CN113755509A

CN113755509A - Lysophospholipase variant, construction method thereof and expression in aspergillus niger strain

Info

Publication number: CN113755509A
Application number: CN202111183145.6A
Authority: CN
Inventors: 赵正阳
Original assignee: Nanjing Zhengyang Biotechnology Co ltd
Current assignee: Nanjing Zhengyang Biotechnology Co ltd
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2021-12-07

Abstract

The invention discloses a sequence of lysophospholipase variant. The invention constructs a recombinant expression cassette containing a lysophospholipase variant sequence by a method for constructing a recombinant Aspergillus niger expression strain, wherein the recombinant expression cassette is a gene fragment containing elements such as a lysophospholipase variant gene sequence, a promoter, a terminator, a screening marker, upstream and downstream homologous sequences and the like. The expression cassette of the lysophospholipase variant sequence is constructed by means of genetic engineering and is introduced into an Aspergillus niger expression host bacterium, so that the high-efficiency secretory expression of the lysophospholipase variant is realized. Compared with the Aspergillus niger recombinant expression strain using the original lysophospholipase sequence before change, the Aspergillus niger recombinant expression strain using the lysophospholipase variant sequence constructed by the method of the invention has the advantages that the enzyme activity of the supernatant is improved by 20.7 percent and the specific enzyme activity per milligram of crude protein is improved by 21.12 percent under the same culture condition.

Description

Lysophospholipase variant, construction method thereof and expression in aspergillus niger strain

Technical Field

The invention belongs to the field of genetic engineering breeding, and relates to a polypeptide which can be expressed in a large amount in Aspergillus niger host bacteria and has higher lysophospholipase specific enzyme activity, and construction and application of an Aspergillus niger recombinant expression strain thereof.

Background

Lysophospholipases are widely used in a variety of industries. However, its relatively high price still limits its wider application. Lysophospholipase is produced essentially by means of microbial fermentation. And the improvement of the expression quantity of the strain in the microbial fermentation process is an excellent mode for improving the industrial production and reducing the industrial production price.

Disclosure of Invention

The invention provides a polypeptide sequence with lysophospholipase activity, which is expressed in a corresponding Aspergillus niger host by using a patent method applied by the company before, and the activity of supernatant and the molecular specific enzyme activity of the polypeptide sequence are respectively improved by 20.7 percent and 21.12 percent compared with the expression of wild-type Aspergillus niger-derived lysophospholipase in the Aspergillus niger host.

The invention provides a sequence of lysophospholipase variant and a method for constructing an Aspergillus niger recombinant expression strain thereof.

The recombinant expression strain can be constructed by a genetic engineering method, and is specifically described as follows:

a method for constructing recombinant Aspergillus niger expression bacteria is to construct a recombinant expression cassette containing a lysophospholipase variant gene sequence, wherein the recombinant expression cassette is a gene fragment containing lysophospholipase variant gene sequence, a promoter, a terminator, a signal peptide, a screening marker and other elements.

The lysophospholipase variant gene has a nucleotide sequence shown in SEQ ID NO. 1; the host cell is Aspergillus niger.

The promoter may be an aspergillus niger endogenous promoter: such as Aspergillus niger glucoamylase promoter, neutral amylase promoter, acid amylase promoter, alpha-glucosidase promoter, etc.; it may also be an exogenous promoter: such as Aspergillus oryzae neutral amylase promoter, Rhizopus oryzae glucoamylase promoter; aspergillus niger glucoamylase promoter or Aspergillus niger neutral amylase promoter is preferred in the present invention.

Linked to the 3' end of the promoter may be regulatory sequences: such as a suitable leader sequence (5' UTR), a nontranslated region of an mRNA that is important for translation by the host cell, such as the Aspergillus oryzae neutral amylase and Aspergillus nidulans triose phosphate isomerase leader sequences.

For secretory expression of a particular protein, signal peptide sequence mediation is required, and commonly used signal peptide sequences in Aspergillus niger are the glucoamylase signal peptide, acid amylase signal peptide, Aspergillus niger phytase signal peptide, Aspergillus oryzae TAKA amylase signal peptide, and in the present invention, the signal peptide encoded by the lysophospholipase variant gene sequence itself is utilized.

