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, and more specifically to a mutant lysophospholipase. The present invention also relates to a gene encoding the enzyme, as well as a vector and a host cell containing the gene. Furthermore, the present invention also relates to uses of the enzyme.

[0002] The present application also relates to mutant Aspergillus niger strains that can be used to express the mutant lysophospholipases of the invention, and to methods for producing exogenous proteins, particularly lysophospholipases of the invention, using the strains. [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, widely distributed in plant products, food, and soil. Its conidial heads are dark brown, radiating, and have a spherical apical capsule. Its conidiophores vary in length, and its hyphae are well-developed and highly branched. Aspergillus niger can grow rapidly by decomposing natural organic matter and absorbing its nutrients. It is classified as Generally Regarded as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) and recognized by the World Health Organization. It is a very commonly used species of filamentous fungus. Its ability to produce enzyme preparations and organic acids has made it an important strain for industrial production.

[0004] Aspergillus niger has efficient protein secretion and expression capabilities. Foreign proteins expressed by Aspergillus niger are characterized by high expression levels, high extracellular secretion rates, and protein molecular folding and modification systems similar to those of higher eukaryotic cells, resulting in native activity of the expressed foreign proteins. Furthermore, Aspergillus niger can also perform various post-translational processes, such as glycosylation modification, protease cleavage, and disulfide bond formation. Therefore, Aspergillus niger is gaining attention and is attractive as an expression strain for expressing homologous and heterologous proteins. Commercial enzyme preparations currently produced using Aspergillus niger include amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, and xylanase, which are used in the food, detergent, textile, and paper industries. Heterologous proteins expressed by Aspergillus niger include lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, and lipase.

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

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

[0007] Jiangke Yang et al. cloned and expressed lipas2 from Aspergillus niger CICC 4009. This lipase 2 is highly homologous to lipase B cloned by Zhu Shu-sen, differing by only two amino acids, but has specific differences in properties. Its optimal substrates are pNPC8 and pNPC10, its optimum pH is below 6.5, its optimum 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, their specific enzymatic activity is very low and their temperature tolerance is not high. Their most suitable substrates are triglycerides of short-chain fatty acids. These enzymes are not as effective as the more widely used lipases TL and RML, which have specific enzymatic activities of 12,000 U / mg and 8,000 U / mg, respectively. TL can withstand temperatures of 60°C for 20 hours without inactivation, and RML can hydrolyze triglycerides of fatty acids with various chain lengths.

[0009] In this patent application, CN2021107079194, the inventors used the gene sequence of lipase B from CBS513.88 from Aspergillus niger GIM 3.24 (AN02) to design primers for the LPL gene AN02-LPL of the above Aspergillus niger strain and cloned it. They discovered that AN02-LPL has extremely high phospholipase A1 and lysophospholipase activities and can be used for oil degumming. It can reduce the phosphorus content of crude oil to 5 ppm without the addition of alkali. It can also be used for enzymatic degumming, eliminating the need for added alkali and reducing soap formation during degumming. Furthermore, because AN02-LPL has extremely high lysophospholipase activity, it can be used in combination with soybean phospholipase A2 to produce glycerol phospholipase (GPC), which has brain-boosting and anti-aging effects and is used in pharmaceuticals and health care products.

[0010] In CN2021107079194, the inventors randomly mutated LPL encoded by the AN02 variant picAN02m1. The mutation sites were L86I, G187D, E209K, and A245D. The thermostability of this variant was 3.2-fold, 31-fold, and 28.5-fold higher than that of wild-type AN02-LPL at 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 for lysophospholipases with high specific enzymatic activity.Furthermore, there remains a need in the art for Aspergillus niger strains that can efficiently express various proteins, particularly lysophospholipases, for food products. [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 the lysophospholipase increased from 12,746 U / mg and 13,473 U / mg of LPL encoded by picAN02 and picAN02m1, respectively, to 30,224 U / mg and 18,973 U / mg, further improving the utilization efficiency of lysophospholipase for producing glycerolphosphorylcholine (GPC).

