WO2023116738A1 - 突变的溶血磷脂酶以及用于表达该酶的突变的黑曲霉菌株 - Google Patents

突变的溶血磷脂酶以及用于表达该酶的突变的黑曲霉菌株 Download PDF

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WO2023116738A1
WO2023116738A1 PCT/CN2022/140539 CN2022140539W WO2023116738A1 WO 2023116738 A1 WO2023116738 A1 WO 2023116738A1 CN 2022140539 W CN2022140539 W CN 2022140539W WO 2023116738 A1 WO2023116738 A1 WO 2023116738A1
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lysophospholipase
strain
aspergillus niger
enzyme
mutated
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PCT/CN2022/140539
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English (en)
French (fr)
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宣姚吉
吴伟
孙佳楠
刘国瑞
戴小军
牛其文
王圣南
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丰益(上海)生物技术研发中心有限公司
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Priority claimed from CN202111598184.2A external-priority patent/CN116376725A/zh
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Publication of WO2023116738A1 publication Critical patent/WO2023116738A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

Definitions

  • the present invention relates to the field of biotechnology. More specifically, a mutant lysophospholipase is involved. It also relates to genes encoding said enzymes, as well as vectors and host cells comprising said genes. Furthermore, the use of said enzyme is also related.
  • the present application also relates to a mutant Aspergillus niger strain, which can be used to express the mutant lysophospholipase of the present invention.
  • the present invention also relates to a method for producing foreign protein, especially the lysophospholipase of the present invention, by using the bacterial strain.
  • Aspergillus niger belongs to the subphylum Ascomycota, the family Aspergillus, and the common species of fungi of the genus Aspergillus. It is also the most common filamentous fungus in nature and is widely distributed in plant products, food and soil. Its conidia heads are dark brown radial, spherical top capsule, conidiophores of different lengths, well-developed hyphae, and multi-branched. Aspergillus niger can grow rapidly by degrading organic matter in nature and absorbing nutrients therein. Aspergillus niger is listed as GRAS (Generally Regards As Safe) by the US Food and Drug Administration (FDA) and recognized by the World Health Organization. It is a very commonly used species in filamentous fungi. Because Aspergillus niger can produce enzymes and organic acids, it has become an important industrial production strain.
  • GRAS Generally Regards As Safe
  • Aspergillus niger has high protein secretion and expression ability.
  • the exogenous protein expressed by Aspergillus niger has the characteristics of large expression amount, high extracellular secretion rate, protein molecular folding and modification system close to higher eukaryotic cells, and the expressed exogenous protein has natural activity.
  • Aspergillus niger can also perform various post-translational processes, such as glycosylation modification protease cleavage and disulfide bond formation. Therefore, the use of Aspergillus niger as an expression strain to express homologous and heterologous proteins has received increasing attention.
  • the commercial enzyme preparations currently produced by Aspergillus niger include amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase and wood enzymes used in food, washing, textile and paper industries. Glycanase, etc.
  • the heterologous proteins expressed through it include lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, etc.
  • Enzyme preparations used in food in China must comply with the national standard of the People's Republic of China GB2760-National Food Standards ⁇ Food Additive Use Standards, in which heterologously expressed food lysophospholipase (derived from Aspergillus niger) that meets the regulations can only be produced by Aspergillus niger for heterologous expression.
  • lipases Two lipases, called lipaseA and lipaseB, or lipase1 and lipase2, have been reported in Aspergillus niger. Among them, the nature of lipaseB is quite special. Zhu Shu-sen cloned and expressed lipaseB from Aspergillus niger A733, and found that the optimum temperature of lipaseB was 15°C, the optimum pH was 3.5-4.0, and it could not tolerate the temperature above 40°C.
  • the enzyme can hydrolyze substrates with pNPC4-pNPC18 chain length, and pNPC12 is the most suitable substrate. However, the specific enzyme activity of lipB is extremely low, and the specific enzyme activity of lipB after purification is only 6.8U/mg.
  • liapse2 or lipaseB derived from Aspergillus niger is not strong.
  • the specific enzyme activity is extremely low, and the temperature tolerance is not high, and the most suitable substrate for use is triglycerides of short-chain fatty acids.
  • This enzyme is not as good as the more widely used lipase TL and RML, and its specific enzyme activity can reach 12000 or 8000U/mg.
  • TL can withstand a temperature of 60°C for 20 hours without inactivation, and RML can hydrolyze triglycerides of various long-chain fatty acids.
  • the inventor designed primers from Aspergillus niger GIM 3.24 (AN02) using the lipaseB gene sequence of CBS513.88 to clone the LPL gene ANO2-LPL of the above-mentioned Aspergillus niger strain, and found that ANO2-LPL has a very high phospholipid content Enzyme A1 activity and lysophospholipase activity can be used for oil degumming, without adding alkali, can reduce the phosphorus content of crude oil to 5ppm, can be used for enzymatic degumming, and can reduce Soap formation during degumming.
  • AN02-LPL has a very high activity of lysophospholipase, it can be used in combination with phospholipase A2, and soybean phospholipase is used as raw material to prepare glycerol phosphorylcholine (GPC), which has the effect of strengthening the brain and anti-aging. It is used in medicine and In health products.
  • GPC glycerol phosphorylcholine
  • the inventor obtained the LPL encoded by the mutant picAN02m1 of ANO2 by random mutation method, the mutation sites are L86I, G187D, E209K, A245D, and the thermal stability under the conditions of pH 5.6, 6.0 and 6.6 is higher than that of the wild type AN02-LPL improved by 3.2 times, 31 times and 28.5 times.
  • Lysophospholipases with higher specific enzyme activity are still desired in the art. Moreover, there is still a need in the art for Aspergillus niger strains capable of efficiently expressing various proteins, especially lysophospholipase for food.
  • the inventor developed a new mutant on the basis of the mutant lysophospholipase developed in CN2021107079194. Specifically, on the basis of picAN02m1, after the threonine mutation at position 255 of LPL is placed in tryptophan or phenylalanine, the specific enzyme activity for lysophospholipase is 12746 U/mg of LPL encoded by picAN02 and picAN02m1 and 13473U/mg, increased to 30224U/mg and 18973U/mg. This further improves the efficiency of using the lysophospholipase to prepare glycerophosphorylcholine (GPC).
  • GPC glycerophosphorylcholine
  • the present invention uses Aspergillus niger CICC2243 as the starting strain to construct an orotate phosphoribosyltransferase auxotrophic strain (pyrE - ) strain, and then through ARTP mutagenesis screening to a strain of lysophospholipase added with uracil
  • the strain whose sedimentation circle was significantly larger compared with the mutagenesis starting strain had its own lysophospholipase (LPL) enzyme activity increased by 10.8 times.
  • Recombinant expression of lysophospholipase LPL found that its expression ability was significantly improved, which was 112% higher than that of the starting strain.
  • the bacterial strain can be used for efficient heterologous expression of various proteins, especially lysophospholipase for food.
  • the present invention relates to the following aspects:
  • the invention relates to a lysophospholipase comprising the amino acid sequence of SEQ ID NO: 14 or 16.
