JP2002510497A - Promoters and recombinant constructs for expression of recombinant proteins in filamentous fungi - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel promoter of the glutamate dehydrogenase (gdh) gene from Aspergillus awamori and related genus aspergilli, and a novel DNA sequence encoding a glutamate dehydrogenase from Aspergillus awamori. The present invention also relates to the use of a promoter of a gdh gene derived from a fungus of the genus Aspergillus for the purpose of expressing a recombinant protein in a filamentous fungus.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to improving the expression of proteins, especially fusion proteins, by recombinant DNA technology using a filamentous fungus as a host. These improvements are mainly due to novel promoters,
And the use of novel DNA constructs containing the same.

BACKGROUND ART [0002] Filamentous fungi are known to naturally produce large amounts of a wide range of homologous proteins. For this reason, filamentous fungi have been regarded as useful hosts for the expression of recombinant proteins. For example, Aspergillus awamori has been used to produce recombinant proteins such as bovine chymosin and human lactoferrin.

[0003] However, some recombinant proteins have proven very difficult to express in filamentous fungi. This is the case, for example, with interleukin-6 and thaumatin. Thaumatine is a protein that has a very sweet taste and has the ability to improve the palatability of food. In industry, these are, at present, the plants Tha
umatoccocus danielii benth is extracted from the provisional coat of the fruit (M. Witty, JD Higginbotham, Tha).
umatin, 1994, CRC press, Boca Ratoon, Florida). Thaumatine is derived from at least five different forms (I
, II, III, b and c), thaumatins I and II
Is the most abundant type among the temporary seed coats. Despite its advantages, industrial use of thaumatin of plant origin is very difficult to obtain the fruits from which it is extracted,
Extremely limited. Attempts have been made to produce thaumatin by genetic manipulation in different hosts such as bacteria, yeast and transgenic plants,
To date, the results have been disappointing, and thaumatin available industrially is very rare and expensive. European Patent EP 684 312 describes a method for preparing recombinant thaumatin in filamentous fungi. One problem with this method is that the yields obtained are low compared to the values required for industrial production of thaumatin.

It is possible to improve the yield of the recombinant protein when the recombinant protein in question is expressed as a fusion with another protein and when the expression of this cassette is driven by a strong fungal promoter It has been known. Other proteins, called "carrier proteins", are normally highly expressed proteins of fungal origin. To date, the most frequently used expression systems include the promoter and gene of glucoamylase from Aspergillus awamori as promoter and carrier protein, respectively (PP Ward et al., Biotechno).
logic 1995, 13, 498-502). However, in some cases, the use of this expression system does not result in high levels of the desired recombinant protein. One of these particularly problematic cases is the expression of recombinant thaumatin in filamentous fungi.

[0005] In view of the foregoing, there is a need to provide a new and more efficient expression system that enables the production of proteins at high concentrations that are difficult to express in filamentous fungi such as thaumatin. Is clear. This object is achieved by the novel promoter and DNA construct provided in the present invention, as described below.

DISCLOSURE OF THE INVENTION [0006] The present invention relates to a fungus of the genus Aspergillus, particularly Aspergillus.
The present invention provides a novel expression system utilizing a promoter derived from a sawamori-derived glutamate dehydrogenase (gdh) gene.

One embodiment of the present invention comprises (a) the sequence of base numbers 1 to 740 in the attached SEQ ID NO: 1, (b) a base sequence similar to that defined in (a), and (c) A) a novel promoter for expression of a recombinant protein in a filamentous fungus containing a nucleotide sequence selected from the group consisting of nucleotide sequences hybridizing under severe conditions or a complementary strand thereof to those defined in (a); . Particularly preferred are the sequences defined in (a), that is, base numbers 1 to 7 in SEQ ID NO: 1, which correspond to the gdhA promoter of the glutamate dehydrogenase A gene from Aspergillus awamori.
A promoter comprising 40 sequences.

[0008] Glutamate dehydrogenase A disclosed herein is a filamentous fungus Aspergillus
Although it is the first glutamate dehydrogenase identified and described in awamori, other glutamate dehydrogenases may be present in Aspergillus awamori. Aspergil shown in SEQ ID NO: 1
rus awamori gdhA promoter and / or a novel nucleotide sequence of a gene, or a part thereof, according to the disclosure of the present invention, Aspergillus
awamori and other organisms, preferably filamentous fungi, more preferably As
pergillus fungi, more preferably Aspergillus awa
mori and Aspergillus niger, especially Aspergi
It can be used as a probe for the identification and isolation of other homologous promoters / genes of glutamate dehydrogenase in L. awamori. As a result, the present invention relates to the Aspergillus awam disclosed herein.
ori-derived gdhA, but not limited to Aspergillus n
The present invention also relates to a promoter of any glutamate dehydrogenase gene derived from a fungus of the genus Aspergillus, which is not derived from idulans. The Aspergil
Examples of li are Aspergillus awamori, Aspergillu
s niger, Aspergillus oryzae and Aspergi
lrus sojae. In a preferred embodiment, the invention relates to Asperg
The present invention relates to a promoter of a glutamate dehydrogenase gene derived from illus awamori or Aspergillus niger. In a more preferred embodiment, the present invention relates to the promoter of the glutamate dehydrogenase gene from Aspergillus awamori. Use of the novel nucleotide sequence shown in SEQ ID NO: 1 or a part thereof as a probe is also an aspect of the present invention. "
The term "part thereof" means any site in the nucleotide sequence of SEQ ID NO: 1 that functions as a probe.

Another aspect of the present invention relates to a method for encoding a glutamate dehydrogenase protein, comprising the steps of: (a) the sequence of base numbers 741 to 2245 in the attached SEQ ID NO: 1;
And (c) a nucleotide sequence selected from the group consisting of nucleotide sequences that hybridize under severe conditions or a complementary strand thereof to those defined in (a). The purified and isolated novel DNA sequence consisting of: In a preferred embodiment, the nucleotide sequence encoding glutamate dehydrogenase comprises (a)
I.e., the sequence of base numbers 741-2245 in SEQ ID NO: 1. However, the present invention relates to the Aspergillus awa disclosed herein.
mori-derived gdhA gene, but not limited to Aspergill
The present invention also relates to any glutamate dehydrogenase gene derived from a fungus of the genus Aspergillus, which is not derived from C. us nidulans. In a preferred embodiment, the present invention relates to Aspergillus awamori or Aspergillus.
The present invention relates to a DNA sequence encoding glutamate dehydrogenase from Niger.
In a more preferred embodiment, the invention relates to Aspergillus awamor
i) a DNA sequence encoding glutamate dehydrogenase.

[0010] Another aspect of the invention is a novel protein encoded by any of the DNA sequences defined above. In a preferred embodiment, the protein has the amino acid sequence disclosed in SEQ ID NO: 2. In the present invention, however, Aspergillus n
any glutamate dehydrogenase derived from a fungus of the genus Aspergillus, not from Aspergillus idulans, more preferably Aspergillus awamo
Glutamate dehydrogenase from ri or Aspergillus niger, more preferably glutamate dehydrogenase from Aspergillus awamori, is also included.

[0011] The present invention relates to the use of the aforementioned glutamate dehydrogenase promoter for the expression of recombinant proteins in filamentous fungi. Certain glutamate dehydrogenases from several microorganisms are already known and their genes, in particular Aspergillus
Glutamate dehydrogenase A (gdhA) gene from S. nidulans has been disclosed (AR Hawkins et al., Mol. Gen. Genet. 1).
989, 218 (1), pages 105-111). However, to the best of our knowledge, to date, A. No expression of a recombinant protein utilizing the gdhA promoter from N. nidulans is disclosed, nor is it disclosed that it may be useful for enhancing the expression of the recombinant protein in filamentous fungi. As shown in the Examples below, the glutamate dehydrogenase promoter from Aspergillus awamori was found to be very potent in promoting transcription of heterologous genes. Thus, this promoter and the related ghd promoter from Asperqilli are expected to induce high level transcription of the gene and are expected to be useful in expressing recombinant proteins in filamentous fungi. Thus, a further aspect of the invention is the use of a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus for the expression of a recombinant protein in a filamentous fungus. Preferably, the gdh promoter is Aspergi, but not from Aspergillus nidulans.
laspus, more preferably Aspergillus
awamori or Aspergillus niger, even more preferably from Aspergillus awamori, and particularly preferably one of the novel gdh promoters described above.

[0012] In principle, there is no restriction on the desired recombinant protein to be expressed. Examples of such desired proteins (the terms used herein include proteins and smaller polypeptides) include, but are not limited to, enzymes, hormones, cytokines,
Includes growth factors, structural proteins, plasma proteins, and others. Infinite examples of proteins that can be expressed include human proteins such as interferons, interleukins, tissue plasminogen activators, serum albumin, growth hormone, and growth factors. Other proteins can be of non-human origin, such as lipases of both fungal and non-fungal origin, proteases, thaumatin, bovine chymosin and the like. Polypeptides that may be of human and non-human origin include calcitonin, glucagon, insulin, nerve growth factor, epidermal growth factor, anticoagulant hirudin, and analogs thereof such as R3-hirulog.

