Mutant lysophospholipase and mutant Aspergillus niger strains for expressing the enzyme

Abstract

A mutant lysophospholipase is provided. A gene encoding the lysophospholipase, as well as a vector and a host cell comprising the gene, are further provided. Furthermore, a use of the lysophospholipase is further provided. The present invention also provides a mutant Aspergillus niger strain and a use thereof. In particular, the present invention provides a mutant Aspergillus niger strain that is an orotate phosphoribosyltransferase auxotroph and has improved ability to produce an endogenous enzyme, preferably a lysophospholipase, compared to a non-mutant strain. The present invention further provides a use of the strain. For example, the strain can be used to express the mutant lysophospholipase of the present invention.

Description

[Technical field]

[0001] Field The present invention relates to the field of biotechnology, more specifically to a mutant lysophospholipase. The present invention also relates to a gene encoding said enzyme, as well as a vector and a host cell containing said gene. Furthermore, the present invention also relates to the use of said enzyme.

[0002] The present application also relates to mutant Aspergillus niger strains that can be used to express mutant lysophospholipases of the invention. The present invention also relates to methods of using said strains to produce exogenous proteins, in particular lysophospholipases of the invention. [Background technology]

[0003] background Aspergillus niger is a common fungus belonging to the subphylum Ascomycota, order Moniliales (Moniliacea), and genus Aspergillus. It is also the most abundant filamentous fungus in nature, and is widely distributed in plant products, food, and soil. Its conidial heads are dark brown radial, spherical apical capsules. Its conidiophores vary in length, and its hyphae are well developed and multi-branched. Aspergillus niger can grow rapidly by decomposing natural organic matter and absorbing its nutrients. Aspergillus niger is classified as GRAS (Generally Regarded as Safe) by the U.S. Food and Drug Administration (FDA) and recognized by the World Health Organization. It is a very commonly used species among filamentous fungi. Aspergillus niger has become an important strain for industrial production due to its ability to produce enzyme preparations and organic acids.

[0004] Aspergillus niger has efficient protein secretion and expression capabilities. Foreign proteins expressed by Aspergillus niger have the characteristics of high expression amount, high extracellular secretion rate, and protein molecular folding and modification system close to that of higher eukaryotic cells, and the expressed foreign proteins have native activity. In addition, Aspergillus niger can also carry out various post-translational processes such as glucosylation modification, protease cleavage and disulfide bond formation. Therefore, Aspergillus niger as an expression strain for expressing homologous and heterologous proteins has attracted increasing attention and is attractive. Currently, commercial enzyme preparations produced using Aspergillus niger include amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase and xylanase, etc., which are used in the food, detergent, textile and paper industries. Heterologous proteins expressed by it include lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, etc.

[0005] Enzyme preparations used in food in China must meet the People's Republic of China National Standard GB2760 - Food National Standard / Food Additives Use Standard. Among them, heterologously expressed lysophospholipase (from Aspergillus niger) for food to meet the regulation can only be heterologously expressed from Aspergillus niger.

[0006] Two kinds of lipases, designated lipase A and lipase B or lipase 1 and lipase 2, have been reported in Aspergillus niger. Among them, lipase B has special properties. Zhu Shu-sen cloned and expressed lipase B from Aspergillus niger A733 and found that the optimum temperature of lipase B is 15°C, the optimum pH is 3.5-4.0, and it cannot tolerate temperatures above 40°C. The enzyme can hydrolyze substrates with chain lengths of pNPC4-pNPC18, with pNPC12 being the optimum substrate. However, the specific enzyme activity of lipB is extremely low, and the specific enzyme activity of purified lipB is only 6.8U / mg.

[0007] Jiangke Yang et al. have cloned and expressed lipas2 from Aspergillus niger CICC 4009. This lipase2 is highly homologous to lipase B cloned by Zhu Shu-sen, with only two amino acid differences, but with certain differences in properties. The optimal substrates are pNPC8 and pNPC10, the optimal pH is below 6.5, the optimal temperature is 50°C, and it cannot tolerate temperatures above 40°C.

[0008] In summary, lipase 2 or lipase B from Aspergillus niger are not very practical. First of all, the specific enzymatic activity is very low and the temperature tolerance is not high. The most suitable substrates are triglycerides of short chain fatty acids. The enzymes are not as good as the more widely used lipases TL and RML, which have specific enzymatic activities of 12000 U / mg and 8000 U / mg, respectively. TL can withstand a temperature of 60°C for 20 hours without inactivation, and RML can hydrolyze triglycerides of fatty acids of various chain lengths.

[0009] In the present application CN2021107079194, the inventors used the gene sequence of lipase B of CBS513.88 from Aspergillus niger GIM 3.24 (AN02) to design and clone the primers of the LPL gene AN02-LPL of the above Aspergillus niger strain, and found that AN02-LPL has extremely high phospholipase A1 activity and lysophospholipase activity and can be used for oil degumming. It can reduce the phosphorus content of crude oil to 5 ppm without adding alkali. It can be used for enzymatic degumming, and can reduce the formation of soap during degumming because there is no need to add alkali. In addition, since AN02-LPL has extremely high lysophospholipase activity, it can be used in combination with phospholipase A2 to use soybean phospholipase as a raw material for producing glycerol phospholipase (GPC), which has brain-healthy and anti-aging effects and is used in medicines and health care products.

[0010] In CN2021107079194, the inventors obtained LPL encoded by AN02 mutant picAN02m1 through random mutation. The mutation sites are L86I, G187D, E209K and A245D. The thermostability is 3.2-fold, 31-fold and 28.5-fold higher than that of wild-type AN02-LPL under pH 5.6, 6.0 and 6.6, respectively. Summary of the Invention [Problem to be solved by the invention]

[0011] There remains a need in the art to obtain lysophospholipases with high specific enzymatic activity.Furthermore, there remains a need in the art for Aspergillus niger strains that can efficiently express various proteins for food products, especially lysophospholipases. [Means for solving the problem]

[0012] Summary of the Invention Based on previous research and the mutant lysophospholipase developed in invention CN2021107079194, the inventors developed a novel mutant. In particular, after mutating threonine at position 255 of LPL to tryptophan or phenylalanine based on picAN02m1, the specific enzyme activity of lysophospholipase was increased from 12746U / mg and 13473U / mg of LPL encoded by picAN02 and picAN02m1, respectively, to 30224U / mg and 18973U / mg. This further improves the utilization efficiency of lysophospholipase to produce glycerol phosphorylcholine (GPC).

