KR101803013B1 - New Lipase Signal Sequences and Expression Method Using The Same - Google Patents

Abstract

The present invention relates to a novel signal sequence for mass-expressing lipase, and more particularly to a novel lipase signal sequence; An expression vector comprising the signal sequence and the lipase gene; A recombinant microorganism into which the expression vector is introduced; And a method for mass-producing lipase by culturing the recombinant microorganism. The use of an expression vector containing the novel signal sequence according to the present invention can induce the mass expression and secretion of not only a specific lipase but also other target proteins, which is very useful for stable mass production of specific lipase and other target proteins.

Description

[0001] The present invention relates to a novel lipase signal sequence and a lipase expression method using the same,

The present invention relates to a novel signal sequence for mass-expressing lipase, and more particularly to a novel lipase signal sequence; An expression vector comprising the signal sequence and the lipase gene; A recombinant microorganism into which the expression vector is introduced; And a method for mass-producing lipase by culturing the recombinant microorganism.

Several kinds of proteins used for industrial purposes have been produced using Saccharomyces , Aspergillus oryzae, Trichoderma reesei , whose stability is well known, In order to express and secrete the target protein, an expression secretion plasmid vector composed of a promoter promoting sequence, a DNA encoding secretory signal sequence, a structural gene of a target protein, and a transcription terminator has been used.

As secretory signal sequences currently used for secretion of target proteins in Aspergillus oryzae, the self-signal sequence of the gene to be expressed and the Aspergillus orygera amylase signal sequence have been used, and strong promoters include glucoamylase glaA) promoter has been used.

However, it is not possible to express a desired protein in large quantities using known signal sequences.

In the case of Aspergillus niger glucoamylase, it is possible to produce high yields of over 30 g / L through industrial process development. There are also many commercially available lipases derived from Aspergillus oryzae, which can induce lipases with traditional mutagenesis and inducing agents to efficiently produce lipases in Aspergillus oryzae. Therefore, there is a need for a method capable of promoting the secretion of lipase proteins out of cells on Aspergillus or its genome. In other words, it is important to search for a signal sequence capable of mass production of a protein, which is a commercially important enzyme, more efficiently, and a recombinant microorganism recombinant microorganism is required to be developed.

Accordingly, the present inventors have made efforts to develop a method for mass-producing lipase using a genetic engineering method in order to overcome the limitations of the above-mentioned expression. As a result, a novel signal sequence for expressing lipase was discovered, Confirming that lipase is expressed in a large amount, thereby completing the present invention.

An object of the present invention is to provide a novel lipase signal sequence for mass-expressing lipase; An expression vector comprising the signal sequence and the lipase gene; And a recombinant microorganism into which the expression vector is introduced.

It is another object of the present invention to provide a method for mass-producing lipase by culturing the recombinant microorganism.

In order to achieve the above object, the present invention provides a method of screening for an antibody that binds to an amino acid sequence selected from the group consisting of An03g06560 (SEQ ID No. 1), An14g00860 (SEQ ID No. 2), An07g00440 (SEQ ID No. 3), An02g09690 (SEQ ID No. 4), An16g08870 ), An16g01880 (SEQ ID No. 7), An12g06560 (SEQ ID No. 8), An09g02270 (SEQ ID No. 9), An15g02210 (SEQ ID No. 10), An13g02820 (SEQ ID No. 11), and Anllg00100 Lipase signal sequence; An03g0860 (SEQ ID NO: 13), An06g00860 (SEQ ID NO: 14), An07g00440 (SEQ ID NO: 15), An02g09690 (SEQ ID NO: 16), An16g08870 The nucleic acid encoding the lipase signal sequence selected from the group consisting of An12g06560 (SEQ ID NO: 20), An09g02270 (SEQ ID NO: 21), An15g02210 (SEQ ID NO: 22), An13g02820 (SEQ ID NO: 23), and Anllg00100 .