A further object of the present invention is to provide (a) a promoter from a glutamate dehydrogenase gene derived from a fungus of the genus Aspergillus, (b) a DNA sequence encoding a protein normally expressed from a filamentous fungus, or a part thereof, c) a DNA sequence encoding a degradable linker peptide, and (d) a DNA construct comprising a DNA sequence encoding a desired protein. In a preferred embodiment, the promoter of (a) is an Aspgillus nidulans-derived Asp
gdh promoter from fungi of the genus Ergillus, more preferably Asp
ergillus awamori or Aspergillus niger
Of origin, and even more preferably, Aspergillus awamori
And more preferably still include any of the novel promoters described above, particularly preferably the nucleotide sequences 1 to 740 in SEQ ID NO: 1. DNA of (b)
The sequence encodes a protein, or a portion thereof, that is normally expressed from a filamentous fungus that is functional, ie, can cause increased secretion of the desired protein. Such (b)
Examples of proteins of Aspergillus awamori, Aspergil
rus niger, Aspergillus oryzae and Asper
glucoamylase, α-amylase and aspartyl protease from G. gillus sojae, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II from Trichoderma sp., glucoamylase from Neurospora and Humicola sp. It contains protein B2 and glutamate dehydrogenase derived from filamentous fungi. In a preferred embodiment, (b)
DNA sequence of (i) Aspergillus awamori, Asper
gillus niger, Aspergillus oryzae or As
glucoamylase from C. pergillus sojae, (ii) Acrem
encoding a protein selected from the group consisting of B2 from C. onium chrysogenum and (iii) glutamate dehydrogenase from a filamentous fungus or a part thereof; more preferably, the DNA sequence of (b) is (i) Aspergillus
sawamori, Aspergillus niger, Aspergil
glucoamylase derived from Aspergillus sojae or Aspergillus sojae; (ii) B derived from Acremonium chrysogenum;
2, and (iii) Aspergillus awamori, Asperg
It encodes a protein selected from the group consisting of glutamate dehydrogenase derived from illus niger or a part thereof. The DNA sequence of (c) encodes a degradable linker peptide; the degradable linker peptide used herein is recognized and cleaved by a sequence adjacent to the degradable linker under certain circumstances, eg, a protease, or A peptide sequence that allows for separation in sequences that are cleaved after exposure to a particular chemical. In a preferred embodiment, the DNA sequence of (c) comprises a KEX2 processing sequence. As mentioned above, the desired protein of (d) can in principle be any recombinant protein. In a preferred embodiment, the DNA sequence of (d) encodes thaumatin; a particularly preferred construct for the preparation of thaumatin is that the DNA sequence encoding (d) thaumatin has an EP684
Thaumatin II obtained from plasmid pThIX, disclosed in US Pat.
And those that are synthetic genes that encode

In an embodiment of the present invention, when expressing a desired protein, g in a fusion construct
Although it is preferable to use a dh promoter, it is also possible to use a gdh promoter for directly expressing a desired protein. Accordingly, a further aspect of the invention is a novel DNA construct comprising a gdh promoter from a fungus of the genus Aspergillus operably linked to a DNA sequence encoding a protein desired to be expressed. In a preferred embodiment, the gdh promoter is
Aspergillus not derived from Spergillus nidulans
s genus, more preferably Aspergillus aw
amori or Aspergillus niger, even more preferably from Aspergillus awamori, even more preferably one of the novel promoters described above, particularly preferably the base in SEQ ID NO: 1. Sequences 1 to 740 are included.

As will be apparent to those skilled in the art of recombinant DNA technology, all such DNA constructs include, but are not limited to, a signal sequence, termination sequence, polyadenylation sequence, selection sequence, DNA replication. Other elements may be included, including enabling arrays and the like. The number and nature of these additional sequences is not limited, and any of the known sequences for performing these functions can in principle be used in the constructs of the present invention. For example, as a signal sequence that functions as a secretory sequence, we use Aspergillus awamori, Aspergillus.
niger, Aspergillus oryzae and Aspergil
a signal sequence from glucoamylase, α-amylase and aspartyl protease from L. sojae, a signal sequence from cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II from Trichoderma sp., Neurospora and Humi
signal sequence from glucoamylase from E. coli species, and Acremo
and a signal sequence from Nium chrysogenum-derived protein B2. Usually, as a signal sequence, those derived from a protein secreted by a filamentous fungus used as an expression host for expression and secretion of a recombinant protein, and, when a fusion construct is used, a protein used as a carrier protein It is also preferable to use those derived from. The termination sequence is a nucleotide sequence that is identified by the expression host and terminates transcription. An example is described in A. nidulan tr
pC gene, A. awamori, A. et al. niger, A .; oryzae or A
. sojae glucoamylase gene; awamori, A .; niger
A. oryzae or A. sojae α-amylase gene, and Sa
It contains a terminator from the cycl1 gene of C. cerevisiae. Selection sequences are sequences that are useful as selectable markers to allow for the selection of transformed host cells. In principle, any of the known selectable markers for filamentous fungi intended for use as hosts can be used. Examples of such selectable markers include genes that confer resistance to drugs such as antibiotics (eg, hygromycin or phleomycin), as well as argB,
Includes auxotrophic markers such as trpC, niaD and pyrG. A polyadenylation sequence is a nucleotide sequence that is recognized by an expression host during transcription and adds a polyadenosine residue to transcribed mRNA. An example is described in A. nidulans
The trpC gene of A. awamori, A. et al. niger, A .; oryzae
Or A. sojae glucoamylase gene, and Mucor mieh
Contains the polyadenylation sequence from the ei carboxyl protease gene.

The present invention also relates to a filamentous fungal culture capable of producing a recombinant protein, transformed with a plasmid containing any of the above-described DNA constructs. Examples of filamentous fungal species that can be utilized as expression hosts include the following genera: Aspergillus
, Trichoderma, Neurospora, Penicillium,
Acremonium, Cephalosporium, Achlya, Pha
neurochaete, Podospora, Endothia, Mucor,
Fusarium, Humicola, Cochliobolus, Rhizo
pus and Phricularia. Particularly preferred is that the filamentous fungus is Asp
ergillus fungi, and more preferably, the filamentous fungus is Aspergillus awamori, Aspergillus ni.
ger, Aspergillus oryzae, Aspergillus n
idulans or Aspergillus sojae. In another preferred embodiment, the recombinant protein produced is thaumatin.

[0017] A further aspect of the present invention comprises (a) preparing an expression plasmid containing the DNA construct as defined above, (b) transforming a filamentous fungal strain with said expression plasmid, (c) transforming Culturing the isolated strain under suitable nutrient conditions to produce the desired protein intracellularly, extracellularly or simultaneously, and (d) optionally separating and purifying the desired protein from the fermentation broth And a method for producing a recombinant protein in a filamentous fungus. Preferred is a method wherein the recombinant protein produced is thaumatin.

The accompanying case illustrates the identification and isolation of the glutamate dehydrogenase A gene from Aspergillus awamori, and its promoter region. This was achieved with a probe from Neurospora crassa. However, Neurospora cra, which should be used as a probe to obtain a homologous gene in Aspergillus awamori
Selection of an appropriate DNA fragment from the glutamate dehydrogenase gene in ssa is not straightforward. In this case, there was no apparent homologous sequence, so the one used was the 2.6 kN gene containing the Neurospora crassa gdh gene.
b BamHI fragment. This is a large piece of DNA and certainly not an optimally sized piece. Ideally, it is desired to use a highly homologous DNA fragment having a length of 200 to 300 bp or less as a probe. Here, a rather large fragment (2600 bp) whose homology was not confirmed was used. The inventor has cloned a sequence which was later found to be gdh from Aspergillus awamori.

The following case also describes the application of the novel promoter and DNA construct described above for the expression of the recombinant protein thaumatin in the filamentous fungus Aspergillus awamori. As shown in these cases, and as illustrated in FIG. 12, the expression system of the present invention offers several advantages over prior art systems. On the other hand, this makes it possible to reach a concentration of the expressed protein of about 100 mg / L, which is an order of magnitude higher than the highest (for example a concentration of about 5 to 10 mg / L using the method described in EP6844312). A. Faus et al., Appl. Microbiol. Biotechnol, 1998, 49th.
Vol., Pp. 393-398). On the other hand, for the same carrier protein and the same fermentation time, the use of the promoter of the invention leads to higher concentrations of the expressed protein. And last but not least, with the constructs of the present invention, it is possible to use a more economical source of nitrogen (ammonium sulfate) than is commonly used (asparagine).

Definitions The term "promoter" refers to a DNA sequence that is functional in a filamentous fungus that is capable of inducing transcription of a coding region when operably linked.

The term “recombinant protein” is one that is not expressed by a host under standard normal conditions, but only as a result of introducing into the host a DNA sequence that allows expression of the recombinant protein. Means a protein expressed by the host. The recombinant protein may be fungal or non-fungal, and may even be found in a non-recombinant host.

Best Mode for Carrying Out the Invention This section describes the application of the novel promoter and construct according to the invention to the preparation of recombinant thaumatin. The teachings of the following examples can be applied to the expression and production of other recombinant proteins, which should not be construed as limiting the scope of the invention in any way.

A; Constructs For all of the constructs prepared in this patent application, the starting point is plasmid p.
ThIX, which is described in European Patent Application EP6844312. This plasmid contains: (i) a sulfanilamide resistance marker; (ii) (a) a synthetic gene encoding thaumatin II, (b) a spacer sequence containing the KEX2 processing sequence in that order, and (c) Aspergillus niger.
A DNA sequence encoding a fusion protein comprising, in this order, the complete glucoamylase gene (genome); (iii) the signal sequence ("pre") and the "pro" sequence of the glucoamylase gene (glaA) of Aspergillus niger;
And finally (iv) the promoter region sequence of the glucoamylase gene (glaA) of Aspergillus niger.

In the case of the present invention, (i) a drug resistance marker (most often a phleomycin resistance marker); (ii) (a) a synthetic gene for thaumatin II, (b) KEX
A spacer sequence comprising two processing sequences, and (c) Acremoniu
D coding for a fusion protein containing, in this order, a cDNA sequence (excluding the carboxy-terminal sequence) encoding most of the B2 protein from M. chrysogenum.
NA sequence; (iii) the signal sequence of the B2 gene of Acremonium chrysogenum, and (iv) three different promoter regions,
Two new expression cassettes were prepared.

In all of the cloning and subcloning operations described in this patent application, Escherichia coli DH5a was utilized as a recipient strain for high frequency plasmid transformation. E. FIG. E. coli WK6 was used as a host to obtain single-stranded DNA from the pBluescript plasmid for sequencing purposes.

A1. Construction of Expression Cassette B2KEX Protein B2 is an extracellular protease produced by the filamentous fungus Acremonium chrysogenum. This protein is Acremonium
Chrysogenum is expressed and secreted late in growth (between 120 and 144 hours after the start of growth).

The plasmid pJE1A (Laboratory of Prof. Juan-
Francisco Martin, Universidad de Leoe
n, Leoen, Spain) are Acremonium chrysogen
It contains a promoter region, a leader peptide (including a signal sequence) and a coding region of the B2 gene derived from um. The gene itself is 1298 base pairs and has two introns. These two introns are, in the sequence subcloned in pJE1A, these subcloned sequences
Not present because it was obtained from DNA. Upstream from the translation start point ATG, 4
There is a 77 base pair promoter region. Acremonium chrysog
When enum grows in a particular medium containing sucrose and glucose as carbon sources and asparagine as a nitrogen source, the gene is expressed to the highest level between 72 and 96 hours of growth.