Preferred terminators are obtained from the genes for the following enzymes: aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

The specific gene is linked to a promoter, a regulatory sequence, a signal peptide sequence and a terminator to form an expression cassette. Can be introduced into the A.niger genome by conventional methods, either randomly inserted into the genome or site-directed integration at one or more loci. Alternative loci are gla (glucoamylase), amya (neutral amylase), amyb (neutral amylase), aa (acid amylase), agda (alpha glucosidase), agdb (alpha glucosidase).

The expression cassette may preferably be linked to one or more selectable markers which allow for easy selection of transformed, transfected, transduced, or the like cells or strains. Selectable markers are genes whose products provide biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs (prototrophy to autotrophs), and the like. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (glufosinate) acetyltransferase, hyg (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase) and equivalents thereof. Preferred for use in an Aspergillus cell is the amdS or hyg of Aspergillus nidulans (Aspergillus nidulans) or Aspergillus oryzae.

The expression cassette may preferably be linked to one or more counter-selectable markers (negative selection markers). Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), pyrG (orotidine-5' -phosphate decarboxylase), hsvTK (herpes simplex virus thymidine kinase).

The expression cassette is introduced into the genome of a host Aspergillus niger by a conventional method and randomly inserted into the genome of the host Aspergillus niger or site-specific integrated into one or more loci of the host Aspergillus niger.

The gene locus is selected from glucoamylase gla, neutral amylase amya, neutral amylase amyb, acid amylase aa, alpha-glucosidase agda and alpha-glucosidase agdb.

The host is Aspergillus niger with the glucoamylase gene, the fungal amylase gene and the acid amylase gene being knocked out.

The invention has the beneficial effects that:

the invention provides a gene sequence of lysophospholipase variant and a polypeptide sequence thereof, and provides an expression method of the lysophospholipase variant in Aspergillus niger. By expressing the variant sequence, the high-yield lysophospholipase variant Aspergillus niger expression strain is obtained. Compared with the strain expressing the lysophospholipase original sequence, under the same condition, the activity of the supernatant and the molecular specific enzyme activity are respectively improved by 20.7 percent and 21.12 percent compared with the expression of the wild type aspergillus niger derived lysophospholipase in an aspergillus niger host.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing comparison of enzyme activities.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Examples

Construction of p-ZYAN02-LPPL-T plasmid

The method mainly comprises the following two steps: 1. an intermediate plasmid p-ZYAN01 was prepared. 2. Linearizing intermediate plasmid p-ZYAN01, and integrating lysophospholipase variant gene expression cassette and upstream and downstream homologous fragments into p-ZYAN01 to form p-ZYAN02-LPPL-T plasmid

The intermediate plasmid p-ZYAN01 was prepared as follows:

the p-ZYAN01 is mainly composed of the following parts and necessary connecting sequences, or is composed of the following parts directly.

(1) A 2305bp fragment obtained after double digestion of pUC57 plasmid XbaI-PscI;

(2) the sequence of the hyg gene expression cassette is shown in SEQ ID NO. 5;

(3) amds expression cassette, sequence shown in SEQ ID No. 6.

The 2305bp fragment obtained by double digestion of pUC57 plasmid XbaI-PciI, the hyg gene expression cassette, and the amds expression cassette, the three gene fragment sequence fragments were recombined by Gibson Master Mix Kit (E2611, New England Biolabs) to obtain recombinant plasmid p-ZYAN 01. Expression of the lysophospholipase variant expression cassette is integrated into the aspergillus niger glucoamylase locus for expression using a glucoamylase promoter and a glucoamylase terminator. Construction of lysophospholipase variant integral expression plasmid p-ZYAN 02-LPPL-T. The construction method of the integration plasmid is as follows: linearizing the p-ZYAN01 plasmid by a method commonly used in the industry; the Aspergillus niger gene fragment SEQ ID NO.7 is taken as a 5 'end homologous fragment, the Aspergillus niger gene fragment SEQ ID NO.8 is taken as a 3' end homologous fragment, and each fragment is 2000bp long. The linearized p-ZYAN01 vector, the homologous fragment and the lysophospholipase variant expression cassette fragment were recombined by GibsoMaster Mix Kit (E2611, New England Biolabs) to obtain an integration plasmid p-ZYAN02-LPPL-T, which contains the lysophospholipase variant expression cassette containing the Aspergillus niger glucoamylase promoter sequence, the Candida antarctica-derived lysophospholipase variant sequence and the Aspergillus niger glucoamylase terminator sequence, and the sequences were confirmed by sequencing.

