JPH0569510B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a DNA sequence containing a yeast glucoamylase gene that imparts starch assimilation ability to yeast and can be used to create expression and secretion vectors in yeast. In general, industrial alcohol fermentation using starch as a substrate is accomplished through two processes: (i) saccharification of starch to glucose by amylases, and (ii) conversion of glucose to ethanol by yeast. Catalyzing these two processes by one organism is necessary for rationalizing the production system.
This is one of the technical systems that has been sought after for many years. Traditionally, a specific type of yeast, Saccharomyces diastateicus,
diastaticus) was known to secrete glucoamylase, which hydrolyzes starch to produce glucose. However, Satsucharomyces
diastateichus has a weak ability to produce ethanol from glucose and to produce and secrete glucoamylase, making it unsuitable as an industrial strain for use in direct alcohol fermentation using starch as a substrate. On the other hand, for alcohol fermentation using glycol as a substrate, Saccharomyces cerevisiae is usually used as an industrial strain. The alcohol tolerance level of Satucharomyces cerevisiae reaches 17% to 21%, but Satucharomyces cerevisiae has no ability to produce glucoamylase and cannot perform direct alcohol fermentation using starch as a substrate. Therefore, the present inventors introduced the gene for glucoamylase, a secreted enzyme, from Satucharomyces diastatias into Satucharomyces cerevisiae, an industrial strain used for alcohol production.
We focused on the possibility of direct alcohol production using yeast as a starch substrate. Furthermore, the reason for selecting Satucharomyces diastateicus as the source of the glucoamylase gene to be introduced is that the gene recipient is Satucharomyces cerevisiae. That is, both belong to the genus Satucharomyces and are genetically closely related, so the glucoamylase gene of Satucharomyces diastateicus is also expressed by Satucharomyces cerevisiae, and it is predicted that the gene product will be produced. be. In addition to Satucharomyces diastateicus, filamentous fungi such as Aspergillus awamori and Rhizopus delemar are considered sources of glucoamylase genes, but they are not closely related biologically.
It is considered extremely difficult to express the glucoamylase genes of these strains in Satucharomyces cerevisiae. As described above, the present inventors have carried out intensive research using genetic recombination techniques to breed industrial strains that can be used directly for alcohol fermentation using starch as a substrate, and have developed S. diastateicus to produce glucoamylase. The gene was successfully cloned, the structure of the gene was determined, and the transformed strain obtained by introducing the gene into Satucharomyces cerevisiae was confirmed to have the ability to directly ferment alcohol from starch. This result represents the first successful cloning of a glucoamylase gene in a yeast system and provides a possible path toward implementing the long-sought technology of directly fermenting alcohol from glucose. In that sense, its significance is extremely large. Another major advantage obtained by the present invention is that the glucoamylase of Satucharomyces diastateicus is an extracellular secreted enzyme. That is, the DNA sequence encoding glucoamylase obtained by the present invention naturally makes it possible to secrete and accumulate glucoamylase itself in large quantities outside the yeast cells, but it also Since it has a promoter region and ribosome binding region that can perform expression, and a region involved in secretion that allows efficient secretion of proteins, a desired heterologous gene such as interferon, growth hormone, etc. can be inserted downstream of this DNA sequence. , interleukin, nerve growth factor, kallikrein, plasminogen activator, or other and bioactive polypeptide, or,
By linking structural genes such as enzymes, it is possible to construct expression and secretion vectors that efficiently secrete and accumulate these proteins outside the bacterial cells using yeast as a host. Moreover, the results obtained by the present invention
The fact that the DNA sequence is effective also in yeast other than the genus Satucharomyces can be confirmed by using the DNA sequence obtained by transforming Schizosaccharomyces pombe as shown in the Examples. The transformed strain is Satucharomyces
This is evident from the production of glucoamylase derived from P. diastaticus. That is, the DNA sequences obtained by the present invention can be used to express heterologous gene products, even in yeast systems other than those of the genus Satucharomyces.
It can be used for constructing secretion vectors. Furthermore, among the DNA sequences of the present invention, the nucleotide sequences of the promoter region and ribosome binding region, which are parts involved in gene expression, and the nucleotide sequences of the region involved in protein secretion may be used alone or in combination. DNA sequences obtained by extracting and using in combination, or by substituting the base sequence of the protein-coding region so that the specified amino acid sequence does not differ, are also included.
Derivatives obtained when a base in any part of the DNA sequence of the present invention is replaced with another, a new base is inserted or deleted, or when a part of the base sequence is transposed, gene expression,
It is envisaged that it will give good results on secretion and secretion, and falls within the scope of the present invention. Hereinafter, specific examples will be given of methods for obtaining the glucoamylase gene of Satucharomyces diastateicus containing the DNA sequence of the present invention and the DNA of the present invention.