[0013] Furthermore, the present invention used Aspergillus niger CICC2243 as the starting strain to construct an orotate phosphoribosyltransferase auxotroph (pyrE-), and then screened the strain through ARTP mutagenesis, which had 10.8-fold increased lysophospholipase (LPL) enzyme activity, and had a significantly larger sedimentation circle compared to the starting strain on lysophospholipase screening plates supplemented with uracil. Recombinant expression of lysophospholipase LPL was carried out with it, and the expression ability was found to be significantly increased, increasing by 112% compared to the starting strain. This strain can be used to efficiently heterologously express various proteins for food, especially lysophospholipase.

[0014] In particular, the present invention relates to the following aspects: In one embodiment, the present invention relates to a lysophospholipase comprising the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16.

[0015] In another aspect, the present invention relates to a nucleic acid molecule comprising, partially or completely: (a) a nucleotide sequence encoding a lysophospholipase as described above; and (b) a nucleotide sequence complementary to the nucleotide sequence described in (a).

[0016] Those skilled in the art will recognize that due to the degeneracy of the genetic code, multiple different nucleotide sequences can encode the same enzyme. It will further be recognized that, using routine techniques, one skilled in the art can make nucleotide substitutions that do not affect the activity of the enzyme encoded by the nucleotide sequence of the invention, and thus reflect the codon bias of any particular host organism in which the enzyme of the invention is expressed.

[0017] The invention also provides a vector comprising said nucleic acid molecule and a host cell comprising said nucleic acid molecule or said vector.

[0018] "Vector" refers to an extrachromosomal element that usually carries genes, which are not part of the central metabolism of the cell and are often in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genomic integrating sequences, phage or nucleotide sequences from any source, linear or circular single- or double-stranded DNA or RNA, where many of the nucleotide sequences have been joined or recombined into specific constructs that can introduce into cells promoter fragments and DNA sequences for selected gene products, along with appropriate 3' untranslated sequences.

[0019] The genes and gene products encoding the lysophospholipases of the present invention can be expressed in heterologous host cells, such as bacterial cells, fungal cells, such as yeast cells, mammalian cells, insect cells and plant cells. The heterologous host cells for expressing the nucleic acid molecules of the present invention can be microbial hosts of the fungal or bacterial families, which grow over a wide range of temperatures, pH and solvent tolerances. For example, it is contemplated that any bacteria, yeast and filamentous fungi can be suitable hosts for the expression of the nucleic acid molecules of the present invention. Examples of host strains include bacterial, fungal or yeast species, such as Pichia, Aspergillus, Trichoderma, Saccharomyces, Phaffia, Kluyveromyces, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, The host cell may be a fungal cell, including, but not limited to, Corynebacterium, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus species. In some embodiments, the host cell is a fungal cell. In some embodiments, the host cell is a Pichia pastoris or Aspergillus niger cell. In some embodiments, the host cell is an Aspergillus niger cell of the present invention, which has the accession number CGMCC No. 40011.

[0020] Vectors useful for transforming the above-mentioned host cells are well known in the art.Typically, vectors contain sequences that direct the transcription and translation of related genes, selectable markers, and sequences that allow autonomous replication or chromosomal integration.Suitable vectors contain the 5' region of the gene that controls transcription initiation and the 3' region of the DNA fragment that controls transcription termination.

[0021] In certain embodiments, the present invention also relates to a method for producing a lysophospholipase comprising expressing a nucleic acid molecule encoding a lysophospholipase of the present invention in a host cell and recovering the resulting polypeptide.

[0022] A variety of culture techniques can be used to produce the enzymes of the invention. For example, large-scale production of specific gene products from recombinant microbial hosts can be carried out in batch, fed-batch and continuous culture techniques.

[0023] Batch and fed-batch culture methods are commonly used and well known in the art, examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989)) and Deshpande, Mukund V., (Appl. Biochem. Biotechnol., 36: 227-234 (1992).

[0024] Commercial production of the enzymes of the invention can also be carried out by continuous culture. Continuous culture is an open system in which conditioned medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous culture generally maintains cells at a constant high liquid density, where the cells are primarily in logarithmic growth phase. Alternatively, continuous culture can be carried out with immobilized cells, where carbon and nutrients are continuously added and valuable products, by-products or waste products are continuously removed from the cell pellet. Cell immobilization can be carried out using a wide range of solid supports made of natural and / or synthetic materials.

[0025] Recovery of the desired enzyme from batch fermentation, fed-batch fermentation or continuous culture can be achieved by any method known to those skilled in the art. For example, when the enzyme is produced intracellularly, the cell slurry is separated from the culture medium by centrifugation or membrane filtration, and optionally washed with water or an aqueous buffer of the desired pH. The cell slurry is then suspended in an aqueous buffer of the desired pH and homogenized to produce a cell extract containing the required enzyme.

[0026] The present invention also relates to compositions comprising fermentation broth, fermentation supernatant and / or fermentation concentrate of the lysophospholipase of the present invention or the host cell of the present invention. The enzyme composition of the present invention may be in any form suitable for use, such as a crude fermentation broth with or without removal of cells, a cell lysate with or without cell debris, a semi-purified or purified enzyme composition or a host cell as the enzyme source. The enzyme composition may be a dry powder or granule, a dust-free granule, a liquid, a stabilized liquid or a stabilized protected enzyme. Liquid enzyme compositions can be stabilized by established methods, such as the addition of stabilizers such as sugars, sugar alcohols or other polyols and / or lactic acid or other organic acids.

[0027] The present invention further relates to a fermentation broth, fermentation supernatant or fermentation concentrate of the host cell of the present invention.