  • the present invention relates to a nucleic acid molecule comprising: (a) a nucleotide sequence encoding the above-mentioned lysophospholipase; and (b) a nucleotide sequence complementary to the nucleotide sequence described in (a), complementary Can be partially or fully complementary.
  • nucleotide sequences of the present invention may encode the same enzyme.
  • skilled artisan can use routine techniques to make nucleotide substitutions that do not affect the enzymatic activity encoded by the nucleotide sequences of the present invention, which may reflect the characteristics of any particular host organism used to express the enzymes of the present invention. Codon bias.
  • the present invention also provides a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector.
  • Vector refers to an extrachromosomal element, usually in the form of a circular double-stranded DNA molecule, carrying genes that are not part of the central metabolism of the cell. Such elements may be autonomously replicating sequences, genomic integrating sequences, bacteriophage or nucleotide sequences, linear or circular, single- or double-stranded DNA or RNA from any source, many of which have been joined or recombined Into a specific construct, the construct is capable of introducing into cells the promoter fragment and DNA sequence of the selected gene product together with the appropriate 3' untranslated sequence.
  • Genes and gene products encoding lysophospholipases of the 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.
  • Heterologous host cells for expression of nucleic acid molecules of the invention may be microbial hosts that exist in the fungal or bacterial family and grow over a wide range of temperature, pH and solvent tolerance.
  • any bacteria, yeast and filamentous fungi are contemplated as suitable hosts for expressing nucleic acid molecules of the invention.
  • host strains include, but are not limited to, bacterial, fungal, or yeast species such as Pichia, Aspergillus, Trichoderma, Saccharomyces, Phaffia , Kluyveromyces, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Phage Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Abnormal Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystosis (Methylocy)
  • the host cell is a fungal cell. In one embodiment, the host cell is a Pichia or Aspergillus niger cell. In one embodiment, the host cell is the Aspergillus niger cell of the present invention having the preservation number of CGMCC No.40011.
  • vectors that can be used to transform the above-mentioned host cells are well known in the art.
  • vectors typically contain sequences that direct the transcription and translation of the associated genes, selectable markers, and sequences that allow autonomous replication or chromosomal integration.
  • Suitable vectors contain a 5' region of the gene containing transcription initiation controls and a 3' region of a DNA fragment that controls transcription termination.
  • the present invention also relates to a method for producing lysophospholipase, comprising expressing a nucleic acid molecule encoding the lysophospholipase of the present invention in a host cell, and recovering the resulting polypeptide.
  • Various culture methods can be used to produce the enzymes of the invention.
  • large-scale production of specific gene products from recombinant microbial hosts can be performed by batch, fed-batch and continuous culture methods.
  • Continuous culture is an open system in which a defined 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 phase density where the cells are primarily in logarithmic phase of growth.
  • continuous culture can be performed with immobilized cells, where carbon and nutrients are continuously added, and valuable products, by-products or waste products are continuously removed from the cell mass.
  • Cell fixation can be performed using a wide range of solid supports consisting of natural and/or synthetic materials.
  • Recovery of the desired enzyme from batch fermentation, fed-batch fermentation, or continuous culture can be accomplished by any method known to those skilled in the art.
  • the cell slurry is separated from the culture medium by centrifugation or membrane filtration, optionally washed with water or an aqueous buffer of the desired pH, and then the cell slurry in the aqueous buffer of the desired pH is suspended for It homogenizes, producing a cell extract containing the required enzymes.
  • the present invention also relates to a composition comprising a lysophospholipase of the present invention or a fermentation broth, a fermentation supernatant and/or a fermentation concentrate of a host cell of the present invention.
  • the enzyme composition of the invention may be in any form suitable for use, for example, a crude fermentation broth with or without depleted cells, a cell lysate with or without cell debris, a semi-purified or purified enzyme composition, or a host cell as a source of enzymes.
  • 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 may be stabilized according to established techniques, for example by adding stabilizers such as sugars, sugar alcohols or other polyols, and/or lactic acid or other organic acids.
  • the invention furthermore relates to a fermentation broth, fermentation supernatant or fermentation concentrate of the host cells of the invention.
  • the present invention also relates to the use of the lysophospholipase of the present invention in oil degumming.
  • the lysophospholipase of the invention is used for oil degumming, compared with the lysophospholipase of the prior art, the specific enzyme activity is significantly improved, and the production cost is reduced.
  • the present invention relates to mutated Aspergillus niger (Aspergillus niger) bacterial strains, which are auxotrophic strains of orotate phosphoribosyltransferase, and which, relative to non-mutated bacterial strains, have higher levels of endogenous enzymes, such as lysophospholipase Increased production capacity. For example, it may be increased by a factor of 10-15, such as by a factor of 10-12, especially by a factor of 10.8.
  • the term "endogenous enzyme” refers to an enzyme expressed by the mutant Aspergillus niger strain itself, for example, including but not limited to lysophospholipase expressed by the mutant Aspergillus niger strain itself.
  • nucleotides TT at positions 64 and 65 of the orotate phosphoribosyltransferase pyrE gene of the mutant Aspergillus niger of the present invention are deleted.
  • the mutant Aspergillus niger strain of the present invention has the deposit number of CGMCC No.40011.
  • mutant Aspergillus niger strains of the present invention can efficiently express heterologous proteins.
  • the mutant Aspergillus niger strains 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 rennet, thaumatin, lipase, etc.
  • the mutant Aspergillus niger strain of the present invention expresses lysophospholipase LPL recombinantly
  • the protein expression level of the starting strain can be increased by more than 100%, such as 100%-150%, especially 100%-120%, more particularly It is 100%-112%. Therefore, this strain has obvious practicability for high-efficiency expression of heterologous proteins.
  • the present invention relates to a recombinant Aspergillus niger strain obtained by introducing a gene encoding a foreign protein into the above-mentioned mutant Aspergillus niger strain.
  • the exogenous proteins are enzymes and other proteins such as amylases, glucose oxidases, catalases, cellulases, pectinases, proteases, phytases, xylanases, lysozyme Enzymes, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, etc., especially lysophospholipase.
  • the present invention relates to a method for producing a target protein, comprising introducing a gene encoding the target protein into the above-mentioned mutant Aspergillus niger strain, and culturing the strain to produce the target protein.
  • it involves culturing the recombinant Aspergillus niger strain described above to produce the protein of interest.
  • the exogenous proteins are enzymes and other proteins such as amylases, glucose oxidases, catalases, cellulases, pectinases, proteases, phytases, xylanases, lysozyme Enzymes, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, etc., especially lysophospholipase.
  • the present invention relates to a biocatalyst comprising the above mutated Aspergillus niger strain into which genes encoding foreign proteins are enzymes and other proteins such as amylase, glucose oxidase, peroxidase Hydrogenase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lipase, etc., In particular lysophospholipase.
  • enzymes and other proteins such as amylase, glucose oxidase, peroxidase Hydrogenase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin-6, human lactoferrin, bovine chymosin, thaumatin, lip
  • the present invention relates to exogenous proteins produced by the strains described above.
  • the exogenous proteins are enzymes and other proteins such as amylase, glucose oxidase, catalase, cellulase, pectinase, protease, phytase, xylanase, lysozyme, interleukin- 6.
