JPH07132089A - New gene, new yeast with the same selectively destroyed and use of the yeast - Google Patents

Abstract

PURPOSE:To provide a new gene useful for producing thermosensitive autolytic yeast advantageous for acquiring yeast extract. CONSTITUTION:The objective gene NLT1 having a base sequence coding protein having an amino acid sequence of the formula. Saccharomyces cerevisiae (FERM P-13410, hereafter referred to as YTS-1 strain), a thermosensitive autolytic mutant, is incorporated with a gene library constituted by incorporating plasmid YEp24 with the chromosome DNA of Saccharomyces cerevisiae X-2180 strain as a wild-type strain, and a YPD medium is inoculated with the transformant followed by culture at 37 deg.C; plasmid pSLY-1 is then extracted from the resultant raised colonies followed by subcloning and then cutting with restriction enzymes Kpn1 and Cla2 to obtain the objective gene NLT1, a fragment of 3Kbp. A Saccharomyces yeast is incorporated with a plasmid destroying the gene NLT1 in a genetic engineering way in yeast to obtain the other objective Saccharomyces cerevisiae DLT11-1B strain (FERM P-13930) or the like, a thermosensitive autolytic yeast.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel gene NLT1, a protein encoded by the gene, and a method for producing a yeast extract and a protein using a novel yeast in which the gene is disrupted.

[0002]

2. Description of the Related Art It is well known that, in the production of heterologous proteins using gene recombination techniques, it is difficult to give sugar chains or fold them correctly, and the original activity is often not exhibited. In particular, when Escherichia coli, which is a prokaryote, is used as a host for production, it has no sugar chain-conferring function and folding is different from the original one. Often not. As one method for solving this problem, it has been attempted to use yeast, which is a eukaryote, and succeeded in producing a hepatitis B vaccine [M. Pasek et al., Nature, vol. ． 282, p575-,
1979]. Also, one of the mite allergens, Der f
In the case of I, the active form was not obtained when E. coli was produced as a host, but the active form was obtained when yeast was used as the host [KY Chua et al., Journal of・ Aragy and Clinical Immunology (J. Allergy Clin. Immun
ol), vol. 89, p95-, 1992]. As described above, the effectiveness of using yeast as a host has been shown,
When yeast was used, it was not easy to crush the cells to recover the target substance, and various measures were required. As one of the methods, a method of adding a signal peptide so that the target protein is secreted outside the bacterium is considered, but it has not yet been established as an effective method for all proteins. In particular, when secretory production of a protein having a large molecular weight was attempted using yeast, it was reported that the secretion of the protein was inhibited by a strong cell wall [Zesevo (KM Zseb
o) et al., Journal of Biological Chemistry (J. Biol. Chem.), Vol. 261,
p5858-, 1986].

It is thought that these problems will be solved if yeasts that can be dissolved under certain conditions are obtained. As a result of cell cycle-related studies, DE Levin et al. Reported that mutations in protein kinases (PKC1 and BCK1) reduce yeast resistance to turgor pressure and cause lysis [The Journal].・ Of Cellbiology (J. Cell Biol., Vol.116, p12)
21-1992) and Molecular and Cellular Biology (Mol. Cell. Biol., Vo).
l. 12, p172-1992)]. Similar reports were made by G. Paravicini and others (Molecular and Cellular Biology (Mo.
l. Cell. Biol. , Vol. 12, p4896
,, 1992). Therefore, mutation or gene disruption to protein kinase is considered to be one of the methods for constructing the target yeast, but the enzyme is one of the key enzymes for many in vivo reactions, and mutations are introduced here. This is likely to affect many yeast reactions. Also,
It has been reported that yeasts having mutations or gene disruptions to these protein kinases have reduced resistance to turgor pressure and require osmotic pressure for growth, which is not suitable as a method for constructing the target yeast. There is a point.

[0004]

[Objective] Therefore, a first object of the present invention is to attempt to isolate a novel gene different from the PKC1 and BCK1 genes for yeast autolysis, and to elucidate the gene. The second object of the present invention is to elucidate the protein encoded by the gene. A third object of the present invention is to provide a novel yeast in which the above gene is disrupted by using a genetic engineering technique. A fourth object of the present invention is to provide a method for producing a yeast extract using the novel yeast. A fifth object of the present invention is to provide a method for producing proteins using the novel yeast.

