JPH0142673B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel hybrid plasmid containing a gene derived from the yeast Candida utilis (hereinafter abbreviated as C. utilis) and a microorganism containing the same. Generally, Escherichia coli is used as a host for genetic manipulation, but it is thought that it is more advantageous to use eukaryotes such as yeast as hosts for microbial production of useful substances produced by higher organisms (such as insulin and growth hormones). It will be done. In addition, there have been reports of cases in which, like the hepatitis B virus surface antigen gene, it is not produced in E. coli, but its production has been confirmed in yeast.
(Atsushi Miyanohara et al. Proc. Natl. Acad. Sci.
USA 80 , 1-5 (1983) Currently, Saccharomyces cerevisiae is used as a host yeast.
Hereafter abbreviated as S.cerevisiae. ) are only allowed. However, yeasts of the genus Candeida are generally used in large-scale industrial production of microbial cells, such as in sulfite pulp waste liquid fermentation and petroleum fermentation. In addition, the genus Candeida has many practical properties that are advantageous compared to S. cerevisiae, such as a wider range of carbon assimilation. It is expected to play a role as a host bacterium when producing substances. Among them, Quyandida utilis (C.
utilis) has already been proven safe as a yeast for food and feed, and today its importance is increasing as a producing bacterium for useful pharmaceuticals and clinical test drugs such as glutathione and coenzyme A (CoA). The present inventors have discovered that C.
First, with the goal of developing a vector system using the utilis host.
As a result of investigating the development of a vector containing a specific gene of C. utilis as a selection marker, the present invention was completed by successfully creating the desired new hybrid plasmid. In the present invention, S.
YRp7, a shared vector between E. cerevisiae and E. coli
(K. Struhl et al. Proc. Natl. Acad. Sci. 76 1035
(1979)) was used. That is, the restriction enzyme of YRp7
E. coli RRI strain (leuB) was transformed with a plasmid mixture in which a partially digested fragment of C. utilis ATTCC9226 DNA was ligated to the BamHI site, and a leu ⁺ strain was selected. Furthermore, E.coli leuB
It has already been confirmed that the mutation is complemented by the LEU2 gene of S. cerevisiae. ((B.Ratzkin
and J.Carbon:Proc.Natl.Acad.Sci. 74 487
(1977)). As a result, it showed ampicillin resistance,
Colonies that complemented the leuB mutation were obtained. The plasmid isolated from this E. coli transformant was
It was named pYKL-30. This plasmid has a molecular weight of 10.7 kilobases (Kb) (6.4 megadaltons (Md)) and contains ampicillin resistance, TRP1 and LEU2 genes.
The restriction enzyme map and general structure are shown in Figure 1. Approximately 4.9Kb LEU2 cloned here
Genes are produced by Southern hybridization (EM
Southern, J.Mol.Biol. 98 503 (1975)), C.
I confirmed that it was derived from utilis. In addition, this plasmid allows S.cerevisiaeDBY-
As a result of transforming 746 (Trp1, leu2), C.utilis
The derived LEU2 gene was found to complement leu2 of S. cerevisiae. The C. utilis gene is S.
This is the first time that it has been expressed in S. cerevisiae. Next, in order to create a plasmid consisting only of the C. utilis gene and pBR322, the Trp1 gene derived from S. cerevisiae was removed. pYKL-30 with restriction enzyme
It was partially digested with EcoRI and ligated again. This mixture was used to transform E. coli JA300 (leuB, TrpC), and ampicillin-resistant and leu ⁺ strains were selected. Next, tryptophan auxotrophy was investigated by the replica method, and trp ^- strains were selected. this
Plasmids were isolated from colonies showing leu ⁺ and trp ^- , the desired Trp1 gene was removed, and C. utilis
Plasmid pYKL consisting of LEU2 and pBR322
-40 was obtained. This plasmid has 9.25Kb (5.55Md) ampicillin resistance and LEU2 gene markers and a second
It has the restriction enzyme map shown in the figure. As a result of subcloning analysis of this plasmid,
The LEU2 gene was found to be located at the site shown in the figure. By cutting out the TRP1 gene part of YRp7 and introducing it into the EcoRI partial digest of this plasmid, pYKL-
30 and 2 μm DNA, etc.
It is also possible to create a new shared vector for yeast and E. coli by ligating fragments that can be propagated in S. cerevisiae. Based on this subcloning analysis data
An approximately 2.5 Kb fragment containing the LEU2 gene was excised from pYKL-40 using the restriction enzyme Hfnd.
Ligation into the Hind site of pBR322 resulted in plasmid pYKL-45, which contained the ampicillin resistance and LEU2 genes and, more conveniently, restored the tetracycline resistance gene. This plasmid has a molecular weight of 6.9 Kb (4.1 Md) and, as shown in Figure 3, has single restriction enzyme sites for Pst and Sal, multiple initiation sites for C. utilis,
It is effective as a cloning better for other genes. As an application example of the plasmid created according to the present invention, plasmid pYKL-60 shown in FIG. 4 was created. Namely, MJCaSadaban et al.
Promoter cloning vector prepared by J. Bact. et al., J. Bact. 143 971-980 (1980)
The lacZ′ part of pMC1403 (8 codon deletion of the bromotor region on the structural gene of β-galactosidase)
Excise with BamH and Sal and extract from pYKL−30.
It was inserted into the 3.9Kb part excised by BamH and Sal. When the new plasmid was transformed into E. coli lacZ' strain and selected on a synthetic medium containing ampicillin and Xgal (5-bromo-4-chloro-3-indolyl-β-galactose), colonies showing blue color were detected. That is, only the lacZ ⁺ strain contained the target plasmid pYKL-60. This indicates that pYKL-30 can express a heterologous gene product using a similar technique, and this plasmid is expected to play a role as an expression vector.
In addition, other pYKL-40 and pYKL-45 can be used as expression vectors in E. coli and cloning vectors for the replication origin of C. utilis DNA. In addition, plasmids pYKL-30, pYKL-40,
pYKL-45 was used in E. coli RR strain, and pYKL-60 was used in E. coli RR strain.
coliMC1061 strain, and pYKL-30 was added to S. coliMC1061 strain.
The strains transferred to the cerevisiaeDBY-746 strain were given accession numbers and
FERM P-6927 No. 6927 (FERM P-6927)
No. 6928 (FERM P-6732), No. 6945 (FERM P-6945), No. 6958 (FERM P-
6958), and the same No. 6944 (FERM P-6944)
Deposited as. E. coli or S. coli transfected with the plasmid of the present invention.
cerevisiae exhibits the same mycological properties as the known parent strain, and also has the characteristics shown in Table 1 below.