  • the exogenous protein can be used in food, preferably an enzyme for food, more preferably lysophospholipase for food.
  • the lysophospholipase is a mutated lysophospholipase of the invention.
  • the present invention also relates to recombinant microbial cells into which the intracellular components derived from the above-mentioned mutated Aspergillus niger strains have been introduced.
  • its intrabacterial components can be isolated by conventional techniques, and the components can be introduced into other microorganisms.
  • the recombinant microbial cells introduced with the components have the excellent properties of the strains of the present invention.
  • the term "bacterial component" refers to the sum of all genetic material of an organism, specifically, including but not limited to: coding DNA and non-coding DNA, mitochondrial DNA.
  • codons for some species may be rare codons in A. niger. Therefore, when introducing the expression vector, the gene encoding the foreign protein can be firstly optimized with codons suitable for Aspergillus niger of the present invention, thereby increasing the expression amount.
  • Aspergillus niger of the present invention can express a variety of exogenous proteins, including enzymes for food, such as lipase for food, pharmaceutical proteins, various enzymes from plants, animals and bacteria, membrane receptor proteins, prosthetic group-containing proteins and Proteins that can be used to study crystal structures, etc.
  • the Aspergillus niger expressing exogenous enzyme components of the present invention can also use the whole cell as a biocatalyst.
  • the mutant Aspergillus niger strain of the present invention such as the Aspergillus niger strain with the deposit number of CGMCC No.40011, can be used to express the mutant lysophospholipase of the present invention, for example comprising SEQ ID NO: 14 or 16 amino acid sequences of lysophospholipase. Due to the combination of both effects, the expression efficiency of lysophospholipase is expected to be further improved.
  • Fig. 1 is the lysophospholipase LPL encoded by picAN02m1 (M1) and pic-AN02-LPL (WT) in CN2021107079194, and the mutant W of the present invention, Y, the lysophospholipase of F is directed against 1-palmitoyl lysophosphatidylcholine specific enzyme activity.
  • Fig. 2 is a schematic diagram of the LPL gene expression vector constructed in the present invention.
  • Fig. 3 is a plate diagram of AN19E anaplerosis transformation.
  • Fig. 4 is a diagram of a screening plate for lysophospholipase LPL activity of AN19E anaplerotic transformants.
  • Fig. 5 is a colony diagram of the mutagenized strain AN19E-13 in the LPL screening medium.
  • Fig. 6 is a comparison chart of the enzyme activity of the endogenous lysophospholipase LPL expressed by the mutant strain AN19E-13 of the present invention and the starting strain AN19E.
  • Fig. 7 is a comparison chart of the enzymatic activity of lysophospholipase LPL recombinantly expressed by the mutant strain AN19E-13 of the present invention and the starting strains CICC2243 and AN19E.
  • Fig. 8 is a protein electrophoresis diagram of lysophospholipase LPL recombinantly expressed by the mutant strain AN19E-13 of the present invention and the starting strains CICC2243 and AN19E.
  • the bacterial strain AN19E-13 of the present invention was preserved on December 20, 2021 in the China General Microorganism Culture Collection Management Center (CGMCC), No. 3, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. .40011, classified as Aspergillus niger (Aspergillus niger).
  • CGMCC General Microorganism Culture Collection Management Center
  • Aspergillus niger strain GIM 3.24 (AN02) was purchased from Guangdong Microbial Culture Collection Center.
  • Aspergillus niger fermentation medium formula is:
  • lysis buffer 100mM Tris-HCl pH 8.0; 50mM Na ⁇ EDTA; 1% SDS.
  • Component A BMMY solid medium: 1% yeast extract, 2% peptone, 100mM citric acid-sodium citrate buffer, pH 6.6, 1.34% YNB, 4 x 10-5% biotin (added before pouring the plate), 2 %methanol (added before pouring the plate), 2% agar was dissolved in 250ml deionized water.
  • Component B soybean lecithin substrate solution 250ml: mixed with 4% soybean lecithin, emulsified with a high-speed homogenizer at 8000rpm for 3 minutes, paused for 1 minute and then emulsified for 3 minutes to prepare a substrate solution.
  • Glass beads were purchased from: American Biospec. DNA Polymerase was purchased from Takara, product number: R010A.
  • SalI, EcoRI and BglII restriction enzymes were purchased from NEB.
  • Lysophospholipid 1-palmitoyl-sn-glycero-3-phosphocholine was purchased from Aladdin, Cat. No. P130493-500mg.
  • NEFA kit was purchased from Wako Pure Chemical Industries, Ltd., Japan.
  • MGYS plate 1.34% YNB, 4x 10-5% biotin (filter sterilized), 2% glycerol, 1M sorbitol.
  • YPD medium 1% yeast extract, 2% peptone, 2% glucose.
  • BMGY shake flask medium 1% yeast extract, 2% peptone, 100mM citric acid-sodium citrate buffer, pH 6.6, 1.34% YNB, 4x 10-5% biotin (sterilized by filtration), 2% glycerol.
  • BMMY-shake flask medium 1% yeast extract, 2% peptone, 100mM citric acid-sodium citrate buffer, pH 6.6, 1.34% YNB, 4x 10-5% biotin (sterilized by filtration), 2% methanol (inoculation when added).
  • Embodiment 1 Cloning of Aspergillus niger lipaseB gene
  • Aspergillus niger strain GIM 3.24 (AN02) was cultured with Aspergillus niger fermentation medium at 30°C for 24 hours. Take the fermentation culture, centrifuge at 4000rpm for 5min, and get the bacteria. Resuspend with 700ul analysis buffer, transfer to cryopreservation tube, add 300ul glass beads, vibrate on mini beadbeater for 40s, centrifuge at 12000rpm for 10min.
  • LPL-1 and LPL-2 were designed according to the lipaseB gene sequence of Aspergillus niger CBS513.88 in NCBI.
  • DNA Polymerase was used to clone the LPL DNA of Aspergillus niger strain GIM 3.24 (AN02) with Mighty TA-cloning Reagent Set for After TA cloning, the kit was transformed into Escherichia coli DH5a, and sent to Sangon Bioengineering Co., Ltd. for DNA sequencing, and finally the LPL DNA sequence of GIM 3.24 (AN02) was obtained, as follows:
  • amino acid sequence of GIM 3.24 (AN02) LPL is as follows:
  • the Sac-II restriction site CCGCGG was added at the 5' end, and the synthetic gene sequence was sent to Shengong Bioengineering Co., Ltd., and cloned into the pA0815 vector (purchased from invitrogen) through the EcoRI restriction site to obtain the pic-AN02 plasmid.
  • the vector was transformed into competent cells of Pichia pastoris GS115 strain (purchased from invitrogen) by electroporation.
  • the transformant was inoculated on the MGYS plate and cultured at 30° C. for 3 days to obtain a Pichia transformant.
  • Pick the single clone on the plate put it on the BMMY-soybean phospholipid medium screening plate, select the clone with a large white precipitation circle, and name it pic-AN02-LPL.