[0028] The present invention also relates to the use of the lysophospholipase of the present invention in oil degumming. When the lysophospholipase of the present invention is used in oil degumming, the specific enzyme activity is significantly improved compared to the prior art lysophospholipases, and the production costs are reduced.

[0029] In another aspect, the present invention relates to a mutant Aspergillus niger strain that is an orotate phosphoribosyltransferase auxotroph and has an increased ability to produce an endogenous enzyme, such as lysophospholipase, compared to a non-mutant strain, for example, 10-15 fold, such as 10-12 fold, in particular 10.8 fold.

[0030] In the present invention, the term "endogenous enzyme" includes enzymes expressed by the mutant Aspergillus niger strain itself, including, for example, but not limited to, lysophospholipase expressed by the mutant Aspergillus niger strain itself.

[0031] In one embodiment, the mutant Aspergillus niger orotate phosphoribosyltransferase pyrE gene of the invention has a deletion of nucleotides TT at positions 64 and 65.

[0032] In one embodiment, a mutant Aspergillus niger strain of the invention has the accession number CGMCC No. 40011.

[0033] In addition to its own endogenous enzymes, the mutant Aspergillus niger strain of the present invention can efficiently express heterologous proteins. The mutant Aspergillus niger strain of the present invention can be used to express a wide range of heterologous proteins, such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, etc. In particular, when the mutant Aspergillus niger strain of the present invention recombinantly expresses lysophospholipase LPL, the protein expression level can be increased by more than 100% compared to the starting strain, for example, 100%-150%, particularly 100%-120%, more particularly 100%-112%. Therefore, it is clear that this strain is useful for efficient expression of heterologous proteins.

[0034] In another aspect, the present invention relates to a recombinant Aspergillus niger strain obtained by introducing a gene encoding a foreign protein into the mutant Aspergillus niger strain described above. In one embodiment, the exogenous protein is an enzyme such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, in particular lysophospholipase, and other proteins.

[0035] In another aspect, the present invention relates to a method for producing a target protein, comprising introducing a gene encoding the target protein into the mutant Aspergillus niger strain and culturing the strain to produce the target protein. Alternatively, the method comprises culturing the recombinant Aspergillus niger strain to produce the target protein. In one embodiment, the exogenous protein is an enzyme and other protein, such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, particularly lysophospholipase.

[0036] In another aspect, the present invention relates to a biocatalyst comprising a mutant Aspergillus niger strain as described above, into which a gene encoding a foreign protein has been introduced, the foreign protein being an enzyme such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, in particular lysophospholipase, and other proteins.

[0037] In another embodiment, the present invention relates to foreign proteins produced by said strains. The foreign proteins are enzymes and other proteins such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, especially lysophospholipase. The foreign proteins can be used in food, preferably as food enzymes, more preferably as food lysophospholipases.

[0038] In certain embodiments, the lysophospholipase is a mutant lysophospholipase of the invention.

[0039] The present invention also relates to recombinant microbial cells into which bacterial components from the mutant Aspergillus niger strains described above have been introduced. After obtaining the strains of the present invention, the bacterial components can be isolated by conventional techniques and introduced into other microorganisms. The recombinant microbial cells into which the components have been introduced have the advantageous properties of the strains of the present invention. In the present invention, the term "bacterial components" refers to the sum of all the genetic material of an organism, including, but not limited to, coding DNA, non-coding DNA, and mitochondrial DNA.

[0040] In particular, the mutant Aspergillus niger strain of the present invention can be used to express foreign proteins. After obtaining the mutant Aspergillus niger of the present invention, a commonly used expression vector can be introduced to express the foreign protein. For example, the expression vector contains a promoter and a terminator with a multiple cloning site between the promoter and the terminator where the gene encoding the foreign protein can be inserted. The promoter can contain one or more copies of an enhancer. Many commercial vectors can be used to express foreign proteins in the mutant Aspergillus niger strain of the present invention.

[0041] When expressing a foreign protein of non-Aspergillus niger origin, certain codons may be rare in Aspergillus niger. Therefore, when introducing an expression vector, the gene encoding the foreign protein may first be codon-optimized for the Aspergillus niger of the present invention, thereby increasing expression.

[0042] The Aspergillus niger of the present invention can express a variety of foreign proteins, including food enzymes, e.g., food-grade lipases, pharmaceutical proteins, various enzymes from plants, animals and bacteria, membrane receptor proteins, proteins containing prosthetic groups and proteins that can be used for crystal structure studies, etc. The Aspergillus niger of the present invention expressing foreign enzyme components can also be used as a biocatalyst via whole cells.

[0043] In one embodiment, a mutant Aspergillus niger strain of the invention, such as the Aspergillus niger strain having accession number CGMCC No. 40011, can be used to express a mutant lysophospholipase of the invention, such as a lysophospholipase comprising the amino acid sequence of SEQ ID NO: 14 or 16. The combined effect of the two is expected to further improve the expression efficiency of the lysophospholipase. [Brief description of the drawings]

[0044] [Figure 1]1 shows the specific enzyme activity of lysophospholipase LPL encoded by picAN02m1 (M1) and pic-AN02-LPL (WT) of CN2021107079194 and the lysophospholipases of mutants W, Y, and F of the present invention against 1-palmitoyl lysophosphatidylcholine.

[0045] [Diagram 2] FIG. 1 is a schematic diagram of the LPL gene expression vector constructed in the present invention.

[0046] [Diagram 3] FIG. 1. Diagram of AN19E complementation and transformation plates.

[0047] [Figure 4] FIG. 1 is a diagram of a plate for screening lysophospholipase LPL activity of AN19E complementation transformants.

[0048] [Diagram 5] FIG. 11 is a diagram of mutant strain AN19E-13 in LPL screening medium.

[0049] [Figure 6] 1 is a comparative chart of the enzymatic activity of endogenous lysophospholipase LPL expressed by the mutant strain AN19E-13 of the present invention and the starting strain AN19E.

[0050] [Figure 7] 1 is a comparative chart of the enzyme activity of lysophospholipase LPL recombinantly expressed by mutant strain AN19E-13 of the present invention and starting strains CICC2243 and AN19E.