The steps involved in the construction of the B2KEX cassette are shown in detail in FIG. 1 (parts AC)
). Plasmid pJE1A was digested sequentially with BamHI and NcoI and
A 560 bp fragment was released which was purified by an 8% agarose gel. This fragment
It contains most of the coding region of the B2 gene, but excludes the active center of the protein. Similarly, plasmid pJL43b (JL Barredo, doctoral dissertation, Univ.
ersidad de Leoen, Leoen, Spain) also BamHI
And NcoI, releasing a large fragment (3740 bp), which was
. Purified by 8% agarose gel. This fragment was 560b from pJE1A.
The pBamHI-NcoI fragment was ligated and the plasmid p43bB2CT (43
00 bp).

Plasmid p43bB2CT was digested with NcoI and DNA polymerase I
(To obtain blunt ends), then digested with StuI,
A 74 bp fragment was generated, which was also purified on a 0.8% agarose gel. Using single-stranded oligonucleotides ThS1 and ThS2 (sequences of which are shown below)
Using the plasmid pThIX as a template, the KEX2-like and thaumatin sequences present in pThIX were amplified by the polymerase chain reaction (PCR). Th
The first 18 bases present in S1 correspond to a KEX2-like sequence.

[0030]

Embedded image A 655 bp DNA fragment was obtained by PCR using plasmid pThIX as a template as template and ThS1 and ThS2 oligonucleotides as primers. This DNA fragment is ligated with the fragment previously obtained from p43bB2CT to give plasmid p43bB2CTTh. This plasmid (about 4530 bp) contains the KEX-2 sequence and a portion of the B2 protein gene fused to a synthetic gene encoding thaumatin II. The transcription termination signal in this construct was derived from Saccharonyces Cerevisiae cyc1.
End sequence from gene.

[0031] Plasmid p43bB2CTTh was digested with BamHI, treated with calf intestinal alkaline phosphatase (CIP), and purified on a 0.8% agarose gel. A 900 bp BamHI-BamHI fragment from pJE1A was also isolated. By subsequently ligating these two DNA fragments, the plasmid pB2KEX (
5430 bp). 900 bp BamHI-Bam from pJE1A
The HI fragment contains the B2 gene promoter sequence (477 bp), the leader peptide sequence (318 bp), and the 107 bp amino terminal sequence of the B2 gene.

Next, the plasmid pB2KEX was digested with XbaI, treated with the Klenow fragment of DNA polymerase I (to obtain a blunt end), and then digested with SalI.
A 400 bp fragment was generated, which was purified on a 0.8% agarose gel. Plasmid pJL43b was digested with HindIII, treated with the Klenow fragment of DNA polymerase I, and then digested with XhoI. The 4500 bp fragment was purified as described above. Finally, the two gel-purified fragments described above are ligated,
Plasmid pB2KTh (6900 bp; FIG. 1C) was generated.

In the last subcloning step, plasmids pB2KTh and pJL43
Both bl were digested with SacI and StuI, giving fragments of 5714 and 1305 bp, respectively, which were purified by 0.8% agarose gel.
These two fragments were then ligated and the plasmid pB2KThb1 (7020
bp; FIG. 1C) was obtained. Plasmid pJL43b1 is
Of the phleomycin resistance gene (Penicillium chr
The promoter for inducing the expression of PpcbC) derived from ysogenum is Asp.
glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter from E. ergillus nidulans (P. Punt et al., Gene 1990).
Year 93, 101-109).

This plasmid contains (i) a phleomycin resistance marker, (ii) a (a) synthetic gene for thaumatin II, (b) a spacer sequence containing a KEX2 processing sequence in that order, and (c) Acremonium chrysogenum.
A DNA sequence encoding a fusion protein comprising, in this order, a cDNA sequence (excluding the carboxy terminal sequence) encoding most of the B2 protein of interest, (iii) A
and a cassette for expressing thaumatin containing the signal sequence of the Cremonium chrysogenum B2 gene, and (iv) the promoter region of the Acremonium chrysogenum B2 gene. In this particular construct, expression of the phleomycin resistance gene (phleo) was determined by Asperg.
Induced by the promoter of glyceraldehyde-3-phosphate dehydrogenase from illus nidulans.

A2. Construction of Expression Cassette GDHTh 2.1 Aspergillus awamori containing the gdhA gene
Cloning of DNA fragment of A. awamori ATCC 22342 was used as a source of DNA and RNA. A. nidulans mutant strain A686 (gdhA1, yA2
, MathH2, galA1) and A.I. nidulans A699 (gdh
A1, biA1) (JR Kinghorn, JA Pateman, J.A.
Gen. Microbiol. 1973, Vol. 78, pp. 39-46).
ngal Genetics Stock Center. aw
amori-derived gdhA gene was used in complementation studies. The partial glutamate auxotrophy of these two strains was confirmed by growth on media using glutamic acid or high ammonium sulfate concentrate (100 mM) as the nitrogen source. Both gdhA mutants grow only marginally in the presence of high ammonium sulfate concentrate, but show normal growth when glutamic acid is used as the nitrogen source. E. FIG. E. coli NM539 is a Lambda GEM12 (Prome
ga Co. , Wis) as a host for phage derivatives.

[0036] The filamentous fungus is treated in a conventional manner with a solid powdered sporulation medium (F. Fierro et al., A.
ppl. Microbiol. Biotechnol. 1996, Volume 43,
597-604) at 30 ° C. for 3 days. A. CM in CM medium (containing 20 g / l malt extract, 5 g / l yeast extract, and 5 g / l glucose) awamor
i. nidulans seed cultures are inoculated at 10 ⁶ spores / l and grown in a rotary G10 incubator (New Brunswick Scientific).
New Brunswick N.M. J. ) Was grown at 28 ° C. for 48 hours. For gdhA transcript isolation and identification studies, MDFA media (YQ Shen et al., J. Antibiot. 1984, 37, 5
03-511). awamori culture with 15% seed culture,
Grow at 30 ° C. for 48-72 hours in a rotary shaker.

A. 2.1.1. Aspergillus awamori genomic library A genomic library of awamori ATCC 22342 total DNA was constructed in the Lambda GEM12 phage vector. Total DNA was extracted and partially digested with Sau3AI to obtain DNA fragments ranging from 17 to 23 kb. This DNA was purified by sucrose gradient centrifugation,
ambda GEM12 phage arm linked to Gigapack III
It was assembled in vitro using a Gold packaging system (manufactured by Stratagene) to give a total of 8 × 10 ⁴ recombinant phages.

In the next step, the gdhA gene of Neurospora crassa (
J. H. Kinnaird, J .; R. S. Fincham, Gene 1983
Using the 2.6 kb BamHI fragment, which contains the phage FAN1 and FAN2, which gave distinct hybridization signals, was purified and purified by three rounds of infection. These two
Restriction maps of the two phages showed that they overlap at 7.2 kb. 2
The total DNA region cloned into one phage spanned 28.7 kb.

The 1.7, 5.5 and 10 kb BamHI fragments were converted to pBluescript
Subcloned into the KS + plasmid, the plasmid p as shown in FIG.
B1.7, pB5.5 and pB10 were generated. Next, these are
Digestion with exonuclease III from the appropriate end using the a-base system (Promega Co., Wis.) Followed by removal of single-stranded DNA using S1 exonuclease By generating a regular deletion. The gdhA gene fragment was sequenced by the dideoxynucleotide chain termination method. For sequencing of cDNA clones containing intron-exon junctions, ABI-PRISM310 was used.
GeneAmp connected to an automatic sequencer (Perkin Elmer)
A reaction with 90 ng of dsDNA was performed using a PSR2400 system. D
Computer analysis of base and amino acid sequences was performed using NASSTAR software (DNASTAR, Inc., UK).

The initial sequencing, as shown in FIG. 3, starts at the right end of the 5.5 kb insert in pB5.5 and extends over the left region of the 1.7 kb BamHI fragment of pB1.7. (ORF1) was present. The 5.5 kb and 1.7 kb BamHI fragments mapped in detail.

The 2.1 kb XbaI-XbaI fragment corresponding to the right end of plasmid pB5.5 was subcloned into the pBluescript SK + plasmid,
Plasmid pBSGh was formed. More specifically, this 2.1 kb XbaI-
An Xbal fragment was generated by digesting pB5.5 at an internal Xbal site and a second Xbal site in the polylinker of pBSKS + (near the BamHI site shown in Figure 3).

A 2570 nt region was sequenced on both strands by the dideoxynucleotide chain termination method. This region starts with the ATG located 740 bp downstream from the left end of the insert in pBSGh, extends to the end of the 5.5 kb BamHI fragment, and has an additional 60 bp in the adjacent 1.7 kb fragment. ORF1 (138
0 bp). ORF1 contains signals necessary for transcription initiation and regulation 74
It is preceded by a zero base region (see SEQ ID NO: 1).

ORF1 is derived from other fungal introns (DJ Ballance, Yea)
at positions 785-850 and 1414-1471 (according to the numbering in SEQ ID NO: 1), showing 5 'and 3' splicing sequences and lariats similar to those of St. 1986, Vol. 2, pp. 229-236. It contained two putative introns. The presence of two introns, (shows the sequence below) using the intron I for oligonucleotide I _A and I _B and intron II and II _A and II _B as primers, A. DNA corresponding to introns I and II obtained by PCR from an awamori cDNA library
The region was confirmed by sequencing.

[0044] The cDNA for these experiments was obtained from total RNA extracted as described above from hyphae grown for 48 hours in MDFA medium. Stratagene (L
a Jolla, Ca) using the cDNA synthesis kit
The NA chain was synthesized. This cDNA was then cycled for one cycle at 94 ° C for 5 minutes, 50 ° C for 1 minute and 72 ° C for 1 minute, followed by 30 minutes at 94 ° C for 1 minute, 50 ° C for 1 minute and 72 ° C for 1 minute. PCR, and finally the fragment containing the exon-exon junction was PCR
Used for amplification.

