JPS62175183A - Heteropolypeptide developing fibrous bacteria, its production and vector producing the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION This application is a continuation-in-part of US Patent No. 771.374, filed August 29, 1985.

(Industrial Application Field) The present invention relates to a heterologous polypeptide expressed and secreted by filamentous fungi, a vector for expressing and secreting the polypeptide, and a process thereof.

In particular, the present invention discloses a transformation vector and its use in expressing and secreting biochemically active calf chymosin and heterologous glucoamylase by filamentous fungi.

BACKGROUND OF THE INVENTION The expression of DNA sequences encoding heterologous polypeptides (eg, polypeptides that are not normally expressed and secreted by the host organism) has advanced to a considerable degree of sophistication.

For example, pharmaceutically desirable polypeptides such as (a) human growth hormone (), (b) human tissue plasminogen activator (2), (c) various human interferons (5), (d) factor 8 (4), (e) human serum albumin (3)) and industrially important enzymes (e.g. (a) chymosin 7), (b) alpha amylase <8), ( C) Alkaline protease (9))
It has been reported that various DNA sequences encoding the peptide have been cloned and expressed in several heterologous expression hosts. These expressions can be expressed in prokaryotes (e.g. E. coli (E, c).
cli) (10), or Bacillus subtilis (B, 5ubtil
is) (11)), or eukaryotes (e.g., Saccharomyces cerevisiae)
s cerevisiae) (7), Kluyveromyces lactis (Kluyverol'1lyce)
s Iactis) (12) or Chinese hamster Ovari cells (2)) with a DNA sequence encoding a heteropolypeptide.

When expressed in a heterologous host, polypeptides expressed in different host organisms do not have the same biological activity as when they are produced naturally. For example, calf chymosin can be found in E. coli' (13) or S. cerevisiae (13).
(7) has very low biological activity when expressed in This low biological activity in E. coli is not due to E. coli's natural inability to glycosylate polypeptides, since chymosin is not normally glycosylated (14). However, such Escherichia coli (IE, coli) or S
Relative inactivity in S. cerevisiae (S. cerevisiae) is due in part to the activation of its polypeptide chains by various manipulations, as shown in part by post-expression activation of polypeptides so expressed. This seems to be caused by improper folding. In such manipulations, the expressed chymosin would be repeatedly denatured and regenerated in several ways such that its biological activity increases.

For example, (a) urea treatment (13), (b) exposure to denaturation/regeneration p++, (c) denaturation and cleavage of disulfide bonds followed by regeneration and regeneration of covalent sulfur bonds (
15). However, such denaturation/regeneration operations are not very efficient, e.g., the recovery of the biological activity of rennin is less than 30% (13), and biologically active polypeptides It takes a considerable amount of time and expense to produce it. Other heterologous polypeptides are successfully expressed in higher eukaryotic hosts (eg, mammalian cells). Typically, such polypeptides are glycopolypeptides that require an expression host capable of recognizing and glycosylating certain amino acid sequences in the heterologous polypeptide. However, such mammalian tissue culture systems often do not secrete large amounts of heterologous polypeptides compared to microbial systems. Moreover, maintaining such a system is technically difficult and, as a result, expensive to operate.

N, Clazza (N,
Transformation and expression in filamentous fungi including complementation of the a r o D mutant of S. crassa has been reported (16). Since then, glutamate
Dehydrogenase-deficient N, Crasosa (N.

Complementation-based transformation of cra, 5sa) mutants has also been developed (17). In each case, the complementation used for complementation was
az) and glutamate dehydrogenase (and the two genes are from N, crassa and therefore also include homologous expression. Other examples of homologous expression in filamentous fungi include △ Nidurans (A, n1
autotrophic marker type (18) in
and argB (1,9) (') complementarity ya, T set-
A. f. Nidurans (A, n1
Δ to the utilization of acetamide or acrylamide by expression of the genetic apex (A. dulans).
ulans) transformation.

Expression of heterologous polypeptides in filamentous fungi is limited to the transformation and expression of fungal and bacterial polypeptides. For example, Δ niturans (
A. , n1dulans) is N.

pyr from crassa (N, crassa) 4
It was transformed by a plasmid containing the DNA sequence encoding the gene. (21,32) A, nigga (
A. niger ) can also be transformed into A. niger by transformation. It expresses a gene encoding acetamidase derived from A. nidulans and utilizes acetamide and acrylamide. (22) Examples of heterologous expression of bacterial polypeptides in filamentous fungi include N,
Crassa (N, crassa) (23), Dectytosterium discoibrum ([]ictyost
ellium discoideum (24) and Cepha ospo'11um acremonium (24).
lo-sporium acremonium) (
25) Expression of bacterial phosphotransferases in

In each of these examples of expression in homologous and heterologous fungi, the expressed polypeptide was maintained within the cells of the filamentous fungus.

It is therefore an object of the present invention to transform filamentous fungi containing vectors for transforming such heterologous polypeptides in order to express and secrete such heterologous polypeptides from such fungi. , to supply the process of secretion.

(Essential Means for Solving the Problems) The present invention is directed to the expression of heterologous polypeptides from filamentous fungi.
Contains a new vector for secretion. The vector is used in a new process to express and secrete the heterologous polypeptide.

The vector used to transform a filamentous fungus to express and secrete a heterologous polypeptide contains a DNA sequence encoding a heterologous polypeptide and a functional secretion system in a given filamentous fungus. and includes a DNA sequence encoding a signal sequence that is operably linked to a sequence encoding the heterologous polypeptide. The signal sequence may be a signal sequence in normal combination with a heterologous polypeptide or may be derived from others.

In addition, the vector has a DN encoding a signal sequence.
It may also include a DNA sequence that is operably linked to the A sequence and encodes a promoter sequence that is functionally recognized by filamentous fungi. A functional polyadenylation sequence is
Desirably, it is operably linked to the 3' end of a DNA sequence encoding a heterologous polypeptide. The polypeptide thus synthesized is then secreted from the filamentous fungus.

The present invention demonstrates that heterologous polypeptides from widely dispersed species can be expressed and secreted from filamentous fungi. Especially calf chymosin, A.spergillus, niger and Humicola, grises.
glucoamylase, derived from Mucor miehei (M
A. carboquine protease derived from A. ucormiehei).

It was expressed and secreted in A. nidulans (formerly A. dulans). In addition, calf chymosin was added to Δ and awamori (A
．． awamori) and Trichoderma reesei (TrICh-oclerma reesei). Biochemically active chymosin was detected in the culture medium without further treatment. This result is
The vector used to transform A. nidulans was constructed to secrete prochymosin, which requires exposure to an acidic environment (approximately p+12) to make biochemically active chymosin. It is surprising in the sense that it is.

Generally, a vector that includes a DNA sequence encoding a functional promoter and a terminator sequence (including a polyadenylation sequence) is operably linked to various internal sequences and DNA sequences encoding a heterologous polypeptide. The thus constructed vector was used to transform filamentous fungi. Surviving transformants are then identified by screening for expression and secretion of the heterologous polypeptide.

Alternatively, an expressible selection characteristic can be used to isolate transformants by incorporating a DNA sequence encoding the selection characteristic into a transformation vector.

Examples of such selection characteristics include different antibody surfaces (e.g., aminoglycosides, benomyl, etc.) and sequences encoding genes that compensate for auxotrophic defects (A pyr 4 deleted).
．． nidulans (A., n1dulans), A.

Awamori (A, atvamori) Trichoderma
A lacking pyr 4 complementation or 8 rg B of Tric oderma reesei =
tulans (A, n1dulans) or A. ndulans. A. awamori 8rgB complementarity) or sequences encoding genes that confer nutritional (eg, acetamidase) or morphological markers to the expression host.

In preferred published embodiments, the transformation vectors of the invention are constructed using Δ.

A N derived from A. n1dulans
Contains a DNA sequence encoding the S-■ sequence.

This sequence increases the transformation efficiency of the vector. However, these sequences are not believed to be absolutely necessary to practice the invention.

In addition, certain DNA sequences derived from the Hatateria plasmid pBR325 reveal the transformation
It forms part of the vector.

Nor are these sequences believed to be necessary for transforming filamentous fungi. Instead, these sequences confer bacterial replication of the vector during vector construction. Other plasmid sequences that can be used during vector construction include pBR322 (ATCC 37017)
, RK-2 (ATCC37125), pMB9 (ATCC37019), psclor (ATCC37
032) is included.

The disclosed preferred embodiments are illustrated by way of example and are not intended to limit the scope of the invention.

(Definitions) "Expression of a polypeptide" means the expression of a DNA sequence encoding a polypeptide.

A "polypeptide" is a polymer of alpha-amino acids covalently linked through peptide bonds.

Polypeptides include low molecular weight polymers as well as high molecular weight polymers, more commonly referred to as proteins. Additionally, polypeptides may be phosphopolypeptides, glycopolypeptides, metallopolypeptides.

moreover. In some cases, one or more polymer chains may be combined to form a single polypeptide chain.

As used herein, a "heterologous polypeptide" refers to a polypeptide that is not normally expressed and secreted by the filamentous fungus used to express the polypeptide. polypeptides (e.g., α-amylase of Bacillus sp., alkaline protease of Bacillus sp., various hydrolytic enzymes of Pseudomonas, etc.), polypeptides of eukaryotic origin (e.g., chymosin of Bacillus sp., human tissue plasminogen activator, human growth hormone, human interferon, urokinase, human serum albumin, factor V, etc.) and polypeptides derived from fungi other than the expression host (e.g., A. ndulans) ), Δ, niger (A, niger > and Humicola
Gricea () lumico! glucoamylase from A. grisea ), Δ, nidulans (A., n1
The carboxyl group derived from Mucor m1ehei, which is expressed in
Contain protease, etc.

Heterologous polypeptides also include a combination of partial or complete polypeptides derived from at least two different polypeptides, each of which is homologous or heterologous to the fungal expression host. Also includes. Examples of such high frit polypeptides include 1) A. D encoding only the signal and prosequences of A. niger curcoa mirrors.
2) a DNA sequence encoding prochymosin fused in a form bound to varying amounts of the amine-terminated mature gel-coamer secotone; and 2) a DNA sequence encoding only a functional shifnal sequence; A DNA sequence encoding a fungal glucoamylase or carboxy protease or a human tissue plasminogen activator or growth hormone fused to various amine-terminated polypeptide futons or mature futons associated with a signal.

Furthermore, the heterologous polypeptides of the present invention include: ■) the natural 7L present or occurring in polypeptide sequences from prokaryotic, eukaryotic and fungal organisms similar to those used to form the hybrid polypeptides described above; / Rick (all
2) Processing changes of the heterologous polypeptide brought about, for example, by site-directed mutagenesis, such that various deletions, insertions, substitutions of one or more amino acids occur in the heterologous polypeptide; A “biochemically active heterologous polypeptide” is a heterologous polypeptide that is secreted in an active form that is clearly mediated by its ability to: 2) in the case of a hybrid polypeptide, a biochemical activity is mediated by at least one natural moiety that constitutes the hybrid polypeptide.

Each heterologous polypeptide defined above is encoded by a heterologous DNA sequence containing a termination signal recognized by the filamentous species upon which expression and secretion occur. Once recognized by the host, the termination signal terminates translation of the mRNA encoding the heterologous polypeptide.

The filamentous fungi of the present invention are eukaryotic microorganisms, and are subcarnivorous Eumycotina (26>
including all fibrous types. These fungi are characterized by a vegetative mycelium consisting of titin, cellulose and other complex polyzarachalides. The filamentous fungi of the present invention are morphologically, physiologically and genetically different from yeast. Vegetative growth is called Haiphal (
due to hyphal, ) elongation, carbon metabolism is obligately aerobic.

In contrast, S. cerevisiae
The vegetative growth of yeasts such as S. iae) is based on the budding of unicellular thallus, and the carbon metabolism is fermentative. S, Cerevisiae has a predominant, very stable diploid phase, and is different from Aspergilli and Neurospora (N
In filamentous fungi such as (Eurospora), diploids are only slightly present before meiosis.

S. Cerevisiae is 17
On the other hand, A. nitourans (A.,
n1clulans) and N, crazza (N, cra
ssa) have 8 and 7, respectively. A recent explanation for the difference between S. cerevisiae and filamentous fungi is 8. Cerevisiae x (S.
Cerevisiae) is Aspergillus (△sper
g i l ] us) and hytolycodel? (Tr]c
These include the lack of in) and Ron of hot3erma) and the inability to recognize many of the transcriptional control factors of filamentous fungi. (27) Various species of filamentous fungi form the following genera.
ra) can be used as expression hosts, including Aspergillus, ) Trichoderma, Nie-0spora (
Neurospora, Podospora
ra), Endothia Mucono [1ndothia
A4ucor), Cochioporus (Cochiobol)
us) and Pyricularia.

Specific expression hosts include A. niturans (A., n
1 dulans) (18, 19, 20, 21, 61
), A, niger (22),
A, awamori (A, awamor i) For example, NRR
L311.2, ATCC22342 (NRI';!L3
112), ATCC/I 4733, ATCC1433
1 and UVK↑43f strains, Δ, oryza x (A, ory
zae), for example, ATCC 11490, N. crassa (16,17,23), Trichoderma reesei
sei), for example, NRRL ], 5709, ΔTC
C1,363], 56764.56765.56466
．． 56767, and Tricoble?・Viride (Trich)
o-derma viride), e.g. ATCC3
2098 and 32086.