[0051] [Figure 8] FIG. 1 is a protein electropherogram of lysophospholipase LPL recombinantly expressed by the mutant strain AN19E-13 of the present invention and the starting strains CICC2243 and AN19E. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Description of deposit The strain AN19E-13 of the present invention was deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 1, West Beichen Road, Chaoyang District, Beijing, China on December 20, 2021, with accession number CGMCC No. 40011, and was classified as Aspergillus niger.

[0053] Detailed Description of the Invention I. Production and functional validation of mutant lysophospholipases of the invention. Experimental materials Aspergillus niger strain GIM 3.24(AN02) was purchased from Guangdong Microbial Culture Collection Center.

[0054] The formulation of Aspergillus niger fermentation medium is: 2% glucose, 10% maltose, 7% sodium citrate, 1.5% ammonium sulfate, 4% tryptic soy broth, 0.1% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 0.07% Tween 80, trace elements (KI 0.83g / L, H3BO3 6.2g / L, MnSO4·4H2O 22.3g / L, ZnSO4·7H2O 8.6g / L, Na2MoO4·2H2O 0.25g / L, CuSO4·5H2O 0.025g / L, CoCl2·6H2O 0.025g / L were added at a ratio of 1 / 1000, FeSO4·7H2O 2.78g / L, and Na2·EDTA 3.73g / L were added at a ratio of 1 / 1000). It was.

[0055] The lysis buffer formulation was 100 mM Tris-HCl pH 8.0; 50 mM Na·EDTA; 1% SDS.

[0056] BMMY-Soy Lecithin Medium: Component A, BMMY solid medium: 1% yeast extract, 2% peptone, 100 mM citric acid-sodium citrate buffer, pH 6.6, 1.34% YNB, 4 × 10-5% biotin (added before inverting the plate), 2% methanol (added before inverting the plate), 2% agar dissolved in 250 ml deionized water.

[0057] Component B, 250 ml of soy lecithin substrate solution: 4% soy lecithin, emulsified in a high speed homogenizer at 8000 rpm for 3 minutes, stopped for 1 minute, then emulsified for 3 minutes to prepare a substrate solution.

[0058] After sterilization, components A and B were mixed, 10 ml of methanol was added and the plate was inverted.

[0059] Glass beads were purchased from Biospec, USA. PrimeSTAR® HS DNA polymerase was purchased from Takara, Cat. No. R010A.

[0060] SalI, EcoRI and BglII restriction enzymes were purchased from NEB.

[0061] Lysophospholipid 1-palmitoyl-sn-glycero-3-phosphocholine was purchased from Aladdin, Cat. No. P130493-500 mg.

[0062] The NEFA kit was purchased from Wako Pure Chemical Industries, Ltd., Japan.

[0063] The Bradford kit was purchased from Sangon (Shanghai) Bioengineering Co., Ltd.

[0064] MGYS plates: 1.34% YNB, 4 × 10–5% biotin (filter sterilized), 2% glycerol, 1 M sorbitol.

[0065] YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

[0066] BMGY shake flask medium: 1% yeast extract, 2% peptone, 100 mM citric acid-sodium citrate buffer, pH 6.6, 1.34% YNB, 4 × 10–5% biotin (filter sterilized), 2% glycerol.

[0067] BMMY shake flask medium: 1% yeast extract, 2% peptone, 100 mM citric acid-sodium citrate buffer, pH 6.6, 1.34% YNB, 4 × 10-5% biotin (filter sterilized), 2% methanol (added during inoculation). EXAMPLES

[0068] Example 1: Cloning of Aspergillus niger lipase B gene Aspergillus niger strain GIM 3.24 (AN02) was grown in Aspergillus niger fermentation medium at 30°C for 24 h. The fermentation culture was centrifuged at 4000 rpm for 5 min, the cells were removed, suspended in 700 μl lysis buffer, transferred to a cryovial, 300 μl glass beads were added, shaken in a mini-bead beater for 40 s, and centrifuged at 12000 rpm for 10 min. 600 μl of the supernatant was removed and 275 μl of 7 M ammonium acetate was added, then placed in a 65°C water bath for 10 min and an ice bath for 5 min. After the addition of an equal volume of phenol, chloroform, and isoamyl alcohol (24:25:1), the mixture was thoroughly vortexed and centrifuged at 12000 rpm for 5 min. The supernatant was removed and an equal volume of chloroform was added, thoroughly vortexed, and centrifuged at 12000 rpm for 5 min. The supernatant was taken, 2 volumes of absolute ethanol was added, placed at -80°C for 20 minutes, and centrifuged at 12000 rpm for 10 minutes to obtain a white DNA precipitate, which was washed twice with 70% ethanol. After the ethanol was completely evaporated, sterile water was added to dissolve the DNA.

[0069] Primers LPL-1 and LPL-2 were designed according to the gene sequence of lipase B of Aspergillus niger CBS513.88 in NCBI. LPL-1 5'-atgtttctccgcagggaatt-3' (SEQ ID NO: 1) LPL-2 5'-ctacgagcattcactaatgt-3' (sequence number 2).

[0070] The LPL DNA of Aspergillus niger strain GIM 3.24(AN02) was cloned with PrimeSTAR® HS DNA polymerase. After TA cloning with Mighty TA-cloning Reagent Set for PrimeSTAR® kit, it was transformed into DH5a E. coli. DNA sequencing was performed by Sangon Bioengineering Co., Ltd., and the following LPL DNA sequence of GIM 3.24(AN02) was obtained: DNA sequence of GIM 3.24(AN02)LPL:

[0071] By using the intron sequence of the DNA sequence of lipase B of CBS513.88, the intron sequence of GIM 3.24 (AN02) was found and removed, and the DNA sequence was translated into an amino acid sequence. The results were as follows: The amino acid sequence of GIM 3.24(AN02)LPL was as follows: MFLRREFGAVAALSVLAHAAPAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDLSNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPLPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS (sequence number 4).

