JPH0379985B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing a novel yeast that has acquired the ability to assimilate cellobiose by introducing a secreted β-glucosidase gene. [Prior art] β-glucosidase (β-D-glucoside
glucohydrolase, EC 3, 2, 1, 21) is β-
It is an enzyme that hydrolyzes glucoside bonds, and is also called cellobiase because it specifically decomposes cellobiose into glucose, and its existence is widely known in nature. On the other hand, cellulose, which is a structural polysaccharide that exists in large quantities in nature, is reduced to a low molecular weight by various cellulases, and a considerable part of it is converted into cellobiose, which is then decomposed into curucose by β-glucosidase. It is thought that it was used by microorganisms. The effective use of cellulose, which is a cheap and abundant biomass resource, is an extremely important issue from the perspective of energy conservation, and attempts to decompose it with cellulose-degrading enzymes and assimilate it to various microorganisms are being considered. It is currently being actively carried out. [Problems to be solved by the invention] However, saccharomyces cerevisiae, which is widely used in various brewing and bread-making processes and is one of the most familiar yeasts to humans, outside β−
Because it does not have the ability to secrete glucosidase, it is unable to assimilate cellobiose and grow, so in order to use cellulose resources as a sugar source using S. cerevisae, it must completely break down the cellulose resources into glucose. It is necessary to decompose it using a complex enzyme system, but conventional cellulase has the disadvantage that cellobiose tends to accumulate. In addition, Saccharomyces cerevise does not have a permease to take cellobiose out of the bacterial body, so it is a non-secreting β-cell.
-The ability to assimilate cellobiose cannot be imparted simply by introducing a glucosidase gene. [Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have conducted intensive studies on imparting the ability to produce secreted β-glucosidase to Satucharomyces cerevisiae using genetic engineering methods, and have found that A specific secreted β- form from Kalomycopsis fibrigera
By introducing the glucosidase gene, the ability to assimilate cellobiose is efficiently expressed, and the enzyme secretes more β-glucosidase, which decomposes cellobiose, in Satucharomyces cerevise than in the parent strain, Satucharomycopsis fibrigera. This discovery led to the present invention. That is, the present invention is derived from Satucharomycopsis fibrigera, and the restriction enzyme map is from the 5' side.
BamH／Sau3A→EcoR→Sal→BamH
→EcoR→Ban→Kpn→Eco→BstE
→Kpn→BstE→EcoR→EcoR→EcoR
→Secreted β arranged in the order BamH/sau3A
- Recombinant DNA obtained by ligating Patsenjar DNA containing the glucosidase gene and vector DNA capable of replicating in yeast.
It has a secretory β-glucosidase gene, which is characterized by introducing it into a host yeast that does not have secretory β-glucosidase activity, and acquires cellobiose assimilation ability by producing β-glucosidase extracellularly. The present invention provides a method for producing yeast. The production of yeast containing the secretory β-glucosidase gene in this way is the first example of S. cerebise growing and effectively carrying out alcoholic fermentation using cellobiose as the sole carbon source, and in the future. This is extremely significant in terms of technological development for fermenting alcohol from cellulose. Another great advantage is that the β-glucosidase of Satucharomycopsis fibrilla is a secreted enzyme. That is, a pharmaceutical agent containing the β-glucosidase gene obtained according to the present invention
DNA contains a strong promoter region and a ribosome binding region that enable large amounts of β-glucosidase to be secreted and accumulated outside the yeast cells.
It is thought that yeast cells have regions involved in the efficient secretion of proteins and proteins. This makes it possible to construct an expression secretion vector that efficiently secretes and accumulates these proteins outside the bacterial cell using the microorganism as a host. Example 1 Satucharomycopsis fibrigera
HUT7212 in YEPD medium (1% yeast extract, 2%
Using 1 liter of polypeptone, 2% glucose), culture was performed at 28°C for 2 days, and the resulting bacterial cells were cultured using a conventional method (Method in Cell Biology, Prescott DM,
Academic Press New York, Vol.12 p39−44,
1975), chromosomal DNA was extracted and DNA0.5
I got mg. 200μg of this chromosomal DNA is digested with restriction enzymes.
Partially disassembled using Sau3A (manufactured by Toyobo),
A fragment length of 5 to 10 Kb was recovered from the entire amount of the reaction product obtained using the sucrose gradient method. This recovered DNA was added to 50mM Tris-HCl buffer (PH).
7.5) Dissolved in 100μ to obtain donor fragment. Yeast-Escherichia coli shuttle vector pY1 (Figure 1) created by the present inventors using this donor chromosomal DNA fragment
[I.Yamashita and S.Fukui, Agric.Biol.chem,
47, 2689-2692 (1983)] at the BamH cleavage site.
After ligation using T4 Ligase (manufactured by Toyobo Co., Ltd.),
162 Kodansha] according to E.coli [E.coli RR1
(Cleu ^- , Anp)], and approximately 30,000 transformed strains exhibiting ampicillin resistance were obtained. About 3 of these
The rapid method (David
S.Holmesand Michael Quigley, Analytical
Plasmid DNA was extracted and mixed to create a gene library using Biochemistry 114, 193, 1981). Using 100μ of this gene library, the conventional protoplast method [Albert Hinnenet et al.
Ptoc.Natl.Acad.Sci., USA 75(4)1929-1933('
78)] according to Saccharomyces cerevisiae YIYD (A, lys7,
his4, leu2-3, IR) were selected using the elimination of ronsin requirement as an indicator. The transformed strain of Satucharomyces cerevisiae YIYD, which has become non-leucine auxotrophic, obtained by the above method is transformed into a minimum amount containing 6 g of paranitrophenol-β-D-glucopyranoside (hereinafter abbreviated as pNPG) and lysine and histidine. The degrading activity of this pNPG was measured by the hydrolysis of the β-glucoside bond of pNPG, which produced paranitrophenol, and the yellowing of the surrounding area of the colony was used as an indicator. As a result of the search, two types of strains with pNPG degrading activity were obtained.
One of these shows pNPG-specific degrading activity but cannot assimilate cellobiose, while the other can grow in a minimal medium with cellobiose as the sole carbon source instead of glucose. Cellobiose decomposition ability was observed. The obtained transformed strain having the ability to degrade cellobiose was named Satucharomyces cerevisiae psfCB1. This strain was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology as part of the Microbiological Research Institute.
No. 8552'.