  • TaKaRa Taq enzyme and primer pair PLPL-1 TCCCCGCGGCGAAACGATGAGATTTCCTTC (SEQ ID NO: 7)/PLPL-2: CCGGAATTCTTAAGAACACTCAGAAATG (SEQ ID NO: 8) to carry out error-prone PCR with pic-AN02-LPL as a template (additionally during PCR 0.3 mM MnCl 2 ), a collection of mutant amplicon fragments with a size of about 1000 bp was obtained. The obtained fragment was cloned into pic-AN02 plasmid through Sac-II and EcoRI restriction sites, and the obtained vector was transformed into Escherichia coli DH5 ⁇ strain.
  • the plate containing the pic-AN02 mutant was washed with 2 ml of sterile water, the plasmid was extracted, linearized with SaiI, and a fragment of about 8.5 kb was recovered as a carrier. Take 500ng of the vector, and transform the vector into competent cells of Pichia pastoris GS115 strain by electroporation. The transformant was inoculated on a BMM-soybean phospholipid screening medium plate, and cultured at 30° C. for 3 days to obtain a Pichia mutant library of pic-AN02-LPL. A mutant pic-AN02m1 was screened.
  • the pic-AN02m1 strain was inoculated in 3ml YPD liquid medium, cultured overnight at 30°C, and the genomic DNA was extracted.
  • genomic DNA of the pic-AN0m1 strain as a template, use DNA polymerase and primer pair AOX-5: GACTGGTTCCAATTGACAAGC (SEQ ID NO: 9) and 3'-AOX1: GGCAAATGGCATTCTGACATCCTC (SEQ ID NO: 10) were used for PCR amplification to obtain the DNA sequence of the ANO2 mutant in the pic-AN02m1 strain.
  • the obtained sequence was sent to Shanghai Sangon Bioengineering Co., Ltd., and AOX-5/3'-AOX1 was sequenced with primers.
  • the DNA sequence of the mutant LPL of the pic-AN02m1 strain and its encoded amino acid are shown below, and the mutation sites are L86I, G187D, E209K, and A245D.
  • Threonine at position 255 of lysophospholipase LPL encoded by pic-AN02m1 was mutated into tryptophan, phenylalanine and tyrosine respectively, and the mutated genes were named W, F and Y.
  • the gene sequence was synthesized by Sendong Bioengineering Co., Ltd., and cloned into the pic-AN02-LPL vector to obtain mutant W, F and Y plasmids. After linearization with SalI, the vector was transformed into competent cells of Pichia pastoris GS115 strain by electroporation. The transformants were inoculated on MGYS plates and cultured at 30°C for 3 days to obtain Pichia transformants of mutants W, F and Y. Pick the single clones on the plate, put them on the BMM-soybean phospholipid medium screening plate, select the clones with large white precipitation circles, and name them as mutants W, F and Y respectively.
  • the obtained fermentation is also concentrated 30 times by ultrafiltration desalting with an ultrafiltration tube with a molecular weight cut-off of 10 kDa.
  • the treated samples were added to buffer (20 mM citric acid-sodium citrate buffer (pH 4.0)).
  • the concentrated enzyme solution was assayed for lysophospholipase activity as follows:
  • 9ml substrate 5ml 1% lysophospholipid, 1ml 20% Triton X-100, 2.5ml 0.1M citric acid-sodium citrate buffer.
  • the initial strain Aspergillus niger was purchased from China Industrial Microorganism Culture Collection Center (CICC for short), and the strain preservation number is CICC2243.
  • the initial strain was first spread on the MM solid medium plate to cultivate the spores, after the spores were washed out, and then subjected to ultraviolet mutagenesis, and finally screened by adding 5-fluoroorotic acid and uracil on the screening plate to obtain milk Clear acid phosphoribosyltransferase auxotrophic strain AN19E.
  • LPL lysophospholipase
  • ARTP is the abbreviation of Atmospheric and Room Temperature Plasma (Atmospheric and Room Temperature Plasma), which specifically refers to particles with high activity (including those in the Excited state of helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc.) concentration of the plasma jet.
  • ARTP mutagenesis refers to the mutagenesis of bacterial strains using atmospheric room temperature plasma technology.
  • the atmospheric room temperature plasma source using helium as the working gas contains a variety of chemically active particle components, such as OH, nitrogen Molecular two positive system, nitrogen molecule one negative system, excited state helium atom, hydrogen atom and oxygen atom, etc.
  • the active energy particles rich in ARTP cause damage to the genetic material of strains/plants/cells, etc., and induce biological cells to start the SOS repair mechanism.
  • the SOS repair process is a high fault-tolerant repair, so a rich variety of mismatch sites will be generated during the repair process, and finally stable genetics will be formed to form mutant strains.
  • the strength of SOS repair is closely related to the degree of DNA damage.
  • Embodiment 4 Obtaining of orotic acid phosphoribosyltransferase auxotrophic Aspergillus niger strain
  • the spores of Aspergillus niger CICC2243 strain were inoculated onto MM solid medium (1% glucose, 0.15% KH 2 PO 4 , 0.6% NaNO 3 , 0.05% KCl, 0.05% MgSO 4 , 2% agar powder), 28°C, Static culture for 5 days to obtain Aspergillus niger spores.
  • Use spore washing solution (0.9% NaCl, 0.05% Tween 80) to elute fresh Aspergillus niger CICC2243 spores, filter through Miracloth (Calbiochem, Cat# 475885) to prepare a spore suspension, wash the thalline twice with sterile water and Adjust to 1 ⁇ 10 7 cells/mL.
  • Embodiment 5 Construction of LPL expression vector
  • LPL Aspergillus niger lysophospholipase
  • the LPL gene (SEQ ID NO: 19, with Aspergillus oryzae ⁇ -amylase signal peptide (NCBI sequence number: XM_001821384. 2, 1-63bp sequence)) inserted into Aspergillus oryzae enolase promoter (NCBI sequence number: D63941.1, 215-734bp; containing 12 copies of enhancer sequence (gtcgtgtcgggcatttatcgggggatggaccaatcagcgtagg, SEQ ID NO: 21) and Aspergillus niger saccharification
  • the enzyme terminator NCBI sequence number: AF214480.1, the terminator sequence part
  • the entire expression frame was inserted into the multiple cloning site of the cloning vector pSP72 with BglII and XhoI, and finally the PyrE derived from Aspergillus niger was expressed
  • the gene (NCBI sequence number: AY840014.1) was inserted into the vector with the Xh
  • Embodiment 6 Aspergillus niger AN19E replenishment experiment
  • Fresh spores of Aspergillus niger AN19E were eluted with spore washing solution, filtered through Miracloth to prepare a spore suspension, and adjusted to 1 ⁇ 10 7 spores/mL. Inoculate 1mL of spore suspension into mycelia medium (2% tryptone, 1% yeast extract, 2% glucose, 0.3% uracil), culture at 28°C, 180rpm for 40 hours, and collect by sterilizing Miracloth growing hyphae.
  • mycelia medium 2% tryptone, 1% yeast extract, 2% glucose, 0.3% uracil
  • the mycelium is transferred to a 100mL Erlenmeyer flask, and every 0.8g of mycelium is resuspended in 20mL of enzymolysis solution (preparation of 1% lyase, 1% cellulase, 0.1% helicase with osmotic pressure stabilizer, 0.22 ⁇ m microporous membrane filtration sterilization), at 30°C, 90rpm, enzymatic hydrolysis for 60-90min.