[0072] Example 2: Expression and random mutagenesis of AN02-LPL in Pichia pastoris The mature peptide of AN02-LPL was selected and its first 10 amino acids were used as the leading peptide to obtain AN02-LPL, whose amino acid sequence was as follows: APAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDLSNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVS SYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS (SEQ ID NO: 5).

[0073] The corresponding DNA sequence was: (Sequence number 6).

[0074] After adding the Sac-II restriction site CCGCGG to the 5' end, the gene sequence was synthesized by Sangon Bioengineering Co., Ltd. and cloned into pAO815 vector (purchased from invitrogen) via EcoRI restriction site to obtain the pic-AN02 plasmid. After linearization with BglII, the vector was transformed into competent cells of Pichia pastoris strain GS115 (purchased from invitrogen) using electroporation. The transformants were inoculated onto MGYS plates and cultured at 30°C for 3 days to obtain Pichia pastoris transformants. One clone on the plate was picked and placed on a BMMY-soybean phospholipid medium screening plate. A clone with a large white precipitated circle was selected and named pic-AN02-LPL.

[0075] Using pic-AN02-LPL as a template, error-prone PCR was performed with TaKaRa Taq enzyme and primer pair PLPL-1:TCCCCGCGGCGAAAACGATGAGATTTCCTTC (SEQ ID NO: 7) / PLPL-2:CCGGAATTCTTAAGAACACTCAGAAATG (SEQ ID NO: 8) (0.3 mM MnCl2 was further added during PCR) to obtain a collection of mutant amplicon fragments of approximately 1000 bp size. The resulting fragments were cloned into the pic-AN02 plasmid via Sac-II and EcoRI restriction sites, and the resulting vector was transformed into E. coli DH5α strain.

[0076] The plate containing the pic-AN02 mutant was washed with 2 ml of sterile water, the plasmid was extracted and linearized with SalI, and an approximately 8.5 kb fragment was recovered and used as a vector. 500 ng of the vector was taken and transformed into competent cells of Pichia pastoris strain GS115 using electrotransformation method. The transformants were inoculated onto BMM-soybean phospholipid screening medium plates and cultured at 30°C for 3 days to obtain a Pichia pastoris mutant library of pic-AN02-LPL. The mutant strain pic-AN02m1 was obtained through screening.

[0077] The pic-AN02m1 strain was inoculated into 3ml YPD liquid medium and cultured at 30°C overnight, and genomic DNA was extracted. Using the genomic DNA of the pic-AN0m1 strain as a template, PCR amplification was performed using PrimeSTAR® HS DNA polymerase and the primer pair AOX-5: GACTGGTTCCAATTGACAAGC (SEQ ID NO: 9) and 3'-AOX1: GGCAAATGGCATTCTGACATCCTC (SEQ ID NO: 10) to obtain the DNA sequence of the AN02 mutant in the pic-AN02m1 strain. The obtained sequence was sent to Shanghai Sangon Bioengineering Company and sequenced with the primer pair AOX-5 / 3'-AOX1. The DNA sequence of the mutant LPL of the Pic-AN02m1 strain and the amino acids encoded by it are as follows, and the mutation sites were L86I, G187D, E209K and A245D.

[0078] Amino acid sequence: APAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSS YPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSTTAHRWYTIYISECS (SEQ ID NO: 11). DNA sequence: (Sequence number 12).

[0079] Example 3: Site-directed mutagenesis of the LPL gene encoded by pic-AN02m1 in Pichia pastoris The threonine at position 255 of lysophospholipase LPL encoded by pic-AN02m1 was mutated to tryptophan, phenylalanine, and tyrosine, respectively, and the mutant genes were named W, F, and Y.

[0080] Amino acid sequence of mutant W APAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSS YPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSwTAHRWYTIYISECS (SEQ ID NO: 13).

[0081] The first 10 amino acids were the leader peptide, so the functional sequence was: DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSwTAHRWYTIYISECS (SEQ ID NO: 14).

[0082] Amino acid sequence of mutant F APAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSS YPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSFTAHRWYTIYISECS (SEQ ID NO: 15).

[0083] The first 10 amino acids were the leader peptide, so the functional sequence was: DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSFTAHRWYTIYISECS (SEQ ID NO: 16).

[0084] Amino acid sequence of mutant Y APAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSS YPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSYTAHRWYTIYISECS (SEQ ID NO: 17).

[0085] The first 10 amino acids were the leader peptide, so the functional sequence was: DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSYTAHRWYTIYISECS (SEQ ID NO: 18).

[0086] The gene sequences were synthesized by Sangon Bioengineering Co., Ltd. and cloned into pic-AN02-LPL vector to obtain mutant W, F and Y plasmids, respectively. After linearization with SalI, the vectors were transformed into competent cells of Pichia pastoris strain GS115 using electroporation. The transformants were inoculated onto MGYS plates and cultured at 30°C for 3 days to obtain Pichia pastoris transformants of mutant W, F and Y. Single clones on the plates were picked and placed onto BMM-soybean phospholipid medium screening plates. Clones with large white precipitated circles were selected and named mutant W, F and Y, respectively.

[0087] The pic-AN02m1, pic-AN02-LPL Pichia pastoris expression strains and mutants W, F and Y Pichia pastoris expression strains were taken and first activated in liquid YPD, then inoculated into BMGY medium and grown overnight at 30°C with shaking at 220 rpm. The cultures were transferred to BMMY medium at an initial OD600 of 6.

[0088] First, 2% methanol was used for induction. 1% methanol was added after 24 and 32 hours, respectively. 1% methanol was added after 48 and 56 hours, respectively, and sampling was performed at 72 hours.

[0089] The resulting fermentation broth was subjected to ultrafiltration, desalting and 30-fold concentration using ultrafiltration tubes with a molecular weight cutoff of 10 kDa. The treated sample was added to a buffer (20 mM citric acid-sodium citrate buffer (pH 4.0)).

[0090] The lysophospholipase activity of the concentrated enzyme solution was determined as follows: 9ml substrate: 5ml 1% lysophospholipid, 1ml 20% Triton X-100, 2.5ml 0.1M citric acid-sodium citrate buffer.