[0013] Furthermore, the present invention constructed an orotate phosphoribosyltransferase auxotroph (pyrE-) strain using Aspergillus niger CICC2243 as the starting strain. Then, through ARTP mutagenesis, a strain was screened for that had a 10.8-fold increase in lysophospholipase (LPL) enzyme activity and a significantly larger precipitation circle compared to the starting strain on uracil-supplemented lysophospholipase screening plates. Recombinant expression of lysophospholipase LPL was then performed, and the expression capacity was found to be significantly increased, by 112% compared to the starting strain. This strain can be used to efficiently heterologously express various proteins, particularly lysophospholipases, for food applications.

[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 16.

[0015] In another aspect, the present invention relates to a nucleic acid molecule comprising, partially or completely: (a) a nucleotide sequence encoding the above-described lysophospholipase; 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 will be expressed.

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

[0018] "Vector" refers to an extrachromosomal element that normally 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, genome-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 capable of introducing 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, e.g., yeast cells, mammalian cells, insect cells, and plant cells. Heterologous host cells for expressing the nucleic acid molecules of the present invention can be microbial hosts of the fungal or bacterial families, capable of growing 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 expressing 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, Examples of host cells include, but are 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 accession number CGMCC No. 40011.

[0020] Vectors useful for transforming the above-mentioned host cells are well known in the art. Typically, a vector contains a sequence that directs the transcription and translation of the relevant gene, a selectable marker, and a sequence that allows autonomous replication or chromosomal integration. A suitable vector contains a 5' region of the gene that controls transcription initiation and a 3' region of the DNA fragment that controls transcription termination.

[0021] In certain embodiments, the present invention also relates to methods 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 of which 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 present 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 variety 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 solution of the desired pH. The cell slurry is then suspended in an aqueous buffer solution of the desired pH and homogenized to produce a cell extract containing the desired enzyme.

[0026] The present invention also relates to compositions comprising fermentation broths, fermentation supernatants, and / or fermentation concentrates of the lysophospholipases of the present invention or host cells of the present invention. The enzyme compositions of the present invention can be in any form suitable for use, such as crude fermentation broths with or without cell removal, cell lysates with or without cell debris, semi-purified or purified enzyme compositions, or host cells as the enzyme source. The enzyme compositions can be dry powders or granules, dust-free granules, liquids, stabilized liquids, or stabilized protected enzymes. 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 cells 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 lysophospholipases of the prior art, 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, by 10-15 fold, e.g., 10-12 fold, particularly 10.8 fold.

[0030] In the present invention, the term "endogenous enzyme" includes enzymes expressed by the mutant Aspergillus niger strain itself, including, 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 present 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, and lipase. 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, by 100% to 150%, particularly 100% to 120%, and more particularly 100% to 112%. Therefore, this strain is clearly useful for the 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 or other protein, such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, particularly lysophospholipase.

[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 described above and culturing the strain to produce the target protein. Alternatively, the method comprises culturing the recombinant Aspergillus niger strain described above to produce the target protein. In one embodiment, the exogenous protein is an enzyme or 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 the mutant Aspergillus niger strain described above, into which a gene encoding a foreign protein has been introduced, such as an enzyme or other protein, including amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, particularly lysophospholipase.

[0037] In another aspect, the present invention relates to foreign proteins produced by the 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, particularly 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 derived 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 using conventional techniques and introduced into other microorganisms. Recombinant microbial cells into which the components have been introduced possess the advantageous properties of the strains of the present invention. In the present invention, the term "bacterial components" refers to the sum of all 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 strains 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, into which a gene encoding the foreign protein can be inserted. The promoter may contain one or more copies of an enhancer. Many commercial vectors can be used to express foreign proteins in the mutant Aspergillus niger strains 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 to increase 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 suitable for crystal structure studies. Aspergillus niger of the present invention expressing foreign enzyme components can also be used as biocatalysts via whole cells.

[0043] In one embodiment, a mutant Aspergillus niger strain of the invention, such as the Aspergillus niger strain with 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 explanation 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 toward 1-palmitoyl lysophosphatidylcholine.