Integration of lysophospholipase variant expression cassettes by transformation

The starting strain in this example is ZYAN05, which is obtained by knocking out glucoamylase gene, fungal amylase gene and acid amylase gene from conventional strains. The gene knockout/knock-in method in Aspergillus niger can be realized by referring to the technical method disclosed in the patent C N103937766A or CN 104962594A example. I.e., the method described with reference to Delmas (apple Environ Microbiol.2014,80(11):3484-7), et al. Specifically, circular DNA vectors are used, which contain the 5 'and 3' homologous sequences, a selectable marker, and an E.coli replication sequence. The circular vector is transferred into Aspergillus niger, and a recombinant strain is obtained by selection.

The protoplast transformation method is adopted to introduce the p-ZYAN02-LPPL-T plasmid into the Aspergillus niger strain ZYAN05, and the specific operation steps are as follows: preparation of protoplast: aspergillus niger mycelia were cultured in a nutrient-rich TZ liquid medium (beef extract powder 0.8%, yeast extract 0.2%, peptone 0.5%, NaCl 0.2%, sucrose 3%, pH 5.8). The mycelium was filtered from the culture broth by mira-cloth (LPPL-Tiochem Co.) and washed with 0.7M NaCl (pH5.8), and after draining the mycelium was transferred to an enzymatic hydrolysate (pH5.8) containing 1% cellulase (Sigma), 1% helicase (Sigma) and 0.2% lywallzyme (Sigma) and enzymatically hydrolyzed at 30 ℃ and 65rpm for 3 hours. Then putting the enzymatic hydrolysate containing the protoplast on ice, filtering the enzymatic hydrolysate by using four layers of mirror paper, carrying out mild centrifugation on the obtained filtrate at 3000rpm and 4 ℃ for 10min, and then removing the supernatant; the protoplasts attached to the vessel wall were washed once with STC solution (1M DSorbitol, 50mM CaCl2, 10mM Tris, pH7.5) and finally the protoplasts were resuspended in the appropriate amount of STC solution.

Adding 10 μ l (concentration: 100ng/μ l) of circular p-ZYAN02-LPPL-T plasmid into 100 μ l of protoplast suspension, mixing, and standing at room temperature for 25 min; then adding 900 mul PEG solution into the mixture for 3 times, mixing evenly, and standing for 25min at room temperature; centrifuging at 3000rpm for 10min at normal temperature, discarding the supernatant, attaching protoplasts to the tube wall, and resuspending them in 1ml of STC solution. Mixing the suspension with a culture medium (acetamide 0.3%, sucrose 20%, agar 0.7%) previously cooled to about 45 deg.C, and plating; after the flat plate is solidified, putting the flat plate into an incubator at 34 ℃ for culture; after 24h, a layer of solid medium (agar 1% and the rest as above) containing 300 ng/. mu.l Hygromycin (Hygromycin) was spread on the plate, and the plate was further placed in an incubator at 34 ℃ for 4-5 days, after which transformants that had grown in the upper medium were called integrative transformants. Randomly selecting several integrative transformants, respectively subculturing the integrative transformants on a solid culture medium containing 300 ng/mu l of Hygromycin (Hygromycin), after culturing for 3 days at a constant temperature of 34 ℃, collecting mycelia, freezing the mycelia by using liquid nitrogen, grinding and crushing the mycelia, then extracting the genomic DNA of the integrative transformants by using a fungus genome extraction kit (Boy science and technology, Ltd. in Hangzhou), and finally performing PCR identification on the genomic DNA of the integrative transformants. Integration into the glucoamylase locus was confirmed by sequencing of the PCR product. And (4) sequencing the positive transformant by using a PCR product and then confirming to obtain a recombinant expression strain.