As used here, ``promoter sequence''
° is a DNA sequence recognized by the filamentous fungus for purposes of expression. It is operably linked to a DNA sequence encoding a polypeptide as defined above. The linkage also includes the positioning of the promoter relative to the initiation codon of the DNA sequence encoding the signal sequence of the published transformation vector.

The promoter sequence contains a signal sequence and transcriptional and translational control sequences that mediate expression of the heterologous polypeptide. Examples include A. niger glucoamylase lid 39.48), Mucor miehei (M.
carboxyl protease of A. ucor mlehei), α-glucosidase of A. niger (28), Trichoderma reesei (Tricbo
derma reesei) cellobiohydrolase I (29), A. nitulans (A., n1dula)
ns ) promoter of trpC (18) and SV4
Contains more advanced eukaryotic promoters such as the early promoter of 0 (24).

Similarly, a "terminator sequence" is a DNA sequence that is recognized by the expression host and terminates transcription. It is operably linked to the 3° end of the DNA encoding the heterologous polypeptide to be expressed. Examples include Δ, nidurans (
A. , n1dulans) trpC(18), Δ
, A. niger (Dg) Ii mycoamylase 39.48), Δ niger
), the α-glucosidase of Mucor miehei (28), and the carboxyl protease of Mucor miehei, but any fungal terminator is functional in the present invention.

The "boriadelation sequence" is a DNA sequence that is recognized by the expression host during transcription and adds a polyadenosine residue to the transcribed mR,NA.

It is a DN encoding the heterologous polypeptide to be expressed.
It is functionally connected to the 3' end of A. Examples include A
tr of niturans (Δ, n1clulans)
pC (18), A. niger glucoamylase (39,48), A. niger glucoamylase (39,48), A. Nigger (A, n
iger) glucondase (28), 2u and the carboxyl polyadenylation sequence of Mucor miehei. but,
Any fungal polyadenylation sequence is likely to be functional in the present invention.

A "signal sequence" is an amino acid sequence that, when operably linked to the amino terminus of a heterologous polypeptide, assists in secretion of the heterologous polypeptide from the host filamentous fungus. The sequence may be a signal sequence normally associated with a heterologous polypeptide (natural signal sequence), or it may be derived from another organism (foreign signal sequence). D that utilizes the native signal sequence or encodes a heterologous polypeptide such that the signal sequence and the heterologous polypeptide are in proper reading frame to allow translation of the heterologous polypeptide.
A heterologous polypeptide is functionally linked by linking a DNA sequence encoding a foreign signal sequence to the NA sequence. Signal sequences useful in practicing the invention include A. preprochymosin (1,5), A. niger glucoamylase (39), Mucor m1ehei carboxyl protease, and A. niger glucoamylase (39). Trichoderma reesei (
Trichoderma reesei) cellulase (29); however, all signal sequences that permit secretion of a heterologous polypeptide are contemplated by the present invention.

A ``propeptide'' or ``prosequence'' is an amino acid sequence located at the amine terminus of a mature, biologically active polypeptide. When so located, the polypeptide is referred to as a zymogen. Generally, zymogens are biologically inert and can be converted to mature active polypeptides by catalytic or autocatalytic cleavage of the propeptide from the zymoken.

In the specific examples of the present invention, the term "transformation vector" refers to a DN encoding a heterologous polypeptide.
A DNA sequence that combines the A sequence and a heterologous or homologous signal sequence operably linked thereto. Additionally, the I-transformation vector may include a DNA sequence encoding a functional promoter and polyadenylation sequence. In addition, each of the -1-8 transformation vectors is also referred to as J-, which contains a sequence encoding a selective characteristic capable of expressing 1J, as well as a sequence that increases the transformation efficiency of the bacterial strain.

Paratransformation is a process in which l-transformation vectors are introduced into filamentous, V, and feral fungi. By the transformation method of the present invention, all or part of the transformation vector is stably integrated into the synthetic genes of filamentous fungi. However, transformations that maintain self-replicating extrachromosomal transformation vectors are also being considered.

(General Method) DNA ``digestion'' is the catalytic cutting of DNA with an enzyme that acts only on certain positions in DNA. Such enzymes are called restriction enzymes, and each specific site is called a restriction site. . "Partial" digestion refers to incomplete digestion by a restriction enzyme, meaning that for a given restriction endonuclease, not all sites of the DNA substrate are digested.
The conditions under which a portion is severed are selected. The various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements established by the enzyme supplier are used. Generally, about 1 unit of enzyme and about 20 microliters of buffer solution are used for about 1 microgram of plasmid or DNA fragment. Typically, incubation times of about 1 hour at 37°C are used, but may vary according to the provider's instructions. After incubation, proteins are removed by extraction with phenol and chloroform and digested nucleic acids are recovered from the wood chips by ethanol precipitation. After digestion with restriction enzymes, the terminal 5' phosphate
to prevent insertion of other DNA fragments at the restriction site during ligation.
Prevents the two ends of the DNA fragment from forming a closed loop.

``Recovery'' or ``isolation'' of a defined DNA fragment from a restriction digest refers to separation of the digest by polyacrylamide gel electrophoresis, identification of the fragment,
It refers to the removal of the gel fraction containing the desired fragments and the separation of the DNA from the gel, generally by electrophoresis.

(30) "Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments. (30) If not stated otherwise, the ligation should be 0.5 micrograms of ligation. , for approximately equimolar amounts of DNA fragments, 1 unit of T4 DNA ligase (
The test is carried out using a known buffer solution under conditions using parigase.

Polyoligonucleotides' are chemically synthesized by the method of Crea et al. (3), and then polyacrylamide oligonucleotides are synthesized using short single-stranded or double-stranded polyacrylamide polymers. is a polydeokine nucleotide.

``Transformation'' is the DN in a certain organism.
A is introduced, so that the DNA is maintained as an extrachromosomal element or a chromosomal integrant. Unless otherwise stated, the method used here for the transformation of B. coli is the chloride method (30).

A. - G19 of A. n1dulans
1 strain (Glasgow University, Culture Collection) is transformed by incubation with the spheroplaston formation vector of A. nidulans. The genotypes of strain 0191 are pabaΔ1 (p-aminobenzoic acid requirement), fwΔ
1 (pigment forming), manA2 (monoamine non-utilization), and ρyrG89 (orotidine phosphate decarboxylase deficient).

Spheroplasts were balanced (Ba
ilance) etc. by the cellophane method.

A. Niturans < A, formerly dulans) cell wall 2
? In order to perform Htj, firstly, Novozym
e) 2 3 4 (Novo of Denmark)
In+jusf:ries) was partially purified. A sample of 10[) to 500mg of Novozyme 234 was added to 2
．． Dissolve in 5 mn of 0.6 Mkl. The 2.5ml fraction was Q6MkCI! ．． PD10 column (Pharmac, Sweden) equilibrated with
ia-11psulla) and the enzyme was eluted with 3.5 mβ of the same buffer. Cellophane desks were incubated in Nopozyme 234 (5 mg/mj2) for 2 hours and further washed with 0.6 MKCp. The digested material and wash water were combined and washed with Calbiochem-Behrink of Miracross (La Jolla, California).
ring) and washed as described (21). Centrifugation was performed at 1000×g for 10 minutes in a 50 or 15 mβ conical tube. Incubate in water for 20 min and 2 mβ polyethylene glycol 40
Add 0.00 solution (250 mg/mp) and incubate for 5 minutes at room temperature, then 4 mff of 0.4 MKl,
A 50mM CaC R 2 solution was added. The transformed pro I plasts were centrifuged and 0.6MKCβ, 5
Resuspend in 0mM CaCβ2 and plate as described further. Zero control is 20μβ207II without plasmid DNAΔ.
M Tris-l(C I!., ],mMEDTAS
It was performed with protoplasts incubated at pH 7.4. Positive controls included transformation with 5 μg of pDJB3, constructed as described herein. Regeneration frequency was determined by blating dilutions onto minimal medium supplemented with 5-10 ppm Paha and 50 Qppm uridine. Playback is 05
It was in the range of 5%.

Due to the low transformation efficiency associated with pDJB1, a derivative containing the Mucor acid propuase gene (pMeJB
]-7) was expected to have very low transformation efficiency. As a result, A. Co-transformation was used to obtain the transformant of A. nidulans pMeJBl-7. First, a vector containing non-selective ΔNS-1 was constructed, and then pMeJBl-7 and AN
This is accomplished by transforming spheroplasts with a mixture with a non-selective vector containing the S-] fraction. The principle for this approach is that ΔNS−1
The vector with pMeJ is integrated into many copies and pMeJ
B ]-7 would provide a homologous region for integration. The ΔNS-1 vector is pDJ
Pst J-Pvu of ANS-1 from B-3
IT7 fragment (Fig. 12Δ, 13B) was transferred to pUc1
Adjust by subcloning to 8. (33)2
Two plasmids (vectors containing pMeJBl-7 and ΔNS-1) were mixed together (2.5 μg each) and the transformation protocol described above was followed.

Vector pGRG1-pGR(l and pDJB-gam
The transformants obtained were incubated at 37° C. for 3 or 4 days and then transferred.

Transformant-derived miserial transfer was inoculated in the center of a minimal medium agar plate supplemented with 5 ppm p-ami7benzoic acid. 3-5 days after inoculation into minimal medium plates, 1 m
Beta distilled water, 0.02% Tween
The spore suspension was prepared by stirring the miserial fragments in -20. Approximately 5X10
' spores of 5 (] mm & 250 containing a medium of the following composition:
mβ shake flask. :(g/
I! ) Maltotextrin M-040 (Iowa,
Muscatine, Grain Processing Coop
g Corp) 50 gXNaN036 g,
Mg5O<・711200.5 g, KCβ0.
52g.

K H2PO, 68g, 1mβ trace element solution, 1mAM
ΔZLI DF-60p Defoamer (Gurnee, IL, Mazef 1 Chemical Medium Ink (
Mazef Chemical Inc. ), 1
0 ppm aminobenzoic acid, and 50 ppm strebtomine sulfate. In place of MAZU, one may also use such as stocked serum albumin or other suitable surfactants. Mucor acid protease secretion is secreted by Aspergillus.
) complete medium (20 g of textrose per II2, 1
g Pep I N, 20 g malto extract).

Aspergillus nitourans
chymosin secretion by transformants was found to be regulated by 1% starch instead of 5% starch.
% fructose, sucrose or dextrose versus similar media supplemented with % fructose, sucrose or dextrose. In all cases,
The culture medium was kept at 37°C in a rotary refrigerator (150 rpm).
Ink 1 was baked. Transformants derived from pDJB3 were used as a control.

Various secreted chymosins and l, Cole minihei (
Western plots of carboxyl proteases of Mucor m1ehei)
(35).

A desalting step is necessary due to the high salt concentration in the chymosin medium and the effect of this salt on gel electrophoresis. Prefabricated G-25 column (Pharmacia
a), P D 10 ) at 50 mM at pH 6.0
Equilibrated with Na28PO4. 2.5 mβ of medium was applied to the column. Proteins were eluted with 3.5 mβ of the same buffer. To detect heterologous polypeptides present on the plot, first nitrocellulose plots were prepared using rabbit anti-chymosin (36) or rabbit anti-Mucor miehei (M
ucor mlehei) carboxyl protease serum (36). Next, the plot was changed to Horseradish peroxidase (California,
The cells were contacted with goat anti-rabbit serum conjugated with Richmond Bio-Rat, Inc. and developed. Before applying to the gel, add 50 μβ of solvent (desalted in case of chymosin) to 2
Mixed with 5μβ of SDS sample buffer. Final concentration is 1%
β-mercaptoethanol was added so that

After treating the sample at 95°C for 5 minutes, 40-50 μp
sample was charged to the gel. Each gel also contains 2pR each.
A 650.65.6.5 μg/mβ chymosin standard material and a molecular weight marker were charged.

pmeDJl-7) Transformant Western
The plots were analyzed in the same manner, except that gel permeation was not performed.

Protease activity was detected by the method described by Sokol et al. (37).

Luria medium with 1-1.5% skim.
Supplemented with milk (Difco), the 30-35 mβ was poured into a 150 mm tube. A 2-5 μβ fraction of the medium was spotted onto a petri dish, and the plate was incubated overnight at 37° C. in a humidified box. Its activity determines the amount of milk clotting that occurs on the plate.
The determination was made based on measurements in meters. The plate was heated to 100'CHII/mβ moji or 16.6 CII.
U/m I! Rennin (CHU-Chr Hansen Unit, Copenhagen, A./S XChr-Hamsen's Laboratory)
Co-spotted with a dilution of Rium). The diameter (mm) of the coagulation zone and the oxygen concentration have a logarithmic relationship.

To distinguish between types of protease, carboxyl
Pepstatin, a chymosin-type inhibitor of proteases, was used for inhibition of chymosin-derived protease activity. Chymosin mutant sample and control medium
IQm in DMSO before subjecting to protease activity analysis.
The plate was incubated with a 100-fold dilution of Mpepscutin for 5 minutes.