The present invention also provides an expression vector comprising a nucleic acid encoding the signal sequence and a gene of a desired protein, and a recombinant microorganism into which the expression vector is introduced.

The present invention also provides a method for producing a recombinant microorganism, comprising: (a) culturing the recombinant microorganism to produce a target protein; And (b) recovering the resulting target protein. The present invention also provides a method for producing a target protein.

The use of an expression vector containing the novel signal sequence according to the present invention can induce the mass expression and secretion of not only a specific lipase but also other target proteins, which is very useful for stable mass production of specific lipase and other target proteins.

Fig. 1 is a schematic diagram of an expression vector of the present invention, showing that the TLA gene can be cloned into Nae I / Not I, following the lipase signal sequence, using the glaA promoter and the pdcA terminator.
Fig. 2 is an image showing the pASP504 vector of Example 1, wherein the pASP504 vector contains a glaA promoter and a pdcA terminator, a hygromycin resistance gene and hygromycin B phosphotransferase, and each lipase signal Is a vector capable of cloning a sequence.
Fig. 3 is an image showing the pASP600s vector of Example 1, and is a typical example of a vector obtained by cloning each lipase signal sequence.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, a lipase signal sequence capable of expressing a large amount of a specific lipase and other target proteins; An expression vector comprising the signal sequence and the gene of the target protein; And a recombinant microorganism into which the expression vector was introduced were prepared. As a result, it was confirmed that a large amount of lipase could be produced by culturing the produced recombinant microorganism.

In one embodiment of the present invention, when a novel lipase signal sequence of the present invention is used using a lipase (TLL) gene derived from Thermomyces lanuginosus as a nucleic acid encoding a target protein, lipase is produced in a large amount . However, this is only one example, and it is obvious to those skilled in the art that the present invention can be applied to other target proteins as well as specific lipases.

Thus, the present invention provides, in one aspect, an amino acid sequence selected from the group consisting of An03g06560, An14g00860, An07g00440, An02g09690, An16g08870, An06g00350, (SEQ ID NO: 7), An12g06560 (SEQ ID NO: 8), An09g02270 (SEQ ID NO: 9), An15g02210 (SEQ ID NO: 10), An13g02820 Signal sequence.

In the present invention, the signal sequence may be derived from Aspergillus niger , but is not limited thereto.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising an amino acid sequence selected from the group consisting of An03g06560 (SEQ ID NO: 13), An14g00860 (SEQ ID NO: 14), An07g00440 (SEQ ID NO: 15), An02g09690 (SEQ ID NO: 16), An16g08870 (SEQ ID NO: 19), An12g06560 (SEQ ID NO: 20), An09g02270 (SEQ ID NO: 21), An15g02210 (SEQ ID NO: 22), An13g02820 To a nucleic acid encoding a signal sequence.

In another aspect, the present invention relates to an expression vector comprising a nucleic acid encoding the lipase signal sequence and a gene of a desired protein.

In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention.

For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site allowing the foreign DNA fragment to be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

In addition, the gene is "operably linked" when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way as to enable gene expression when a suitable molecule (e. G., Transcriptional activator protein) is attached to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation.

Generally, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

In the present invention, the target protein may be lipase, and the lipase may be a lipase derived from Thermomyces lanuginosus (TLL), but is not limited thereto.

In the present invention, the expression vector may further comprise a glucoamylase promoter, and the glucoamylase promoter may be an Aspergillus niger glucoamylase (glaA) promoter. It is not.

In the present invention, the expression vector may be selected from the group consisting of pASP601TLL, pASP602TLL, pASP603TLL, pASP604TLL, pASP605TLL, pASP606TLL, pASP607TLL, pASP608TLL, pASP609TLL, pASP611TLL, pASP612TLL, no.

In another aspect, the present invention provides a recombinant microorganism into which a gene encoding a lipase signal sequence and a gene of a target protein are introduced; Or a recombinant microorganism into which the expression vector is introduced.