  • the enzymatically hydrolyzed protoplast mixture was filtered with Miracloth, and the filtrate was collected, centrifuged at 1000g at 4°C for 10min, resuspended with 5mL pre-cooled 1.0mol/L sorbitol solution, centrifuged at 800g, 4°C for 10min, and discarded. clear.
  • the colonies grown on the plate were transferred to the lysophospholipase LPL screening medium and cultured at 28°C for 3 days.
  • composition of LPL screening medium is as follows:
  • Solution A 2% maltose, 1.34% YNB, citric acid 6.88g/500mL, sodium citrate 5.07g/500mL, 5mM CaCl2, add water to 200mL.
  • Solution B Add 200ml of water to 1% lecithin and emulsify 2% agarose with a homogenizer, add water to 300ml with 0.02% Triton-x-100
  • Transformation found that Aspergillus niger AN19E can well realize the pyrE gene complementation experiment, and the transformant showed the activity of lysophospholipase LPL in the lysophospholipase LPL screening medium, as shown in Figure 3 and Figure 4, which further proves that the following Orotate phosphoribosyltransferase auxotrophic Aspergillus niger AN19E as the host Aspergillus niger expression system.
  • Embodiment 7 Aspergillus niger AN19E ARTP mutagenesis experiment
  • the fresh Aspergillus niger AN19E spores were eluted with the spore washing solution, filtered through Miracloth to prepare a spore suspension, and adjusted to 2 ⁇ 10 7 spores/mL.
  • Instrument parameter setting radio frequency
  • the power range is 120W
  • the amount of helium is 10SLM (99.999% high-purity helium)
  • the irradiation distance is 2mm.
  • Embodiment 8 Screening of mutagenic strains
  • Example 7 The spores that had been mutagenized in Example 7 were spread on a lysophospholipase screening plate added with uracil for screening, and a strain with a significantly larger sedimentation circle was found, as shown in Figure 5, named AN19E-13 .
  • the assay method for lysophospholipase LPL activity is as follows:
  • 9mL substrate 5mL 1% soybean lecithin, 1mL 20% Triton X-100, 2.5mL 0.1M pH4.0 citric acid-sodium citrate buffer.
  • the strain AN19E-13 was deposited on December 20, 2021 at the China General Microorganism Culture Collection Center (CGMCC), No. 3, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.40011 , classified as Aspergillus niger (Aspergillus niger).
  • CGMCC General Microorganism Culture Collection Center
  • Embodiment 9 the investigation of the expression ability of Aspergillus niger AN19E-13
  • Aspergillus niger AN19E transformant in Example 6 was used as a control, and pANE-LPL was transformed into Aspergillus niger CICC2243 in addition, because pANE-LPL cannot use pyrE as a screening marker, so p3SR2 (BCCM/LMBP: Accession number: 2363) was used, Contains the acetamidase (amdS) gene for transformation selection.
  • the regeneration medium needs to be depleted of sodium nitrate and supplemented with 15mM acetamide and 20mM cesium chloride.
  • Polyacrylamide gel electrophoresis analysis Use a 0.22 ⁇ m filter membrane to filter the supernatant, concentrate the same amount of supernatant to the same volume using a Milipore 10KDa ultrafiltration concentration tank, and take the same volume of concentrated enzyme solution for polyacrylamide coagulation Gel electrophoresis analysis. The results of electrophoresis are shown in Figure 8.