Glucoamylase secretion by the pDJB-gam-1,) transformant in 5% starch medium was
Nitrophenol-α-glucopyranone (PNPΔG
) (38) was assayed by a method based on the activity of glucoamylase, which catalyzes the conversion of free glucose to p-nitrophenoxide. Substrate, PNPΔG, is DMS
Dissolved at 150 mg/mp in O, the 3-15 μp fraction was adjusted to pH
It was diluted with 200 μβ of 4,30,2M sodium acetate and 1mM calcium chloride solution. Place 25 μp samples into the wells of the microcuicou plate. Equal amounts of 0 to 10 sik-y (5igma) A nigma (Δ nig
er) -+-knit/mβ (MO, St. Louis,
Sigma Chemical Co.
Standard substances ranging from o, )) are placed in separate wells. 2.25 to 11.25 mg/m R in each well
Add 200 μβ of PNPΔG solution. The reaction was carried out at 60°C for 30 minutes to 1 hour.

The time depends on the enzyme concentration. The reaction is 50μ1)
2tVl) was stopped by adding Luzmarbase. The plate was measured at 4051 m, and the enzyme concentration was calculated from the calibration curve.

Unless otherwise stated, chromosomal DNA was extracted from filamentous fungi by the following procedure. Filamentous fungi were grown for 3 to 4 days in a suitable medium. Mycelia were harvested by filtering the medium through fine cheesecloth and supplemented with 50 mM Tris.
- Washed with a buffer of HCl, pH 7, 5, 5mMEDTA. Excess liquid was removed by pressing the Mycelia through cheesecloth.

Approximately 3 to 5 grams of wet Mycelia was mixed with an equal amount of sterilized, acid-washed sand in a mortar and pestle. The mixture was ground for 5 minutes to form a fine paste. Another 10m
1 of 50 m Mtris-HCl, pH 7,5,5 m
MEDTΔ was added and the mixture was triturated for 5 minutes. Pour the slurry into a 50 mβ centrifuge tube with a cap,
Extracted with 25 mA phenol-chloroform (same volume of 50 m Mtris-HCA, pH 7,5,
(equilibrated with 5mM EDTA). The phases were separated by low-speed centrifugation, and the aqueous layer was extracted three times. The aqueous layers were combined (total volume approximately 20 mf!) and diluted with 2 mβ of 3M acetic acid in a sterile centrifuge tube, pH 5.4.
mixed with. Ice-cold isopropanol (25 mβ) was added, and the centrifuge tube was left at −20° C. for 1 hour. The centrifuge tube was centrifuged at high speed to pellet the nucleic acids, and the supernatant was discarded. The pellet was air-dried for 30 minutes, then 400 pR
10 m 1 Vltris-HCR, pHV, 5.
Suspended in 1mM EDTA (TE buffer). Pancreatic ribonuclease (Sigma Chemical Co., St. Louis, MD)
is added to a concentration of 10 μg per mA and incubated for 30 minutes at room temperature (30). Thereafter, ribonuclease' is removed by phenol-chloroform extraction. Carefully remove the aqueous layer and transfer to a test tube containing 40 μβ of 3M Sodium Acetate, p++ 5.4. The solution is layered with water-cooled ethanol, and DNA precipitates at the interface.
Spread it with a glass rod. This DNA is dried and dissolved in a small amount of TE buffer (100-200μj2).
NA concentration was determined by absorption at 260 nm.

Southern hybridization was performed to confirm the chromosomal integration of chymosin DNA sequences in selected transformants (30).

The transformant spore suspension was inoculated into Aspergillus complete medium and incubated at 37°C in a rotary shaker.
Incubated for 24-48 hours. The medium is non-selective, supplemented with 5 ppm p-aminobenzoic acid, and contains sufficient uracil for growth of the autotrophic parent strain. In fact, these Southerns also tested the stability of the transformants. After filtration of Mycelia, it was ground with sand and the DNA was purified as described above. Furthermore, the transformant) DNA was digested with various restriction enzymes, and the fragments were separated by agarose gel electrophoresis. Control lanes contain digested pDJB3 transformant DNA and undigested DNA. The gel was stained with ethidium furomite, photographically rubbed, and nitrocellulose or Nidoran (NH, Keene (K)
eene), Shu L/Icha and Shu Iru (Sc
The cells were probed with blots, radiolabeled plasmids, or specific fragments (manufactured by Hleicher and Chuel 11).

Example 1 Aspergillus nidulans (Aspergillus nidulans)
Expression and secretion of glucoamylase from Aspergillus niger (A. snidulans) and construction of pGA1.
s niger ) (Culture number 7
#7),, CΔ, Zaus Zanfrancisco (
South San Francisco), Culture Collection Genencar (Culture
[:olect,+on Gcnencor) )
were grown for 3 days at 30°C under strict aeration. Chromosome D
NA is done in the same way as mentioned above.

Synthetic oligonucleotides were used as hyphritic probes to detect Aspergillus niger (Asp.
The culcoamylase gene derived from S. ergillus niger was detected. The length of the oligonucleotide is 28 bases (28 mar) and corresponds to the first 9173 futons of the published glucoamylase bihood sequence. (39) MetSerPheArgS
TGTC to erLeuLeu81aLeuSer5'
GTT to CGATCTCTACT biGCCroT683
'The oligonucleotides were tested in BioSearch (Bi) using reagents and methods directed by the manufacturer.

Synthesis was performed using an automatic DNA synthesizer (CΔ, manufactured by San Rafael, manufactured by Bio 5earch). A.spergillus nig
The presence or absence of a glucoamylase sequence was analyzed using the Southern method on the genomic DNAΔ of er) (30). Aspergillus niger (A. spergillus)
lus niger) 10 μg of BcoR1 DNA
Digest with restriction endonuclease.

Digested DNA is subjected to 1% agarose Kel electrophoresis according to standard methods (30). DNA 10x S SC
Plotting in (1,5 M NaC, i!, 0.15 M trisodium citrate) showed that the gel to nitrocellulose membrane (NH, Keene (1), Si, (Neucher and N: w, x /l/ (Schl
(manufactured by Eicher & Chuell) (30
). DNA is immobilized on nitrocellulose by baking in a vacuum oven at 80°C and low salt (2,40) hybridization is performed using radiolabeled oligonucleotide probes. Radioactive labeling of synthetic oligonucleotides was performed using 7 Q m M Tris-HCl.
(pH 7,5) 10 mM MgCA2.5 mtv
l dithiothreitol, 3 Q pmole synthetic oligonucleotide, 2 Q pmole gamma (32p)
ΔTP (Ip, Chicago, 7 Fushiyam (Amersha)
m), specific activity 5000 ci/mmol), and 5 units of T4 polynucleotide, Kinase, New England, Biolab (New-England).
-Biolabs) in a 50 μn reaction solution containing
Performed at 7°C. After hybridization, the filters are washed for 15 minutes in 2X SSC 10,1% lauryl sulfate (SDS) and twice in 2X SSC at 37°C. After air drying the filter, wrap it in saran wrap (
Autoradiographic images were obtained by wrapping in Dow Chemical and applying to Kodak X Omat-AR X-ray film at -70°C. After the autoradiograph image, the hybridization band was 3.
It corresponds to the 5 kilobase pair BcoR1 fragment and can be clearly identified.

Aspergillus niger
iger ) was digested with EcoR1 and size-fractionated by polyacrylamide gel electrophoresis according to standard methods (30). DNA fragments of size 3 to 4 kb were cut and eluted from the gel (
30). This DNA fragment was transferred to Escherichia coli (Esche
ric:hia col i) cloning vector pBR322 (ATCC37017),
It was used to make i1-line life library. The cloning vector was cut with 1EcoR] and treated with Hatateria alkaline phosphatase (Bethesda Research Rough).
A typical dephosphorylation reaction is 5.
0 μp of 50 m Mtris-11cp (pH 8,0
), 1 μg of digested vector DNA in 50 mM NaCl.
and 1 unit of alkaline phosphatase.

The reaction was incubated at 65°C for 1 hour. Osphacose was removed by phenol-chloroform extraction. Then selected Aspergillus niger by BCOR1.
er) DNA was cleaved with 1EcoR] and ligated with dephosphorylated pBR322. A typical ligation reaction includes: each 1100n of vector and insert DNA.
A, I unit T4 DNA ligase (Bethesda)
Research Lab (Bethesda Research)
Labs)), 25 m M Tris-11c, f
7 (pH 75), 10mM MgCn2.10mM
Dithiothreitone and lmMATP are present in 0 μβ volume. The ligation reaction is incubated for 18 to 24 hours at 16°C. The ligated DNA was prepared using Morrison (Mo
E. coli 294 competent cells (AT
CC31446) was used to transform.

Transformants were selected on LB agar plates (30) containing carbenecillin at a final concentration of 50 μg/mβ. Transformants carrying the glucoamylase gene sequence were identified by colony hybridization (30) using a glucoamylase-specific 28-mer as a probe. Hybridized colonies were incubated at 14M m, and plasmid DNA was transferred to alkaline-3D.
isolated from each of them by the S-miniscreen procedure (30). All plasmids selected in this manner hybridize to the synthetic lucoamylase probe3,5.
It contained a k b EcoR1 fragment. Such a plasmid, designated pGaj2, was selected for further analysis. 1.1 k from insert in pGap
EcoR1-Bgβ■ fragment of b is Ml, 3
The cloned DNA was subcloned into mp9 (42) and partially sequenced by the guideokine chain combination method (43), confirming that the cloned DNA encodes the glucoamylase gene. A restriction endonuclease cleavage map of the 3.5 kb BcoRl fragment contained in pGa,gl is shown in FIG. It was created by single and double restriction digests to obtain orientation with respect to the known restriction sites of pBR322. (44) Construction of BypGa5 The nucleotide sequence and restriction map of pGap showed that pGap contains the entire glucoamylase coding region and 221 nucleotides of 5′ DNA. The sequence of this 5' region is surprisingly present "first stream" of the start codon of ΔTG. Typical eukaryotic bromo-cou sequence occurrence with TATΔAΔT and CΔΔT boxes (48).

However, Aspergillus niger
The activation site far upstream of the glucoamylase gene of M. usniger is responsible for the final transformation.
To confirm that it is included in the vector, at least 1. ．． A large genomic fragment containing 000 bp of DNA was cloned. By the same Southern plotting experiment as previously described, the 6.5 kb Cla hybridized with the radiolabeled EcoR1 culcoamylase fragment from pGap.
The l fragment was identified.

The BcoR1 fragment is alpha[:32P
:] -dCTP (Amar-Jam, specific activity 3000
ci/m not ) by nick translation (30). Label reactions were performed using nicks according to the instructions provided by the manufacturer.
Transformer Kit (Bethesda Research Lab)
) was used. Filters were hybridized and washed under high salt conditions (30).

6.5kb C identified by hybridization
The la I fragment was cloned as previously described. Aspergillus niger (Aspergi
llus niger)'s I) N A [:] a
It was digested with I and fractionated according to size by polyacrylamide gel electrophoresis. DNA fragments that migrated between 55 and 3 kb were excised and flowed out of the gel. This fraction was cleaved with C1a I and dephosphorylated p
BR325 (45). This ligation mixture was used to transform competent cells of E. coli >294. Transformants were selected on LB agar plates containing carhenenrin (50 μg/mn).

Colonies containing the glucoamylase gene sequence were identified by co-hybridization (30). Plasmid DNA extracted from hybridized colonies
A is 3,5k previously cloned into pGaj2
It contained the BcoR1 fragment of b. These recombinant plasmids were identified by supercoiled DNA sequencing using synthetic oligonucleotides (28-mer) as sequence primers.
Aspergillus niger
iger ) glucoamylase gene. A restriction endonuclease cleavage map of the 5.5 kb Cla I fragment was constructed by single and double digests of DNA cloned into pBR325.

The restriction sites in vector DNAΔ were used as reference points to orient the fragment. This restriction map is shown in Figure 1. The location of the glucoamylase gene was determined by comparing the restriction site of pGa5' with that of the previously published glucoamylase gene (39.47
．． 48). From the map data, approximately 3,3 kb of 5' flanking DN was found within the cloned fragment.
It was confirmed that A and about 1 kb of 3' flanking DNA were included.

On August 28, 1985, plasmidyl Ga5 was introduced into Escherichia coli (
E. coli) of 294, deposited with ATCC, 5
It was named number 3249.

C. Aspergillus niger
The C]aI7 fragment of b was added to 6.5 from pGa5 containing the glucoamylase gene, a vector for expression and secretion of glucoamylase in E. coli (E. coli).
) - Aspergillus nido'silans (Aspe
rgillus nidulans) into the shuttle vector pDJB3 as shown in FIG. The pDJB3 shuttle vector was derived from Escherichia coli (E,
Selectable beta-lactamylase gene and origin of replication from plasmid pBR325 of A. coli, A. spergillus
pyr from Newsporacrassa rescues nutritional requirements for uridine in the G191 strain of N. n1clulans.
4 genes, Aspergillus nidurans ((A. spe.
Aspergillus nidulans (Aspergillus n1dulans), which has a high frequency of stable integrated transformants,
The sequence known as ANSI from C. n1dulans), the only BcoR1 and Cβa of the cloning pool
It has an I restriction site. pDJB was constructed as shown in Figure 14. Plasmid pFB6 (32) was transformed into Bgl
It was completely digested with m and then partially digested with old nd■.

fragment containing the pyr 4 gene (approximately 2 kb)
B was purified by gel electrophoresis, and the former ndl]J and Bam
It was ligated with previously digested pBR,325 (fragment A) to generate plasmid pDJB1.

pFB6 digested with EcoR1 and A. n1clulans genome DNA (strain 0191 or FGSC#4-derived strain)
The ANS-1 sequence was cloned by ligating. The resulting EcoR1 fragment pool in pFB6 was used to transform ura3-3 cerebinae (S.