In the present invention, the recombinant microorganism may be Aspergillus niger , but is not limited thereto.

The recombinant microorganism is a host cell in which the efficiency of introducing DNA is high and the expression efficiency of the introduced DNA is high. Usually, all microorganisms including prokaryotic and eukaryotic cells are usable as bacteria, yeast, fungi, Aspergillus oryzae were used in the Examples of the present invention, but the present invention is not limited thereto, and any kind of microorganism may be used as long as the lipase can be sufficiently expressed.

Of course, not all vectors function equally well in expressing the DNA sequence of the present invention, and likewise all hosts do not function equally in the same expression system. However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without departing from the scope of the present invention without undue experimentation. For example, in selecting a vector, a host should be considered, since the vector must be replicated within it, the number of copies of the vector, the ability to control the number of copies, and other proteins encoded by the vector, The expression of antibiotic markers should also be considered.

The transformed recombinant microorganism can be produced according to any transformation method. "Transformation" of the present invention refers to a phenomenon in which DNA is introduced as a host and DNA can be cloned as a chromosome factor or by chromosome integration completion, introducing an external DNA into a cell to cause an artificial genetic change .

As a method for inserting the gene into the chromosome of the host cell, a commonly known gene manipulation method can be used in the present invention. Examples of the method include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex A virus vector, a poxvirus vector, a lentivirus vector or a nonviral vector may be used.

In addition to the method of using an expression vector, a method of directly inserting it into a chromosome of a host cell may be used as a transformation method.

In general, the methods of electroporaton, lipofection, ballistics, virosomes, liposomes, immunoliposomes, polyvalent cations or lipid: nucleic acid conjugates, naked DNA, artificial virens, chemical promoting DNA inflow, calcium phosphate (CaPO4) , Calcium chloride (CaCl 2) precipitation, microinjection, lithium acetate-DMSO method and the like can be used.

Other representative nucleic acid delivery systems are available from Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), And BTX Molesular Syetem (Holliston, MA). Ripofection methods are described in U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787 and U.S. Pat. No. 4,897,355, and lipofection reagents are commercially available, for example, TRANSFECTAM ^™ and LIPOFECTIN ^™ . Cation or neutral lipids suitable for effective receptor-recognition lipofection of polynucleotides include Felgner's lipid (WO 91/17424 and WO 91/16024) and can be delivered to the cell via in vitro introduction, into the target tissue via in vivo introduction have. Methods of making lipid: nucleic acid complexes comprising target liposomes, such as immunological lipid complexes, are well known in the art (Crystal, Science, 270: 404-410, 1995; Blaese et al., Cancer Gene Ther., 2: 291 Gene et al., Gene Therapy., 2: 710-9, 1995; Behr et al., Bioconjugate Chem., 5: 382389,1994; Remy et al., Bioconjugate Chem., 5: 722, 1995; Ahmad et al, Cancer Res, 52:.. 4817-4820, 1992; U.S. Patent No. 4,186,183; U.S. Patent No. 4,217,344; U.S. Patent No. 4,235,871; U.S. Patent No. 4,261,975; U.S. Patent No. 4,485,054 U.S. Patent No. 4,501,728; U.S. Patent No. 4,774,085; U.S. Patent No. 4,837,028; U.S. Patent No. 4,946,787).

In another aspect, the present invention provides a method for producing a recombinant microorganism, comprising: (a) culturing the recombinant microorganism to produce a target protein; And (b) separating the resulting target protein. The present invention also relates to a method for producing a target protein.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Example  1: Preparation of expression vector pASP600s

The signal sequences of SEQ ID NOS: 13 to 24 (Table 2) encoding the amino acid sequences (Table 1) of the following SEQ ID NOS: 1 to 12 were chemically synthesized to secrete TLLs into aspergillus or the outside. Single chain oligonucleotides were chemically synthesized to have recognition sites of restriction enzymes ClaI and NaeI at both ends of the signal sequence base sequence, respectively. From the single chain oligonucleotides, double-stranded oligonucleotides Annealing reaction was carried out in order to prepare a polyimide precursor. Each of the signal sequence DNA fragments was recovered.