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Abstract

提供了突变的溶血磷脂酶。还提供了编码所述酶的基因，以及包含所述基因的载体以及宿主细胞。此外，还提供了所述酶的用途。本发明另外提供了突变的黑曲霉菌株及其应用。具体地，本发明提供了突变的黑曲霉(Asporgillus niger)菌株，其是乳清酸磷酸核糖基转移酶营养缺陷型菌株，并且其相对于未突变的菌株，内源酶，优选溶血磷脂酶的生产能力提高。本发明还提供了所述菌株的各种应用。例如，所述菌株可以用于表达本发明的突变的溶血磷脂酶。

Description

突变的溶血磷脂酶以及用于表达该酶的突变的黑曲霉菌株 技术领域
本发明涉及生物技术领域。更具体地，涉及一种突变的溶血磷脂酶。还涉及编码所述酶的基因，以及包含所述基因的载体以及宿主细胞。此外，还涉及所述酶的用途。
本申请还涉及一种突变的黑曲霉菌株，其可以用于表达本发明的突变的溶血磷脂酶。本发明还涉及用该菌株生产外源蛋白，特别是本发明的溶血磷脂酶的方法。
背景技术
黑曲霉(Aspergillus niger)属于子囊菌亚门、丛梗孢科、曲霉属真菌的常见种，也是自然界中最为常见的丝状真菌，在植物性产品、粮食和土壤中广泛分布。其分生孢子头为黑褐色放射状，球状顶囊，分生孢子梗长短不一，菌丝发达，多分支。黑曲霉能够通过降解自然界中的有机质并吸收其中的营养物质而快速生长。黑曲霉被美国食品与药品管理局(FDA)列为GRAS(Generally Regards As Safe)级别并得到了世界卫生组织的认可，是丝状真菌中应用非常普遍的种属。由于黑曲霉能够产生酶制剂和有机酸，因而成为重要的工业生产菌株。
黑曲霉具有高效的蛋白分泌表达能力。黑曲霉表达外源蛋白具有表达量大、胞外分泌率高、蛋白质分子折叠和修饰系统接近高等真核细胞等特点，且表达的外源蛋白具有天然活性。另外，黑曲霉还能进行各种翻译后加工，如糖基化修饰蛋白酶切割和二硫键的形成等。因而，利用黑曲霉作为一个表达菌株来表达同源和异源蛋白日益受到重视。目前利用黑曲霉生产的商业酶制剂包括食品、洗涤、纺织和造纸等工业中用到的淀粉酶、葡萄糖氧化酶、过氧化氢酶、纤维素酶、果胶酶、蛋白酶、植酸酶和木聚糖酶等，通过其表达的异源蛋白有溶菌酶、白细胞介素-6、人乳铁蛋白、牛凝乳酶、奇异果甜蛋白、脂肪酶等。
在中国应用于食品中的酶制剂必须符合中华人民共和国国家标准GB2760-食品国家标准\食品添加剂使用标准，其中符合法规的异源表达食品用溶血磷脂酶(黑曲霉来源)只能由黑曲霉来进行异源表达。
在黑曲霉中已报道了有两种脂肪酶，称为lipaseA和lipaseB，或者称为lipase1和lipase2。其中lipaseB的性质比较特殊。Zhu Shu-sen从黑曲霉A733中克隆表达了lipaseB，发现lipaseB的最适温度为15℃，最适pH在3.5-4.0，不能耐受超过40℃以上的温度。该酶能够水解pNPC4-pNPC18链长的底物，其中pNPC12为最适底物。但lipB的比酶活极低，经过纯化后的lipB比酶活仅为6.8U/mg。
Jiangke Yang等人从黑曲霉CICC 4009中克隆表达了lipas2，虽然这个lipase2和Zhu Shu-sen克隆的lipaseB高度同源，仅有两个氨基酸的差别，但是性质有一定程度的差异，最适底物为pNPC8和pNPC10，最适pH小于6.5，最适温度为50℃，不能耐受超过40℃以上的温度。
综上所述，黑曲霉来源的liapse2或者lipaseB的实用性不强。首先比酶活极低，温度耐受性不高，适用的最适底物是短链脂肪酸的甘油三脂。该酶不如用途较为广泛的脂肪酶TL和RML，其比酶活能够达到12000或8000U/mg。TL能够耐受60℃的温度保持20小时不失活，RML能水解各种长链脂肪酸的甘油三脂。
在发明CN2021107079194中，发明人从黑曲霉GIM 3.24(AN02)中利用CBS513.88的lipaseB的基因序列设计引物克隆了上述黑曲霉菌株的LPL基因AN02-LPL，并发现AN02-LPL具有非常高的磷脂酶A1活力和溶血磷脂酶活力，可以用于油脂脱胶，在不添加碱的条件下，可以将毛油的含磷量降低至5ppm，可以用于酶法脱胶，并且由于不需要加碱可以减少脱胶时皂的形成。另外由于AN02-LPL具有极高的溶血磷脂酶活力，所以，可以与磷脂酶A2合用，利用大豆磷脂酶为原料制备甘油磷酸酰胆碱(GPC)，具有健脑防衰老的作用用于医药及保健品中。
在CN2021107079194中，发明人通过随机突变的方法得到AN02的突变体picAN02m1编码的LPL，突变位点为L86I，G187D，E209K，A245D，在pH5.6，6.0和6.6的条件下热稳定性较野生型AN02-LPL提高了3.2倍，31倍和28.5倍。
本领域仍然期望得到比酶活更高的溶血磷脂酶。并且，本领域仍然需要能够高效表达各种蛋白，尤其是食品用溶血磷脂酶的黑曲霉菌株。
发明内容
本发明人基于前期工作，在发明CN2021107079194中开发的突变溶血磷脂酶基础上，开发了新的突变体。具体地，在picAN02m1的基础上将LPL的255位的苏氨酸突变位色氨酸或或者苯丙氨酸后，针对溶血磷脂酶的比酶活，由picAN02和picAN02m1编码的LPL的12746U/mg和13473U/mg，提高至30224U/mg和18973U/mg。这进一步提高了利用该溶血磷脂酶制备甘油磷酸酰胆碱(GPC)的效能。
此外，本发明以黑曲霉CICC2243为出发菌株，构建了乳清酸磷酸核糖基转移酶营养缺陷型菌株(pyrE -)菌株，再通过ARTP诱变筛选到了一株在添加了尿嘧啶的溶血磷脂酶筛选平板上，与诱变出发菌株相比沉淀圈明显变大的菌株，其自身溶血磷脂酶(LPL)酶活提高了10.8倍。进行重组表达溶血磷脂酶LPL发现其表达能力有明显的提升，相对于出发菌株，提高了112％。该菌株可以用来高效异源表达各种蛋白，尤其是食品用溶血 磷脂酶。
具体地，本发明涉及以下方面：
一方面，本发明涉及溶血磷脂酶，其包含SEQ ID NO：14或16的氨基酸序列。
另一方面，本发明涉及核酸分子，其包含：(a)编码上述溶血磷脂酶的核苷酸序列；和(b)与(a)所述的核苷酸序列互补的核苷酸序列，互补可以是部分或完全互补。
技术人员将会认为，由于遗传密码的简并性，多种不同的核苷酸序列可以编码同样的酶。另外，可以认识到，技术人员能够使用常规技术进行不影响本发明的核苷酸序列所编码的酶活性的核苷酸取代，这样可以反映表达本发明的酶所用的任何特定的宿主生物体的密码子偏倚性。
本发明还提供了包含所述核酸分子的载体，以及包含所述核酸分子或所述载体的宿主细胞。
“载体”是指通常携带有不属于细胞中心代谢的部分的基因的染色体外元件，并且常常是环状双链DNA分子的形式。此类元件可为得自任何来源的自主复制序列、基因组整合序列、噬菌体或核苷酸序列、线性或环状的单链或双链DNA或RNA，其中许多核苷酸序列已经被接合或重组到特定构建体中，该构建体能够将所选基因产物的启动子片段和DNA序列连同合适的3′非翻译序列一起导入细胞。