Liquid fermentation production of lysophospholipase variant by recombinant expression strain

Slant culture: inoculating one strain of the Aspergillus niger recombinant expression strain to a PDA solid inclined plane, and culturing at constant temperature of 35 ℃ for 60 h; and (3) shake flask culture: inoculating one strain of the strain obtained by slant culture into seed culture medium, and culturing at initial pH of 5.5 and 35 deg.C and shaking table rotation speed of 200rpm for about 110 h. The slant culture medium is as follows: 20g of sucrose, 32 g of NaNO, 40.5 g of MgSO40, 0.5g of KCl, 40.01g of FeSO40, 41 g of K2HPO and 20g of agar, and the components are dissolved in 1000mL of water, and the pH is adjusted to 5.5,121 ℃ for sterilization for 20min for later use. The shake flask seed culture medium is as follows: 200mL of wort and 5g of bean cake powder, adjusting the pH value to 5.5,121 ℃, and sterilizing for 20min for later use.

The following table shows the fermentation of 2 batches in a 250ml shake flask, and a comparison with the original lysophospholipase fermentation.

Table 1: comparison of the enzyme production activity of lysophospholipase original sequence and lysophospholipase variant sequence in the same host and under the same culture conditions

According to the content, compared with the original lysophospholipase gene, the lysophospholipase variant gene provided by the invention has 21.12% higher molecular specific enzyme activity and 20.7% higher enzyme activity of fermentation supernatant (figure 1)

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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tgccgcggtt ctgcagcttt ccttggcccg taaaattcgg cgaagccagc caatcaccag 480