The recovered fragment was cut into pASP503 vector with restriction enzymes ClaI and NaeI and cloned into pASP601, pASP602, pASP603, pASP604, pASP60pASP605, pASP606, pASP607, pASP608, pASP609, pASP610, pASP611 and pASP612 as the sequence numbers respectively.

The pASP504 vector contains the glaA promoter and the pdcA terminator and includes the hygromycin resistance gene, hygromycin B phosphotransferase (FIGS. 1 and 2). Hydromycin B is inserted into the peptidyl-tRNA in the ribosome to interfere with the translation of the peptidyl-tRNA. The hygromycin B phosphotransferase enzyme inactivates hygromycin B, so that recombinant microorganisms can be selected.

Lipase signal sequence Gene ID enzyme Signal sequence (SS) amino acid
(AA) SS No. N-region H-region C-region An03g06560 triacyl
glycerol lipase maimkallwlslslavwa
(SEQ ID NO: 1) 562 18 1-5 6-15 15-18 An14g00860 triacyl
glycerol lipase masallwlsllggstla
(SEQ ID NO: 2) 582 17 One 2-12 13-17 An07g00440 triacyl
glycerol lipase myipsvlllaaslfhgata
(SEQ ID NO: 3) 418 19 1-2 3-14 15-19 An02g09690 triacyl
glycerol lipase mlklavalfsllavgna
(SEQ ID NO: 4) 561 17 1-3 4-12 13-17 An16g08870 triacyl
glycerol lipase miffhlapffflfglvvs
(SEQ ID NO: 5) 538 19 1-5 6-14 15-19 An06g00350 triacyl
glycerol lipase mvalltsfawagltalalg
(SEQ ID NO: 6) 529 19 1-2 3-14 15-19 An16g01880 triacyl
glycerol lipase mfsgrfgvlltalaalsaa
(SEQ ID NO: 7) 298 19 1-5 6-14 15-19 An12g06560 triacyl
glycerol lipase mfvsslallaliaplia
(SEQ ID NO: 8) 580 17 One 2-12 13-17 An09g02270 triacyl
glycerol lipase mlnarsialaslpvllllfaqqlas
(SEQ ID NO: 9) 592 25 1-7 8-19 20-25 An15g02210 triacyl
glycerol lipase mkgmlptlswlalgmaslatc
(SEQ ID NO: 10) 564 21 1-4 5-13 14-21 An13g02820 triacyl
glycerol lipase mrlsslalassailpalg
(SEQ ID NO: 11) 277 18 1-2 3-14 15-18 An11g00100 triacyl
glycerol lipase mgvmrktadqalgltatalalvsaaqa
(SEQ ID NO: 12) 581 27 1-10 11-22 23-27