[Table] Example 2 The transformed strain psfCB1 of Satucharomyces cerevisiae that can grow using cellobiose as the sole carbon source obtained in Example 1 was added to 10 ml of YEPD medium.
[A.
C.Birbaim and J.Doly, Necleic Acid.Res.7,
1513-1523 (1979)]. Example 1 using this plasmid DNA
Transform E. coli RR1 using the same method as
A transformed strain exhibiting ampicillin resistance was obtained. This transformed strain was grown using 5 ml of LB medium (0.5% yeast extract, 1% polypeptone, 0.5% NaCl, PH7.0).
Plasmid DNA was isolated and purified by the rapid method from the bacterial cells obtained by culturing at ℃ for 16 hours, and this plasmid DNA was used to transform Saccharomyces cerevisiae YIYD by the protoplast method. Convertible stock was obtained. Examination of the restriction enzyme map of the obtained plasmid DNA revealed that the vector used
At the BamH site of pY1, the approximately
It contained 9.1kb of Patsenjar DNA. Example 3 Three strains of Satucharomyces cerevisiae psfCB1, Satucharomyces fibrigera HUT7212, and Satucharomyces cerevisiae YIYD were grown on a plate in which 2 g/100 ml of cellobiose was added instead of glucose in the minimal medium shown in Table 1 above.
℃ for 3 days and the growth status was observed, and the results shown in Table 2 below were obtained.