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

[0046] [Figure 3] FIG. 10 is a 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] [Figure 5] FIG. 10 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 enzymatic 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 INVENTION

[0052] Deposit description 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 added at a 1 / 1000 ratio, FeSO4·7H2O 2.78g / L, Na2·EDTA 3.73g / L added at a 1 / 1000 ratio). 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 × 10⁻⁵5% biotin (added before inverting the plates), 2% methanol (added before inverting the plates), 2% agar dissolved in 250 ml deionized water.

[0057] Component B, 250 ml of soybean lecithin substrate solution: 4% soybean 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 x 10–5% biotin (filter sterilized), 2% methanol (added during inoculation). [Example]

[0068] Example 1: Cloning of the Aspergillus niger lipase B gene Aspergillus niger strain GIM 3.24 (AN02) was grown in Aspergillus niger fermentation medium at 30°C for 24 hours. The fermentation culture was centrifuged at 4000 rpm for 5 minutes, and the cells were harvested and suspended in 700 μl lysis buffer. The cells were transferred to a cryovial, 300 μl glass beads were added, and the mixture was shaken in a mini bead beater for 40 seconds and centrifuged at 12000 rpm for 10 minutes. 600 μl of the supernatant was removed, 275 μl of 7 M ammonium acetate was added, and the mixture was then placed in a 65°C water bath for 10 minutes and an ice bath for 5 minutes. After adding an equal volume of phenol, chloroform, and isoamyl alcohol (24:25:1), the mixture was thoroughly vortexed and centrifuged at 12000 rpm for 5 minutes. The supernatant was then removed, an equal volume of chloroform was added, thoroughly vortexed, and centrifuged at 12000 rpm for 5 minutes. The supernatant was removed, and 2 volumes of absolute ethanol was added. The mixture was then placed at -80°C for 20 minutes and centrifuged at 12,000 rpm for 10 minutes to obtain a white DNA precipitate, which was washed twice with 70% ethanol. After the ethanol had 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' (SEQ ID NO: 2).

[0070] The LPL DNA of Aspergillus niger strain GIM 3.24 (AN02) was cloned using PrimeSTAR® HS DNA polymerase. After TA cloning with the Mighty TA-cloning Reagent Set for the PrimeSTAR® kit, the DNA 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] The intron sequence of the DNA sequence of lipase B of CBS513.88 was used to find and remove the intron sequence of GIM 3.24 (AN02), and the DNA sequence was translated into amino acid sequence. The results were as follows: The amino acid sequence of GIM 3.24(AN02)LPL was as follows: MFLRREFGAVAALSVLAHAAPAPAPMQRRDISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDLSNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS (SEQ ID NO: 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 a 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 as follows: (SEQ ID NO: 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 the pAO815 vector (purchased from Invitrogen) via the EcoRI restriction site, yielding 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. Transformants were inoculated onto MGYS plates and cultured at 30°C for 3 days to obtain Pichia pastoris transformants. Single clones on the plates were picked and placed on BMMY-soybean phospholipid medium screening plates. A clone with a large white precipitated circle was selected and designated pic-AN02-LPL.

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

[0076] The plates containing the pic-AN02 mutant were washed with 2 ml of sterile water, and the plasmid was extracted and linearized with SalI. An approximately 8.5 kb fragment was recovered and used as a vector. 500 ng of the vector was transformed into competent cells of Pichia pastoris strain GS115 using electrotransformation. The transformants were inoculated onto BMM-soybean phospholipid screening medium plates and cultured at 30°C for 3 days to obtain the 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 3 ml of YPD liquid medium and cultured overnight at 30°C, 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 resulting sequence was sent to Shanghai Sangon Bioengineering Company for sequencing using 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, with the mutation sites being L86I, G187D, E209K, and A245D.

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

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

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

[0081] Its first 10 amino acids were a leader peptide, and its functional sequence was therefore: DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSwTAHRWYTIYISECS (SEQ ID NO: 14).

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

[0083] Its first 10 amino acids were a leader peptide, and its functional sequence was therefore: DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSFTAHRWYTIYISECS (SEQ ID NO: 16).

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

[0085] Its first 10 amino acids were a leader peptide, and its functional sequence was therefore: DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYEAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTDGNDGTLLDSYTAHRWYTIYISECS (SEQ ID NO: 18).