Lipase signal sequence sequence Gene ID Signal sequence (SS) base sequence An03g06560 atgtggaacgccgcggtattctgcaggatgaactcatatgtcccgttgcggaaa (SEQ ID NO: 13) An14g00860 atggcgagcgcactcctctggctctccctgctgggtggcagcaccctagcc (SEQ ID NO: 14) An07g00440 atgtatatcccctcggtgctgcttctggccgcgagcctgttccatggcgcaacg (SEQ ID NO: 15) An02g09690 atgctaaagctcgctgttgctcttttttcgttacttgccgtgggcaatgca (SEQ ID NO: 16) An16g08870 atgattttctttcatctggcaccgttcttctttctctttggtctagtagtatcttcta (SEQ ID NO: 17) An06g00350 atggtggccttgctcacttcatttgcctgggcagggcttactgct (SEQ ID NO: 18) An16g01880 atgttctctggacggtttggagtgcttttgacggcgctcgctgcgctgagtgctgcg (SEQ ID NO: 19) An12g06560 atgtttgtttcctcgcttgctctattagcacttattgctcctttgatcgca (SEQ ID NO: 20) An09g02270 atgctgaatgcacgctccattgctctggcctcgttgccagttcttctcctactattcgcccagcaacttgcctct (SEQ ID NO: 21) An15g02210 atgaagggaatgctcccgacgcttagttggttggcactgggcatggccagcctggcaacctgc (SEQ ID NO: 22) An13g02820 tcacctgccgggagatcccagcgagcagacgaactcacgcttcggcgtagagtc (SEQ ID NO: 23) An11g00100 atgggcgtgatgcgcaagactgctgaccaggccttgggcctgaccgccaccgccctggctctggtcagtg ccgcccaggcc (SEQ ID NO: 24)

Example 2: Preparation of TLL expression vector

TLL encodes 291 amino acids and includes a 17-amino acid signal sequence and a 5-amino acid free sequence. The mature TLL is composed of 269 amino acids and has a molecular weight of 29.3 kD.

The TLL DNA that does not contain the signal sequence has a primer (TLLF: atgaggagctcccttgtgctg) of SEQ ID NO: 25 which does not have a restriction enzyme recognition site at the 5 'end of the sequence and a primer 26 primers (TLLNotR: gcggccgcctaaagacatgtcccaattaac) were chemically synthesized and polymerase chain reaction (PCR) was carried out using the two primers to obtain a 2145 bp fragment. PASP600TL, pASP603TLL, pASP605TLL, pASP606TLL, pASP607TLL, pASP608TLL, pASP609TLL, pASP610TLL, pASP611TLL, pASP612TLL were named as the sequence numbers by cutting the pASP600s vector with NaeI and NotI, respectively.

Example 3: Preparation of recombinant microorganism into which an expression vector was introduced and selection method of recombinant microorganism

The expression vector of Example 2 was introduced into Aspergillus oryzae and transformed (Tilburn et al., Gene. , 26: 205-221, 1983). For the transformation, the mycelia cultured in liquid phase were treated with cell wall degrading enzyme to make protoplast, and pASP600s DNA was inserted into the genome. For the selection of recombinant microorganisms, subculture was performed by selecting from agar medium supplemented with hygromycin.

Example 4: Flask culture of recombinant microorganism

Among the recombinant microorganisms of Example 3, the recombinant microorganisms resistant to hygromycin B are first inoculated by inoculation in the same agar medium. After 4 days, the recombinant microorganism spores are uniformly dispersed in agar medium and incubated at 30 ° C for 5-6 days until spores are uniformly formed.

Unspiked spores are harvested at 1 × 10 ⁶ cells / ml with 0.1% Tween 80 from the culture dishes cultured for 5 days, and this dilution is inoculated into 1 ml of complete medium. And cultured in a shaking incubator at 28 DEG C, 200 rpm for 4 days. The culture was centrifuged at 10,000 g for 10 minutes to remove the cells and the activity was confirmed in the recovered culture supernatant. Aspergillus of each recombinant vector or culture of this recombinant microorganism flask was carried out in each of 20 pieces.

Example 5: Measurement of activity of TLL enzyme

1U is the amount of 1 mole of butyric acid liberated enzyme per minute. 1% solution Tributyrin (13.5 ml), 2% calcium chloride and 1.0 ml of 1 M sodium chloride (NaCl) are added to the acacia solution to make the solution at pH 10. Add 21 ml of the above solution and 1 ml of the fermentation broth to the pH STAT (STAT), and react at 30 ° C for 15 minutes. Prepare the titration curve per minute for the consumption ml of 0.05 M NaOH solution added during the reaction.