[0005]

[Structure] The first aspect of the present invention relates to a novel gene NLT1 involved in yeast autolysis. This gene is a temperature-sensitive autolytic mutant, Saccharomyces cerevisiae [(Sa
ccharomyces cerevisiae) Accession No. FERM P-13410 (hereinafter referred to as YTS-1 strain)] was used to isolate a gene that suppresses this mutation. First, the chromosomal DNA of the wild-type strain Saccharomyces cerevisiae X-2180 strain was transformed into plasmid Y.
The gene library constructed by incorporating it into Ep24 was introduced into the YTS-1 strain. The transformant was planted in YPD medium, cultured at 37 ° C., and plasmid pSLY-1 was extracted from the grown colonies. A DNA fragment of about 6 Kbp was inserted into this plasmid. Subcloning was performed, and it was determined that a fragment of about 3 Kbp cut by restriction enzymes Kpn I and Cla I had an activity of suppressing the temperature-sensitive autolytic mutation of the YTS-1 strain (FIG. 4). The gene that suppresses the temperature sensitivity of the YTS-1 strain was named NLT1 and its nucleotide sequence was examined.
And the base sequences shown in 2 (for convenience of electronic filing, one figure is shown separately in FIG. 1 and FIG. 2). We investigated homology with other genes, especially with protein kinases reported to be associated with lytic mutations. As a result, NLT1 was a novel gene with no homology to any previously reported gene.

The second aspect of the present invention relates to a protein having the amino acid sequence shown in FIG. 3 which is encoded by the gene NLT1. The protein encoded by the NLT1 gene predicted from the determined nucleotide sequence has 493 amino acids and an estimated molecular weight of 56000. This protein may be used as an inhibitory agent for yeast autolysis.

A third aspect of the present invention is a temperature-sensitive autolytic yeast which is genetically modified by introducing a plasmid prepared for the purpose of genetically disrupting the gene NLT1 into yeast into Saccharomyces yeast. In particular, it relates to the yeast Saccharomyces cerevisiae DLT11-1B strain with the accession number FERM P-13930. The genetically modified temperature-sensitive autolytic yeast is obtained as follows. A plasmid was prepared by substituting the protein-encoding portion in the sequence of the gene NLT1 that suppresses the temperature-sensitive autolytic mutation with a genetic marker such as the gene URA3 that makes the host non-auxiliary. This plasmid was introduced into Saccharomyces cerevisiae KA31, which is a wild-type diploid, and a transformant DLT11 strain was selected using uracil non-auxotrophy as a marker. By sporulating the DLT11 strain, the NLT1 gene disrupted strain FERM P-13930 (hereinafter referred to as DLT1
1-1B strain) was obtained.

The fourth aspect of the present invention relates to a method for producing a yeast extract using the DLT11-1B strain. Specifically, it is a method of producing a yeast extract by culturing, collecting and washing the yeast, leaving it at 37 ° C. or higher for 10 hours or more, and removing the bacterial cell residue if necessary.

The fifth aspect of the present invention relates to a method for producing a protein using the DLT11-1B strain. In particular,
The present invention relates to a method for producing a protein by treating the DLT11-1B strain under a temperature condition suitable for secreting the intracellular proteins into the medium.

[0010]