[0086] The gene sequences were synthesized by Sangon Bioengineering Co., Ltd. and cloned into the pic-AN02-LPL vector to obtain the 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 for mutants W, F, and Y. Single clones on the plates were picked and placed on BMM-soybean phospholipid medium screening plates. Clones with large white precipitated circles were selected and designated mutants W, F, and Y, respectively.

[0087] The pic-AN02m1, pic-AN02-LPL, and W, F, and Y Pichia pastoris expression strains were first activated in liquid YPD and then inoculated into BMGY medium and grown overnight at 30°C with shaking at 220 rpm. The cultures were transferred to BMGY 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 an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The treated sample was added to a buffer solution (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 and 90 μl of substrate were 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 removed, and 80 μl of Reagent A from the NEFA kit was added. The mixture was reacted at 37°C for 10 minutes. 160 μl of Reagent B was added and the mixture was reacted for 10 minutes. The absorbance at 550 nm was determined, and the enzyme activity was calculated.

[0092] Protein concentrations were determined using a Bradford kit. The specific enzyme activities of the lysophospholipases of the LPL encoded by PicAN02m1, the LPL encoded by Pic-AN02-LPL, and the mutants W, Y, and F were calculated by calculating the enzyme activity and protein concentration. As shown in Figure 1, the specific enzyme activities of the lysophospholipases of the mutants W and F were 30,224 U / mg and 18,973 U / mg, respectively; the specific enzyme activities of the lysophospholipases of picAN02m1 (M1) and pic-AN02-LPL (WT) were 13,473 U / mg and 12,746 U / mg, respectively. Compared with the starting sequence M1, i.e., picAN02m1, which had a specific enzyme activity of 13,473 U / mg, the specific enzyme activities 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) under accession number CICC2243. The starting strain was first spread onto MM solid medium plates to culture spores. After elution, the spores 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] Strain AN19E-13 was then screened using ARTP mutagenesis. The sedimentation circle of AN19E-13 was significantly larger than that of the starting strain AN19E on a lysophospholipase screening plate supplemented with uracil. The test revealed that the lysophospholipase (LPL) enzyme activity of AN19E-13 itself was 10.8-fold higher than that of the starting strain AN19E.

[0095] Attempts to perform heterologous recombinant expression of lysophospholipase (LPL) in Aspergillus niger strain AN19E-13 revealed a 112% increase in the ability of this strain to express LPL, confirming its distinct advantages as a protein expression system, particularly for lysophospholipases used in food.

[0096] In this invention, the term "ARTP" stands for ambient pressure and room temperature plasma, specifically referring to a plasma jet capable of producing high concentrations of active particles (including excited helium atoms, oxygen atoms, nitrogen atoms, and OH radicals) at atmospheric pressure and temperatures between 25 and 40°C. The term "ARTP mutagenesis" refers to the use of ambient pressure and room temperature plasma technology for strain mutagenesis. In particular, ambient pressure and room temperature plasma sources using helium as the working gas contain a variety of chemically active particle components, such as OH, nitrogen ions (positive and negative), nitrogen ions (negative), excited helium atoms, hydrogen atoms, and oxygen atoms. The abundance of active energy particles in ARTP causes damage to the genetic material of strains, plants, and cells, which then initiates the SOS repair mechanism. The SOS repair process is highly error-tolerant, producing a wide variety of mismatch sites during the repair process, ultimately stabilizing the genetic code and forming mutant strains. The intensity of SOS repair 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. The cells were washed twice with sterile water and diluted to 1 × 10 7The spore suspension was adjusted to cells / mL. 2 mL of the spore suspension was evenly spread on the surface of a Petri dish and placed under a UV lamp on a clean workbench for 90 seconds. 100 μL of the suspension was coated onto MM solid medium supplemented with 0.3% uracil and 1 mg / mL 5-fluoroorotic acid (5-FOA) and incubated at 28°C in the dark for 7 days (the entire process was carried out under a red light to prevent back mutation). Single colonies grown on MM solid medium in the previous step were transferred to MM solid medium and MM-uracil solid medium. A strain that could only grow on MM-uracil solid medium was selected, resulting in the orotate phosphoribosyltransferase auxotroph AN19E. Sequencing of the pyrE gene of Aspergillus niger AN19E strain revealed a deletion of nucleotides TT at positions 64 and 65, resulting in 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 carried out in accordance with 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 Figure 2, was constructed as follows: The LPL gene (SEQ ID NO: 19, containing the Aspergillus oryzae α-amylase signal peptide (NCBI SEQ ID NO: XM_001821384.2, 1-63 bp sequence) obtained by total gene synthesis by Sangon (Shanghai) Bioengineering Co., Ltd.) was inserted into an expression cassette containing the Aspergillus oryzae enolase promoter (NCBI SEQ ID NO: D63941.1, 215-734 bp; containing 12 copies of the enhancer sequence (gtcgtgtcgggcatttatcgggggatggaccaatcagcgtagg, SEQ ID NO: 21)) and the Aspergillus niger glycosylase terminator (NCBI SEQ ID NO: AF214480.1, terminator sequence portion) via the SphI and HindIII enzyme sites. The entire 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 Prepare a spore suspension by eluting fresh spores of Aspergillus niger AN19E with spore wash solution and filtering through Miracloth. 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 hours at 28°C and 180 rpm. The vegetative mycelia were 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 0.8 g of mycelia were suspended in 20 mL of enzymatic hydrolysis solution (1% lytic enzyme, 1% cellulase, and 0.1% helicase prepared 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 1,000 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. The supernatant was discarded. The protoplasts were then diluted to 1 × 10 in pre-chilled STC solution (1.0 M sorbitol, 50 mM CaCl, 50 mM Tris-HCl, pH 7.5). 7 / mL and kept in an ice bath until use.