Cerevisiae) (e.g. ATCC447
69,44770, etc.) were transformed. The self-replicating plasmid plntA is
erevisiae). plntA to Bc
The ANS-1 fragment was purified by gel electrophoresis and ligated with pDJBl digested with 3coRl to generate plasmid pDJB2. EC pDJB2
Plasmid pDJB3 was generated by partial digestion with OR1, treatment with DNA polymerase 1 (Klenow), and religation. The partial nucleotide sequence and restriction map of the ANS-1 fragment are shown in Figures 13Δ and 13B.

plasmid) pGa5 was digested with Cf1a1 and the larger fragment (fragment Δ) was separated from the vector by agarose gel electrophoresis.

This fragment was digested with βaI and ligated with dephosphorylated pDJB3 (fragment B).

Using this ligation mixture, Escherichia coli (E. coli)
294 competent cells were transformed. The transformants were selected on LB agar plates supplemented with carbenenrin (50 μg/mβ). Analysis of plasmid DNAΔ from these transformants showed that the glucoamylase fragment had been inserted, as expected. Glucoamylase fragments oriented in both directions were obtained by screening various transformants. pDJB
- One plasmid, named gam 1, was added to the Aspergillus nitourans (A.
n+dulans) protoplasts were randomly selected.

D Expression and secretion of glucoamylase As mentioned above, Aspergillus nitourans (As
pergillus nidulans) G191
The strain was transformed with pI) JB-gaml.

As previously described, pDJB-gam-174,9,1
Five transformants named 0, 11 and 13 were analyzed for glucoamylase activity. The results are shown in Table 1.

As shown in Table 15, each pI) J B -gam -1)
The transformants produced glucoamylase activity greater than that of the control, indicating that biologically active glucoamylase was expressed and secreted from the transformed fungi.

Example 2 Aspergillus nidulans (Δspergillus
Expression and secretion of calf chymosin from the natural calf chymosin precursor (preprochymosin),
Aspergillus niger
An expression vector was constructed to encode a fusion precursor in which the DNA sequences for glucoamylase and prochymosin of P. us niger are precisely fused. The strategy for constructing these vectors includes the following steps. First, a DNA sequence containing a part of the glucoamylase promoter and a part of the 5' cod region of glucoamylase was converted into a DNA sequence corresponding to the amine terminal part of preprochymosin.
cloned upstream of the sequence. Next, the nucleotides between the DNA fragments are paired with a specific primer sequence, M1. 3 was deleted by site-directed mutagenesis (40). Finally, the DNA segment containing the fused sequence, along with the remainder of the procymonone sequence, is transferred to Aspergillus nig.
er) into an expression vector containing the 5' and 3' control sequences of the glucoamylase gene. These steps are outlined in FIGS. 3-7.

A. Construction of mp19GAPR Plasmid pGa. 5 was used to generate a 337 bp EcoRI-RsaID with a portion of the glucoamylase promoter and an amine-terminal segment of the coding region.
An NΔ fragment (fragment Δ) was induced.

Fragment Δ was ligated with M13ml11.9RF-DNA digested with ECOR 1 and 3ma J (fragment B).

Using the ligation mixture, Escherichia coli (E. coli) J
Mlol (ΔTCC33876) was transformed. Clear plaques were analyzed for fragment Δ by restriction analysis of the corresponding RF-DNA. mpl
The fragment Δ, named 9 R -Rsd, was
One isolate containing Pst ] and Xba I was digested with Xba I and the larger fragment (fragment C) was isolated. The smaller xba■-Pst■ sequence (fricmen) D) is pR3 containing the 5' preprochymosin sequence.
(49), purified by electrophoresis, and then ligated with fragment C to create the phage template mp19cΔPR shown in Figure 3.

B1 site-directed deletion mutation As shown in Figure 8, m1119GAPRΔC1 was generated from mp19GAPR by site-directed mutation by deleting the nucleotide between the signal peptide tocodon of glucoamylase and the codon for prochymosin. guided. Thus, in m p 1.9GΔPRΔC1, the signal peptide tocodon of glucoamylase is precisely fused with the first codon of prochymosin. Site-directed mutagenesis was performed in a single-stranded M 1.3 ten boot as previously described (40), except that only one oligonucleotide was used to initiate synthesis of the second strand. (Figure 4) (40). mp19GΔPRΔC1
The synthetic oligonucleotide used to induce 5′GCTCGGGGTTGGCAGCTG8G8T
CACCAG3'. Plaques in which the desired deletion occurred were identified by hybridization with radiolabeled primers as previously described.

In mp19GAPRΔC3, the nucleotide immediately before the start codon of glucoamylase and the nucleotide between the ATC start codon of preprochymosin are linked by site-directed mutagenesis using a synthetic oligonucleotide (Fprimer-3).

5 'ACTC mouth CCC 8CCG AATGAGGT
GTCTCGT 3'As shown in FIG. 8, the resulting mutation causes the glucoamylase promoter region to be precisely linked to the start codon of preprochymoson.

Construction of a Vector for Expression and Secretion of C9 Calf Cymonin As shown in Figure 4, the glucoamylase sequence and
'Each fusion of prochymone sequence (mp19GAPRΔC
1 and mp19GAPRΔC3) are Aspergillus
Aspergillus nidulan
s ) transformation vector pDJB3
3' prochymosin sequence and Saccharomyces cerevisiae x (Saccharomyces cerevisiae)
isiae) phosphoglycerate kinase (PGK)
) is combined with the Couminator. mp19GAPR
ΔC1 and mp19GAPR8C3 replication type all C3oR1
and PSt1. The smaller fragment (fragment 1) was isolated. Plasmid pBR32
2 was also digested with EcoR1 and Pst1 and its larger vector fragment (fragment 2) was isolated.

Fragments 1 and 2 were ligated, and E. coli
) 294 was used to transform.

Plasmid pBR322GAPRΔC1 or pBR
A tetracycline resistant colony containing 322GAPR8C3 was isolated. Fragment 2 is also derived from Escherichia coli (E, co
l i ) with polymerase 1 (Klenow fragment). The resulting plant end fragment was circularized by ligation and transformed into Escherichia coli (E. coli) 29
4 was used to transform. Plasmid pB
One tetracycline resistant colony containing R322ΔRP was isolated and digested with old nd ■ and Sal I. The larger vector fragment (formant 3) was isolated. Plasmid pCR160 was transformed into the former ndI [and P
After digestion with st I, fragment 5 (containing the yeast PGK couminator fused to the 3' prochymonin codon) was isolated. Fragments 3.4 and 5 were ligated and used to transform E. coli 294. Plasmid pBR, 322GAPR△
The tetracycline-resistant transformant plasmid pCR 160 containing C1 or pBR322GΔPRΔC3 has a yeast 2 μm replication origin that allows its maintenance as a plasmid in yeast, and a yeast TR as a yeast selection marker.
It contains the P-1 gene, the E. coli (E. coli) replication origin and ampicillin resistance gene from plasmid pBR322, and the prorennin expression unit. The prorennin expression unit is the yeast P
It contains a promoter derived from the GK gene, a prorennin coding region, and a couminator derived from the PCK gene. This plasmid was constructed in the following manner as shown in FIG. Plasmid YEplPT (50)
was partially digested with old ndIII and then completely digested with tEcoRl to isolate vector fragment Δ. Second
Plasmid pPGK-1600 (51) of EcoR1
and old ndllI, and PGK promoter fragment B was isolated. Fragments Δ and B were ligated to create the intermediate pintl, which was again partially digested with P, coRI and 11ndl to isolate vector fragment C. PGK couminator fragment D was isolated and plasmid pB1 was combined with old ndlJJ and 3an
3A (52). PR, 1 (49>DNA 1
Prorennin fraction E was isolated by cleavage with 3coR1 and BCβ1. Fragment C1D
and E to create yeast expression plasmid pCR16
0 was generated. The nucleotide sequences of the PGK promoter, structural gene and terminator have been reported (5
3).

Plasmids pBR322GAPRΔC1 and pBR322
GAPRΔC3 contains a complete transcription unit for each prochymonin. This transcription unit contains the sequence for the precursor prochymosin, the glucoamylase promoter, and the yeast PGK complex. However, these plasmid transfectants and plasmids pBR322GΔPRΔC2 and pBR322GΔPRΔ4 (pint I C1-
4).

Figure 5) is A, n1dulan.
When transformed into G191 of s ), it did not produce detectable cymonone. The reason why these derivative plasmids are unable to express and secrete chymosin is
I don't understand. However, in light of subsequent results, it appears that the PKG or short glucoamylase promoter sequences in these plasmids are
Nidulans (A, n1dulans) G 19
1 does not seem to be recognized. Based on these results, the pBR322GAPRΔC plasmid was further modified.

The transcription unit is transferred to A. spergillus niturans in the following steps.
clulans) transformation vector pDJB3. Additionally, the 5' flanking sequences of glucoamylase were incorporated just 5' to its promoter, ensuring the presence of a significant upstream activation site involved in controlling expression.

Furthermore, PKG Koumineku is A derived from 5 of PG.

The terminator of Δ niger glucoamylase was substituted. In particular, in Figure 5, each plasmid (
pBR322GAPRΔC1 or pBR322GAPR
ΔC2> was digested with la l and fragment 6 was isolated. Plasmid pDJB3 was also digested with C1al and treated with bacterial alkaline osphatase.
Prevented self-connection.

This digested plasmid (fragment 7) was ligated with fragment 6 and plasmid pINTI-1 or pIn
An ampicillin-resistant colony containing tl-3 was isolated. These plasmids were digested with Xho T and Nsi I and the larger vector fragment (fragment 8) was isolated. Plasmid pGa5, containing the entire Curcoamylase gene as well as extensive 5' and 3' flanking sequences, was digested with Xho and N511 to generate a smaller fragment (fragment 9, approximately 1.70
(containing the 5' sequence of 0'bp) was isolated. Fragments 8 and 9 were ligated and E. coli (B, coli)
294 was transformed. plasmid plnt
Ampicillin-resistant colonies containing pint 2-1 or pint 2-3 were isolated.

These plasmids differ critically from the final vector in that they contain the yeast PGK terminator rather than the glucoamylase terminator (Fig. 7
reference).

Additional steps in constructing the chymosin expression vector are outlined in Figure 6. Plasmid pR1 (49) was used to generate a small cDNA containing the 3′ end of prochymonin cDNAΔ.
Fragment A) was released by IJ. Pocket fragment Δ and PO
cloned into Cl8, Δsp7], 8 and Bam H
Digested with I (fragmen) B). Similarly, plasmid p
C1a I-Asp 71.8 12kb from Ga5
The DNA 7 tract (fragment D) was digested with single jl, 8ccl and 8sp7], POCl cut with 8
2 (fragment C). The resulting intermediate plasmids, PUC-inll and PUC-int2
was digested with Sal 1 and Hindll, and fragments E and F were separated. These fragments were further ligated to create a PU containing the 3' end of prochymosin, followed by HindIII-Δ5p718 fragment) (fragment H)''-, a curcoamylase terminator sequence.
C-int, 3 was generated.

A new cloning vector, named pBR-1ink, was created by inserting a synthetic oligonucleotide into the unique Bam H1 site of pBR322. This linker contains the following array:

5' GATCCATCGATCTCG8G8T[
:To G'rC3to3' GTAGCTAGAGCT
CTAGCTACCT8G5' The larger fli dllT-Xho I fragment (fragment G) of this vector was purified by electrophoresis. Similarly, Xho of plasmids plnt2-] and plnt2-3
I-ASp7], 8 restriction fragments were electrophoretically purified. In a series of three ligation reactions, different ■-fragments and fragments G and l were ligated to generate intermediates plnt 3-1 and p'tnt 3-3. Propeptide fusions to the various internal and prochymosin (or preprochymosin) sequences of the promoter region, followed by the final glucoamylase terminus region, all within convenient Cffa I restriction sites.

Some la I sites, such as the one in the linker of pBR-1ink, are linked to the E. coli D
Plasmid pin is damaged by NA methylation.
Those from t 3-1 to plnt 3-4 are 0M4
E. coli (fE, named 8 (ΔTCC39099)
The plasmid was transformed into a dam- strain of E.coli, from which the plasmid was reisolated. C,i! unmethylated DNA! a Digested with I and electrophoresed,
Fragment J was released. Subsequently, fragment J from each crucoamylase prochymosin fusion was inserted into the unique la of pDJB3 (fragment K).
cloned into the final expression vector pcRc
1 and pGRC3 were generated.