[0091] 10 μl of diluted enzyme solution + 90 μl of substrate was reacted at 50°C for 10 minutes, inactivated at 95°C for 5 minutes, and centrifuged at 7000 rpm for 5 minutes. 1 μl of the supernatant was taken, and 80 μl of Reagent A in the NEFA kit was added, reacted at 37°C for 10 minutes, and 160 μl of Reagent B was added, and reacted for 10 minutes. The absorbance value at 550 nm was determined to calculate the enzyme activity.

[0092] Protein concentration was determined using Bradford kit. The specific enzyme activities of lysophospholipase of LPL encoded by PicAN02m1, LPL encoded by Pic-AN02-LPL and mutants W, Y and F were calculated by calculating enzyme activity and protein concentration. As shown in FIG. 1, the specific enzyme activities of lysophospholipase of mutants W and F were 30224 U / mg and 18973 U / mg, respectively; the specific enzyme activities of lysophospholipase of picAN02m1 (M1) and pic-AN02-LPL (WT) were 13473 U / mg and 12746 U / mg, respectively. Compared with the starting sequence M1, i.e., picAN02m1, which had a specific enzyme activity of 13473 U / mg, the specific enzyme activities were increased by 124% and 40%, respectively.

[0093] II. Preparation and functional validation of the mutant Aspergillus niger strains of the present invention. The starting Aspergillus niger strain was purchased from the China Center of Industrial Culture Collection (CICC) with the accession number CICC2243. The starting strain was first spread on MM solid medium plates to culture spores. After elution of the spores, they were subjected to UV mutagenesis, and finally screened by adding 5-fluoroorotic acid and uracil to the screening plate to obtain the orotate phosphoribosyltransferase auxotroph AN19E.

[0094] Then, strain AN19E-13 was screened using ARTP mutagenesis. On lysophospholipase screening plates supplemented with uracil, the sedimentation circle of AN19E-13 was significantly larger than that of the starting strain AN19E. The test revealed that the lysophospholipase (LPL) enzyme activity of AN19E-13 itself was 10.8 times higher than that of the starting strain AN19E.

[0095] Attempts were made to perform heterologous recombinant expression of lysophospholipase (LPL) in Aspergillus niger strain AN19E-13 and the ability of this strain to express LPL was found to be increased by 112%, confirming its distinct advantages as a protein expression system, especially for lysophospholipases used in food.

[0096] In the present invention, the term "ARTP" is an abbreviation of ambient pressure and room temperature plasma, which specifically refers to a plasma jet that can produce high concentrations of active particles (including excited helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc.) at atmospheric pressure and a temperature of 25-40°C. The term "ARTP mutagenesis" refers to the use of ambient pressure and room temperature plasma technology for strain mutagenesis. In particular, the ambient pressure and room temperature plasma source using helium as the working gas contains a variety of chemically active particle components, such as OH, nitrogen molecule 2 positive system, nitrogen molecule 1 negative system, excited state helium atoms, hydrogen atoms and oxygen atoms, etc. The abundance of active energy particles in ARTP causes damage to the genetic material of strains / plants / cells, etc., and biological cells initiate the SOS repair mechanism. The SOS repair process is a highly error-tolerant repair process, and a wide variety of mismatch sites are produced during the repair process, and the genetic material is finally stabilized to form mutant strains. The SOS repair strength is closely related to the degree of DNA damage.

[0097] Example 4: Obtaining an Aspergillus niger strain auxotrophic for orotate phosphoribosyltransferase Spores of Aspergillus niger strain CICC2243 were inoculated onto MM solid medium (1% glucose, 0.5% KH2PO4, 0.6% NaNO3, 0.05% KCl, 0.05% MgSO4, 2% agar powder), coated, and statically cultured at 28 °C for 5 days to obtain Aspergillus niger spores. Fresh spores of Aspergillus niger strain CICC2243 were washed with spore washing solution (0.9% NaCl, 0.05% Tween 80) and filtered through Miracloth (Calbiochem, Cat#475885) to prepare a spore suspension. Cells were washed twice with sterile water and diluted to 1 × 10 7The spore suspension was adjusted to 100 cells / mL. 2 mL of the spore suspension was spread evenly on the surface of a Petri dish and placed under a UV lamp in a clean work bench for 90 seconds. 100 μL was taken and coated onto MM solid medium supplemented with 0.3% uracil and 1 mg / mL 5-fluoroorotic acid (5-FOA), and cultured at 28 °C in the dark for 7 days (the whole process was operated under a red lamp to prevent back mutation). A single colony that had grown on MM solid medium in the previous step was transferred to MM solid medium and MM-uracil solid medium, and a strain that could only grow on MM-uracil solid medium was selected to obtain the orotate phosphoribosyltransferase auxotroph AN19E. Sequencing of the pyrE gene of Aspergillus niger AN19E strain revealed that the 64th and 65th nucleotides TT were deleted, leading to the inactivation of the pyrE gene.

[0098] Example 5: Construction of LPL expression vector The sequence of the exogenous Aspergillus niger lysophospholipase (LPL) gene was as follows: Nucleic acid sequence:

[0099] Amino acid sequence: APAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDLSNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSS YPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS (SEQ ID NO: 20).

[0100] The specific procedures were performed according to the method described in "Molecular Cloning Experimental Guide" (Third Edition, New York: Cold Spring Harbor Laboratory Press, 1989). The LPL gene expression vector pANE-LPL shown in FIG. 2 was constructed as follows: the LPL gene (SEQ ID NO: 19, containing Aspergillus oryzae α-amylase signal peptide (NCBI SEQ ID NO: XM_001821384.2, 1-63 bp sequence) obtained from total gene synthesis by Sangon (Shanghai) Bioengineering Co., Ltd.) was inserted into an expression cassette containing Aspergillus oryzae enolase promoter (NCBI SEQ ID NO: D63941.1, 215-734 bp; containing 12 copies of enhancer sequence (gtcgtgtcgggcatttatcgggggatggaccaatcagcgtagg, SEQ ID NO: 21)) and Aspergillus niger glycosylase terminator (NCBI SEQ ID NO: AF214480.1, terminator sequence portion) via SphI and HindIII enzyme sites. The whole expression cassette was inserted into the multiple cloning site of the cloning vector pSP72 via BglII and XhoI, and finally the PyrE expression gene from Aspergillus niger (NCBI sequence number: AY840014.1) was inserted into the vector via the XhoI restriction site to construct the LPL gene expression vector pANE-LPL (see Figure 2).