[0103] To 200 μL of the protoplast suspension, 10 μL of the LPL expression vector pANE-LPL (1 μg / μL) was added, followed by 50 μL of PTC solution (40% PEG4000, 50 mM CaCl2, 50 mM Tris-HCl, pH 7.5), mixing thoroughly, and maintaining the mixture in an ice bath for 30 minutes. Next, 0.2 mL of PTC solution was added, mixing thoroughly, and then 0.8 mL of PTC solution was added, mixing thoroughly, and maintaining the mixture 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 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 was able to successfully complete the pyrE gene complementation experiment, and as shown in Figures 3 and 4, the transformants were 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 Prepare a spore suspension by eluting fresh spores of Aspergillus niger AN19E with spore wash solution and filtering through Miracloth. 7 The concentration was adjusted to spores / mL. After mixing the spore suspension and 10% glycerol at a 1:1 ratio, 10 μL of the mixed solution was placed on an iron sheet and treated with an ARTP device (Wuxi Tmaxtree Biotechnology Co., Ltd., device model: ARTP-M) for 100 seconds. The device parameters were: radio frequency power range 120 W, helium volume 10 SLM (99.999% high-purity helium), and irradiation distance 2 mm.

[0110] After treatment, the iron sheet was removed and transferred to a centrifuge tube filled with 1 mL of sterile water. A pipette tip was then used to repeatedly aspirate the cells to wash them off. The cells were then diluted to approximately 100 colonies per screening plate and finally coated onto uracil-supplemented lysophospholipase screening plates. The plates were 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 designated AN19E-13.

[0112] Strain AN19E-13, which had a significantly larger sedimentation circle, and the 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 CaCl, 1% lecithin) autoclaved at 115°C for 15 minutes. The fermentation conditions were 28°C, 200 rpm, 5 days, and an inoculum volume of 1 x 10. 7 Spores / 50 mL. Lysophospholipase LPL activity was determined.

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

[0114] 10 μL of appropriately 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 removed, and 80 μL of Reagent A from the NEFA kit (Wako: 294-63601) was added. The mixture was reacted at 37°C for 10 minutes. 160 μL of Reagent B was added and the mixture was reacted for 10 minutes. The absorbance at 550 nm was measured.