[0045] Oligonucleotide

[0046]

Embedded image A. 2.1.2 O encoding putative NADP-dependent glutamate dehydrogenase
RF1 ORF1 encoded a protein of 460 amino acids (see SEQ ID NO: 2) with an estimated molecular weight of 49.4 kDa and a pI of 5.62. A comparison between the protein encoded by ORF1 and other proteins in the SWISS-PROT database showed that the encoded protein was A.R. nidulans (84.7% amino acid homology); crassa (74.4% homology), Saccharo
myces cerevisiae (65.5% homology) and Schwan
NAD of N. myomysococdentalis (66.9% homology)
It was shown to have high homology with P-dependent glutamate dehydrogenase. Homology is spread throughout the protein. All of these proteins are NADP-dependent glutamate dehydrogenases that catalyze the reductive amination of α-ketoglutamic acid in the presence of ATP to produce L-glutamic acid. The protein encoded by ORF1 shows 9% of glutamate dehydrogenase from other fungi and yeasts.
Contains two conserved motifs. One of the conserved regions (amino acids 108-12
1) corresponds to a portion related to the catalytic mechanism of the enzyme. The sequence common to this part is [LIV] -X (2) -GG- [SAG] -KX- [GV] -X (3)-
[DNS]-[PL] (PROSITE PS00074). Glycine-
Lysine residue K ¹¹⁴ is located in the rich region GGGK ¹¹⁴ GG is, Glu / Leu / P
He / Val (GLFV) corresponds to lysine at the active center of dehydrogenase. Therefore,
According to standard fungal gene nomenclature, the gene encoded by ORF1 is gdh
It was named A.

A. 2.1.3. A. by cloned gene nidulans
Complementation of gdhA mutant strain nidulans A686 and A699 strains have a 2570 bp Xba
A. in the I-XbaI fragment plasmid p containing the awamori gdhA gene
Using GDHaw (7.1 kb), a known method (MM Yelton et al.,
Proc. Natl. Acad. Sci. USA 1984, 81, 14
70-4). This fragment is ORF1 (gdhA gene)
740 bp of the upstream promoter region, and a 322 bp region downstream. The 2570 bp XbaI-XBaI fragment was prepared as described in A.C. Under the control of the awamori gdhA promoter, it was inserted into the XbaI site of the fungal vector p43gdh containing the phleomycin resistance marker.

A. A. harboring the awamori gdhA gene. nidulans A68
6 and 7 transformants Fifteen transformants of A. nidulans A699 were analyzed in minimal medium supplemented with ammonium sulfate at different concentrations (10, 50 and 100 mM) as a nitrogen source, and their growth was determined by wild-type A. nidula
ns growth. As a control, growth was also tested on a medium containing 10 mM glutamic acid. As shown in FIG. The nidulans mutants A686 and A699 grow very little on plates containing 100 mM ammonium sulfate, but the three randomly selected transformants also grow very well on this medium. Even in ammonium sulfate as a nitrogen source,
A. Residual growth of Nidulans gdhA mutants A686 and A699 is known (JR Kinghorn, JA Pateman, Hered).
(1973, 31: 427) and also due to the presence of a second glutamate dehydrogenase activity which allows partial growth of these mutants.

A. 2.1.4. Glutamate dehydrogenase activity in transformed strains Nicotinamide-adenine dinucleotide phosphate (NADP) -specific glutamate dehydrogenase (NADP-GDH) activity was determined by reducing α-ketoglutamic acid in the presence of ammonium and NADPH. Tested by following amination, expressed as units of enzyme activity per mg of protein. The initial reaction rate was estimated from the change in light intensity at 340 nm in a Hitachi U-2001 spectrophotometer. One unit of glutamate dehydrogenase was defined as the activity of catalyzing the oxidation of 1 nmol of NADPH per minute.

To verify the results of complementarity, nidulans gdhA mutant A6
86 and A699; In three randomly selected transformants complemented with the awamori gdhA gene, NADP-dependent glutamate dehydrogenase activity was measured. The results are shown in Table 1, where the glutamate dehydrogenase activity in strains A686 and A699 is clearly lower than the lower limit of detection. Transformants harboring the awamori gdhA gene clearly showed that significant levels of glutamate dehydrogenase activity were obtained, especially at 24 and 48 hours of growth. Some transformants, such as A699-4, exhibited relatively high levels of glutamate dehydrogenase activity, probably due to the integration of more than one copy of the gdhA gene in the genome of this transformant.

[0051]

[Table 1] A. 2.1.5. Identification of the promoter region of the gdhA gene Analysis of the nucleotide sequence upstream from the ATG translation initiation codon revealed that the transcription initiation codons may correspond to the putative TATA and CAAT boxes involved in the regulation of gene expression, -316 and -316, respectively. GTATA, C at positions 61 and -17
Revealed the presence of TATA and TCAATC sequences (see SEQ ID NO: 1)
.

The identification of the transcription start site was performed by “primer extension” using 2 μg of mRAN obtained from hyphae grown in MDFA for 48 hours as shown in FIG.

Oligonucleotide “Pe” 5′-GGGGTTCTTC as primer
Primer extension analysis using TGGAAGAGGGT-3 ′ (corresponding to the nucleotide sequence 70 bp downstream from ATG) showed a single band in the extension reaction (FIG. 8).
). The thymine (T) located 86 bp upstream of the ATG start codon corresponds to the 5 'end of the mRNA.

A. 2.1.6. To study the 1.7 kb monocistronic transcript transcribed from gdhA and its expression regulated by the nitrogen source. awamori total RNA was phenol-S
According to the DS method, 55.5 mM glucose and 1 as a carbon source and a nitrogen source were obtained.
In an MDFA medium containing 0 mM ammonium sulfate, 12, 24,
Hyphae were grown for 48, 60 or 72 hours. For studies of regulation by the nitrogen source, MDFA basal medium (without ammonium sulfate) was supplemented with glutamic acid, L-glutamine, sodium nitrite, sodium nitrate and L-asparagine at a final concentration of 10 mM.

For Northern analysis, total RAN (5 μg) was run on a 1.2% agarose-formaldehyde gel. Gels were run on nylon filters (NYTRAN 0.45; Schleicher and schuell) by standard methods.
Blotted on top. RNA was analyzed using a UV-Stratalinker 2400 lamp (manufactured by Stratagene, La Jolla, Calif.).
Specified by irradiation.

For slot blots, Bio-Dot SF Microfiltra
RNA (5 μg) was loaded by suction onto a filter (NYTRAN 0.45) in a Tion device; (Slot Blotting, Bio-Rad). R
NA was identified by UV irradiation as above. The filters were prehybridized in 50% formamide, 5 × Denhardt's solution, 5 × SSPE, 0.1% SDS, 500 μg denatured salmon sperm DNA per ml for 3 hours at 42 ° C. The internal DNA fragment of the awamori gdhA gene (0.694 k
b PvuII) was used as a probe to hybridize in the same buffer containing 100 μg of denatured salmon sperm DNA per ml. Filter 2 × S
Wash once in SC, 0.1% SDS at 42 ° C. for 15 minutes, 0.1 × SSC, 0
. Wash once in 1% SDS at 42 ° C. for 15 minutes, 0.1 × SSC, 0.1% S
Another wash for 20 minutes at 55 ° C. in DS was followed by radiography using Amersham X-ray film. MRNA was purified from total RNA using a Poly (A) Quick mRNA isolation kit (Stratagene, La Jolla, Calif.).

Northern analysis of the gdhA gene transcript revealed that it was strongly expressed as a 1.7 kb transcript (mRNA) slightly larger in size than the β-actin gene mRNA, as shown in FIG. Since ORF1 contains 1380 bases, this size of the transcript suggests that the gdhA gene is expressed as a monocistronic transcript.

Since the same amount of total RNA was used in all lanes of FIG. 7, gd in cells
It was concluded that the constant transcription level of hA was higher than the β-actin gene (arrow),
This suggests that glutamate dehydrogenase A is expressed from a very effective promoter.

A. In order to determine the pattern of expression of the gdhA gene during the growth of awamori, gdhA-hybridizing RNA was converted to MDF using ammonium sulfate.
Compared to β-actin hybridizing RNA in A medium (FIG. 8A),
-Expressed as a ratio of the count number in the gdhA hybridized band to the actin hybridized count number (Fig. 8B). The results show that the expression of both genes (gdhA and β-actin) was A. Although associated with awamori growth, low steady-state levels of β-actin nRNA were maintained in cells up to 96 hours of growth, whereas levels of glutamate dehydrogenase mRNA dramatically increased after 48 hours. It indicates that it has decreased.

In a MDFA medium containing ammonium sulfate (10 mM) as a nitrogen source, A. aw
Glutamate dehydrogenase activity detected at different times of culture when amori was grown is shown in FIG. 8C. Between 24 and 48 hours after the start of growth, there is a rapid decrease in glutamate dehydrogenase activity, which is in good agreement with the decrease in transcript level at this time of culture, as shown in FIG. 8B.

Glutamate dehydrogenase can be obtained from A. Playing a central role in awamori nitrogen utilization, it is also important to study whether gdhA expression is regulated by different nitrogen sources. As shown in FIG. 9, very high levels of gdhA transcription (m2) were observed in media containing NH ₄ ⁺ or asparagine as the sole nitrogen source.
RNA) was obtained. Glutamate repressed the transcription of the gdhA gene, but intermediate levels of expression (normalized for the β-actin gene) resulted in nitrate as a nitrogen source,
This was observed in media containing glutamine or nitrite. These results indicate that NADP-dependent glutamate dehydrogenase is subject to strong nitrogen source control at its transcriptional level.

Glutamate dehydrogenase activity in 24-hour cultures grown in MDFA media containing different nitrogen sources, all at a concentration of 10 mM, is shown in Table 2. In a medium containing NH ₄ ⁺ or asparagine as a nitrogen source, the highest activity (1 ml of medium)
Hit) was observed. Further, these two sources of nitrogen are awamori
Also preferred for strong growth. When the results were expressed per mg of protein in the cell extract, the highest specific activity was observed in MDFA medium using nitrate as sole nitrogen source. This is because A.I. Due to the fact that awamori grows very slowly. MD containing glutamic acid as a nitrogen source
The lowest activity is observed in the FA medium, confirming the results previously observed at that transcription level.

[0063]

[Table 2] A. 2.2. Construction of Expression Cassette GDHTh Having identified the promoter region of the gdhA gene, a thaumatin expression cassette similar to that described above was constructed. Using the plasmid pBSGh as a template, a 750 bp DNA fragment corresponding to the promoter region of the gdhA gene was obtained. This fragment was obtained by DNA amplification using oligonucleotides gdh1 and gdh2, and Pfu enzyme (Stratagene).

[0064]

Embedded image This amplified DNA fragment was digested with SalI and NcoI, and
Purified by 8% agarose gel.