D. As mentioned in the expression and secretion destination of regulated chymosin, Aspergillus nidulans (A
G19 of Spergillus n1dulans)
1 was transformed with pGRGl and pGRC,3.

Five pGRGI and five pGRC3) transformants were analyzed. Wescone analysis (not shown) showed that each transformant secreted a protein that reacted with anti-chymosin and migrated to a location with the same or slightly higher molecular weight than calf chymosin. Higher molecular weight species may be due to improper processing, bare ground effect or glucosylation. Integration was confirmed in one transformant by Sauzane Hybridization Solution (results not shown). Each transformant was also assayed for cymonin activity. The test results are shown in Table H.

Table ■ p D J 83 1. 0-0.13pGRG
1 5 0-1.5 pGRG3 5 0.05-7.0 These results indicate that both pcRcl and pGRC3 secrete varying levels of protease above the pDJB3 control.

By chance, background proteolytic activity was detected in the pDJB3 control medium. As shown below, the proteolytic activity of this transformant is associated with the aspartate family of carboxyl proteases, of which chymosin is one member.

Example 3 Aspergillus nithurans
Expression and Secretion of Fusion Polypeptides from A. nidulans Two fusion polypeptides were constructed to be expressed and secreted from A. nidulans. One fusion polypeptide contains the amine-terminal region containing the prosequence and first 10 amino acids of Aspergillus niger glucoamylase, and the carboquine-terminal region constituting calf prochymosin. There is.

The second fusion polypeptide is derived from Aspergillus niger (Δ
It contains only the amino-terminal region of the pro-sequence of glucoamylase (Spergillus niger) and the carboquine-terminal region of calf prochymosin.

Δ, Vector for expressing and secreting the fusion polypeptide The specific sequence was extracted from mp19GAPR by following the same procedure as when constructing the vector encoding the fusion polypeptide described above for pcp, cl, and pGRG3. It was constructed by deletion. As shown in Figure 8, m
In p19GAPRΔC2, the nucleotide between the glucoamylase propeptide codon and the prochymosin codon was deleted by the site-directed mutagenesis described above. Oligonucleotide synthesized for this mutation′
The sequence of (primer?) is 5'TG8T to TCC88G[:GCTGAG
ATCACC863'.

This mutation is intended to fuse the glucoamylase promoter, the ngnal peptide, and the propeptide codon to the first codon of prochymosin. m
In p19GAPRΔC4, the nucleotide between the 10th codon of mature glucoamylase and the codon of prochymosin was converted into a synthetic oligonucleotide (primer 4
) was deleted by site-directed mutagenesis of M1.3.

5'To TG8G to AACGΔAGCGGCTG8G to TCAC8G' This deletion results in the deletion of the glucoamylase promoter region, the signal peptide sequence, the propeptide sequence, and the codons up to the 10th position of the mature glucoamylase. It was fused to the codon of prochymosin as shown in . These expression and secretion vectors are pcR2 and pGR.
Named G4, Δ, nidula (Δ, n1dula
ns) was used to transform.

B, Aspergillus nidulans transformed with pGR2 and pGR4 Δspergillus old+
pGRG2 and pGRG4) transformants were cultured as described for expression and secretion of chymosin from 1lulans). The culture medium was assayed for chymosin activity by Western blotting, and pGRG I and pG,
It gave the same results as those obtained with RG3. pGRG2
) Transformant incorporation was confirmed by Southern analysis (results not shown). The results of the chymosin assay are shown in Table ■.

Table ■ pDJB3 1 0-0.13 pGRG2 1. 0.001-0.42pGRG4
6 0.004-0.75 Again, each transformant showed protease activity greater than that of the pDJB3 control, indicating that protease was expressed and secreted by the transformant. pGRG
As in the case of pGRG3 and pGRG3, these proteases belong to the aspartate family of carboxyl proteases, as confirmed by pepscutin inhibition. Importantly, these results demonstrate that hybrid polypeptides are expressed in filamentous fungi.

Example 4 Pepsuctin Inhibition Studies Three of the above vectors containing various constructs containing chymosin were subjected to pepsuctin inhibition assay analysis as previously described. The results are shown in Table ■.

Table ■ Samples preincubated with pepstatin showed a significant decrease in activity, indicating that the protease produced by the transformant belongs to the aspartate family of acid proteases, of which chymosin is a member.

This data, together with data from Weskoon analysis, shows that biologically active chymosin is present in pGRGl, pctmaG
2, Δ transformed with pGRG3 and pGRG4.

G19 of A. n1dulans
1.

The degree of chymosin activity detected for the different vector constructs in Example II and Example III may reflect differences in the degree of recognition of the various chymosin proteins incorporated within each transformation vector. unknown.

In a particular construct, changes in chymosin activity are likely to be related to the number of copies of the vector integrated into the fungal genome or the location of such integration.

Example 5 Carbon source study One vector pcRc4 with Δ, nidulans (A,
n1dulans) and then grown on the various carbon sources mentioned above.

The test results are shown in Table 5.

Table V Chymosin activity produced by various carbon sources (μg/m
β) Starch Cucrose Fructose Sucrose pD
JB3 0 0 0 0p
GRG4 3.5 3.5 0.9
1.75 These results clearly indicate that chymosin is secreted independently of the carbon source.

This indicates that transcriptional control of the glucoamylase promoter is not induced by starch, unlike in Δ, niger.

Example 6 Expression and secretion of the carboxyl protease of Mucor m1ehei A, genomic probe for carboxyl protease The partial-substructure (54) of the acid protease of Mucor m1ehei shows low genetic redundancy The area was investigated. try-try of residues 187-191 (using the porcine pepsin numbering system)
-phe -trp -asp was selected. Oligonucleotide 5'-GC (G/A>TCCC8 (G/8)AA) complementary to the coding sequence corresponding to this amino acid
(G/A>TA (G/8) To synthesize TA-3'3
1), gamma-1:32P)-ATP and T4 polynucleotide kinase (30).

B. Mucor m1ehei
) carboxyl protease cloning kit,
Mucor m1ehei
(Netherlands 370.75.
Central Bur
eau Voor Schimmel cultures
)) was prepared as follows. YM
E medium (3g/p yeast extract), 3g/,
+2 malt extract, 5g/β peptone, 10g
Cells grown in /β-curcose) were collected by centrifugation, washed twice with 0.5M NaCp, and lyophilized.

Cell membranes were further disrupted by adding sand to the cells and grinding the mixture with a mortar and pestle. The resulting powder is 2
5% sucrose 50 mM Tris-HCII]88.
0 ), suspended in a solution containing 1.0 mM EDTA (per dry weight crumb], 5 mff). SDS to final concentration 0
The suspension was extracted once with half the amount of phenol and three times with half the amount of chloroform. The final wood chips were 10 m M Tris-DCI
! .

(pt18.0) was dialyzed against 1mM EDTA. The DNA was then purified with sodium acetate (p115.
5) was added to give a concentration of 0.3 M, and further precipitated by adding 25 times the volume of cold ethanol. This DNA fraction was digested with various restriction nucleases according to the manufacturer's instructions and analyzed for sequences complementary to that of the probe described above using the Sauthern method. A positive hybridizing band of approximately 2,5 kb (kilobases) was identified in the 11indlTI digested 1) NA. ) lindlll-digested genomic DNA was separated by polyacrylamide gel electrophoresis, and 2.0 to 3.
(The gel fragment containing the lkb DNA was electroflowed as described above. Mucor-Miehei)
This electroflow DNA, which is thought to be enriched in sequences corresponding to the acid protease gene of Mlehei), was ethanol precipitated. Cloning vector pBR32
2 (ATCC37017) was digested with old ndlJJ and dephosphorylated with bacterial alkaline phosphatase.

Typically, 1.0 mg of vector and 100 mg of uniformly sized DNA are combined in a 10 μp reaction with ATP and T4.
DNA was ligated in the presence of IJ gauze. The reactant was purified by E. coli (
E. coli) 294. Approximately 2
Anpinlin-resistant colonies of , OXl, 04 were obtained.

Approximately 98% of these contain cloned inserts, as shown by their failure to grow in medium containing tetrazycline. About these colonies,
The presence or absence of a complementary sequence to the DNA probe sequence was tested by standard colony hybridization procedures. Positive hybridizing colonies containing plasmid pMe 5'muc were found to contain the expected 2.5 kb size tlin d"■ insert.

The ends of this fragment were cloned into the M13 sequencing vector (33), and the sequence was determined by the guideoxy chain coomi sequence method.

One end contained a sequence corresponding to the known amine-terminal amino acid sequence of an acid protease gene.

The adjacent 3' region was sequenced to obtain the more C-terminal coding sequence. The sequencing strategy is shown in FIG. Thus, the entire coding sequence for the mature form of the protein was obtained.

The 5' end of the fragment was found to occur 112 bp (base pairs) upstream of the codon corresponding to the mature amine end. This "-stream region does not contain an in-frame start codon, so it is considered to be part of the propeptide. D containing the start codon as well as the 5' untranslated sequence
To obtain NA, the more 5' clone was isolated as follows.

Old nd of pMe5'CRatlind In insert
The III-Cla I 813 bp 5' subfragment was isolated and labeled by nick translation (30).

This labeled fragment was labeled with Cla by the Sauthern method.
Digested Mucor m1e
hei) Used to lift genomic DNA. A single band of hybridization corresponding to a molecular weight of approximately 1300 bp was gBHed in this experiment. This DNA of uniform size was isolated, digested with C1aI, and cloned into dephosphorylated pBR322 as described above.

Approximately 9000 ampicillin resistant colonies were obtained. Approximately 90% of them contained loned inserts as demonstrated by their inability to grow on tetrazycline-containing media. These colonies were assayed for the presence of sequences complementary to those of the nick-translated probe by standard colony hybridization procedures. Positively hyphridizing colonies containing plasmid pMe2 contain the expected 1.3 kb C1a
It was found to contain an I insert. Sequencing of the ends of this fragment revealed that one end was pMe 5'C
This corresponds to the sequence near the Cla 1 site of the old ndllI fragment in la, indicating that the fragment had the orientation shown in FIG. 10. Furthermore, C1a
Sequencing of the I fragment revealed the initiation codon and 5' untranslated sequences. The entire coding sequence and the 5' and 3' flanking sequences are shown in Figure 10. A comparison of the derived primary structure with that determined directly by amino acid sequencing revealed that Mucor-/l/ (Mucor
) It was found that the protein is produced as a precursor having a length of 69 amino acids. Based on the structural characteristics generally present in the rieter sequence, residues -21 to -1 encompass the leak-peptide and residues 21 to 69 are found in the zymogenic forms of other acid proteases, including chymosin and pepsin. It appears to contain a propeptide similar to that found in . (55) C. Mucor m1ehei
Expression and secretion vector of carboxyl protease of Mucor m1ehei
A vector for expressing and secreting carboxyl propase consists of a coding sequence, a 5' flanking sequence (
promoter), and all original Mucor m1ehei sequences, including the 3' flanking sequences (terminal and polyadenylation sites).
) contains an acid protease transcription unit.

The entire strategy for creating this vector is shown in Figure 12. Aspergillus n1dulans transformation vector pDJB1 was transformed into Cl
aI and BcoR1 and the larger vector fragment (fragment I) was isolated. Plasmid pMe 5'Cla was digested with EcoR1 and Cla I and fragment 2 was isolated. This formant contains the 5' codon of the acid protease, along with approximately 500 bp of 5' flanking sequence. Fragments 1 and 2 were ligated and E. coli (B, coli) 294 was transformed into A L. Plasmid pMe J Bi
An ampicillin-resistant colony containing nt was isolated. This plasmid was digested with Cla T, treated with Bacterium alicaris osphacosus to prevent self-ligation, and designated Formant 3. Plasmid pMe2
was digested with Cla I and a smaller fragment (fragment 4) was isolated. This fragment contains the 3' codon of the Jucor mlehei acid protease and approximately 1800 pb of 3' flanking sequence. Fragments 3 and 4 were ligated and E. coli >294 was transformed. Anpinlin-resistant colonies containing plasmid pMe J 131-7 were isolated.
7 ohms.

D, Aspergillus nido 1 crans (Δspergi
Expression and secretion of carboxyl proteases of Ueyama + 1ans) and Mucormiehei (Southern Prop) of 6 transformants
Analysis LiAspergillus nidulans (Asperg
illus n1dulans) genome, all Mucor m1ehei acid proteins are present in the genome.
showed the presence of a tease gene (results not shown). Furthermore, each transformant was subjected to Western blot analysis (results not shown) and acid protease activity analysis. Table of results of protease assay■
It was shown to.

Table Vl 1 0. OO320, 007 40, OO5 50, 005 60,012 These experiments were carried out by Mucor Miehely.
rmiebei), indicating the expression and secretion of a protein with milk coagulation activity. The protein is
The original Mukormihei
It was found by electrophoretic analysis that the protein had an apparent molecular weight slightly larger than that of the protein derived from (Ehci). This is Aspergillus nitourans (
Δspergillus Snidulans )gakono/
” IJ Coprotinwo, Mukor Miehei (Muc
This may indicate a different level of criconization than (or m1ehci). Aspergillus niturans (△spcrgillusnidu)
lans) derived from Mucor miehei (Mu co
rmicl) ei) Since the acid protease appears to have the same specific activity as the native one, it seems to have been processed (either by the cell or autocatalytically) into the mature form. Other non-prosensing forms of acid proteases, such as chymosin and peptin, are zymogens that become active (autocatalytically) upon prosensing. Different levels of expression in different transformants are expressed in Aspergillus nidulans (Δs
pergillus nidulans) likely reflects the location or copy number of the protease gene in the genome. However, the expression and secretion of biologically active carboquine proteases is limited to Δ, nidulans (Δ,
n1dulans) is Mucor 9 mini hei Uucor
mlehei) recognizes the promoter, signal, and couminator signals of the carboquine proteinase.