Determination of base sequence, direct alcohol fermentation of starch by Satucharomyces cerevisiae having starch assimilation properties obtained by transformation using the DNA of the present invention, and transformation example of yeast other than the genus Satucharomyces using the DNA of the present invention I will explain about it. Example 1 Preparation of gene library of Saccharomyces diastaticus chromosomal genes Saccharomyces diastaticus 5106-9A strain (STA1) (FERMP-7641) was grown in YEPD medium (1%
Yeast extract, 2% peptone, and 2% glucose)2 were cultured at 28°C for 2 days, and the resulting bacterial cells were cultured using the method of RWDavis et al.
Davis et al;Advanced bacteriol genetics,
Chromosomal DNA was extracted using Cold Spring Harbor Laboratory (1980) to obtain 1.0 mg of DNA. 200 μg of this chromosomal DNA was partially digested at 37°C using the restriction enzyme Sau3AI (manufactured by Takara Shuzo), and DNA of approximately 5 kb or more was fractionated from the total amount of the reaction product by sucrose density gradient (10-40%) centrifugation. , recovered. The recovered DNA was added to 50mM Tris-HCl buffer (PH7.5).
It was dissolved in 100μ and used as a donor chromosome fragment. This donated stained fragment was used as yeast-Escherichia coli shuttle vector pKS2-20 (K. Suzuki, Y.
Imai, I. Yamashita and S. Fukui; Journal of
Bocteriology 155 , 747-754, (1983))
_T4 ligase (manufactured by Takara Shuzo) at the BmaHI cleavage site
The method described in the literature (M.Carlson and D.
Botstein; Cell, 28 , 145 (1982)). The obtained fusion plasmid was transferred to a method using 100mM calcium chloride (SNCohn ACYChang).
and L.Hsu；proc.Natl.Acad.Sci.USA69,
E.ColiJA221 (Leu ^- , by 2110-2114 (1972)
Amp ^S ) (NRRL No. 15014), and approximately 20,000 transformed strains exhibiting ampicillin resistance were obtained.
From each of these 20,000 transformed strains, the rapid method (David S. Holmes and Michael
Quigley; Analytical Biochemistry 144 , 193,
(1981), plasmid DNA was extracted and mixed to create a gene library. Example 2 Plasmid containing STA1 gene
Isolation of pSTA1 100μ of gene library DNA obtained in Example 1
using Satscharomyces cerevisiae 338
( ^Leu- , ^Sta- ) (Saccharomyces cerevisiae,
FERMP−7637) to the JDBeggs method (Nature
(London) 275 , 104-109 ^, 1978) and plated on selective medium ^.
A transformed strain showing the following was obtained. From about 30,000 transformants exhibiting Leu ⁺ , five transformants (Sta ⁺ ) were obtained that produced a halo around the colony indicative of glucoamylase production. The composition of the selective medium is 1% glucose, 2% soluble starch, 1.2M sorbitol, 20mg/ml histidine, 30mg/ml lysine,
It is 2% agar. One of the five transformed strains that produced a halo around the colony was placed in YEPD medium.
The method described in the literature (HC Birnboim and J. Doly;
When the plasmid was extracted by Nucleic Acids Res.), 1 μg of plasmid was extracted from the transformed strain.
DNA was obtained. Using this plasmid DNA and the same method as described in Example 1, E. coli
The JA221 strain was transformed to obtain a transformed strain exhibiting Leu ⁺ , Am ^r , and Tc. This transformed strain is LB
(0.5% yeast extract, 1% peptone, 0.5% NaCl)
Plasmids were extracted from the bacterial cells obtained by culturing them at 37°C for 16 hours using 5 ml of the above-mentioned Birnboim and
After isolation and purification using Doly's method, 1 μg of plasmid DNA was obtained. This plasmid
Using DNA, Saccharomyces cerevisiae 338 (Leu ^- ,
Sta ^- ), a transformant strain exhibiting Sta ⁺ was obtained, and this plasmid was named pSTA1. The restriction enzyme map of pSTA1 is shown in FIG. As shown in Figure 1, pSTA1 has an inserted DNA of 2.8 kb, with one restriction enzyme Bam HI site, two Sal I sites, and two Pvu sites.
Location, Hind site, Eco RI site, Pst I
There was one site each. Example 3 Secretory enzyme glucoamylase gene
Determination of the nucleotide sequence of STA1 Transformed strain having pSTA1 obtained in Example 2 (E.coli JA221-pSTA1, FERMP-7640)
The plasmid was prepared by the method shown in Example 2 from the bacterial cells cultured in LB medium 2 at 37°C for 16 hours, and as a result, 1 mg of purified plasmid was obtained. This plasmid contains restriction enzymes Sal I and
Partially digested with Bam HI and subjected to agarose electrophoresis.