编码本发明的溶血磷脂酶的基因和基因产物可在异源宿主细胞，例如细菌细胞、真菌细胞，例如酵母细胞、哺乳动物细胞、昆虫细胞和植物细胞中表达。用于表达本发明的核酸分子的异源宿主细胞可以是存在于真菌或细菌家族中并在宽温度、pH值和溶剂耐受性范围内生长的微生物宿主。例如，预期任何细菌、酵母和丝状真菌可为表达本发明核酸分子的合适宿主。宿主菌株的实例包括但不限于细菌、真菌或酵母物种例如毕赤酵母属(Pichia)、曲霉属(Aspergillus)、木霉属(Trichoderma)、糖酵母属(Saccharomyces)、发夫酵母属(Phaffia)、克鲁维酵母属(Kluyveromyces)、耶氏酵母属(Yarrowia)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、沙门氏菌属(Salmonella)、芽孢杆菌属(Bacillus)、不动杆菌属(Acinetobacter)、发酵单胞菌属(Zymomonas)、土壤杆菌属(Agrobacterium)、赤细菌属(Erythrobacter)、绿菌属(Chlorobium)、着色菌属(Chromatium)、黄杆菌属(Flavobacterium)、噬纤维菌属(Cytophaga)、红细菌属(Rhodobacter)、红球菌属(Rhodococcus)、链霉菌属(Streptomyces)、短杆菌属(Brevibacterium)、棒状杆菌属(Corynebacteria)、分支杆菌属(Mycobacterium)、异常球菌属(Deinococcus)、埃希氏菌属(Escherichia)、欧文氏菌属(Erwinia)、泛菌属(Pantoea)、假单胞菌属(Pseudomonas)、鞘氨醇单胞菌属(Sphingomonas)、甲基单胞菌属(Methylomonas)、甲基细菌属(Methylobacter)、甲基球菌属(Methylococcus)、 甲基弯菌属(Methylosinus)、甲基微菌属(Methylomicrobium)、甲基孢囊菌属(Methylocystis)、产碱菌属(Alcaligenes)、集胞蓝细菌属(Synechocystis)、聚球蓝细菌属(Synechococcus)、鱼腥蓝细菌属(Anabaena)、硫杆菌属(Thiobacillus)、甲烷杆菌属(Methanobacterium)、克雷伯氏菌属(Klebsiella)和粘球菌属(Myxococcus)物种。在一个实施方案中，所述宿主细胞是真菌细胞。在一个实施方案中，所述宿主细胞是毕赤酵母或黑曲霉细胞。在一个实施方案中，所述宿主细胞是本发明的具有CGMCC No.40011的保藏号的黑曲霉细胞。
可用于转化上述宿主细胞的载体是本领域熟知的。通常，载体包含指导相关基因转录和翻译的序列、可选标记和允许自主复制或染色体整合的序列。合适的载体包含含有转录起始控制的基因5′区域和控制转录终止的DNA片段3′区域。
一方面，本发明还涉及生产溶血磷脂酶的方法，包括在宿主细胞中表达编码本发明的溶血磷脂酶的核酸分子，并且回收得到的多肽。
可应用多种培养方法以制备本发明的酶。例如，从重组微生物宿主中大规模生产特定基因产物可通过分批、分批补料和连续培养方法进行。
分批和补料分批培养方法在本领域内是常用的且众所周知，并且实例可见于如下文献：Thomas D.Brock in Biotechnology：A Textbook of Industrial Microbiology，第二版，Sinauer Associates，Inc.，Sunderland，MA(1989))，以及Deshpande，Mukund V.，(Appl.Biochem.Biotechnol.，36：227-234(1992)。
本发明的酶的商业生产也可通过连续培养进行。连续培养是一种开放式系统，其中将设定好的培养基连续加入生物反应器中，并同时移出等量条件培养基用于加工。连续培养一般使细胞维持在其中细胞主要处于对数生长期的恒定高液相密度。或者，连续培养可以用固定化细胞来进行，其中连续添加碳和营养素，且连续从细胞团块中取出有价值的产物、副产物或废弃物。细胞固定可使用范围广泛的固体载体进行，所述固体载体由天然材料和/或合成材料组成。
从分批发酵、分批补料发酵、或连续培养中回收期望的酶可通过本领域技术人员已知的任何方法完成。例如，当在细胞内生产酶时，通过离心或膜过滤从培养基中分离细胞浆液，任选地用水或期望pH的含水缓冲液洗涤，然后将期望pH的含水缓冲液中的细胞浆液悬浮使其匀化，生产出包含需要的酶的细胞提取物。
本发明还涉及组合物，其包含本发明的溶血磷脂酶或本发明的宿主细胞的发酵液、发酵上清和/或发酵浓缩液。本发明的酶组合物可为任何适于使用的形式，例如，去除或未去除细胞的粗发酵液，含或不含细胞碎片的细胞裂解物，半纯化或纯化的酶组合物，或宿主细胞作为酶的来源。所述酶组合物可为干粉或颗粒、无粉尘的颗粒、液体、稳定 化液体或稳定化受保护的酶。液体酶组合物可根据确立的工艺，例如通过添加稳定剂如糖、糖醇或其他多元醇，和/或乳酸或其他有机酸来稳定化。
本发明另外涉及本发明的宿主细胞的发酵液、发酵上清或发酵浓缩液。
本发明还涉及本发明的溶血磷脂酶在油脂脱胶中的用途。将本发明的溶血磷脂酶用于油脂脱胶时，相对于现有技术的溶血磷脂酶，比酶活有显著提高，降低了生产成本。
另一方面，本发明涉及突变的黑曲霉(Aspergillus niger)菌株，其是乳清酸磷酸核糖基转移酶营养缺陷型菌株，并且其相对于未突变的菌株，内源酶，例如溶血磷脂酶的生产能力提高。例如，可以提高10-15倍，例如10-12倍，特别是10.8倍。
在本发明中，术语“内源酶”指的是突变的黑曲霉(Aspergillus niger)菌株自身表达的酶，例如，包括但不限于突变的黑曲霉菌株自身表达的溶血磷脂酶。
在一个实施方案中，本发明的突变的黑曲霉的乳清酸磷酸核糖基转移酶pyrE基因第64和65位的核苷酸TT缺失。
在一个实施方案中，本发明的突变的黑曲霉菌株具有CGMCC No.40011的保藏号。
除了自身的内源酶，本发明的突变的黑曲霉菌株能够高效表达异源蛋白。本发明的突变的黑曲霉菌株可以用于表达广泛的异源蛋白，如淀粉酶、葡萄糖氧化酶、过氧化氢酶、纤维素酶、果胶酶、蛋白酶、植酸酶、木聚糖酶、溶菌酶、白细胞介素-6、人乳铁蛋白、牛凝乳酶、奇异果甜蛋白、脂肪酶等。特别地，本发明的突变的黑曲霉菌株在重组表达溶血磷脂酶LPL时，可以比出发菌株的蛋白表达水平提高100％以上，例如100％-150％，特别是100％-120％，更特别是100％-112％。因此，该菌株具有显而易见的高效表达异源蛋白的实用性。
另一方面，本发明涉及重组黑曲霉菌株，其通过在上述突变的黑曲霉菌株中引入编码外源蛋白的基因而获得。在一个实施方案中，所述外源蛋白是酶和其它蛋白，例如淀粉酶、葡萄糖氧化酶、过氧化氢酶、纤维素酶、果胶酶、蛋白酶、植酸酶、木聚糖酶、溶菌酶、白细胞介素-6、人乳铁蛋白、牛凝乳酶、奇异果甜蛋白、脂肪酶等，特别是溶血磷脂酶。
另一方面，本发明涉及生产目标蛋白的方法，包括将编码所述目标蛋白的基因引入上述突变的黑曲霉菌株，并且培养所述菌株以产生目标蛋白。或者，包括培养上述重组黑曲霉菌株以产生目标蛋白。在一个实施方案中，所述外源蛋白是酶和其它蛋白，例如淀粉酶、葡萄糖氧化酶、过氧化氢酶、纤维素酶、果胶酶、蛋白酶、植酸酶、木聚糖酶、溶菌酶、白细胞介素-6、人乳铁蛋白、牛凝乳酶、奇异果甜蛋白、脂肪酶等，特别是溶血磷脂酶。
另一方面，本发明涉及生物催化剂，其包含上述突变的黑曲霉菌株，其中引入了编 码外源蛋白的基因，所述外源蛋白是酶和其它蛋白，例如淀粉酶、葡萄糖氧化酶、过氧化氢酶、纤维素酶、果胶酶、蛋白酶、植酸酶、木聚糖酶、溶菌酶、白细胞介素-6、人乳铁蛋白、牛凝乳酶、奇异果甜蛋白、脂肪酶等，特别是溶血磷脂酶。
另一方面，本发明涉及上文所述菌株生产的外源蛋白。所述外源蛋白是酶和其它蛋白，例如淀粉酶、葡萄糖氧化酶、过氧化氢酶、纤维素酶、果胶酶、蛋白酶、植酸酶、木聚糖酶、溶菌酶、白细胞介素-6、人乳铁蛋白、牛凝乳酶、奇异果甜蛋白、脂肪酶等，特别是溶血磷脂酶。所述外源蛋白可用于食品，优选是食品用酶，更优选是食品用溶血磷脂酶。