ctaggcacca gctaaaccct ataattagtc tcttatcaac accatccgct cccccgggat 540

caatgaggag aatgaggggg atgcggggct aaagaagcct acataaccct catgccaact 600

cccagtttac actcgtcgag ccaacatcct gactataagc taacacagaa tgcctcaatc 660

ctgggaagaa ctggccgctg ataagcgcgc ccgcctcgca aaaaccatcc ctgatgaatg 720

gaaagtccag acgctgcctg cggaagacag cgttattgat ttcccaaaga aatcggggat 780

cctttcagag gccgaactga agatcacaga ggcctccgct gcagatcttg tgtccaagct 840

ggcggccgga gagttgacct cggtggaagt tacgctagca ttctgtaaac gggcagcaat 900

cgcccagcag ttagtagggt cccctctacc tctcagggag atgtaacaac gccaccttat 960

gggactatca agctgacgct ggcttctgtg cagacaaact gcgcccacga gttcttccct 1020

gacgccgctc tcgcgcaggc aagggaactc gatgaatact acgcaaagca caagagaccc 1080

gttggtccac tccatggcct ccccatctct ctcaaagacc agcttcgagt caaggtacac 1140

cgttgcccct aagtcgttag atgtcccttt ttgtcagcta acatatgcca ccagggctac 1200

gaaacatcaa tgggctacat ctcatggcta aacaagtacg acgaagggga ctcggttctg 1260

acaaccatgc tccgcaaagc cggtgccgtc ttctacgtca agacctctgt cccgcagacc 1320

ctgatggtct gcgagacagt caacaacatc atcgggcgca ccgtcaaccc acgcaacaag 1380

aactggtcgt gcggcggcag ttctggtggt gagggtgcga tcgttgggat tcgtggtggc 1440

gtcatcggtg taggaacgga tatcggtggc tcgattcgag tgccggccgc gttcaacttc 1500

ctgtacggtc taaggccgag tcatgggcgg ctgccgtatg caaagatggc gaacagcatg 1560

gagggtcagg agacggtgca cagcgttgtc gggccgatta cgcactctgt tgagggtgag 1620

tccttcgcct cttccttctt ttcctgctct ataccaggcc tccactgtcc tcctttcttg 1680

ctttttatac tatatacgag accggcagtc actgatgaag tatgttagac ctccgcctct 1740

tcaccaaatc cgtcctcggt caggagccat ggaaatacga ctccaaggtc atccccatgc 1800

cctggcgcca gtccgagtcg gacattattg cctccaagat caagaacggc gggctcaata 1860

tcggctacta caacttcgac ggcaatgtcc ttccacaccc tcctatcctg cgcggcgtgg 1920

aaaccaccgt cgccgcactc gccaaagccg gtcacaccgt gaccccgtgg acgccataca 1980

agcacgattt cggccacgat ctcatctccc atatctacgc ggctgacggc agcgccgacg 2040

taatgcgcga tatcagtgca tccggcgagc cggcgattcc aaatatcaaa gacctactga 2100

acccgaacat caaagctgtt aacatgaacg agctctggga cacgcatctc cagaagtgga 2160

attaccagat ggagtacctt gagaaatggc gggaggctga agaaaaggcc gggaaggaac 2220

tggacgccat catcgcgccg attacgccta ccgctgcggt acggcatgac cagttccggt 2280

actatgggta tgcctctgtg atcaacctgc tggatttcac gagcgtggtt gttccggtta 2340

cctttgcgga taagaacatc gataagaaga atgagagttt caaggcggtt agtgagcttg 2400

atgccctcgt gcaggaagag tatgatccgg aggcgtacca tggggcaccg gttgcagtgc 2460

aggttatcgg acggagactc agtgaagaga ggacgttggc gattgcagag gaagtgggga 2520

agttgctggg aaatgtggtg actccatagc taataagtgt cagatagcaa tttgcacaag 2580

aaatcaatac cagcaactgt aaataagcgc tgaagtgacc atgccatgct acgaaagagc 2640

agaaaaaaac ctgccgtaga accgaagaga tatgacacgc ttccatctct caaaggaaga 2700

atcccttcag ggttgcgttt ccag 2724

<210> 7

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctaccaatgc tctcgaggat tgcctgaaca ttgacattcg gcgtccggcc gggaccaccg 60