The plasmid pJL43 (a derivative of pJL43b, Dr. Josei Louis
Barredo, Doctoral Dissertation, Universidad de Leoen, L
eoen, Spain) was digested with SalI and NcoI, and the large fragment (3740 bp) was purified on a 0.8% agarose gel. This DNA fragment is then ligated with the previously amplified SalI-NcoI fragment to generate plasmid p43gdh (4500 bp), where Penicilliu
The chrysogenum-derived pcbC promoter is Aspergill.
Us awamori had replaced it with the gdhA promoter.

In the next step, plasmid p43gdh was digested with NcoI and
Treatment with the Klenow fragment of A polymerase I followed by calf intestinal phosphatase (C
IP). In parallel, the 1140 bp fragment containing the B2 protein gene was
Amplification was performed by PCR using plasmid pJE1A as a template and NTB2b and CTB2b (sequences are shown below) as primers. This 1149 bp fragment was digested with BamHI, followed by DNA polymerase I.
Klenow fragment. From this reaction mixture, a 425 bp DNA fragment containing the amino terminal sequence of the B2 gene was purified by 1.0% agarose gel. This D
The NA fragment was ligated to the aforementioned p43gdh DNA fragment by blunt-end ligation to give the plasmid p43gdhB2, in which the BamHI site shown in FIG. 10 was regenerated. This plasmid is 4925 bp long and contains the gdhA promoter fused "in frame" with the amino-terminal portion of the B2 gene.

The next step in the construction of the complete expression cassette is the second part of the B2 gene, KE
X2 sequence and the addition of a synthetic thaumatin II gene. The plasmid pB2KEX was used as a component for this preparation.

PB2KEX was in turn digested with XbaI and Kl from DNA polymerase I
It was treated with the enow fragment and finally digested with BamHI. The 4637 bp fragment contained 0.
Purified by 8% agarose gel. In parallel, the plasmid p43gdhB2 was in turn digested with SalI, treated with the Klenow fragment from DNA polymerase I, and finally with BamHI. The 1173 bp fragment was purified on a 0.8% agarose gel. The ligation of these two fragments results in the plasmid pGDHTTh (
5810 bp), where a new SalI site was created. This allows excision of the complete GDHTh cassette as a 2670 bp SalI-SalI fragment.

Starting from plasmid pGDHTTh, two new plasmids were constructed. The first is p43GDTh, which was constructed as follows. Plasmid pJL43 was linearized by digestion with SalI and the plasmid from pGDHTTh
Ligation with a 170 bp SalI-DraI fragment (see FIG. 10, part B).

Similarly, plasmid pGD71 was constructed as follows: plasmid pA
N7-1 (PJ Punt et al., J. Biotecnol. 1990, No. 1)
7, pages 19-34), in turn, were digested with XbaI, treated with the Klenow fragment from DNA polymerase, finally digested with HindIII, and purified by 0.8% agarose gel. In parallel, plasmid pGDHTTh is constructed with Ec1136
II (or SacI ^* , a SacI mutant from fermented yeast (Fermentas) that recognizes a standard SacI restriction site but leaves blunt ends), HindII
Digested with I and DraI. The 2175 bp fragment was purified by agarose gel. Ligation of these two fragments resulted in plasmid pGD71 (
(See FIG. 10, Part C).

The plasmids p43GDTh and pGD71 are: (i) (a) thaumatin II
(B) a spacer sequence containing a KEX2 processing sequence in that order, and (c) a cDNA sequence encoding most of the B2 protein (excluding the carboxyl-terminal sequence) from Acremonium chrysogenum in this order. A DNA sequence encoding the fusion protein; (ii) Acremoniu
M chrysogenum B2 gene signal sequence, (iii) Aspe
rguilus awamori glutamate dehydrogenase A gene, and (iv) a cassette for expressing thaumatin comprising a drug resistance gene that can be used as a transformation marker. Plasmid p43GDTh contains the phleomycin resistance gene (phleo) driven by the pcbC promoter from Penicillium chrysogenum.
). Plasmid pGD71 is Aspergillus nidula
It has a hygromycin B resistance gene driven by the glyceraldehyde-3-phosphate dehydrogenase promoter from ns.

A. 3. Construction of Expression Cassette GPDTTh The expression cassette GPDTTh is prepared by using a promoter from the glyceraldehyde-3-phosphate dehydrogenase (hereinafter, referred to as “gpd”) gene derived from Aspergillus nidulans, by Acremonium chrysogen.
Expression cassette B2KE, except that the B2 promoter from num was replaced.
Same as X.

The complete promoter region of the gpd gene was constructed using plasmid pAN52-1 (PJ
. Punt et al. Biotecnol. 1990, Vol. 17, 19-34
Page). The SacI-NcoI fragment from pAN52-1 (880 bp
) Is subcloned to generate pJL43b1.

Plasmid pJL43b1 was digested with NcoI, first treated with the Klenow fragment of DNA polymerase I, and then with calf intestinal phosphatase (CIP), as shown in FIG. In parallel, a 1140 bp fragment of DNA is
JE1A, and oligonucleotides NTB2b and CT as primers
It was obtained by DNA amplification using PCR technology using B2b. This DN
The fragment of A was digested with BamHI and treated with a Klenow fragment from DNA polymerase to give a 425 bp fragment, which was purified on a 0.8% agarose gel. This last ligation produces the plasmid pB1B2 (see FIG. 11).

[0075]

Embedded image As with the GDHTh cassette, the next step in the construction of the full expression cassette is the addition of the second part of the B2 gene, the KEX2 sequence and the synthetic thaumatin II gene. As this production part, the plasmid pB2KEX was used again.

PB2KEX was in turn digested with XbaI and Kl from DNA polymerase I
It was treated with the enow fragment and finally digested with BamHI. The 4637 bp fragment contains 0
. Purified by 8% agarose gel. In parallel, plasmid pb1B2 was digested with BamHI and Ec1136II (or SacI ^* ) in turn (leaving blunt ends), and the 1130 bp fragment was purified on a 0.8% agarose gel. The ligation of these two fragments resulted in the plasmid pGPDTTh (5800b
p) was produced.

In the next step, the GPDTh cassette was loaded with Ec1136II (or Sac
I ^* ), by digestion with HindII and DraI, is isolated from pGPDTTh to obtain a DNA fragment 2800 bp in length. In parallel, plasmid p
B2KThb1 was in turn digested with BamHI and digested with DNA polymerase I.
Treated with the Klenow fragment and finally digested with HindIII. The 4500 bp fragment was isolated on an 08% agarose gel. The plasmid resulting from the ligation of these two fragments was named pGPThb1.

This plasmid contains gpd from Aspergillus nidulans
Acremonium chrysogenu by promoter from gene
m, except that the promoter from the B2 gene has been replaced.
Includes a cassette for expression of thaumatin that is the same as KEX.

B. Strain used and transformation protocol Aspergillus awamori NRRL312 strain was obtained from Ameri
Obtained from can Type Culture Collection (ATCC). Using a standard mutagenesis technique with nitrosoguanidine (NTG), a variant of this strain was obtained and named LpR66. This mutant strain is expressed in the growth medium in an inactive exoprotease Aspergillulopepsin A (hereinafter referred to as
(Called "pepA"). In all the transformation experiments described below, the strain used was this Aspergillus awamori LpR66 strain.

[0080] The three expression cassettes described above were used for Aspergillus awamor.
i LpR66 strain was used for transformation.

In all single transformation experiments, the antibiotic phleomycin was used as a selectable marker. The LpR66 strain can grow on plates containing 20 μg / ml phleomycin. Therefore, all transfectants were selected in plates containing 25 μg / ml antibiotic. The regeneration medium used was TSAS and contained 30 g / l Triptone-Soya (Difco), 103 g / l sucrose and 1.5% agar (Difco).

The transformation protocol, with some improvements, has been
(See above). On the plate containing the enriched medium,
10 ⁷ spores were inoculated. The plate was incubated at 30 ° C. for 72 hours, at which point the spores were scraped off the plate and incubated in 100 ml of CM medium (500 ml shake flask). Incubation is at 250C at 250C.
Performed for 18 hours at rpm. The mycelium obtained from this growth was filtered through a 30 μm nylon filter (Nytal), containing 1% 0.6M magnesium sulfate.
The plate was washed with a 0 mM sodium phosphate buffer (pH 5.8). 1 g of mycelium was resuspended in "protoplast buffer" (10 mM sodium phosphate buffer (pH 5.8) also containing 1.2 M magnesium sulfate). Enzyme “Lysing” (Sigma
Was added to bring the final concentration of enzyme to 3 mg / ml. The incubation of the mycelium solution was continued at 30 rpm and 100 rpm for 3-4 hours until protoplasts were formed. Protoplast formation was monitored by visual observation using an optical microscope. Protoplasts were filtered, washed and finally resuspended in STC solution to a final concentration of 10 ⁸ protoplasts / ml.

100 μl of protoplast solution is mixed with 10-20 μg of DNA, and
Left for 0 minutes. After this time interval, 500 μl of PTC was added and left at room temperature for another 20 minutes. Next, 600 μl of STC medium was added and the transformation mixture was dispensed into different tubes. Finally, a phleomycin antibiotic solution and
TSAS medium containing agar was added. The contents of the test tube were slowly homogenized and added to a TSAS plate containing phleomycin. Plates were incubated at 30 ° C. until transformants became visible as individual colonies. A similar protocol was used when hygromycin B was used as a selectable marker.

Linearization of all plasmids described herein increased the efficiency of transformation by a factor of 4 compared to transformation performed with non-linearized plasmids. Therefore, in most transformation experiments, the plasmid was used linearized.

[0085] Several transformants were obtained and analyzed. Initial screening was performed on plates containing 25 μg / ml phleomycin. Next, 200 μg /
Confirmatory screening was performed using a high concentration of ml of phleomycin.

The transformants were prepared substantially as described (see EP6844312).
The thaumatin II gene was analyzed by PCR to detect whether it was integrated into its genome. The positive transformants were then further analyzed for thaumatin expression by immunoblot analysis and ELISA (enzyme-linked immunoassay) as described (EP 684,312).

C: Recombinant strain producing thaumatin 1. Materials and Methods 1.1. Medium CM medium: malt extract 5 g / l; yeast extract 5 g / l; glucose 5 g
/ L.

