeolli.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> An engineered yeast reducing ethyl carbamate in makgeolli and a preparation method of makgeolli using it <130> YP-21-119 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SDM-gRNA-CAR1-F <400> 3 ggtttgaaca gttttagagc tagaaatagc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SDM-gRNA-CAR1-R <400> 4 cattaggaat gatcatttat ctttcactgc 30 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SDM-gRNA-GZF3-F <400> 5 gaacgtaagt gatcatttat ctttcactg 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> SDM-gRNA-GZF3-R <400> 6 ccaagttata gttttagagc tagaaatag 29 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Hyg-insert-F <400> 7 atcgacagag attgtactga gagtgcag 28 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Hyg-insert-R <400> 8 cggccagcct ccttacgcat ctgtgc 26 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Cas9-NAT-backbone-F <400> 9 taaggaggct ggccgggtga cccgg 25 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Cas9-NAT-backbone-R <400> 10 agtacaatct ctgtcgattc gatactaacg ccgccatcc 39 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Donor-CAR1-F <400> 11 gaaacaacaa caacaactat atcaataaca ataactacta tcaagtttat atcatcatcc 60 60 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Donor-CAR1-R <400> 12 ataaaaagag aatgcttatt ttgataaaag ggatgatgat ataaacttga tagtagttat 60 60 <210> 13 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Donor-GZF3-F <400> 13 gagaacatat tgcaagcggt tgaagctata atactagata tacgaatgta tgcatatata 60 60 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Donor-GZF3-R <400> 14 gttttgcaac tgattatgct actatgtatt tatatatgca tacattcgta tatctagtat 60 60 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Check-CAR1-F <400> 15 catcagggtt atgagcc 17 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Check-CAR1-R <400> 16 ggataacgta ccagtgg 17 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Check-GZF3-F <400> 17 agctcgttcc cgtca 15 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Check-GZF3-R <400> 18 ctgctttagt aaaaatcaat 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DUR1, 2-F <400> 19 ggtgtcccta ttgctgttaa g 21 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DUR1, 2-R <400> 20 ccgtgtgccg actaatcc 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DUR3-F <400> 21 actgcctgtg ggtgttgttg 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DUR3-R <400> 22 cgtctactgg atgcctcttg g 21 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CAR1-F <400> 23 gctgtcccgt gtcattcc 18 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CAR1-R <400> 24 gaccttcacc gtttgtttct g 21 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ACT1-F <400> 25 ttattgataa cggttctggt atg 23 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ACT1-R <400> 26 ccttggtgtc ttggtctac 19

Claims (7)

To reduce arginase activity, the CAR1 gene was deleted,
Makgeolli, characterized in that it was manufactured using a recombinant Saccharomyces cerevisiae with a deletion of the GZF3 gene as a fermentation strain to relieve the inhibition of DUR 1, DUR 2 and DUR 3 gene expression.
According to paragraph 1,
The recombinant Saccharomyces cerevisiae ,
Makgeolli characterized by increased DUR 1, DUR 2, and DUR 3 gene expression due to a deletion of the GZF3 gene.
According to paragraph 2,
The recombinant Saccharomyces cerevisiae ,
Due to the increase in DUR 1 and DUR 2 gene expression, the elements accumulated in the bacterial cell are decomposed, and due to the increase in DUR 3 gene expression, urea from outside the bacterial cell flows into the bacterial cell, increasing the amount of urea present outside the bacterial cell. Makgeolli characterized by reduction.
A fermentation strain using recombinant Saccharomyces cerevisiae , in which the CAR1 gene was deleted to reduce arginase activity, and the GZF3 gene was deleted to relieve the inhibition of DUR 1, DUR 2, and DUR 3 gene expression. A method of producing makgeolli with reduced ethyl carbamate content.
delete delete According to paragraph 1,
The makgeolli,
Makgeolli characterized by reduced ethyl carbamate content.