[0115] The results of the enzyme activity measurement 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 times 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] The Aspergillus niger AN19E transformant from Example 6 was used as a control, and pANE-LPL was transformed into Aspergillus niger CICC2243. Because pANE-LPL cannot use pyrE as a screening marker, p3SR2 (BCCM / LMBP: Accession Number: 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 from each of the three transformants 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 minutes. The fermentation conditions were 28°C, 200 rpm, 8 days, and an inoculum size of 1 x 10. 7 The spores were collected per 50 mL of the culture medium. 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 with that of 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 supernatant were concentrated to the same volume using a Millipore 10 KDa ultrafiltration concentration tank, and 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 the AN19E-13 transformants was significantly higher than that of the control. [1] A lysophospholipase comprising the amino acid sequence of SEQ ID NO: 14 or 16. [2] A method for producing a lysophospholipase, comprising expressing a nucleic acid molecule encoding the lysophospholipase of [1] above in a host cell and recovering the resulting polypeptide. [3] A mutant Aspergillus niger strain for expressing a food-grade lysophospholipase, which is an orotate phosphoribosyltransferase auxotroph and has increased endogenous enzyme, preferably lysophospholipase, productivity compared to a non-mutant strain. [4] A mutant Aspergillus niger strain of the above [3], which has 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 [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 [6]. [8] A host cell comprising the nucleic acid molecule of [6] or the vector of [7]. [9] The host cell according to [8] above, 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] The host cell of [9], wherein the host cell is a fungal cell, preferably a Pichia pastoris cell or an Aspergillus niger cell, preferably an Aspergillus niger cell having accession number CGMCC NO. 40011.

[11] A composition comprising the lysophospholipase of [1] above or a fermentation broth, fermentation supernatant, and / or fermentation concentrate of the host cell of any of [8] to

[10] above.

[12] A fermentation broth, fermentation supernatant, or fermentation concentrate of the host cell according to any one of [8] to

[10] above.

[13] Use of the lysophospholipase of [1] above, the composition of

[11] above, or a fermentation broth, fermentation supernatant, and / or fermentation concentrate of a host cell of any of [8] to

[10] above in oil degumming.

[14] A recombinant Aspergillus niger strain obtained by introducing a gene encoding a foreign protein into any of the strains described in [3] to [5] above.

[15]

[14] The recombinant Aspergillus niger strain according to

[14] , wherein the foreign protein is an enzyme, preferably a lysophospholipase, preferably the lysophospholipase according to [1].

[16] A method for producing a target protein, comprising introducing a gene encoding the target protein into any of the strains [3] to [5] above and culturing the strain to produce the target protein, or culturing the recombinant Aspergillus niger strain

[14] or

[15] above to produce the target protein.

[17] The method according to

[16] above, wherein the target protein is an enzyme, preferably a lysophospholipase, preferably the lysophospholipase of [1] above.

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

[19] A foreign protein produced by any of the strains [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 enzyme, more preferably a food lysophospholipase.

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

Claims

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

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

4. (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:

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. 7. The host cell of claim 6, 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.

8. 8. The host cell of claim 7, 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.

9. A composition comprising the lysophospholipase of claim 1.

10. A fermentation broth, fermentation supernatant or fermentation concentrate of the host cell of any one of claims 6 to 8, comprising the lysophospholipase of claim 1.

11. 11. Use of the lysophospholipase of claim 1 or the composition of claim 9 or the fermentation broth, fermentation supernatant and / or fermentation concentrate of claim 10 in oil degumming.

12. A recombinant Aspergillus niger strain obtained by introducing a gene encoding a foreign protein into the strain of claim 3.

13. 13. The recombinant Aspergillus niger strain of claim 12, wherein the foreign protein is an enzyme, preferably a lysophospholipase, preferably a lysophospholipase of claim 1.

14. 14. A method for producing a target protein, comprising introducing a gene encoding the target protein into the strain of claim 3 and culturing the strain to produce the target protein, or culturing the recombinant Aspergillus niger strain of claim 12 or 13 to produce the target protein.

15. 15. The method of claim 14, wherein the target protein is an enzyme, preferably a lysophospholipase, preferably a lysophospholipase of claim 1.

16. 4. A biocatalyst comprising the mutant Aspergillus niger strain of claim 3, into which a gene encoding an enzyme, preferably a lysophospholipase, preferably the lysophospholipase of claim 1, has been introduced.

Images