cggactcgaa gctgcctgtg ctggtctgga tctttggcgg aggctttgaa cttggttcaa 120

aggcgatgta tgatggtaca acgatggtat catcgtcgat agacaagaac atgcctatcg 180

tgtttgtagc aatgaattat cgcgtgggag gtttcgggtt cttgcccgga aaggagatcc 240

tggaggacgg gtccgcgaac ctagggctcc tggaccaacg ccttgccctg cagtgggttg 300

ccgacaacat cgaggccttt ggtggagacc cggacaaggt gacgatttgg ggagaatcag 360

caggagccat ttccgttttt gatcagatga tcttgtacga cggaaacatc acttacaagg 420

ataagccctt gttccggggg gccatcatgg actccggtag tgttgttccc gcagaccccg 480

tcgatggggt caagggacag caagtatatg atgcggtagt ggaatctgca ggctgttcct 540

cttctaacga caccctagct tgtctgcgtg aactagacta caccgacttc ctcaatgcgg 600

caaactccgt gccaggcatt ttaagctacc attctgtggc gttatcatat gtgcctcgac 660

cggacgggac ggcgttgtcg gcatcaccgg acgttttggg caaagcaggg aaatatgctc 720

gggtcccgtt catcgtgggc gaccaagagg atgaggggac cttattcgcc ttgtttcagt 780

ccaacattac gacgatcgac gaggtggtcg actacctggc ctcatacttc ttctatgacg 840

ctagccgaga gcagcttgaa gaactagtgg ccctgtaccc agacaccacc acgtacgggt 900

ctccgttcag gacaggcgcg gccaacaact ggtatccgca atttaagcga ttggccgcca 960

ttctcggcga cttggtcttc accattaccc ggcgggcatt cctctcgtat gcagaggaaa 1020

tctcccctga tcttccgaac tggtcgtacc tggcgaccta tgactatggc accccagttc 1080

tggggacctt ccacggaagt gacctgctgc aggtgttcta tgggatcaag ccaaactatg 1140

cagctagttc tagccacacg tactatctga gctttgtgta tacgctggat ccgaactcca 1200

accgggggga gtacattgag tggccgcagt ggaaggaatc gcggcagttg atgaatttcg 1260

gagcgaacga cgccagtctc cttacggatg atttccgcaa cgggacatat gagttcatcc 1320

tgcagaatac cgcggcgttc cacatctgat gccattggcg gaggggtccg gacggtcagg 1380

aacttagcct tatgagatga atgatggacg tgtctggcct cggaaaagga tatatgggga 1440

tcatgatagt actagccata ttaatgaagg gcatatacca cgcgttggac ctgcgttata 1500

gcttcccgtt agttatagta ccatcgttat accagccaat caagtcacca cgcacgaccg 1560

gggacggcga atccccggga attgaaagaa attgcatccc aggccagtga ggccagcgat 1620

tggccacctc tccaaggcac agggccattc tgcagcgctg gtggattcat cgcaatttcc 1680

cccggcccgg cccgacaccg ctataggctg gttctcccac accatcggag attcgtcgcc 1740

taatgtctcg tccgttcaca agctgaagag cttgaagtgg cgagatgtct ctgcaggaat 1800

tcaagctaga tgctaagcga tattgcatgg caatatgtgt tgatgcatgt gcttcttcct 1860

tcagcttccc ctcgtgcaga tgaggtttgg ctataaattg aagtggttgg tcggggttcc 1920

gtgaggggct gaagtgcttc ctccctttta gacgcaactg agagcctgag cttcatcccc 1980

agcatcatta cacctcagca 2000

<210> 8

<211> 2000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acaatcaatc catttcgcta tagttaaagg atggggatga gggcaattgg ttatatgatc 60