【Example】

Example 1 (Cloning of gene NLT1 involved in autolysis) Temperature-sensitive autolysis mutant YTS-1 strain (FERM P
-13410) was used to clone a gene involved in autolysis. Chromosomal DNA of Saccharomyces cerevisiae X-2180 is a restriction enzyme Sau3
The gene library prepared by inserting the fragment partially digested with AI into the BamHI site of the plasmid YEp24 was transformed into the temperature sensitive autolytic mutant yeast YTS-1 strain. The transformant selected without uracil requirement was replaced with YPD medium, and the plasmid pSLY-11 was extracted from the colonies grown by culturing at 37 ° C. The obtained plasmid pSLY-11 had a size of about 6 Kbp.
DNA fragment was inserted. The results of subcloning are shown in FIG. It was judged that a gene that suppresses the temperature sensitivity of YTS-1 is present in a fragment of about 3 Kbp that is cleaved with the restriction enzymes Xba I and Cla I. This gene was named NLT1 and its DNA base sequence was determined. The determined nucleotide sequences are shown in FIGS. The protein encoded by the NLT1 gene predicted from the determined nucleotide sequence was composed of 493 amino acids and a molecular weight of 56000. This gene had no homology with other reported genes or yeast genes PKC1 and BCK1 which exhibit autolysis due to mutation.
From the above results, the NLT1 gene cloned this time was a completely novel gene different from any of the genes reported so far.

Example 2 (Construction of NLT1 gene-disrupted strain) A plasmid pDNLT1 for gene disruption of the gene NLT1 involved in yeast autolysis was prepared as shown in FIG.

Restriction enzyme Sma of plasmid pUC119
I, Kpn I digestion fragment, and plasmid pSLY-1
Plasmid pSL11-SK2 was prepared by inserting a DNA fragment of about 3 Kbp containing the NLT1 gene cleaved with Sma I and Kpn I of 1. Plasmid p
SL11-SK2-Δ36 is the same as pSL11-SK2,
Cleavage with restriction enzymes SacI and SmI, followed by digestion with nucleolytic enzyme ExoIII, nucleolytic enzyme Mung Be
treated with an Nuclease, and the nucleic acid synthase Kle
It is created by smoothing with now Fragment and then ligating. pSL11-SK2-
The NLT1 gene inserted in Δ36 is represented by A in FIG.
Is the part shown in.

Further, a 1.9 Kbp DNA containing the NLT1 gene cleaved with the Bam HI and Hind III of the plasmid pSLY-11 in the restriction enzyme Bam HI and Hind III cleaved fragment of the plasmid pBR322.
Plasmid pDNLT1-HB with the fragment inserted
Created 1. NL inserted into pDNLT1-HB1
The T1 gene is the portion indicated by B in FIG.
Restriction enzyme EcoR of plasmid pDNLT1-HB1
I, Hind III cleavage fragment, and plasmid pSL1
1-SK2-Δ36 EcoRI, about 1.3 Kbp DN containing the NLT1 gene cleaved with Hind III
The A fragment was inserted into the plasmid pDNLT1-E
B1 was created. N inserted into pDNLT1-EB1
The LT1 gene is the part shown by A and B in FIG. 4, and the part shown by C is missing. Furthermore, the generated plasmid pDNLT1-EB1 had a Hi
Hin containing the yeast Saccharomyces cerevisiae URA3 gene of the plasmid YEp24 at the ndIII site
The dIII fragment 1.1 Kbp was inserted to insert NL
A plasmid pDNLT1 for T1 gene disruption was prepared.
The created plasmid pDNLT for disrupting the NLT1 gene
1 has a portion containing A and B in FIG. 4, and the portion indicated by C is u of yeast Saccharomyces cerevisiae.
The ra3 gene is inserted instead. As a result, by introducing a DNA fragment obtained by treating the plasmid pDNLT1 with the restriction enzyme Cla I into the ura3 mutant strain of the yeast Saccharomyces cerevisiae, homologous DNA
Was obtained, and a transformant lacking the protein-encoding portion of the NLT1 gene could be obtained.

Therefore, the Cla I-treated DNA fragment of the plasmid pDNLT1 was inserted into the diploid yeast Saccharomyces cerevisiae KA31 strain homozygous for the ura3 mutation for transformation. By this transformation, the uracil non-auxotrophic strain DLT11 strain was obtained. This DLT11 strain is sporulated to give uracil non-auxotrophic haploid yeast DLT1.
The 1-1B strain was selected. The obtained DLT11-1B strain has the NLT1 gene disrupted. FIG. 6 shows NL
It is a graph which shows the relationship between the viable cell count and time of DLT11-1B strain which is a T1 gene-disrupted strain and its parent strain KA311A. The vertical axis represents the time after the temperature shift of 37 ° C., and the horizontal axis represents the viable cell count in logarithmic form. The wild-type strain KA311A grows normally even after 37 ° C. temperature shift, whereas the NLT1 gene-disrupted strain DLT11-.
The 1B strain had a decreased viable cell count after a temperature shift of 37 ° C.
It decreased to about 1% 48 hours after the shift.