U / ml = (Slope determination by titration in ml / min * DF * 0.05 / W

DF: dilution factor, the specified concentration (M) of 0.05-NaOH solution,

W: Amount of sample (g) contained in 1 ml of sample solution

An activity of up to 7 times higher in the lipase signal sequence was shown in Table 3 than in the TLL self-sequence (Table 3).

Identification of the activity of the expression vector Expression vector Signal sequence (SS) Active U / ml pASP43TLL TLL SS 850 pASP601TLL An03g06560 2.130 pASP602TLL An14g00860 5.029 pASP603TLL An07g00440 6.012 pASP604TLL An02g09690 1.020 pASP605TLL An16g08870 310 pASP606TLL An06g00350 5.050 pASP607TLL An16g01880 1.009 pASP608TLL An12g06560 3.251 pASP609TLL An09g02270 10 pASP610TLL An15g02210 2.669 pASP611TLL An13g02820 532 pASP612TLL An11g00100 9 Host cell - 11

As a result of SDS PAGE analysis, TLL molecular weight was 29.3 kD, but glycosylation revealed that the actual size was 31 kD. As a result, it was confirmed that the recombinant gene was secreted into the aspergillus or the outside by the aspergillus or the lipase signal sequences of the present invention.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Genofocus, Inc. <120> New Lipase Signal Sequences and Expression Method Using The Same <130> P14-B176 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> PRT <213> Aspergillus niger <400> 1 Met Ala Ile Met Lys Ala Leu Leu Trp Leu Ser Leu Ser Leu Ala Val   1 5 10 15 Trp Ala         <210> 2 <211> 17 <212> PRT <213> Aspergillus niger <400> 2 Met Ala Ser Ala Leu Leu Trp Leu Ser Leu Leu Gly Gly Ser Thr Leu   1 5 10 15 Ala     <210> 3 <211> 19 <212> PRT <213> Aspergillus niger <400> 3 Met Tyr Ile Pro Ser Val Leu Leu Leu Ala Ala Ser Leu Phe His Gly   1 5 10 15 Ala Thr Ala             <210> 4 <211> 17 <212> PRT <213> Aspergillus niger <400> 4 Met Leu Lys Leu Ala Val Ala Leu Phe Ser Leu Leu Ala Val Gly Asn   1 5 10 15 Ala     <210> 5 <211> 18 <212> PRT <213> Aspergillus niger <400> 5 Met Ile Phe Phe His Leu Ala Pro Phe Phe Phe Leu Phe Gly Leu Val   1 5 10 15 Val Ser         <210> 6 <211> 19 <212> PRT <213> Aspergillus niger <400> 6 Met Val Ala Leu Leu Thr Ser Phe Ala Trp Ala Gly Leu Thr Ala Leu   1 5 10 15 Ala Leu Gly             <210> 7 <211> 19 <212> PRT <213> Aspergillus niger <400> 7 Met Phe Ser Gly Arg Phe Gly Val Leu Leu Thr Ala Leu Ala Ala Leu   1 5 10 15 Ser Ala Ala             <210> 8 <211> 17 <212> PRT <213> Aspergillus niger <400> 8 Met Phe Val Ser Ser Leu Ala Leu Leu Ala Leu Ile Ala Pro Leu Ile   1 5 10 15 Ala     <210> 9 <211> 25 <212> PRT <213> Aspergillus niger <400> 9 Met Leu Asn Ala Arg Ser Ale Leu Ala Ser Leu Pro Val Leu Leu   1 5 10 15 Leu Leu Phe Ala Gln Gln Leu Ala Ser              20 25 <210> 10 <211> 21 <212> PRT <213> Aspergillus niger <400> 10 Met Lys Gly Met Leu Pro Thr Leu Ser Trp Leu Ala Leu Gly Met Ala   1 5 10 15 Ser Leu Ala Thr Cys              20 <210> 11 <211> 18 <212> PRT <213> Aspergillus niger <400> 11 Met Arg Leu Ser Ser Leu Ala Leu Ala Ser Ser Ala