The approximately 2.8 kb inserted DNA fragment was purified and isolated. The method of Maxam and Gilbert (A.Maxam and W.Galbert, proc.Natl.Acad.
SciU.SA 74 , 560 (1977)), and the entire nucleotide sequence was determined using the strategy diagram for nucleotide sequence determination shown in FIG. As a result, the following DNA base sequence was obtained, and this base sequence includes the RNA polymerase binding site, ribosome binding site (RBS), and signal sequence necessary for the expression and secretion of the glucoamylase gene of Satucharomyces diastateicus. (Signal sequence), a DNA sequence corresponding to the secreted mature protein, a stop sequence (Stop), and a polyA signal (Stop) further downstream.
signal). Example 4 Transformed strain Satucharomyces cerevisiae 338-
pSTA1 is Saccharomyces diastateicus
Secretes the same glucoamylase as strain 5106-9A (STA1). Example 2 using pSTA1 obtained in Example 2
By the same method as shown in
Strain Satucharomyces cerevisiae 338-pSTA1 obtained by transformation of S. cerevisiae 338 strain
(Saccharomyces cerevisiae, FERMP−7638)
were cultured at 28°C for 3 days using YPS medium (1% yeast extract, 2% peptone, 3% soluble starch). The glucoamylase activity of this culture supernatant was measured using the method described in the literature (I. Yamashita and S.
Fukui, Agric. Biol. Cmem; 47 , 2689, 1983).
It was compared with glucoamylase derived from S. diastaticus 5106-9A strain. As a result, Satsucharomyces
As shown in Table 1, the S. cerevisiae 338-pSTA1 strain produces glucoamylase that exhibits exactly the same properties as those derived from the S. diastaticus 5106-9A strain in terms of optimal temperature, optimal PH, and thermostability.
secreted, and the production amount is 1.0×10 ⁶ units 1
reached, Satucharomyces diastateicus 5106
The production and secretion amount was more than 10 times that of the −9A strain. Furthermore, purified enzymes derived from both strains were treated with endoglycosidase H (Endo H, manufactured by Seikagaku Corporation),
When the molecular weights of the obtained peptides were compared by SDS-polyacrylamide gel electrophoresis, all glucoamylases showed 41,000 daltons. In addition, when we prepared a monoclonal antibody that uses the peptide portion of glucoamylase secreted by S. diastaticus 5106-9A strain as an antigen and reacted it with glusoamylase of S. cerevisiae 338-pSTA1, a strong precipitation reaction was observed. It was done.

[Table] *1 No decrease in activity was observed over at least 2 hours.
Example 5 Satucharomyces cerevisiae 338−
Ethanol production using starch as a substrate by pSTA1 Satucharomyces cerevisiae 338-pSTA1
Using 5 ml of YPS medium (1% yeast extract, 2% peptone, 3% soluble starch), static culture was performed at 20°C for 3 days, and the amount of ethanol and glucose in the medium was measured using high-performance liquid chromatography ( Pump, Hitachi; Column, Showdex: Ionpak801; Buffer,
Measured using a 5% phosphoric acid solution; detector, refractometer RI-8 Toyo Soda). As a result, 9.76 g of ethanol and 0.32 g of glucose were found to be produced in medium 1, and almost all of the consumed starch was converted to ethanol. Example 6 Production and secretion of glucoamylase from starch by a transformed strain of the fission yeast Schizosatucharomyces pombe using the prammid pSTA1 containing the DNA of the present invention Example 2 Schizosaccharomyces pombe HM123 (h ^- , leu1) (Schizosaccharomyces pombe) FERMP−
The transformed strain obtained by transforming 7639 was cultured by the method described in Example 4, and the properties of the glucoamylase produced and secreted were examined ^. It was confirmed that glucoamylase derived from the STA1 gene was produced and secreted. This result shows that the DNA sequence of the present invention and pSTA1 function also in Schizosatucharomyces, a yeast other than the genus Satucharomyces, and that the DNA sequence of the present invention can be used to construct expression and secretion vectors in yeast. It means possible.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme map of pSTA1. FIG. 2 shows a strategy diagram for determining the base sequence of the inserted DNA of pSTA1.

Claims (1)

[Scope of Claims] 1. A promoter region involved in the expression of the glucoamylase gene of Satucharomyces diastateicus, a ribosome binding region, a region involved in the secretion of proteins to the outside of the bacterial cell, and a region encoding the amino acid sequence of an enzyme protein. A DNA sequence characterized in that the DNA sequence contained in the base sequence is the following base sequence.