SMM medium: 8% sodium citrate; 1.5% (NH ₄ ) ₂ SO ₄ ; 0.13% NaH ₂ PO ₄ .2H ₂ O; 0.2% MgSO ₄ .7H ₂ O; 1% Tween 80; 0.1% uridine, 0.1% suds AF and 7% soy milk. The carbon source (glucose, sucrose, maltose, etc.) is present at a final concentration of 15%. The pH of the medium is adjusted to 6.2 with H ₂ SO _4.

MDFA medium: 1.2% L-asparagine; 0.8% salt solution I [2% Fe (
NH ₄ ) ₂ (SO ₄ ) ₂ . 6H ₂ O]; and 14.4% salt solution II [10.4% K ₂ HPO ₄ ; 10.2% KH ₂ PO ₄ ; 1.15% Na ₂ CuSO ₄ .5H ₂ O; 2% MgSO ₄ .7H ₂ O; 0.02% ZnSO ₄ .7H ₂ O; 0.005% Cu
_{SO 4 · 5H 2 O; 0.05} % CaCl 2 · 2H 2 O]. The carbon source used is maltose (usually 6.5%) or sucrose (3.6%) and glucose (2.7%)
Is a mixture with Other amounts of carbon source are indicated in each of the experiments described. The initial pH of this medium is 6.5.

C. 1.2. Fermentation analysis Growth and expression studies were performed in SMM and MDFA media, first in shake flasks, and then equipped with a system for measuring and controlling variables such as agitation, dissolved oxygen, pH, defoaming and culture levels. In a fermenter.

The experiment was performed in a 1 liter shake flask with a working volume of 150 ml.
Inoculation was performed to a final concentration of 3 × 10 ⁵ spores / ml. Agitation was 150 rpm and incubation temperature was 30 ° C. The medium used was SMM or MDFA.

The experiments performed in the fermenter showed that the pH of the medium was kept constant at a preset value,
% Except that adjusted by automatic addition of NaOH or 0.5 N H ₂ SO ₄ were similar to those in shake flasks.

C. 1.3. Analytical Methods Two to ten ml samples were taken from the fermentation medium at different times and processed to determine the dry weight, thaumatin, maltose and glucose concentrations contained.

[0094] The dry weight was determined by using a pre-filter (manufactured by Nucleopore, Cat.
No. 211114). The biological material remaining in the prefilter was washed with 40 ml of pure ethanol and 50 ml of distilled water. This was then incubated at 90 ° C. until the weight was constant. The filtrate was dispensed and frozen for further analysis.

The thaumatin concentration in the culture broth was determined by enzyme-linked immunoassay (ELISA) and immunoblot (Western blot) analysis using an anti-thaumatin polyclonal antibody, essentially as described (EP684312). In immunoblots, samples were optionally concentrated as follows: 500 μl of the filtrate was mixed with an equal volume of 10% trichloroacetic acid (TCA) and frozen for 12 hours. Next, the sample was returned to room temperature and centrifuged (15,000 rpm; 20 minutes, 4 ° C.) using a tabletop centrifuge. The recovered pellet contains all the proteins present in this sample. Next, the pellet is described (E
P6844312), resuspended in protein impregnation buffer, boiled for 5 minutes and subjected to SDS-PAGE.

For the determination of glucose / maltose, about 1 ml of the filtrate was used. SIGMA
DIAGNOSTICS kit (Procedure number 510
) Was used to determine glucose levels.

The maltose concentration in the culture broth was determined as follows: 250 μl of the sample filtrate was placed in a pre-cooled test tube; 1.250 ml of anthrone solution (2 g of anthrone in 50 ml of absolute ethanol) And then 7
(Prepared by adding 950 ml of 5% H ₂ SO ₄ ) was added and the sample was cooled for 5 minutes. Next, the samples were transferred to a boiling water bath and incubated for 10 minutes. Finally, the sample was cooled again and the absorbance at 625 nm was read. Maltose concentration was determined by comparison with a calibration curve created by measuring the absorbance of a maltose solution of known concentration (range: 0-0.2 g / 1).

C. 2. Thaumatine producing strain C. 2.1. Strain TB2b1-44 This strain is a derivative of LpR66 obtained by transforming the Lpr66 strain with the expression plasmid pB2KTh-b1. This expression cassette contains A
It contains a synthetic thaumatin II gene under the control of the promoter of the B2 protein from C. cremonium chrysogenum. In shake flask culture with MDFA medium, this strain secretes 6-8 mg / l thaumatin.

[0099] Further optimization studies were performed in a 5 liter New Brunswick fermenter. Seed cultures were obtained by growing the strain in a CM medium at 30 ° C. for 40 hours. Then, using 450 ml of this seed culture, a 5-liter fermenter (
(Operating volume: 4.5 liters). RPM is between 250 and 500,
It was varied according to the oxygen status of the system which was always set to 30%.

Different parameters were tested, such as medium pH, carbon and nitrogen sources. A representative experiment is described in FIG.

1. MDF using 6.0% sucrose and L-asparagine as nitrogen source
Growth in A medium. The pH was set at 6.2 and a feed batch system was provided. 36 from fermentation start
, 48, 60 and 72 hours later, feeds were made. 0.5g /
45 ml of the sucrose solution was added.

[0102] 2. The conditions are the same as those described in 1 above, except that L-asparagine is replaced by ammonium sulfate (molar amount is the same in both experiments) as nitrogen source.

Using asparagine as a nitrogen source and 6% sucrose as a carbon source, 36
The highest productivity was obtained under the conditions described in 1 above, wherein the sucrose was "fed" four times every 12 hours after hours of fermentation. Under these conditions, 100 mg /
A thaumatin yield of 1 was obtained.

C. 2.2. Strain TGDTh-4 This strain has been accorded accession no.
. It was deposited with the following organization (CECT) as CECT20241.

[0105]

Embedded image This is a derivative of Lpr66 obtained by transforming the above Lpr66 strain using the expression cassette p43GDTh. This expression cassette contains Asp
and a synthetic thaumatin II gene under the control of the promoter of the gdhA gene from E. ergillus awamori. In shake flask cultures using MDFA medium (containing 6.0% sucrose), this strain secretes 6-8 mg / l thaumatin.

As described above for strain TB2b1-44, experiments were performed with 5 liter New
The experiment was also performed in a controlled environment of a Brunswick fermenter. As a nitrogen source, ammonium sulfate was used in place of asparagine at the same molar level. In this experiment, as also shown in FIG. 12, the following conditions were tested: In MDFA medium supplemented with 6% sucrose and ammonium sulfate as nitrogen source, strain TGDT was used.
h-4 was grown. The pH set value was 6.2 and a feed batch system was provided. Feeding was performed 36, 48, 60 and 72 hours after the start of fermentation. For each feed, 45 ml of a 0.5 g / ml sucrose solution was added.

The results (FIG. 12) also show that although thaumatin production is on the order of 100 mg / 1, there is the added advantage of faster production and more economical nitrogen sources available. . Therefore, the glutamate dehydrogenase promoter from Aspergillus awamori is Acremonium chrysogen.
It is concluded that it is more efficient than the um-derived B2 protein promoter.

C. 2.3. Strain TGP-3 This strain is a derivative of Lpr66 obtained by transformation of the Lpr66 strain using the expression cassette pGPThb1. This expression cassette is
and a synthetic thaumatin II gene under the control of the promoter of the gpd gene from G. nidulans. In shake flask culture on MDFA medium, this strain secretes 9-10 mg / l thaumatin.

C. 2.4. The double transformant strains TB2b1-44 and TGP-3 are The thaumatin gene under the control of the glutamate dehydrogenase promoter from awamori was retransformed with the expression plasmid pGD71 containing the hygromycin B resistance gene as a selectable marker for transformation experiments. One set of different transformants (see Table 3)
Were analyzed in shake flask experiments. Retransformation of strain TGP-3 was shown to not result in a better producer strain. However, retransformation of TB2b1-44 resulted in better producing strains when cultured in shake flasks under the standard conditions.

[0110]

[Table 3] D. Purification of recombinant thaumatin Two procedures were used for the purification of recombinant thaumatin. In the first step,
The fermentation broth was simply clarified, concentrated, diafiltered, and obtained a concentrated, clearer extract, which was used in sensory experiments to confirm the sweetness of the recombinant thaumatin. The second procedure involved a conventional purification protocol for pure thaumatin.

D. 1. Clarification, concentration and diafiltration of fermentation broth Biomass was removed by filtration through filter paper. The filtrate was collected in a filtration flask submerged in ice. Next, the clarified broth was centrifuged at 6000 rpm for 15 minutes at 4 ° C.

The clarified fermentation broth is ProFlux ^™ M12 Tangential F
Further concentration was achieved by ultrafiltration using an illumination system. The system configuration is: main unit, level switch, 2.5 L storage tank, cooling coil, inlet and outlet pressure transducers, secondary pump, one spiral wound thin film cartridge S1Y3 (3000 dalton molecular weight cut off).

The system was operated as follows: (1) Calibration of the pressure sensor. (2) Adjustment of alarm setting: lower limit of inlet pressure 3.0 bar, upper limit of inlet pressure 3.5 bar, pressure difference 0.3 bar. (3) Washing the system and cartridge with deionized distilled water. (4) Filling the storage tank with the processing solution; circulation of cold water through a cooling coil (HAAK
E, DC1-K20 cooling circulator) to maintain the solution at 8-10 ° C. (5)
Level switch setting to desired concentration volume (1/4 to 1/5 of initial volume). (6
) Operation of the circulation pump at 75%. (7) Adjustment of the back pressure valve to obtain an inlet pressure of 3.0 bar. Back pressure was reduced during operation as needed.

Once the fermentation broth was concentrated to the desired volume, the solution was subjected to diafiltration to remove low molecular weight solutes (salts, sugars, etc.).

The system configuration is capable of operating in “pump diafiltration with automatic safe stop” mode. When a command is given by the level switch,
The dialysate (5 volumes of deionized water) is transferred stepwise by a secondary pump. When the dialysate supply is depleted, the system and the secondary pump are automatically shut off.

The diafiltered solution was withdrawn from the system and filtered (St
reicup, 0.22 μm, Millipore) and stored at 4 ° C.

D. 2. Purification of recombinant thaumatin to homogeneity Recombinant thaumatin was purified to homogeneity using a four-step purification scheme detailed in Table 4. The starting point for the specific purification protocol described here is:
500 ml fermentation broth obtained from the growth of strain TGDTh-4, with thaumatin present at a concentration of 50 mg / 1.