[Table] *The present invention will be specifically explained with reference to examples below. Example 1 Preparation of plasmid pYKL-30 Method already known for S. cerevisiae (JR
Broach et al., Gene8 121-133 (1979)), a hybrid plasmid was created by ligating a partial DNA digest of C. utilis ATCC9226 and plasmid YRp7, and the result was transformed into E. coli (leu ^- strain). ,
A strain exhibiting leu ⁺ was isolated, and the target plasmid pYKL-30 was extracted from the strain. That is, 10 μg of plasmid YRp7 was injected with restriction enzyme Bam.
Digestion with H140 units for 1 hour at 37°C followed by alkaline phosphatase treatment (1 unit, 60°C,
30 minutes) to remove the phosphorus at the 5′ end. Separately C.
utilisDNA is partially digested with 0.5 units of restriction enzyme Sau3A, the fragment and the above plasmid are mixed, and a ligation reaction is performed at 15°C for 16 hours using 0.1 unit of T4 ligase. On the other hand, E. coli RR strain (leuB ^- ), which had been previously cultured in ordinary bouillon medium to mid-logarithmic growth phase,
The ligation mixture was added to the suspension in a 50mM calcium chloride aqueous solution and left at 0°C for 30 minutes.
°C for 30 min to complete transformation. The transformants were cultured on ordinary bouillon agar medium containing 100 μg/ml of ampicillin at 37°C for 16 to 24 hours, and the resulting colonies were cultured using the replica method on Davis minimal medium containing 25 μg/ml each of proline and thiamine. Transfer to a cell and select the resulting colonies. The plasmid isolated from the colony thus obtained is
pYKL-30. Example 2 Preparation of plasmid pYKL-40 Plasmid pYKL-305 μg obtained in Example 1
is partially digested with restriction enzyme Eoc R5 units at 37°C for 15 minutes and ligated with T4-ligase (0.05 units, 15°C, 16 hours). This mixed plasmid was used to transform E. coli JA-300 strain (leuB, trpC) in Davis minimal medium (1) containing 100 μg/ml ampicillin, 25 γ/ml each of tryptophan, threonine, thymine, and thiamine. °C, 24
Incubate for ~48 hours. Culture medium (2) is obtained by removing tryptophan from culture medium (1) using the replica method of the resulting colonies.
Investigate growth in. As a result, pYKL-40 was a plasmid isolated from a colony that grew only in medium (1) and did not proliferate in medium (2). Example 3 Preparation of plasmid pYKL-45 Plasmid pYKL-40 obtained in Example 2 10μ
g was digested with the restriction enzyme Hind40 unit at 37°C for 1 hour, and each fragment was fractionated on a 0.8% agarose gel. After staining with ethidium bromide, a fragment of approximately 2.5 Kb was cut out under ultraviolet irradiation, and the DNA in the gel was cut out.
The fragments are extracted by the freeze-thaw method. On the other hand, 2 μg of pBR322 was heated at 37°C in Hind8 units.
After digestion for 1 hour, it was treated with alkaline phosatase (0.2 units, 60°C, 1 hour), mixed with the 2.5 Kb fragment, and ligated together using T4-ligase.
Transform into E. coli RR (leu ^- ) strain. next,
Spread on Davis minimal medium containing 100 γ/ml ampicillin, 25 γ/ml each of proline and thymine, and incubate at 37°C.
Incubate for 24-48 hours. Regarding the colonies that emerged,
The plasmid pYKL-45 was isolated from a colony that showed resistance after being transferred to a normal bouillon medium containing 25 γ/ml of tetracycline. Reference example 1 Preparation of plasmid pYKL-60 Plasmid pYKL- obtained in Example 1
30 10 μg each of restriction enzymes BamH and Sal
Digest at 37°C for 1 hour, and fractionate each fragment using 0.8% agaroma gel. On the other hand, plasmid
10 μg of pMC1403 was added to BamH and Sal
Digest with 0.8% agaroma gel and fractionate with 0.8% agaroma gel.
In the former case, 6.9Kb fragment, in the latter case
Each 6.2 Kb fragment is extracted and ligated together using T4-ligase. Next, E. coli MC1061 (lacZ') is transformed and cultured at 37° C. for 16 to 24 hours on a normal bouillon medium containing 100 γ/ml ampicillin. The resulting colonies were treated with Xgal (5-bromo-4-chloro-
The cells were streaked onto Davis minimal medium containing 25 γ/ml each of alanine and leucine (3-indolyl β-galactose), and blue colonies indicating β-galactosidase production were isolated. The plasmid obtained from that colony is pYKL-60. Example 4 Expression of hybrid plasmid pYKL-30 in S.cerevisiae S.cerevisiaeDBY with plasmid pYKL-30
-746 transformation was performed. For transformation, the lithium acetate method (Murata et al., Japanese Society of Agricultural Chemistry, 1957 Mizo Abstracts, P568 (1982)) was used. That is, YEPD
Cultivate to around OD1.0 with 10ml of culture solution, and after harvesting, suspend in 0.5ml of 0.2M lithium acetate, and store at 28℃.
Shake for 1 hour, then take 0.1 ml and add 10 μg of
Add pYKL-30 and leave at 30℃ for 30 minutes. Add 0.1ml of 70% polyethylene glycol 4000 and heat at 30℃ for 1
Leave it for a while. After treatment at 42℃ for 5 minutes, when spreading on yeast amino acid-requiring minimal medium containing 25γ/ml each of uracil and histidine, 30℃ for 2 to 2 minutes.
After 4 days, target colonies transformed with plasmid pYKL-30 were obtained.