atgtatgtag tgggtgtgca taatagtagt gaaatggaag ccaagtcatg tgattgtaat 120

cgaccgacgg aattgaggat atccggaaat acagacaccg tgaaagccat ggtctttcct 180

tcgtgtagaa gaccagacag acagtccctg atttaccctt gcacaaagca ctagaaaatt 240

agcattccat ccttctctgc ttgctctgct gatatcactg tcattcaatg catagccatg 300

agctcatctt agatccaagc acgtaattcc atagccgagg tccacagtgg agcagcaaca 360

ttccccatca ttgctttccc caggggcctc ccaacgacta aatcaagagt atatctctac 420

cgtccaatag atcgtcttcg cttcaaaatc tttgacaatt ccaagagggt ccccatccat 480

caaacccagt tcaataatag ccgagatgca tggtggagtc aattaggcag tattgctgga 540

atgtcggggc cagttggccc ggtggtcatt ggccgcctgt gatgccatct gccactaaat 600

ccgatcattg atccaccgcc cacgaggcgc gtctttgctt tttgcgcggc gtccaggttc 660

aactctctct gcagctccag tccaacgctg actgactagt ttacctactg gtctgatcgg 720

ctccatcaga gctatggcgt tatcccgtgc cgttgctgcg caatcgctat cttgatcgca 780

accttgaact cactcttgtt ttaatagtga tcttggtgac ggagtgtcgg tgagtgacaa 840

ccaacatcgt gcaagggaga ttgatacgga attgtcgctc ccatcatgat gttcttgccg 900

gctttgttgg ccctattcgt gggatgcgat gccctcgctg tgcagcagca ggtactgctg 960

gatgaggagc catcggtctc tgcacgcaaa cccaacttcc tcttcattct cacggatgat 1020

caggatctcc ggatgaattc tccggcgtat atgccgtata cgcaggcgag aatcaaggaa 1080

aagggtaccg agttcttgaa ccatttcgtc actaccgcgc tttgctgtcc gtcgcgcgtg 1140

agtctttgga cgggaagaca ggctcataat actaatgtga cggatgtgaa cccgccttat 1200

ggtatggaca ctgcttcgat cggtcttgat tcttcagcgt ggttacaatt gctaatgcgg 1260

cataggcgga taccccaaat tcgtcgctca aggcttcaac gaaaacttcc tccccgtttg 1320

gctgcagtcc gccggttaca atacctacta cacggggaag ctgttcaact cgcacagtgt 1380

cgctacctat aacgcgccct ttgtgaacgg tttcaatggc tccgacttcc tcctcgaccc 1440

ccacacatat tcctactgga atgcgacata ccagcgaaac catgagcctc cgcggagtta 1500

cgagggacaa tatactacgg atgtgatgaa ggagaaggca tcgggattgt tggcagatgc 1560

gctggacagt gacgcgccat tcttcctgac ggtcgcgccg atcgcaccgc acacgaacat 1620

cgatgtggag gggctgagcg gtgcgggtgg accgaagatg acagagccgc tgcctgcacc 1680

gagacatgcg catttgtttg ctgatgcaaa ggtgccgcgg acgcctaatt tcaatccgga 1740

caaggtgtgt gatatcctga cacagtggtg gggacgggca ctgacaagag taggattctg 1800

gtgcggggtg gatccaaacc atggaactac agaaccagac cgtcatcgac tacgaagacc 1860

atctttatcg ccagcgtctg cgcactttgc aagccgtcga tgagatggtg gatgcgctga 1920

tcacgcagct ggaagaaagt gggcagatcg acaataccta catcatttac agtgctgata 1980

acggctacca cattggccat 2000

Claims

1. A variant lysophospholipase sequence based on a lysophospholipase from aspergillus.

2. The sequence of claim 1, wherein the nucleotide sequence is shown as SEQ ID NO.1, and other sequences having 90% or more homology with SEQ ID NO. 1.

3. The sequence of claim 1, wherein the amino acid sequence is shown in SEQ ID NO.2, and other sequences having homology of 90% or more with SEQ ID NO. 2.

4. The sequence of claim 1, wherein the signal peptide is derived from lysophospholipase from Aspergillus niger when expressed in a host bacterium, and the nucleotide sequence is shown in SEQ ID NO.3, and other sequences having homology of 90% or more with SEQ ID NO. 3.

5. The sequence of claim 4, wherein the amino acid sequence is shown in SEQ ID NO.4, and other sequences having 90% or more homology to SEQ ID NO. 4.

6. The method of claim 1, wherein the construction is carried out using Aspergillus niger as a host, from which the glucoamylase gene, the fungal amylase gene and the acid amylase gene are deleted.

7. The method of claim 6, wherein the promoter used in the strain construction process is selected from the group consisting of an Aspergillus niger endogenous promoter or an exogenous promoter; the Aspergillus niger endogenous promoter is an Aspergillus niger glucoamylase promoter, a neutral amylase promoter, an acid amylase promoter or an alpha-glucosidase promoter; the exogenous promoter is an aspergillus oryzae neutral amylase promoter or a rhizopus saccharidase promoter; the control sequence connected with the 3 'end of the promoter is a leader sequence 5' UT.

8. The method according to claim 7, wherein the terminator is obtained from the following genes for enzymes: aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, or Fusarium oxysporum trypsin-like protease.

9. The method of construction according to claim 7, wherein the selectable marker element is selected from the group consisting of a selectable marker and/or a counter-selectable marker; said selectable marker is selected from the group consisting of acetamidase amdS, ornithine carbamoyltransferase argB, glufosinate bar, acetyltransferase, hygromycin phosphotransferase hyg, nitrate reductase niaD, orotidine-5' -phosphate decarboxylase pyrG, sulfate adenyltransferase sC, anthranilate synthase trpC, or equivalents thereof, preferably used in a cell of Aspergillus is amdS, hyg of Aspergillus nidulans or Aspergillus oryzae; the counter selection marker is selected from the group consisting of a selectable marker for filamentous fungal host cells or hsvTK, preferably acetamidase amdS, orotidine-5' -phosphate decarboxylase pyrG.

10. An expression cassette for a lysophospholipase variant gene, wherein the lysophospholipase variant sequence of claim 1 is introduced by conventional methods, randomly inserted into the genome of a host A.niger, or site-directed integration into one or more loci of a host A.niger; the locus is selected from any one of glucoamylase gla, neutral amylase amya, neutral amylase amyb, acid amylase aa, alpha glucosidase agda or alpha glucosidase agdb.