Example 7 Δ, av+amori and Trichoderma reesei
pG from a pyrG auxotrophic mutant strain
Expression and secretion of chymosin encoded by RGl-4 Plasmids from pcRc 1 to pGRG4 (pGR
Gl-4), A, Awamori (Δaν) total or +)
and iJ) Lycoderma reesei (Trichode)
An orticine-5'-phosphate decarboquine race (OMPCase) deletion mutant of S. rma reesei) was used to transform. N encoded by the pGRGl-4 plasmid.

The pyr 4 gene from Crassa (N, Crassa) is able to express these OMP Ca in the absence of uridine.
We successfully isolated transformants that did not complement the 5e mutant. Such Δ, Awamo IJ (A, av
+amori) and T, reesei (T, re
The transformed mutant strain of E. esei ) secreted detectable amounts of biologically active chymosin into its medium.

A, pyrG auxotrophic adult The method used to obtain pyrG auxotrophic mutants of A. awamori (A. awamori) and T. reesei (T. reesei) including the selection for orotic acid (FOA) (56). The mechanism by which FOA kills wild strains is unknown. However, OMP for FOA
In terms of resistance of the Ca5e-deletion mutant, it appears that its toxicity occurs through the conversion of FOA to 5-fluoro-UMP. It is not clear whether cell death is due to fluorination or to bonucleotides or deoquine ribonucleotides. The following is T, l/Isey (T
, reesei) and A, a'7% (Δ, awa
mori) OMPCase-deletion (FOA-resistant)
This is a method for isolating mutant strains.

1. Trichoderma reesei
P37 strain (N
A fresh spore suspension of RRL 15709) was washed with sterile distilled water containing 0.01% Tween 80. 15ml of this spore suspension (1
1 per milliliter. X107 spores) were placed in a sterile petri dish (1,000 spores) together with a sterilized magnetic cooler rod.
OX20mm). I took off the lid, and in the dark,
The spores were irradiated with 254% m ultraviolet light (LIV) (7000 microwatts per square centimeter) at a distance of 25 cm from a red light source. The spores were constantly dispersed.

UV irradiation was continued for 3 minutes (this is enough time to kill 70% of the spores). Irradiated spores were collected in 50 mp centrifuge tubes and kept in the dark for 1 hour to prevent photorecovery. The spores are pelletized by centrifugation, and the pellet is 0.00%.
The suspension was suspended in 200μβ of sterile water containing 01% Tween-80.

The suspension was diluted with 0.15% FOA (Frog, Gainsville, S.
CM Specialty, Chemicals
YNB agar medium containing (yChemicals))
0.7% yeast nitrogen base excluding amino acids, 10
56). 37℃, 4 days ink” After vane-con, 75
2 colonies were picked on fresh YNB medium containing I''OA. 62 out of 75 colonies grew and were picked on minimal agar medium (6 g/RNaNO2, 0,52 g/I2
KcI! , 1.52g/β K112PD,
, 1 ml' β (quantitative element solution, 1% glucose,
0.1% Mgs[], 20 g/I2 agar) and 1
Uridine auxotrophy was examined by pinching and planting in a minimal medium supplemented with mg/mβ uridine. All 62 mesophylls grew on minimal agar medium containing uridine, but 9 of them could not grow on minimal agar medium alone. These nine strains were again planted in minimal medium and minimal medium with uridine. Two of the strains were supplemented with uridine. It grew only on minimal agar medium.

Among these, 'F reesei (T, reesei)
The species named pyr G 29 could not grow on minimal medium alone, but grew well on minimal medium containing uridine. 2,7′ Supergillus awamori Δspe
rgillusawamori) (i) A, 77mori (Δ, awamori)
Production A of hyperproducer of glycoamylase of UVK 143[- strain, NR of Awamori (Δ, a+vamori)
The spores of the RL3112 strain were grown at 30°C (potato dextrose)
-S agar medium (PDΔ, Difco) for 5 to 7 days. Spores were harvested by washing the plate surface with sterile 01% Tween distilled water solution and gently peeling off the spores. The spores are washed by centrifugation and finally
The cells were resuspended in the same buffer to a concentration of 2 x 108 spores/mβ from XlO7. Preparations were stored at 4°C.

Spores 2mβ were added to a sterile petri dish. The lid of the petri dish was removed and the spores were exposed to an ultraviolet (UV) lamp (15 watts, for sterilization). Conditions such as exposure time and distance from the lamp were adjusted to kill 90 to 99.9% of the spores. Surviving spores were plated onto PDΔ medium and allowed to form individual independent colonies.

Spores from individual mutagenized colonies were mixed with 5% cornmeal, 0.5% yeast inostruct, and 2% corn staple liquor after adjusting to p1145.
Q+njl! Inoculate into sterile 5QmA screening medium in a flask. However, any medium containing corn or corn scooch as the carbon source will give similar results. Cultivation was carried out at 30-35° C. for 4-5 days with constant stirring. For assay, samples were taken daily or at the end of the run.

Estimation of glucoamylase activity using a colorless substrate (hala-nitro-phenylated alpha glucoside, PNPΔG)
The release of color-forming groups (para-nitro-phenol) from

The following method was used.

Substrate - 180mg PNPAC at 0.1M at pf+4.7
Dissolve in 25 [] nl of sodium acetate buffer and store at 4°C.

Assay: 1 ml of substrate is equilibrated in a water bath at 40°C. 02mI2. sample (or diluted sample), and
Incubate for 30 minutes at °C. 9mβ of 0. I
Add M Na2Co3 while stirring and leave at room temperature for 15 minutes until color appears. The mixture is filtered through Wattman 42 filter paper and the absorption at 420 nm is measured with a suitable spectrophotometer. The P N PΔG 1−′ levels of all mutant strains are compared to the nominal f value produced by the parent strain and are reported as a percentage of PNPΔG hydrolysis of the parent strain.

[One glucoamylase hyperproducing strain, named JVK143f, was selected by auxotrophic mutagenesis.

(11) Auxotrophic mutation A a'7%υ (A, avtamari) tJ
Preparation of spores from VK I 431 strain, UV mutation,
and mutant analysis according to the following modifications: T, reesei (T
, reesei).

a, 25 minutes were required for 70% killing by LIV light.

b, Minimal medium was used instead of YNB agar medium.

c, FOΔ concentration was set to 0.1%.

Fifteen pyr G mutant strains were found. Three of these isolates were pyr4-5, pyr4-7, pyr4
-8 and selected for transformation experiments.

B, Transformation of pyr auxotrophic strains of Δ, avtamorl and T, reesei. The auxotrophic strain was transformed into Δ, n1dulans using a modification of the procedure described previously. Approximately 1 x 108 spores were grown in 2% glucose, 0.
Yeast inotract glucose (YE) supplemented with 1 mg/mβ uridine in 5% yeast inotract
G) Inoculated into culture medium. The culture was carried out at 37°C in an acre (20 rpm) for 12-LL for 5 hours (T, reesei, 30
). Harvest germlinks by centrifugation,
Washed once with sterilized YEC medium. After that, sterilized 2
0 Qmβ plus deck bin (N, Y, Corning,
Corning, Inc.) in 0.6 M KCl.

0.5% Novozyme 234 (
Denmark, Novo Industries (Nova!ndustrie)
s) ), 0.5% Mg5Oa 7I]20.
Incubation was carried out at 30° C. using 50% YEG medium containing 0.05% calf growth albumin. After stirring at 150 rpm for 30 minutes, the protoplast suspensions were incubated as above for an additional hour and incubated with sterile Miracloth (Calbio Chem Bering, La Jolla, California) moistened with 0.6 MKCβ. (Calbiochem
Behring Corp.). Filtered protoplasts were centrifuged, washed, and transformed with each of the plasmids pGRG1-4 described above. A. The following modifications were made to the transformation section of Awamori (Δ, av+amori).

1,, 0.6 M KC,i! 0.7MKC instead of
R was used.

2. Transformation and regeneration medium 1.2M sorbitol was used in place of 0.6MKCβ for total osmotic stabilization.

CA, Awamori (Δ, awamori) and T
, reesei (T, reesei) Transformant analysis A, 7 Wamori (Δ, alI + amor
Both T. i ) and T. reesei transformants secreted chymosin polypeptides into the culture medium. This was determined by analyzing culture filtrates for chymosin polypeptides and enzymatically active chymosin that reacted with specific chymosin antibodies (results not shown) (Western
immunoblotting techniques and enzyme immunoassays).

Example 8 Expression and secretion of a heterologous polypeptide from an arg B auxotrophic mutant of Aspergillus sp.

arg of Aspergillus sp.
Expression and secretion of heterologous polypeptides from B. auxotrophs was also performed.

This example is A. nidulans (Δ old dulans
) derived arg B gene and plasmid p G RG
Δ, nidulans (Δ.

n1dulans) and Δ Awamori (A. a+・)
A complementation of the arg B auxotrophic strain of A. amori ) is described. The arg B gene is ornithine trans hamylase (OT
C) is coded.

Genetic marker used here, bi8 arg B2
, Δ nidulans containing metG 1 (Δ, n1
arg B auxotrophs of Borland, Warunyawa, and Al Uzijasudowski (A.l.
11 jasdov7sk ie ) 400-47
8, Department of Genetics, University of Warsaw, P.

It was developed by Dr. Weglenski (P.). Δ, Awamori (A.aν+a
mori ) arg B mutant strain was induced as follows.

A, 7 Supergillus awamori (Δspergillus
A fresh suspension of spores of the arg B auxotrophic mutant of Awamori (A, av+amori) JVK 143 was prepared and the exposure time was only sufficient to kill 95% of the spores. , UV mutagenesis was performed similarly to that described previously.

The spores were then centrifuged, washed with sterile water, and resuspended in 25 mβ sterile minimal medium. These suspensions were inked and baked in a 37°C oven with vigorous aeration. Under these conditions, wild-type spores germinate and develop into vegetative Myceria, but auxotrophs do not. The medium was aseptically filtered through sterile Miracloth every 6 to 8 hours for 3 days. At this stage, most wild-type Mycelia are removed, while non-budding auxotrophs pass through the Miracloth filter (filter lane enrichment). At each filtration step, the filtered spores were centrifuged and resuspended in fresh minimal medium. 3
After 8 hours of concentration, the spores were diluted and plated onto 50 ml V4 of minimal agar supplemented with citrulline. The plates were incubated at 37°C for 2-311 days. Individual colonies were transferred from these plates to two screening plates with toothpicks. One plate is 1. On minimal agar medium containing OmM ornithine,
The other is a minimal agar medium containing 5 []mM torulin. The transfer of CoD knee to these screening plays 1 is based on the following principle. OTC (arg B gene product) catalyzes the conversion of ornithine to citrulline in the arginine biosynthetic pathway.

Thus, the arg B mutant (lacking OTC)
grows on minimal medium supplemented with citrulline, but cannot grow on minimal medium supplemented with ornithine.

By this method, approximately 4000 colonies were screened and 15 arg B mutants were obtained. Δ
, Awamori (A, awamori) arg B
One strain, designated No. 3, shows no growth on minimal medium, but grows well on minimal medium supplemented with arginine or citrulline. ○Assay to determine TC activity level (
(57) showed that the argB3 mutant strain produced at least 30 times less ○TC activity than the wild-type strain. According to these data, Δ, awamori (Δ, awam
ori ) arg B 3 strains were selected for transformation experiments.

B. Construction of an argB-based prochymosin expression vector for transformation of Aspergillus species. In this construction (see Figure 15), the first step is
The goal is to combine the transformation promoting sequence ANS-1 and the selectable arg B gene on the same plasmid. To do this, we need A niturance〈Δ,
pBB116 (59) containing the arg B gene derived from PstI and Bam
After digestion with HI, the desired fragment Δ containing the arg B structural gene was isolated. Plasmid pDJB2
(59) was digested with BCOR1 and Pst1, and ΔNS-
1 sequence was isolated. Fragments Δ and B were transferred to plasmid vector PUC18 (3
3) was ligated with fragment C containing the larger EcoR1-Bam HI fragment to create a plasmid pΔRG-DJB in which the three parts were ligated.

As a second step in this construction, a synthetic NA polylinker containing a C1a I site was inserted into pΔRG-DJB, allowing insertion of C1a I fragments containing various prochymosin expression units. Plasmid pAR (,
DJB was digested with BamHI and further dephosphorylated with bacterial alkaline phosphatase. The indicated synthetic NA polylinker was phosphorylated and cleaved with T4 polynucleotide kinase p A RG -, -D J B
and ligated to generate pcJl,61-. This plus
, l- was found to be resistant to the digestion of [Iar, so we first used the E. coli dam- mutant GM to prevent methylation at the Cla I Kg position.
48 was used to transform. A plasmid from 0M48) transformant) was isolated, successfully excised, and dephosphorylated with Cutilia alkaline phosphatase.