Ile Leu Pro Ala   1 5 10 15 Leu Gly         <210> 12 <211> 27 <212> PRT <213> Aspergillus niger <400> 12 Met Gly Val Met Arg Lys Thr Ala Asp Gln Ala Leu Gly Leu Thr Ala   1 5 10 15 Thr Ala Leu Ala Leu Val Ser Ala Ala Ala Gln Ala              20 25 <210> 13 <211> 54 <212> DNA <213> Aspergillus niger <400> 13 atgtggaacg ccgcggtatt ctgcaggatg aactcatatg tcccgttgcg gaaa 54 <210> 14 <211> 51 <212> DNA <213> Aspergillus niger <400> 14 atggcgagcg cactcctctg gctctccctg ctgggtggca gcaccctagc c 51 <210> 15 <211> 54 <212> DNA <213> Aspergillus niger <400> 15 atgtatatcc cctcggtgct gcttctggcc gcgagcctgt tccatggcgc aacg 54 <210> 16 <211> 51 <212> DNA <213> Aspergillus niger <400> 16 atgctaaagc tcgctgttgc tcttttttcg ttacttgccg tgggcaatgc a 51 <210> 17 <211> 58 <212> DNA <213> Aspergillus niger <400> 17 atgattttct ttcatctggc accgttcttc tttctctttg gtctagtagt atcttcta 58 <210> 18 <211> 45 <212> DNA <213> Aspergillus niger <400> 18 atggtggcct tgctcacttc atttgcctgg gcagggctta ctgct 45 <210> 19 <211> 57 <212> DNA <213> Aspergillus niger <400> 19 atgttctctg gacggtttgg agtgcttttg acggcgctcg ctgcgctgag tgctgcg 57 <210> 20 <211> 51 <212> DNA <213> Aspergillus niger <400> 20 atgtttgttt cctcgcttgc tctattagca cttattgctc ctttgatcgc a 51 <210> 21 <211> 75 <212> DNA <213> Aspergillus niger <400> 21 atgctgaatg cacgctccat tgctctggcc tcgttgccag ttcttctcct actattcgcc 60 cagcaacttg cctct 75 <210> 22 <211> 63 <212> DNA <213> Aspergillus niger <400> 22 atgaagggaa tgctcccgac gcttagttgg ttggcactgg gcatggccag cctggcaacc 60 tgc 63 <210> 23 <211> 54 <212> DNA <213> Aspergillus niger <400> 23 tcacctgccg ggagatccca gcgagcagac gaactcacgc ttcggcgtag agtc 54 <210> 24 <211> 81 <212> DNA <213> Aspergillus niger <400> 24 atgggcgtga tgcgcaagac tgctgaccag gccttgggcc tgaccgccac cgccctggct 60 ctggtcagtg ccgcccaggc c 81 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TLLF <400> 25 atgaggagct cccttgtgct g 21 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TLLNotR <400> 26 gcggccgcct aaagacatgt cccaattaac 30

Claims (14)

delete delete delete An expression vector for expression of lipase comprising a nucleic acid encoding a lipase signal sequence An07g00440 represented by the amino acid sequence of SEQ ID NO: 3, a gene encoding lipase, and a glucoamylase promoter.
delete 5. The expression vector according to claim 4, wherein the lipase is lipase (TLL) derived from Thermomyces lanuginosus .
delete 5. The expression vector according to claim 4, wherein the glucoamylase promoter is an Aspergillus oryzae glucoamylase (glaA) promoter.
delete A recombinant microorganism into which the expression vector of any one of claims 4, 6, and 8 has been introduced.
11. The recombinant microorganism according to claim 10, which is Aspergillus niger .
delete A method of producing a lipase comprising the steps of:
(a) culturing the recombinant microorganism of claim 10 to produce a lipase; And
(b) separating the resulting lipase. delete

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