Example 3 (Yeast extract production method using NLT1 gene-disrupted yeast strain DLT11-1B) After culturing parent strain KA311A strain and NLT1 gene-disrupted strain DLT11-1B at 25 ° C. for 2 days , Collect and wash, suspend in distilled water, 37 ℃ or 52 ℃
Left for 18 hours. Then, centrifugation was performed to remove the bacterial cell residue to obtain a yeast extract. As a control, the KA311A strain, which is the parent strain of the disrupted strain, was used. Extract extraction rate is
It was determined according to the method of Nakanishi et al. (Japanese Patent Application No. 1-249502). The extraction rate of the control KA311A strain was 10.7% at 37 ° C and 34.1% at 52 ° C, while the extraction rate of the NLT1 gene-disrupted strain DLT11-1B strain was 20% at 37 ° C. At 3% and 52 ° C., it was 50.7%, and improvement of the extract extraction rate was recognized due to NLT1 gene disruption (Table 1).

[Table 1]

Example 4 (Secreted production of intracellular protein using NLT1 gene-disrupted yeast strain DLT11-1B) Parent strain KA311A and NLT1 gene-disrupted yeast strain DLT11-1B were cultured at 25 ° C. for 2 days. After that, the cells were collected and washed, suspended in distilled water, the temperature was raised to 37 ° C., and left for 18 hours. Then, the mixture was centrifuged, and the protein content secreted in the supernatant was determined by the method of OH Lowly et al. [J. Biol. Chem. Vo1, 193, p26.
5-, 1951)]. The results are shown in Table 2. The NLT1 gene-disrupted strain DLT11-1B strain was found to leak 9.32 mg of protein per dry cell weight, whereas the wild-type strain KA311A strain contained 2.14 mg of protein per dry cell weight. A leak was noted. By the temperature treatment at 37 ° C., the protein leakage amount of the NLT1 gene-disrupted strain increased by about 4-fold.

[Table 2]

[0017]

【effect】

(1) In the present invention, a gene that functions to inhibit yeast dissolution at 37 ° C was found, and its nucleotide sequence was confirmed. (2) A novel yeast in which the above gene was disrupted by genetic engineering was created. (3) Use of this yeast enables efficient production of yeast extract.

[Brief description of drawings]

FIG. 1 shows a part of the nucleotide sequence of the gene NLT1 of the present invention.

FIG. 2 shows a part of the nucleotide sequence of the gene NLT1 of the present invention, which is a continuation of FIG. 1.

FIG. 3 shows the amino acid sequence of the protein encoded by the gene NLT1 of the present invention.

FIG. 4 shows a process of constructing a plasmid in the process of subcloning the cloned gene NLT1 of the present invention.

FIG. 5 shows a process for preparing a plasmid for preparing the yeast of the present invention in Example 2.

FIG. 6 is a graph showing changes in the viable cell count of the temperature-sensitive yeast DLT11-1B strain of the present invention with respect to time after temperature shift to 37 ° C. The horizontal axis represents the time after the temperature shift, and the vertical axis represents the viable cell count in logarithmic form.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 21/02 C 9282-4B // (C12N 1/19 C12R 1: 865) (C12P 21/02 C12R 1: 865)

Claims (6)

[Claims] 1. A gene having the nucleotide sequences shown in FIGS. 1 and 2. 2. A protein having the amino acid sequence shown in FIG. 3, which is encoded by the gene according to claim 1. 3. A yeast obtained by introducing a plasmid prepared for the purpose of disrupting the gene according to claim 1 by genetic engineering into a yeast of the genus Saccharomyces. 4. The yeast is yeast Saccharomyces cerevisiae DLT11-1B (accession number FERM P-13).
930), The yeast according to claim 3. 5. A method for producing a yeast extract using the yeast according to claims 3 and 4. 6. A method for producing a protein using the yeast according to claim 3 or 4.