In the final step of this construction, the Cla I-cut p
cJ16L vector into plasmids pcRc1 to pGR
Each (':1a I prochymonin expression fragment from G4 was combined with the resulting four plasmids 1-8pCJ
::GR,G1~pCJ: :GRG4 for prototrophic strains, A, 2F urans (Δn1dulans) and Δn1dulans
, was used to transform the arg B mutant strain of Awamori (Δ, av+amori ). The resulting transformants were analyzed for expression of prochymosin polypeptide.

C9A. Nidulans (A, n1dulans)
and △.

Analysis of transformants of Awamori (Δ awamori) pCJ・・GRG1 to pCJ::GRG4
Δ, awamori (A, awama) transformed with
ri) and Δ, niturans (A, n1dul
The chymosin polypeptides secreted from the ans) were detected by milk clotting assays and Enzyte Im/'R' test and Western Im/Flowering techniques. In each case (results not shown), the transformed fungi were Biologically active chymosin was secreted into the medium.

Example 9 Humicola gris from A, n1dulans
Expression and secretion of glucoamylase in the fungus Humicola gricea
The glucoamylase gene was isolated from S. sea and talonized. This gene was then ligated into the argB expression plasmid pcJ16r-. The generated vectors, pCJ:R8HI and pCJ:R3H2, were transformed into Humicola grisea (llumicola grisea).
In order to express and secrete glucoamylase, argB
Deletion A. Nidulans (Δ, n1dulans) (
Example 8) was used to transform.

A. Humicola gris
Isolation and cloning of the glucoamylase gene of ea ).

1, 7 Mycola ψ Grisea □ Iumicola gri
sea ) of glucoamylase! '+'r correct H, grisea (H, grisea) (variant of thermoidea 7 / NRRL
1.5219) glucoamylase from Δ, E, 5taley Company (Δ, E, 5taley Company)
It was staffed from The enzyme is 4.6mm x 250mm
SynchromPak C
It was purified to homogeneity by chromatography on a 4 reverse phase column. Initially, the column was washed with 0.05% triethylamine and 0.05% trifluoroacetic acid (solution Δ).
Equilibration was performed at a flow rate of 5 mβ/min. After glucoamylase sample injection, the column was washed with solution Δ for 2 minutes, then
A gradient of 5% solution B was run per minute until 40% solvent B was reached. The gradient was then changed to 0.5% solvent B per minute, and the glucoamylase was flowing out at approximately 55% solvent B. At this point, glucoamylase is
It was determined to be homogeneous by DS polyacrylamide gel electrophoresis.

2. Amino acid sequence of H. grisea (Jf, grisea) glucoamylase The amino terminal sequence of purified H. grisea (H. grisea) glucoamylase was obtained by the method described above 60). The array is as follows.

Eight people VDTFINTEKPSAXNSL shown here,
In these and other letter peptide sequences, each letter refers to the amino acid corresponding to the next amino acid.

Amino acids Three-letter code/One-letter code To obtain the necessary peptide fragments for additional amino acid sequencing, purified curcoamylase (1 mg/
mβ) was digested with 2% acetic acid for 2 hours at 108°C. The material was directly applied to a Synchrom-pak C4 column (
4.8 mm x 100 mm). After a 2 min wash with 100% solvent Δ (see above), the peptide was eluted with a linear gradient of solvent C (1% per minute). Solvent C was 0.05% triethylamine and 0.05% in propatool.
Contains trifluoroacetic acid. At this point, three peptides were selected for further analysis. One peptide (CA3) was directly sequenced. A mixture of two other peptides (GAI and CA2) was prepared as follows in 4.
, 8mm x 250mm Synch
-rompak) Further purification was performed on a C4 column. G
The mixture of Al and CA2 was diluted with 3 volumes of solvent Δ and injected onto the column. After washing for 2 minutes, the peptide was eluted with a linear gradient of solvent (0.5% per minute).

The solvent contained 0.05% triethylamine, 0.05% trifluoroacetic acid in 35% propanol-65% acetonitrile. The separated GAl and CA2 were purified again in the same manner, and the amino acid sequences were determined as described in -L. The sequences of peptides GΔ1, CA2, and CA3 are as follows.

GAI PI, lN5ITVPIKATGXAVO
YKYIKVXQLGA2 AAVRPLINPE
KPIAWNXLK8NIGPNG83 1NTIE
To K to IAWNKL to ANIGPNGKAAPGAAAG
VVI85PSRTD3, synthetic oligonucleotide probe H, gene 1. DNA was cloned as follows. A synthetic mixture of 48 oligonucleotides was used as a hybridization probe to detect the glucoamylase gene. The length of the oligonucleotide is 17 bases (17 mer), and H
, corresponds to the sequence of six amino acids derived from H. grisea glucoamylase (the underlined portion of the GΔ1 peptide above).

Gln Tyr Lys Tyr lle Lys5
' CAA TAT to A TAT ATT
A mixture of 48 AA 3 'CGCC oligonucleotides was synthesized in 6 boules, each containing 8 different synthetic 17-mers.

Boolean 1: 5 'CATATAAAATATTATT
AA 3' CG Boolean CG Boo/Shiheto: 5' CA8T to T AATAT
ATCAA 3' CG Boole CG Boole Hehe G Boole CG Oligonucleotides were synthesized on a Biosearch automated DNA synthesizer (C
A, Zan Rahuanil, Pio Search).

4゜Selection of the correct oligonucleotide probe 1-(
, Genol derived from grisea (i-1, grisea),
DNA was extracted by the Southern method (30) and
The presence or absence of the r-mylase sequence was analyzed. Briefly, 1
-i, grisea (H, grisea) DNA to Ba
mHI restriction endonuclease. Six fractions of this digested I)NA (one for each probe pool) were size-fractionated by 1% agarose gel electrophoresis. After plotting the DNA on nitrocellulose as previously described, the DNA was fixed on nitrocellulose-L in a vacuum oven at 80'j. The filter was added to Ram l (I Digested DN
It was cut into 6 pieces according to the 6 fractions of A, and each piece was hybridized (2.40) at low salt concentration with one of the pools of synthetic oligonucleated cytoprobes at 18:00. (The probe is a gamma (32plΔTP
and radioactively labeled using T4 polynucleotide kinase. ) After hybridization, the filter was incubated at 37°C for 15 minutes using 2x SSC, 0.1% SDS.
It was washed for 1 minute, and then washed twice with 2XSSC at the same temperature. Air dry the filter, wrap it in Zaran wrap, and wrap it in Kodak's X Omat-AR.
Autoradiographic images were obtained by applying X-ray film.

After development of the autoradiograph, 3.7 kb Bam H
The faint hand corresponding to the T fragment is pool 3.
could be seen in small pieces that had been hybridized.

To improve the hybridization signal,
Pool 3 was resynthesized as 8 individual oligonucleotides. The Sauerne hybridization experiment was repeated using each of the eight oligonucleotides as probes. One of those 17-mer profiles is H, 3.7 k of HgriSea genome DN△.
It was found to hybridize with the bBam HI fragment. The composition of the oligonucleotide IJI 5'
C8 GTAC 88 GTATATC 88. This 17
The oligonucleotide was used as a hybridization probe for the cloning of the grisea (It, gr1sea) curcoamylase gene.

5 Cloning of glucoamylase gene sequence 1-(,
Genomic DNA of Grisea (l(, gTisea)
am )-i I and size fractionated by polyacrylamide gel electrophoresis according to standard methods (30). DNA fragments ranging in size from 2 to 4 kb were excised and flowed out of the cell. This DNA fraction was transferred to the Escherichia coli (E. coli) cloning vector pB.
It was used to create a clone library in R322 (ΔRCC37017). The cloning vector was digested with Bant(I) and dephosphorylated with bacterial alkaline phosphatase. The phosphatase was removed by extraction with phenol-chloroform (lv/v). , sized I (, grisea (H.

grisea) DNA was cut with Bam HI and ligated into dephosphorylated pBR322. The DNA thus ligated was prepared using Morrison (4
Escherichia coli (E, coli) 2 prepared by method 1)
94 (ΔTCC31446) competent cells were used to transform. 50 g/mj of Transformant! L containing carbenecillin at a concentration of
B agar play) (30). Transformants carrying the glucoamylase gene sequence were identified by colony hybridization (30) using a specific 17-mer as a probe. The hybridized colonies were purified and each was purified using an alkaline-8DS miniscreen (30).
The plasmid was isolated by. The Blasmi l = selected in this way were all 3.7 kb B that hybridized to the glucoamylase-specific 17-mer probe.
It contained the amHI fragment. One plasmid, designated pRS H1, was selected for further analysis.

The 600 bp 5an3A fragment from pR8) (I was subcloned into grouper radiophage M13mp (33) and partially sequenced by the Guideoquin-Dene termination method, and the cloned DNA contained the glucoamylase gene. It was confirmed that the 3.7 kb B code contained in pR8Hl
The restriction enonuclease cleavage map of the am fi 17 lacment is shown in FIG. In pBR'322, single and double restriction digests occur with the orientation of the DNAΔ fragment relative to known restriction sites (44). Based on the DNA sequence data we obtained and the restriction map, there is a high probability that the entire coding sequence of the alcohol coamylase gene is contained within the 3.7 kb Baml-11 fragment in pRS Hl. I understand.

B Humicola grisea (1-1umicola, g
arg containing the glucoamylase gene of
Construction of the B vector The 3.7 kb flam HI fragment from pRS H1 was isolated from selectable, Δ, nidulans (Δ).

pc containing the arg B gene derived from
Jl, 6■-cloned in (including plane orientation) (
Figure 17). The resulting vector, pcJiR3l(1 and p
CJ 1R3H2 was transformed into arg deletion Δ, niturans (A.
n1clulans).

The expression and secretion of glucoamylase from CH, grisea (1-1, grisea) was purified and inoculated into minimal medium containing starch as the sole carbon source (
This medium is the same as that described for the production of Kimonun, except that the p++ was adjusted to 50). For the culture filtrate, I-1, Grisea (f(, grisc
The glucoamylase activity in a) was assayed. Figure 18
are Δ, Nidulans (Δ) transformed with pcJjR8H1.

I” by Grisea (H, g
risea) shows extracellular production of curcoamylase. Negative controls included untransformed arg B deletion Δ, nidulans (
A. n1dulans) was used.

Although particularly preferred embodiments have been described above, it will be understood that the invention is not limited thereto. For those who are familiar with this field, in response to published concrete examples,
It will be obvious that various modifications may be made and such modifications do not depart from the scope of the invention.

The following bibliography is summarized and each reference is incorporated by reference into the preceding text with referenced numbers in parentheses.

Literature Transcript 1, Hitchman, R.A.
9th place, 1984 ``Integrated DNA products'', inulin-interferon-growth hormone, Godel (G
oeddel), D, 11. et al., 1979, Nature, vol. 281, 5/1.4. -5
48 p. 2, Llaynes, J., et al., 1
983 Nucleic Acids Research
s,) Volume 11, pp. 687-706 Penni
ca), D. et al., 1983, Nature (Na
ture) Volume 301, 2+, 4. -221 page 3,
o-7 (Lawn), R, et al., 1981 Nucleic acid research (Nucl, 8 cicls Res,) vol. 9, 61
03-Old 14 p. 4, Wood, J 1 et al., 1984, Nature, 31
Volume 2, pp. 330-337 5 Heinecker
ker), H, L, et al., J.P. Patent Office Publication No. 0092182, 1.983/1/0/2 Published 6
, Tuite, M, F, etc., 1
982 EMBO Journal (EMBO・J) Volume 1,
603-608, 7 Meteor, Jo et al., 1982 Gene 24, 1-148. Shiz East r7 (S+baknv), M, etc. 19
In 1983, FIEMS Micro Hiorref-(Mi
crobiol Lett) Vol. 17, pp. 81-85, 9, U. Welts, , 1. A. 2nd prize,
1982 Nuclear research (Nucl, 8cids Re5)
1 ] Volume, 7911 pages
, y), D, , et al., 1.981, Gen 7 (Gen
e), vol. 16, pp. 79-87] 1 Shiva, :] 7
(S+bakov) 0M, , etc. 1984 ``Bacillus genetics and high technology PA AT, Ganesa 7
(Ganesan), , J.A., Hock, Aya New York, Academic Press Edition 12, tEdens, L., et al.
European Patent Office Publication 1984 [EPO12926
8A2 No. 13, 7-Sudo 7 (Marston)
, F, A, 0. .

et al. 1984, Bio-te
chnol, ) vol. 2, pp. 800-804 14, 7
Ortho77 (Foltman), B, , etc.

1979, Biochemistry Journal (J, Biol, Chem)
.

Volume 254, pp. 8447-8456 15, Ha, yenga, K.
J., etc.

1984 European patent application BPO114507
16, Case, M, E, etc., 1
979, Proc. Natl., 8ca.
d, sci, ).

U.S.A., Vol. 76, pp. 5259-5263 Lambowitz, U.S. Pat. , and J. A. Lambosek 1.98
4th year, “Molecular and Cell Biology”
and Cellular Biology) Volume 4, Il'l-122 Page 18, Yelton, M.,
et al., 1984.