From this broth, the protein was precipitated with ammonium sulfate (range 20-50%). Next, the precipitate was resuspended in 25 mM phosphate buffer of H7.0.

Next, the mixture was passed through a Sephadex G-25 column (for desalting) and eluted with the same buffer. Finally, the sample was CM-S at a flow rate of 0.5 ml / min.
Loaded on an epharose column. The column was washed with 25 mL phosphate buffer pH 7.0 to remove proteins in the effluent fraction. Thaumatine was eluted with a linear gradient of NaCl (0-400 mM). Thaumatine, Coomas
It is eluted from this column in almost pure state as detected by sie Blue staining.

[0120]

[Table 4] Although the above illustrative examples are directed to the production of recombinant thaumatin, the production of other recombinant proteins by the novel methods provided by the present invention, and in particular the novel promoters and DNA constructs disclosed herein, It is included in the scope of the present invention. SEQ ID NO: 1 shown in the sequence listing shows the 2570 bp nucleotide sequence present in plasmid pBSGh together with the 5 ′ end of plasmid pB1.7. a
wamori gdhA gene. The numbers on the right indicate base numbers. A.
The promoter region of the awamori gdhA gene is located before the start codon ATG at position 741 in the sequence. The start of transcription is-with respect to the translation initiation codon.
It is the 86th T (655th in the sequence). The portion of the gene encoding the protein starts at position 741. The numbers below the amino acids indicate the numbers. There are a total of 460 amino acids. The two introns that are present are also shown.

[0121] SEQ ID NO: 2 is the sequence of A. awamor
1 is an amino acid sequence of a glutamate dehydrogenase A (gdhA) protein derived from i.

[0122]

[Sequence list]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A, 1B and 1C schematically show the steps involved in the construction of a B2KEX expression cassette.

FIG. 2. A. containing the gdhA gene. It is a restriction map of 28.7 Kb area | region of awamori DNA. It is a map of phage FAN1 and FAN2. The bold line is
The overlap region between two phages containing the gdhA gene is shown. pB10, pB5
. 5 and PB1.7 are DNA subcloned into the corresponding plasmids
Shows a fragment. B = BamHI, S = SalI.

FIG. 3. 2.1 Kb XbaI-BamH from sequenced pB5.5 plasmid.
It is a restriction map of I fragment. The 3 'end of the gdhA gene was included in the left region of the insert of pB1.7. B = BamHI, E = EcoRI, EV = EcoRV
, P = PstI, S = SalI, X = XbaI.

FIG. 4 shows parts A and B of FIG. awamori, A. et al. nidulans (G
enebank accession number P18819); crassa (P00369),
S. cerevisiae (P07262); occidentalis (
p29507); bisporus (P54387), S.P. typhimu
rium (P15111); coli (P00370) and C. coli. glut
FIG. 4 is a diagram showing a relative match of the deduced amino acid sequence of NADP-specific glutamate dehydrogenase of Amicum (P31026). Identical amino acids are shaded. A line is drawn above the motif ai with several consecutive conserved residues.

FIG. The gdhA mutation in two strains of A. nidulans, A. awa
FIG. 4 is a diagram showing complementarity of the mori gdhA gene. Part A: 1. n
idulans A686 mutant strain; 2, transformant A686-4; 3
, Transformed strain A686-6; 4, transformed strain A686-7. Part B: 1
A. nidulans A699 mutant strain; 2, transformed strain A699-
2; 3, transformed strain A699-3; and 4, transformed strain A699-4
.

FIG. 6 is a diagram showing identification of primer extension at the 5 ′ end of gdhA gene transcription. In the lane corresponding to the extension reaction, one protected band (arrow) is observed (
Lane Pe). G, A, T, C lanes correspond to the sequencing reaction of M13 phage from the -40 primer.

FIG. 7 shows Northern blot analysis of transcripts of gdhA and β-actin gene. A: Hybridization with a probe internal to the gdhA gene (0.694 kb PvuII fragment). B: A. as control. nidulans
Hybridization with β-actin gene.

FIG. 8: A. during fermentation in MDFA medium with 1% glucose and 10 mM ammonium sulfate. FIG. 4 is a diagram of a slot blot analysis of a transcript of the awamori gdhA gene (part A). For comparison purposes, β in the same RNA sample
-The actin gene transcription was also studied. Part B: g for β-actin gene
FIG. 4 is a diagram showing the relative level of expression of dhA. Part C: NDAP-dependent glutamate dehydrogenase activity in the same culture from which mRNA was extracted.

FIG. 9: A. during fermentation process in MDFA medium using different nitrogen sources. awamor
FIG. 4 shows a slot blot analysis of a transcript of the idghA gene (part A
). The medium contained 10 mM ammonium sulfate as a control and glutamic acid, glutamine, sodium nitrite, sodium nitrate and aspartic acid as nitrogen sources, all at a concentration of 10 mM. For comparative purposes, the transcription of the β-actin gene was also studied. Part B: shows the relative level of gdhA expression relative to the β-actin gene.

FIGS. 10A, 10B, and 10C schematically show the steps involved in the construction of a GDH expression cassette.

FIGS. 11A and 11B schematically show the steps involved in the construction of a GPD expression cassette.

FIG. 12. A. in the study of fermented substances. awamori TB2b1-44 and TGD
FIG. 7 is a diagram showing production of thaumatin from the TH-4 strain (concentration CT of secreted protein is expressed as mg / l). The medium used was MDFA supplemented with the components described below. Hollow square: TB2b1-44 strain; 6.0% sucrose, pH 6.2, fed batch with added asparagine. Hollow circle: TB2b1-44 strain; 6.0% sucrose, pH 6.2, feed batch to which ammonium sulfate was added. Solid triangle: TGDT
h-4 strain; 6.0% sucrose, pH 6.2, fed batch with added ammonium sulfate.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] June 5, 2000 (2006.5.5)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0003

[Correction method] Change

[Correction contents]

[0003] However, some recombinant proteins have proven very difficult to express in filamentous fungi. This is the case, for example, with interleukin-6 and thaumatin. Thaumatine is a protein that has a very sweet taste and has the ability to improve the palatability of food. In industry, these are, at present, the plants Tha
umatoccocus danielii benth is extracted from the provisional coat of the fruit (M. Witty, JD Higginbotham, Tha).
umatin, 1994, CRC press, Boca Ratoon, Florida). Thaumatine is derived from at least five different forms (I
, II, III, b and c), thaumatins I and II
Is the most abundant type among the temporary seed coats. Despite its advantages, industrial use of thaumatin of plant origin is very difficult to obtain the fruits from which it is extracted,
Extremely limited. Attempts have been made to produce thaumatin by genetic manipulation in different hosts such as bacteria, yeast and transgenic plants,
To date, the results have been disappointing, and thaumatin available industrially is very rare and expensive. European Patent EP 684 312 describes a method for preparing recombinant thaumatin in filamentous fungi. One problem with this method is that the yields obtained are low compared to the values required for industrial production of thaumatin.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction contents]

It is possible to improve the yield of the recombinant protein when the recombinant protein in question is expressed as a fusion with another protein and when the expression of this cassette is driven by a strong fungal promoter It has been known. Other proteins, called "carrier proteins", are normally highly expressed proteins of fungal origin. To date, the most frequently used expression systems include the promoter and gene of glucoamylase from Aspergillus awamori as promoter and carrier protein, respectively (PP Ward et al., Biotechno).
logic 1995, 13, 498-502). However, in some cases, the use of this expression system does not result in high levels of the desired recombinant protein. One of these particularly problematic cases is the expression of recombinant thaumatin in filamentous fungi. Gene (1983) 26: 253-260 lists the complete nucleotide sequence of the Neurospora crassa NADP-specific glutamate dehydrogenase gene . Appl. Microbiol. Biotechnol. (1997) Vol. 47, 1 to 11 pp., Discloses the effective production of minute泌蛋 white matter caused by Aspergillus. Particular focus is on the means of gene fusion. EP0684312A2 relates to a method for preparing thaumatin, a natural protein sweetener . Said document discloses a novel nucleotide sequence encoding thaumatin, which allegedly utilizes optimized codons for expression in filamentous fungi .

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction contents]

[0005] In view of the foregoing, there is a need to provide a new and more efficient expression system that enables the production of proteins at high concentrations that are difficult to express in filamentous fungi such as thaumatin. Is clear. This object is achieved by the novel promoter and DNA construct provided in the present invention, as described below.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction contents]

DISCLOSURE OF THE INVENTION [0006] The present invention relates to a fungus of the genus Aspergillus, particularly Aspergillus.
The present invention provides a novel expression system utilizing a promoter derived from a sawamori-derived glutamate dehydrogenase (gdh) gene.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction contents]

[0007] One form of the present invention is the sequence, and (b) is not a gdh gene promoter from Aspergillus nidulans sequence of (a) the attached SEQ ID NO: 1 in the nucleotide numbers 1 to 740 a novel promoter for the expression of recombinant proteins in filamentous fungi comprising the nucleotide sequence or its complementary strand is selected from the group consisting of nucleotide sequence hybridizing to the harsh conditions to those defined in (a). Particularly preferred is the sequence defined in (a), ie Aspergil.
a promoter comprising the sequence of base numbers 1 to 740 in SEQ ID NO: 1, which corresponds to the gdhA promoter of the glutamate dehydrogenase A gene derived from L. awamori.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

[0008] Glutamate dehydrogenase A disclosed herein is a filamentous fungus Aspergillus
Although it is the first glutamate dehydrogenase identified and described in awamori, other glutamate dehydrogenases may be present in Aspergillus awamori. Aspergil shown in SEQ ID NO: 1
rus awamori gdhA promoter and / or a novel nucleotide sequence of a gene, or a part thereof, according to the disclosure of the present invention, Aspergillus
awamori and other organisms, preferably filamentous fungi, more preferably As
pergillus fungi, more preferably Aspergillus awa
mori and Aspergillus niger, especially Aspergi
It can be used as a probe for the identification and isolation of other homologous promoters / genes of glutamate dehydrogenase in L. awamori. As a result, the present invention relates to the Aspergillus awam disclosed herein.
ori-derived gdhA, but not limited to Aspergillus n
The present invention also relates to a promoter of any glutamate dehydrogenase gene derived from a fungus of the genus Aspergillus, which is not derived from idulans. The Aspergil
Examples of li are Aspergillus awamori, Aspergillu
s niger, Aspergillus oryzae and Aspergi
lrus sojae. In a preferred embodiment, the invention relates to Asperg
The present invention relates to a promoter of a glutamate dehydrogenase gene derived from illus awamori or Aspergillus niger. In a more preferred embodiment, the present invention relates to the promoter of the glutamate dehydrogenase gene from Aspergillus awamori. Use of the novel nucleotide sequence shown in SEQ ID NO: 1 or a part thereof as a probe is also an aspect of the present invention. "
The term "part thereof" means any site in the nucleotide sequence of SEQ ID NO: 1 that functions as a probe.