[0101] Example 6: Aspergillus niger AN19E complementation experiments Fresh spores of Aspergillus niger AN19E were eluted with spore wash solution and filtered through Miracloth to prepare a spore suspension at a concentration of 1 × 10 7 The concentration was adjusted to spores / mL. 1 mL of the spore suspension was inoculated into mycelial culture medium (2% tryptone, 1% yeast extract, 2% glucose, 0.3% uracil) and grown for 40 h at 28°C and 180 rpm. The vegetative mycelium was collected by sterile Miracloth filtration.

[0102] The collected mycelia were washed three times with sterile osmotic stabilizer (0.6 M MgSO4, 10 mM NaH2PO4, pH=5.8) and pressure dried. The mycelia were transferred to a 100 mL Erlenmeyer flask and every 0.8 g of mycelia was suspended in 20 mL of enzymatic hydrolysis solution (adjust the enzymatic hydrolysis solution of 1% lytic enzymes, 1% cellulase and 0.1% helicase using an osmotic stabilizer and sterilized with a 0.22 μm microporous membrane filter) and subjected to enzymatic hydrolysis at 30 °C and 90 rpm for 60-90 min. The enzyme-digested protoplast mixture was filtered through Miracloth. The filtrate was collected and centrifuged at 1000 g for 10 min at 4 °C. The protoplast pellet was dissolved in 5 mL of pre-chilled 1.0 mol / L sorbitol solution and centrifuged at 800 g for 10 min at 4 °C and the supernatant was discarded. The protoplasts were then diluted to 1 × 10 in pre-chilled STC solution (1.0 M sorbitol, 50 mM CaCl2, 50 mM Tris-HCl, pH = 7.5). 7 / mL and kept in an ice bath until use.

[0103] To the 200μL protoplast suspension, 10μL LPL expression vector pANE-LPL was added at 1μg / μL concentration, then 50μL PTC solution (40% PEG4000, 50mM CaCl2, 50mM Tris-HCl, pH=7.5) was added, mixed well, and kept in ice bath for 30 minutes. Then 0.2mL PTC solution was added, mixed well, then 0.8mL PTC solution was added, mixed well, and kept at room temperature for 30 minutes.

[0104] The mixture was coated onto regeneration medium (1% glucose, 0.6% NaNO3, 0.15% KH2PO4, 0.05% KCl, 0.05% MgSO4, 0.001% FeSO4, 1 M sucrose, 2% agar powder) and cultured at 28°C for 7 days until colonies grew.

[0105] Colonies grown on the plate were transferred to lysophospholipase LPL screening medium and cultured at 28°C for 3 days.

[0106] The components of the LPL screening medium were as follows: Solution A: 2% maltose, 1.34% YNB, citric acid 6.88 g / 500 mL, sodium citrate 5.07 g / 500 mL, 5 mM CaCl2, add water to 200 mL.

[0107] Solution B: 1% lecithin, add 200 ml of water, homogenize with a homogenizer, 2% agarose, 0.02% Triton-x-100, add water up to 300 ml. After sterilization, solutions A and B were mixed and the plate was inverted.

[0108] After transformation, Aspergillus niger AN19E could well achieve the pyrE gene complementation experiment, and as shown in Figures 3 and 4, the transformant was found to exhibit lysophospholipase LPL activity in the lysophospholipase LPL screening medium, further proving the successful establishment of an Aspergillus niger expression system using orotate phosphoribosyltransferase auxotrophic Aspergillus niger AN19E as the host.

[0109] Example 7: Aspergillus niger AN19E ARTP mutagenesis experiment Fresh spores of Aspergillus niger AN19E were eluted with spore wash solution and filtered through Miracloth to prepare a spore suspension, with a concentration of 2 × 10 7 The concentration was adjusted to spores / mL. After mixing 1:1 spore suspension and 10% glycerol, 10 μL of the mixed solution was taken on an iron sheet and treated with an ARTP instrument (Wuxi Tmaxtree Biotechnology Co., Ltd., instrument model: ARTP-M) for 100 seconds. The instrument parameter settings were: radio frequency power range 120 W, helium volume 10 SLM (99.999% high purity helium), and irradiation distance 2 mm.

[0110] After the treatment was completed, the iron sheet was removed and transferred to a centrifuge tube filled with 1 mL of sterile water, and then a pipette tip was used to repeatedly aspirate to wash off the cells on the iron sheet. The cells were diluted to about 100 colonies / screening plate, and finally coated onto a uracil-added lysophospholipase screening plate. The plate was then placed in a 30°C incubator for 3 days.

[0111] Example 8: Screening of mutant strains The spores mutated in Example 7 were coated onto uracil-supplemented lysophospholipase screening plates for screening. A strain with a significantly larger sedimentation circle was observed (FIG. 5) and was named AN19E-13.

[0112] Strain AN19E-13, which had a significantly larger sedimentation circle, and starting strain AN19E were fermented in shake flasks supplemented with fermentation medium (2% maltose, 1.34% YNB, 1.38% citric acid, 1% sodium citrate, 5 mM CaCl2, 1% lecithin) that had been autoclaved for 15 min at 115 °C. Fermentation conditions were 28 °C, 200 rpm, 5 days, and an inoculum volume of 1 × 10 7 Spores / 50 mL. Lysophospholipase LPL activity was determined.

[0113] The method for determining lysophospholipase LPL activity was as follows: 9mL Substrate: 5mL 1% soy lecithin, 1mL 20% Triton X-100, 2.5mL 0.1M pH 4.0 citric acid-sodium citrate buffer.