Proceedings of the National Academy of Sciences 77. (Proc, Natl, Acad, Sci
) United States, Vol. 81, pp. 1470-1474.

Mull, E., J., et al., 1985.

``Molecular and general genetics'' (Mol, Gen, Gene
t, ) Volume 199, pp. 37-45 19, J:, 7 (John), M, A,
and J.F., Peberdy, I.
, 1984, Enzyme Microb, techno
6, pp. 386-389 20, Tilburn et al., 1982. Gy
(Gene) Volume 26, pp. 205-221 21, Balance, D, J, etc.
1983 Biochem Biophys, Research Communication
Res, Comm, 112, pp. 284-289 22, Kelly, J., M., and M., Llynes, 1985, EM.
Bo, 4 volumes.

475-479 pp. 23, BLIII, J, H, and J, C, Wooton 1984, Nature, 31 Q, 701-7
04 page 24, Barclay,
S, L and E Meller 198
3rd year 1 Molecular and Cell Biology
d Ce1lular Biology), 3 volumes,
21.17-2130 pages 25, Ingolia, P, L
, ,etc.

1984, 28 pages ASM Conference, Microbial Genetics and Molecular Biology (Gen
et, and Mol, Bio, lnd, Mic
roorganisms).

(Summary) 26. Alexopoulos
), c, J, .

1962, Industry, Mycology (Indu
ctory Mycology) 2: L-York, John Wiley & Sons, Inc.
Wiley & 5ons, Inc.) 27, Innis, M.A.
et al., 1985.

Sc+ence, Volume 228,
21-26 pages 28, to/Penttila,
M, E, etc.

1984 Single molecule and general genetics (Mol, Gen.

Genet, ), vol. 194, p. 494-4992
9, 'y unique car (Shoemaker)
, S, P.

et al., 1984, European Patent Application EPO 137280Δ1 30, Maniatis, T. et al.
1982.

Molecular Cloning
g), New York, Cold Splinter Harno\- Cold Spring Noe-no\-Publishing (
Cold.

Spring, Harbor Press) 31,
Crea, R. et al., 1978, Proceedings of the National Academy of Sciences (Proc, Natl., Acad, Sci.) USA, vol. 75, pp. 5765-5769 32, Buxton, F. and Radford A. ,,1.9
1983 7 Molecular and General Genetics (Nlal, Gen,
Gent, > 190 volumes.

403-105 pp. 33, Yani Sch -P
erron).

C1, Vieira, J., and Messing, J., 1985, Gene, 33, pp. 103-119, 34, Clutterbuck.
), J, tl,.

1974 Genetics Handbook
of Genetics)” 7 Sherki 5 S. nitourans (Δspergillus n1cl
ulans), King, R,C,$
L P447-510 New York, Plenum Publishing 5 35, Towbin, H., et al., 197
9 years.

Proceedings of the National Academy of Sciences (Proc, Natl, Human CAD,
Sci, ) USA, Vol. 76, pp. 4350-4354, 36, Iichiyama, H.
, et al., 1981.

Biochemistry Journal (J, Biochem), Vol. 90, pp. 483-437 37 Sokol, PΔ, 1, etc.
1.979 years.

"Pacific Clinical and Microbiology Journal" (J, CIinoMicr
Cllno, ) vol. 9, pp. 538-540 38 Pollock, M. R.
1961 ``General Microbiology Journal'' (J, Gen,
Microbiol, Volume 26, Pages 239-253, 39, Nunberg, J.
H, etc.

1984 Single molecule cell biology (Mol, Ce1l.

Bias, ) Volume 4, pp. 2306-2315 40,
Azoluyn (Adellman), J, Pl
l etc.

1983, DNA, vol. 2, p. 183-1.93 41,
Mori 7: / (Morrison), D, A,
, 1979.

Methods in Enzymology
nzymol) Volume 68, Pages 326-331 42,
Messing, J., 19
1983.

Methods in Enzymology (MethodsB
zymol) vol. 101, p. 20-78 43, Sanger, F., et al., 1977.
Proceedings of the National Academy
Science (Proc, Natl, 8 cad.

3ci, ) United States, Vol. 74, pp. 5463-5467 44
, Bolivar, F., et al. 1977, Gene, vol. 2, 95-
113 page 45, Bolivar,
S., 1978.

Gene, 4 volumes, 121136 pages 46
, '7Wallace, R,B, ,
etc.

1981 Nucleic acid research (Nucl, 8cids Re5
) Volume 9.

3647-3656 pp. 47, Nunberg, J.
H, etc.

1984 1 International Patent Publication WO341020921.

48, Boer, E., et al., 1984
Year.

EMB○ Journal, Volume 3, Pages 1581-1585 49, European Patent Office Publication, No. 0116778, Published August 29, 1984.

50, Hitzeman (formerly Tzeman), R,
A, , etc.

1983, Science',
219, pp. 620-625, 51, Hitzeman, R.
, Δ, 1 etc.

1983 Nucleic acid research
, > Volume 11, Page 27451763, 52, Hitzeman
, R, A, .

1980, Journal of Biochemistry (J, Biol, Ch.
em, ).

Volume 255, page 12073 53, Hira 7 x 77 (Hitzeman)
, R, Δ, 9 etc.

1982 Nucleic acid research
, ) Volume 10, pp. 7791-7808 54, Bech, Δ9M, and Foltman, B, , 1
981, Ness Milk Diary Journal (Ne
th, MilkDairy J, ), Volume 35,
275180 pages 55, 7,? /l/door 7 (F
oltmann), B., and Pe'delsev (
Pedersen), V.B., 1977
Year.

New York Plenum Publishing, J. Tang.
+Nilt, "Acid Protease" P3-22゜5
6. Boeke, J.D.
, etc., 1.984.

Molecular and general genetics (Mol, Gen, Genet
,).

197, pp. 345-346 57 Hazabe, J. R., et al., 1.979.

Analytical Biochemistry (Anal, Bi
ochem,) Volume 94, Pages 356-360 58,
Berse, B., et al., 1983
Year.

Gene vol. 25, pp. 109-117 59
, Balance , D.
J., etc.

1985, Gene 36, 321-3
31 pages 60.0 Rodriguez
H, etc.

1984 Analytical Biochemistry (A
．． na1. Biochem, ) Volume 134, 53L-
547 pages 61, Johnston (Johnstorl),
I, I-, etc.

1985, EMBO Journal, 4 volumes.

1, pp. 307-1311

[Brief explanation of drawings]

FIG. 1 is a restriction map in which Aspergillus niger glucoamylase was inserted into pGal and pGa5. FIG. 2 shows the structure of pD J B -gam-]. Figure 3 shows the composition of ml)19GAPR. Figures 4.5.6 and 7 show pGRGl, pGRG2, pG
The structure of RG3 and pGRG4 is shown. Figure 8 is ILI) 19GAPR to mp 19GAPR
The strategy used to generate ΔC1-C4 is shown. FIG. 9 shows the configuration of pcR160. Figure 10 shows the 5' and 3'7-77 kings (f Iank
Mucor miehei (Mucor ming) sequences containing
m1ehei) Partial restriction map of the carboxyl protease gene. Figure 11 Δ, B and C are Mucor mixhei (Mu) containing the complete coding sequence and 5' and 3' flanking sequences.
cor mjehei) carboxyl◆protease DNA sequence. FIG. 12 shows the structure of pMeJB-7. To Figure 13 and B are partial nucleotides and ANS-
This is the 1st restriction map. FIG. 14 shows the structure of pDJB-3. Figure 15 shows plasmids pCJ:GRGI to pCJ:GR
The configuration of G4 is shown. Figure 16 shows the 3.7 kb Ram from pR3H1.
Restriction endonuclease cleavage map of the HI fragment. Figure 17 shows the structures of pCJ:R3HI and pCJ:R3H2. Figure 18 shows A. Nidulans (Δ, n1dulans
) shows the expression of H, grisea (, grisea) glucoamylase. 8 LO ・h From) To the bowl-← zu< LDo t-t← Okucf
−I (D← P−1← 2 mouths 〇 −〇＞〇 −υ 慴＜ −t−s＜
ct. 1← Mouth υ Ilj← 0 (Hook○
NaE Nugogo 2g ni8 Ogo illusion ○ ■← Hook O-O 飄← xObuH toichiku illusion ← +C＞υE−1Ω
Bartome0 -01') C1s
-+o s←
Sniff test; Ro t8 Nijigen 〇 -c) cl:○ Koro 0
0 work: Trial:〉 Two, publicly two and fumi! 5 Umami Snake

Claims (25)

[Claims] (1) A DNA sequence encoding a heterologous polypeptide, which can be expressed in filamentous fungi and can promote secretion of the heterologous polypeptide. (2) A vector for transforming filamentous fungi, which contains a DNA sequence encoding a heterologous polypeptide and a signal sequence that functionally binds thereto, wherein the signal sequence is functional in the secretion system of the filamentous fungus. A vector that is (3) The vector according to claim 2, wherein the signal sequence is natural to the heterologous polypeptide. (4) The above natural signal sequence and the above hetero sequence are (
b) Calf priprochymosin, (b) Mucor miehei priprocarboxyl protease, and (c) A. niger (A.nige)
r) DNA encoding preproglucoamylase
4. A vector according to claim 3, which is selected from the group consisting of sequences. (5) The vector according to claim 2, wherein the signal sequence is foreign to the heterologous polypeptide. (6) The foreign signal sequence is (a) calf priprochymosin, (b) A. A. niger glucoamylase, and (c) Mucor miehei (Mu
6. The vector of claim 5, wherein the vector is selected from the group comprising a DNA sequence encoding a signal sequence of carboxyl protease of Cor miehei. (7) A claim further comprising a DNA sequence encoding a promoter sequence functionally recognized by the filamentous fungus, which includes a transcription and translation control sequence functionally linked to the DNA sequence encoding the signal sequence. The vector described in Section 2. (8) The above DNA sequence encoding the above promoter sequence is (a) A. A. niger glucoamylase and Mucor mie
8. The vector according to claim 7, which is selected from the group consisting of DNA sequences encoding the promoter sequence of carboxyl protease of A.hei). (9) The vector according to claim 2, further comprising a DNA sequence encoding a functional polyadenylation sequence operably linked to the DNA sequence encoding the heterologous polypeptide. (10) The polyadenylation sequence is A. A. niger glucoamylase or mucor
10. The vector of claim 9, which is selected from the group consisting of DNA sequences encoding the polyadenylation sequence of the Mucor miehei protease. (11) The vector according to claim 2, further comprising a DNA sequence encoding a secretory property expressible in the filamentous fungi. (12) The above-mentioned secretory properties are determined by (a) N. Kurasa Pill 4
(N. crassa pyr4) (b) A. (c) A. nidulans acetamidase; (c) A. nidulans acetamidase; A. nidulans arg B
argB) and (d)A. Nidulans tripe C (
A. DNA encoding S. nidulans trpC)
12. The vector of claim 11 selected from the group consisting of sequences. (13) The vector according to claim 2, further comprising a DNA sequence capable of increasing the transformation efficiency of the vector into the filamentous fungi. (14) The vector according to claim 13, wherein the DNA sequence for increasing transformation efficiency into fungi is ANS-1. (15) The vector according to claim 2, wherein the secreted heterologous polypeptide is an enzyme. (16) The above enzyme is (a) chymosin, (b) prochymosin, (c) priprochymosin, (d) A. Nigger (A.
niger) glucoamylase, (e) Humicola
Glucoamylase of Humicola grisea and Muco
16. The vector of claim 15, wherein the vector is selected from the group consisting of carboxyl proteases of R. miehei. (17) The vector according to claim 2, wherein the secreted heterologous polypeptide is a mammalian polypeptide. (18) The vector according to claim 2, wherein the secreted heterologous polypeptide is biochemically active. (19) A filamentous fungus comprising the DNA sequence according to claim 1. (20) Filamentous fungi capable of expressing and secreting heterologous polypeptides. (21) The above filamentous fungi are (a) Aspergillus (As)
pergillus) species, (b) Trichoderma (Tri
choderma) species, and (Ha) Muco
21. The filamentous fungus of claim 20, which is selected from the group comprising: r) species. (22) The above filamentous fungi are A. Nidurans (A.ni)
dulans), A. Awamori (A.awamori)
, or Mucor species. (23) The above heterologous polypeptide is (a) chymosin, (
b) Prochymosin, (c) Priprochymosin, (d) A
．． A. niger glucoamylase, (e) Humicola grisea
21. The filamentous fungus according to claim 20, which is an enzyme selected from the group consisting of glucoamylase of a) and carboxyl protease of Mucor miehei. (24) The filamentous fungus according to claim 20, wherein the secreted heterologous polypeptide is a mammalian polypeptide. (25) (a) transforming a filamentous fungus with a vector containing a DNA sequence capable of expressing a heterologous polypeptide and promoting secretion of the heterologous polypeptide from the filamentous fungus; and (b) transforming the heterologous polypeptide into a filamentous fungus; A method for producing a heterologous polypeptide comprising expressing and secreting the polypeptide.