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction contents]

Another aspect of the present invention relates to a glutamate dehydrogenase protein, which comprises (a) the sequence of base numbers 741 to 2245 in the attached SEQ ID NO: 1, and ( b) the gdh gene derived from Aspergillus nidulans. is a sequence not, severe conditions comprising a nucleotide sequence or its complementary strand is selected from the group consisting of hybridizing nucleotide sequence to be isolated and purified to those defined in (a) A novel DNA sequence. In a preferred embodiment, the nucleotide sequence encoding glutamate dehydrogenase has the sequence defined in (a), ie, SEQ ID NO: 1.
This is the sequence of base numbers 741-2245 in the figure. However, the present invention is not limited to the particular gdhA gene from Aspergillus awamori disclosed herein, except that A gene not derived from Aspergillus nidulans,
It also relates to any glutamate dehydrogenase gene from a fungus of the genus spergillus. In a preferred embodiment, the present invention relates to Aspergillus awam
The present invention relates to a DNA sequence encoding glutamate dehydrogenase from ori or Aspergillus niger. In a more preferred embodiment, the present invention provides
The present invention relates to a DNA sequence encoding glutamate dehydrogenase from S. spargillus awamori.

[Procedure amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction contents]

[0010] Another aspect of the invention is a novel protein encoded by any of the DNA sequences defined above. In a preferred embodiment, the protein has the amino acid sequence disclosed in SEQ ID NO: 2. In the present invention, however, Aspergillus n
any glutamate dehydrogenase derived from a fungus of the genus Aspergillus, not from Aspergillus idulans, more preferably Aspergillus awamo
Glutamate dehydrogenase from ri or Aspergillus niger, more preferably glutamate dehydrogenase from Aspergillus awamori, is also included.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C07K 14/43 4H045 // C07K 14/43 C12R 1:66) (C12N 15/09 ZNA ( C12N 1/15 C12R 1:66) (C12N 1/15 (C12N 9/06 C12R 1: 665) (C12N 9/06 (C12N 9/06 C12R 1: 665) C12R 1: 685) (C12N 9/06 C12N 15/00 ZNAA C12R 1: 685) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Casqueiro Branco, Francisco Javier Spain I-24195 Vizijahobis Po de las Regueras Jie Menendez Pidar 4 2nd floor-A (72) Inventor Moralejo Lorensou, Francisco Jose I-49800 Toulo Cardi e Rehadourada 7 (72) Inventor Martin Martin, Houan Francisco I-24004 Leon Ave Niida de la Facturer 13 4th floor-Ah (72) Inventor Gutierrez Martin, Santiago Spain i-24195 Vizillahobis Po de las Regeras Cargier José Bergamin 5 3rd floor-Sea (72) Inventor Hihalbia Ibraim, Maria Jose Spain I-09200 Miranda de Ebrow Cardier Estacion 32 8th floor-Be (72) Inventor Del Rio Pericacho, Jose Luis Spain -08225 Terrasse Car Gie Soumentent Castageya 118 3rd floor-1st (72) Inventor Faus Sa Tussana, Ignacio Spain -08022 Barcelona Cardier Ricardo Carbo 8-10 2nd floor-1st (72) Inventor Caldousa Silva, Rosa Elena Spain-24195 Viziyahobis Po de las Regeras Cardier Jose Bergamin 5th floor-Sea F Term (Reference) 4B024 AA05 AA11 AA20 BA08 BA80 CA03 CA05 CA07 CA09 DA11 EA04 FA02 FA20 GA11 GA19 HA12 4B050 CC03 DD03 LL02 LL05 4B063 QA18 QA20 QQ07 QQ42 QQ44 QR32 QR55 QS34 4B064 AG01 CA05 AAAAAXA6A6A6A6A6A6A6A6A6AAX AB01 AC14 BA02 CA28 CA32 CA41 4H045 AA10 AA20 BA10 BA41 CA15 CA30 EA02 FA73 FA74 GA06 GA10 GA15 GA22 HA05

Claims (35)

[Claims] 1. (a) a sequence of base numbers 1 to 740 in the attached SEQ ID NO: 1, (b) a base sequence similar to that defined in (a), and (c) a base sequence defined in (a) A promoter for expression of a recombinant protein in a filamentous fungus containing a nucleotide sequence selected from the group consisting of a nucleotide sequence that hybridizes under severe conditions, or a complementary strand thereof. 2. The promoter according to claim 1, which has a sequence of base numbers 1 to 740 in SEQ ID NO: 1 or a complementary strand thereof. 3. An isolated promoter for a glutamate dehydrogenase gene derived from a fungus of the genus Aspergillus, which is not derived from Aspergillus nidulans. 4. The method according to claim 1, wherein the fungus is Aspergillus awamori or As.
4. The isolated promoter of claim 3, which is pergillus niger. 5. The isolated promoter according to claim 4, wherein the fungus is Aspergillus awamori. 6. A nucleic acid encoding a glutamate dehydrogenase protein, (a) a sequence of base numbers 741 to 2245 in the attached SEQ ID NO: 1, (b) a base sequence similar to that defined in (a), And (c) a purified and isolated DNA sequence comprising a base sequence selected from the group consisting of: a base sequence hybridizable under severe conditions to the one defined in (a), or a complementary strand thereof. . 7. The DNA sequence according to claim 6, which has a sequence of base numbers 741 to 2245 in SEQ ID NO: 1 or a complementary strand thereof. 8. An isolated DNA sequence encoding a glutamate dehydrogenase derived from a fungus of the genus Aspergillus, which is not derived from Aspergillus nidulans. 9. The method wherein the fungus is Aspergillus awamori or As
9. An isolated DNA sequence according to claim 8 which is pergillus niger. 10. The isolated DNA sequence of claim 9, wherein the fungus is Aspergillus awamori. 11. A protein encoded by any of the DNA sequences according to claim 6. 12. A protein having the amino acid sequence of SEQ ID NO: 2. 13. An isolated glutamate dehydrogenase derived from a fungus of the genus Aspergillus, which is not derived from Aspergillus nidulans. 14. The method according to claim 14, wherein the fungus is Aspergillus awamori or A.
14. The isolated glutamate dehydrogenase of claim 13, which is spergillus niger. 15. The isolated glutamate dehydrogenase according to claim 14, wherein the fungus is Aspergillus awamori. 16. An Asper for expression of a recombinant protein in a filamentous fungus.
Use of a promoter from a glutamate dehydrogenase gene from a fungus of the genus gillus. 17. Use according to claim 16, wherein the promoter is the promoter according to any of claims 1 to 5. 18. A promoter from a glutamate dehydrogenase gene derived from a fungus of the genus Aspergillus, (b) a DNA sequence encoding a protein normally expressed from a filamentous fungus or a part thereof, and (c) a degradable linker. A DNA recombinant construct comprising a DNA sequence encoding a peptide, and (d) a DNA sequence encoding a desired protein. 19. The DNA construct according to claim 18, wherein the promoter of (a) is the promoter according to any one of claims 1 to 5. 20. The DNA sequence of (b) comprising: (i) Aspergillus awamori, Aspergillus
niger, Aspergillus oryzae or Aspergill
19. The DNA according to claim 18, which encodes a protein selected from the group consisting of glucoamylase derived from C.us sojae, (ii) B2 derived from Acremonium chrysogenum, and (iii) glutamate dehydrogenase derived from a filamentous fungus, or a part thereof. Construct. 21. The DNA sequence of (b) is Aspergillus awa
mori, Aspergillus niger, Aspergillus o
21. The DNA construct according to claim 20, which encodes glucoamylase derived from Ryzae or Aspergillus sojae or a part thereof. 22. The DNA sequence according to (b), wherein the DNA sequence is Acremonium chry.
21. The DNA construct according to claim 20, which encodes a sogenum-derived B2 protein or a part thereof. 23. The DNA construct according to claim 20, wherein the DNA sequence (b) encodes a glutamate dehydrogenase derived from a filamentous fungus or a part thereof. 24. The DNA construct according to claim 18, wherein the DNA sequence of (c) contains a KEX2 processing sequence. 25. The DNA sequence of (d) encodes thaumatin.
25. The DNA construct according to any one of-24. 26. The DNA construct according to claim 25, wherein the DNA sequence of (d) is a thaumatin II synthetic gene from plasmid pThIX. 27. A DNA construct comprising a promoter from a glutamate dehydrogenase gene from a fungus of the genus Aspergillus that is operably linked to a DNA sequence encoding a protein desired to be expressed. 28. The DNA construct according to claim 27, wherein the promoter is the promoter according to any one of claims 1 to 5. 29. A filamentous fungal culture capable of producing a recombinant protein transformed with a plasmid containing the DNA construct according to any one of claims 18 to 28. 30. The culture of claim 29, wherein the filamentous fungus is a fungus from the genus Aspergillus. 31. The filamentous fungus is Aspergillus awamori, A
spergillus niger, Aspergillus oryzae,
Aspergillus nidulans and Aspergillus s
30. The culture of claim 29 selected from the group consisting of Ojae. 32. The culture of claim 29, wherein the plasmid comprises the DNA construct of claim 25 or 26. 33. (a) preparing an expression plasmid containing the DNA construct according to any one of claims 18 to 28; (b) transforming a filamentous fungus strain with the expression plasmid; (c) Culturing the transformed strain under suitable nutrient conditions to produce the desired protein intracellularly, extracellularly or simultaneously, and (d) optionally separating and purifying the desired protein from the fermentation broth Producing a recombinant protein in a filamentous fungus. 34. The method according to claim 33, wherein the recombinant protein is thaumatin and the expression plasmid comprises the DNA construct according to claim 25 or 26. 35. A DNA sequence derived from the nucleotide sequence according to claim 1 or 6 as a probe for identifying and isolating the glutamate dehydrogenase gene and / or the promoter sequence of the glutamate dehydrogenase gene. use.