[Table] Example 4 Yeast extract 1 for the three strains shown in Example 3
%, polypeptone 2%, and cellobiose 2% for 3 days at 28°C with shaking, the entire liquid medium was centrifuged at 3000 rpm for 5 minutes, and the resulting supernatant was diluted with deionized water for 2 hours. Table 4 shows the results of diluting the daily dialyzed product to a certain amount and measuring cellobiose decomposition activity as a crude enzyme solution using the method described in Table 3. For Satucharomycopsis fibrigera, the culture solution was prepared by filtration with a membrane filter instead of centrifugation. Table 3: Cellobiose decomposition activity measurement method 0.02M cellobiose solution 10μ 0.1M Macluvain buffer 30μ Enzyme solution 10μ ↓ 30℃, 10 minutes reaction Boiling bath treatment 1 minute ↓ Cooling Fujisawa Glucostat 200ml ↓ 37℃, 20 minutes 500nm absorbance measurement ( OD500) (Note) In this measurement method, the enzyme activity that produces a color equivalent to glucose OD1.0 is defined as 1 unit.

[Table] Example 5 Cellobiose decomposition of the culture solution of Satucharomycopsis cerevisiae psfCB1 and Satucharomycopsis fibrigera cultured at 28°C for 3 days in a medium consisting of 1% yeast extract, 2% polypeptone, and 2% cellobiose. When the relationship between activity and PH was investigated using the method listed in Table 3, as shown in Figure 3, the optimum PH for both was around PH5.0.
The PH dependence of enzyme activity also showed similar behavior. this
When the psfCB1 strain was cultured in a normal medium consisting of 1% yeast extract, 2% polypeptone, and 2% glucose and cured to restore the leucine requirement, the ability to assimilate cellobiose also disappeared. From the above knowledge, this
It is thought that the psfCB1 strain is derived from the β-glucosidase gene of Satucharomycopsis fibrigera transformed into Satucharomyces cerevisiae in a plasmid-dependent manner. In addition, pNPG was added to the culture medium.
and cellobiose degrading activity.
It was found that the psfCB1 strain also secretes β-glucosidase activity outside the bacterial cells of S. cerevisiae. The transformed strain produced by the method of the present invention was found to secrete greater β-glucosidase activity than the gene-donating bacterium Satucharomycopsis fibrigera. Furthermore, as for the thermal stability, it was found from Fig. 5 that all of them were stable up to 45°C.
Furthermore, the optimum temperature for the action of the transformed strain was around 50°C, as is clear from Figure 4. These findings strongly suggest that the transformed strain and the gene donor bacterium produce β-glucosidase as well. Therefore, it is highly likely that the passenger DNA of plasmid psfCB1 encodes the β-glucosidase gene of the gene donor bacterium.

[Brief explanation of drawings]

Figure 1 is a restriction enzyme map of vector pY1, Figure 2 is a restriction map of transformed strain psfCB1 passenger DNA, and Figure 3 is cellobiose-degrading activity of β-glucosidase produced by transformed strain psfCB1 and S. fibrilla. Figure 4 is a diagram showing the temperature dependence of the cellobiose decomposition activity of β-glucosidase produced by the transformed strain psfCB1. Figure 5 is a diagram showing the PH dependence of the transformed strain psfCB1.
FIG. 2 is a diagram showing the thermostability of psfCB1 and the cellobiose decomposition activity of β-glucosidase produced by Satucharomycopsis fibrilla.

Claims (1)

[Claims] 1. Derived from Satucharomycopsis fribrigera, the restriction enzyme map is BamH/Sau3A from the 5' side.
→EcoR→Sal→BamH→EcoR→Ban
→Kpn→EcoR→BstE→Kpn→BstE
→EcoR→EcoR→EcoR→BamH/
A recombinant DNA obtained by ligating Patsenjar DNA containing a secretory β-glucosidase gene arranged in the order of sau3A and a vector DNA that can be replicated in yeast is used as a recombinant DNA that does not have secretory β-glucosidase activity. A method for producing yeast that has a secreted β-glucosidase gene, which is characterized by being introduced into a host yeast, and that has acquired zero biose assimilation ability by producing β-glucosidase extracellularly. 2. A method for producing yeast having a secreted β-glucosidase gene according to claim 1, which uses Saccharomyces cerevisiae as the host yeast.