[Brief explanation of drawings]

Figure 1 - Restriction enzyme map of plasmid pYKL-30, Figure 2 - Restriction enzyme map of plasmid pYKL-40, Figure 3 - Restriction enzyme map of plasmid pYKL-45, Figure 4 - Restriction enzyme map of plasmid pYKL-60. map. In the figure pBR322, S.CerevisiaeTRP1
gene,. utilisLEU2 gene, E.coli
lacZ′ gene.

Claims (1)

[Scope of Claims] 1. A hybrid plasmid consisting of at least bacterial plasmid DNA and the LEU2 gene, which is derived from a fragment partially digested with the restriction enzyme Sau3AI of the yeast Candida utilis ATCC9226 and complements the LEU2 gene of Satucharomyces cerevisiae. 2. The hybrid plasmid according to claim 1, which also contains the TRP1 gene of the yeast Saccharomyces cerevisiae. 3. The hybrid plasmid of claim 1, wherein the plasmid is pYKL-30. 4. The hybrid plasmid of claim 2, wherein the plasmid is pYKL-40. 5 Consists of at least bacterial plasmid DNA and the LEU2 gene, which is derived from a fragment partially digested with the restriction enzyme Sau3AI of the yeast Candida utilis ATCC9226 and complements the LEU2 gene of Satucharomyces cerevisiae, and further contains the TRP1 gene of the yeast Satucharomyces cerevisiae. Yeast Candida utilis ATCC9226 excised with the restriction enzyme Hind from a hybrid plasmid
Plasmid pBR322 fragment containing the LEU2 gene
A hybrid plasmid characterized in that the tetracycline resistance gene is restored by inserting it into the Hind site of . 6. The hybrid plasmid according to claim 5, wherein the plasmid is pYKL-45. 7. Escherichia coli containing a hybrid plasmid selected from pYKL-30, pYKL-40, and pYKL-45. 8 Yeast Saccharomyces cerevisiae containing plasmid pYKL-30.