[0114] 10μL of appropriately diluted enzyme solution + 90μL substrate was reacted at 50℃ for 10 minutes, inactivated at 95℃ for 5 minutes, and centrifuged at 7000rpm for 5 minutes. 1μL of supernatant was taken, and 80μL of Reagent A from NEFA kit (Wako:294-63601) was added, reacted at 37℃ for 10 minutes, and 160μL of Reagent B was added, and reacted for 10 minutes. The absorbance value at 550nm was measured.

[0115] The results of the enzyme activity measurements are shown in FIG.

[0116] The results showed that the activity of lysophospholipase LPL expressed by strain AN19E-13, which had a significantly larger sedimentation circle, was 1058 U / mL, 10.8-fold higher than that of the starting strain AN19E (98 U / mL).

[0117] Strain AN19E-13 was deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 1, West Beichen Road, Chaoyang District, Beijing, China on December 20, 2021, with accession number CGMCC No. 40011, and was classified as Aspergillus niger.

[0118] Example 9: Investigation of the expression ability of Aspergillus niger AN19E-13 The transformation procedure of Aspergillus niger AN19E-13 was the same as in Example 6. The LPL expression vector pANE-LPL was transformed into the strain for recombinant expression.

[0119] Aspergillus niger AN19E transformants from Example 6 were used as controls, and pANE-LPL was transformed into Aspergillus niger CICC2243. Because pANE-LPL cannot use pyrE as a screening marker, p3SR2 (BCCM / LMBP: Accession No.: 2363), which contains the acetamidase (amdS) gene, was used for transformation screening. The regeneration medium required the removal of sodium nitrate and the addition of 15 mM acetamide and 20 mM cesium chloride.

[0120] Forty transformants for each of the three transformations were selected for shake flask fermentation. The fermentation medium (2% glucose, 15% maltose, 7% sodium citrate, 1.5% ammonium sulfate, 4% TSB, 0.1% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 0.07% Tween 80, trace elements) was autoclaved at 115°C for 15 min. The fermentation conditions were 28°C, 200 rpm, 8 days, inoculum size 1 × 10 7 The spores / 50 mL were cultured. The activity of lysophospholipase LPL was measured.

[0121] The lysophospholipase LPL activity was measured as described in Example 8.

[0122] The results of the enzyme activity measurement are shown in FIG.

[0123] The results show that the enzymatic activity of lysophospholipase LPL recombinantly expressed by AN19E-13 was 25920 U / mL, an increase of 112% and 100% compared to the starting strains CICC2243 (12230 U / mL) and AN19E (12990 U / mL), respectively.

[0124] Polyacrylamide gel electrophoresis analysis: The supernatant was filtered using a 0.22 μm filter membrane. Equal amounts of the supernatant were concentrated to the same volume using a Millipore 10 KDa ultrafiltration concentration tank, and then the same volume of concentrated enzyme solution was used for polyacrylamide gel electrophoresis analysis. The electrophoresis results are shown in Figure 8.

[0125] Protein electrophoresis results showed that the LPL protein band concentration of AN19E-13 transfectants was significantly higher than that of the control.

Claims

1. A lysophospholipase comprising the amino acid sequence of SEQ ID NO: 14 or 16.

2. 13. A method for producing a lysophospholipase comprising expressing a nucleic acid molecule encoding the lysophospholipase of claim 1 in a host cell and recovering the resulting polypeptide.

3. A mutant Aspergillus niger strain for expressing a food grade lysophospholipase, said mutant being auxotrophic for orotate phosphoribosyltransferase and having increased endogenous enzyme, preferably lysophospholipase, producing capacity compared to a non-mutated strain.

4. 4. The mutant Aspergillus niger strain of claim 3, having a deletion of nucleotides TT at positions 64 and 65 of the orotate phosphoribosyltransferase pyrE gene.

5. The mutant Aspergillus niger strain has accession number CGMCC No. 40011.

6. (a) a nucleotide sequence encoding the lysophospholipase of claim 1; and (b) a nucleotide sequence complementary to the nucleotide sequence described in (a). A nucleic acid molecule comprising:

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. 9. The host cell of claim 8, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a mammalian cell, an insect cell and a plant cell.

10. 10. The host cell of claim 9, wherein the host cell is a fungal cell, preferably a Pichia pastoris cell or an Aspergillus niger cell, preferably an Aspergillus niger cell with accession number CGMCC NO.40011.

11. A composition comprising the lysophospholipase of claim 1 or a fermentation broth, fermentation supernatant and / or fermentation concentrate of the host cell of any of claims 8 to 10.

12. A fermentation broth, fermentation supernatant or fermentation concentrate of the host cell of any of claims 8 to 10.

13. Use of a lysophospholipase according to claim 1 or a composition according to claim 11 or a fermentation broth, a fermentation supernatant and / or a fermentation concentrate of a host cell according to any of claims 8 to 10 in oil degumming.

14. A recombinant Aspergillus niger strain obtained by introducing a gene encoding a foreign protein into the strain of any of claims 3 to 5.

15. 15. The recombinant Aspergillus niger strain of claim 14, wherein the foreign protein is an enzyme, preferably a lysophospholipase, preferably a lysophospholipase of claim 1.

16. A method for producing a target protein comprising introducing a gene encoding the target protein into the strain of any of claims 3 to 5 and culturing said strain to produce the target protein or culturing a recombinant Aspergillus niger strain of claim 14 or 15 to produce the target protein.

17. 17. The method of claim 16, wherein the target protein is an enzyme, preferably a lysophospholipase, preferably a lysophospholipase of claim 1.

18. A biocatalyst comprising a mutant Aspergillus niger strain according to any of claims 3 to 5, into which a gene encoding an enzyme, preferably a lysophospholipase, preferably the lysophospholipase of claim 1, has been introduced.

19. The foreign protein produced by the strain of any of claims 3 to 5 and 14 to 15, wherein the foreign protein is an enzyme, preferably a lysophospholipase and / or the foreign protein can be used as a food, preferably a food grade enzyme, more preferably a food grade lysophospholipase.

20. A recombinant microbial cell into which a bacterial component derived from any one of the strains of claims 3 to 5 has been introduced.

Images