在一个实施方案中，所述溶血磷脂酶是本发明的突变的溶血磷脂酶。
本发明还涉及重组微生物细胞，其中引入了源自上述突变的黑曲霉菌株的菌内成分。在获得本发明的菌株后，可以通过常规技术分离其菌内成分，并且将所述成分引入其它微生物。引入所述成分的重组微生物细胞具有本发明所述菌株的优良性能。在本发明中，术语“菌内成分”指的是指生物体所有遗传物质的总和，具体而言，包括但不限于：编码DNA和非编码DNA、线粒体DNA。
具体地，本发明的突变黑曲霉菌株能够用于表达外源蛋白。在获得了本发明的突变黑曲霉后，可以将常用的表达载体引入其中，用于表达外源蛋白。例如，表达载体中可以包含启动子和终止子，在启动子和终止子之间具有多克隆位点，可以插入编码外源蛋白的基因。启动子中可以包含一个或多个拷贝的增强子。许多商业载体都可以用于本发明的突变黑曲霉菌株中进行外源蛋白的表达。
在表达非黑曲霉来源的外源蛋白时，某些物种的密码子可能在黑曲霉中是稀有的密码子。因此，在引入表达载体时，可以先对编码外源蛋白的基因进行适合本发明的黑曲霉的密码子优化，从而增加表达量。
本发明的黑曲霉可以表达多种外源蛋白，包括食品用酶，如食品用脂肪酶、药用蛋白、来自植物、动物和细菌的各种酶、膜受体蛋白、含辅基的蛋白以及可用于研究晶体结构的蛋白等。本发明的表达外源酶组分的黑曲霉还可以以全细胞作为生物催化剂。
在一个实施方案中，本发明的突变的黑曲霉菌株，例如具有CGMCC No.40011的保藏号的黑曲霉菌株，可以用于表达本发明的突变的溶血磷脂酶，例如包含SEQ ID NO：14或16的氨基酸序列的溶血磷脂酶。由于二者效果的组合，溶血磷脂酶的表达效率预期可以进一步提高。
附图说明
图1是CN2021107079194中picAN02m1(M1)和pic-AN02-LPL(WT)编码的溶 血磷脂酶LPL，以及本发明的突变体W，Y，F的溶血磷脂酶针对1-棕榈酰基溶血磷脂酰胆碱的比酶活。
图2是本发明构建的LPL基因表达载体的示意图。
图3是AN19E回补转化平板图。
图4是AN19E回补转化子溶血磷脂酶LPL活性筛选平板图。
图5是LPL筛选培养基中诱变菌株AN19E-13的菌落图。
图6是本发明的突变菌株AN19E-13与出发菌株AN19E表达的内源溶血磷脂酶LPL的酶活对比图。
图7是本发明的突变菌株AN19E-13与出发菌株CICC2243和AN19E重组表达的溶血磷脂酶LPL的酶活对比图。
图8是本发明的突变菌株AN19E-13与出发菌株CICC2243和AN19E重组表达的溶血磷脂酶LPL的蛋白电泳图。
关于保藏的说明
本发明的菌株AN19E-13于2021年12月20日保藏于中国普通微生物菌种保藏管理中心(CGMCC)，北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏号CGMCC No.40011，分类命名为黑曲霉(Aspergillus niger)。
具体实施方式
一.本发明的突变的溶血磷脂酶的制备和功能验证。
实验材料
黑曲霉菌株GIM 3.24(AN02)购自广东省微生物菌种保藏中心。
黑曲霉发酵培养基配方为：
2％葡萄糖，10％麦芽糖，7％柠檬酸钠，1.5％硫酸铵，4％Tryptic soy broth，0.1％磷酸二氢钠，0.1％硫酸镁，0.07％Tween 80，微量元素(KI 0.83g/L，H 3BO 36.2g/L，MnSO 4·4H 2O 22.3g/L，ZnSO 4·7H 2O 8.6g/L，Na 2MoO 4·2H 2O 0.25g/L，CuSO 4·5H 2O 0.025g/L，CoCl 2·6H 2O 0.025g/L按1/1000比例添加；FeSO 4·7H 2O 2.78g/L，Na 2·EDTA 3.73g/L按1/100比例添加)。
lysis buffer配方为：100mM Tris-HCl pH 8.0；50mM Na·EDTA；1％SDS。
BMMY-大豆磷脂培养基：
组分A，BMMY固体培养基：1％yeast extract，2％peptone，100mM柠檬酸-柠檬酸钠缓冲液，pH 6.6，1.34％YNB，4 x 10-5％biotin(倒平板前加)，2％methanol(倒平板前加)，2％琼脂溶于250ml去离子水中。
组分B，大豆磷脂底物溶液250ml：配4％大豆磷脂，用高速匀浆机8000rpm乳化3min，暂停1min后再乳化3min，制备底物溶液。
灭菌后将A和B组分混合，加入10ml甲醇，倒平板。
glass beads购自：美国Biospec。
Figure PCTCN2022140539-appb-000001
DNA Polymerase购自Takara，货号：R010A。
SalI、EcoRI和BglII限制性内切酶购自NEB。
溶血磷脂1-棕榈酰-sn-甘油-3-磷酸胆碱购自阿拉丁，货号P130493-500mg。
NEFA试剂盒购自日本和光纯药工业株式会社。
Bradford试剂盒购自生工(上海)生物工程有限公司。
MGYS平板：1.34％YNB，4x 10-5％biotin(过滤除菌)，2％甘油，1M山梨醇。
YPD培养基：1％yeast extract，2％peptone，2％葡萄糖。
BMGY摇瓶培养基：1％yeast extract，2％peptone，100mM柠檬酸-柠檬酸钠缓冲液，pH 6.6，1.34％YNB，4x 10-5％biotin(过滤除菌)，2％甘油。
BMMY-摇瓶培养基：1％yeast extract，2％peptone，100mM柠檬酸-柠檬酸钠缓冲液，pH 6.6，1.34％YNB，4x 10-5％biotin(过滤除菌)，2％甲醇(接种时加入)。
实施例1：黑曲霉lipaseB基因克隆
黑曲霉菌株GIM 3.24(AN02)用黑曲霉发酵培养基30℃培养24小时。取发酵培养物，4000rpm离心5min，取菌体。用700ul lysis buffer重悬，转移至冻存管中，加入300ul glass beads，上mini beadbeater振动40s，12000rpm离心10min。取600ul上清加入275ul 7M醋酸铵，65℃水浴10min，冰浴5min，加入等体积的酚氯仿异戊醇(24∶25∶1)，充分涡旋，12000rpm离心5min，取上清加入等体积的氯仿，充分涡旋，12000rpm离心5min，取上清加入2倍体积的无水乙醇，-80℃放置20min，12000rpm离心10min，得到白色DNA沉淀，用70％乙醇洗2次，待乙醇充分挥发后加入无菌水溶解DNA。
根据NCBI中的黑曲霉CBS513.88的lipaseB的基因序列设计引物LPL-1和LPL-2。
LPL-15’-atgtttctccgcagggaatt-3’(SEQ ID NO：1)
LPL-25’-ctacgagcattcactaatgt-3’(SEQ ID NO：2)。
利用
Figure PCTCN2022140539-appb-000002
DNA Polymerase克隆黑曲霉菌株GIM 3.24(AN02)的LPL DNA，用Mighty TA-cloning Reagent Set for
Figure PCTCN2022140539-appb-000003
试剂盒进行TA克隆后转化DH5a大肠杆菌，送生工生物工程有限公司进行DNA测序，最终得到GIM 3.24(AN02)的LPL DNA序列，如下：
GIM 3.24(AN02)LPL的DNA序列：
Figure PCTCN2022140539-appb-000004
Figure PCTCN2022140539-appb-000005
根据CBS513.88的lipaseB的DNA序列的内含子序列，找出GIM 3.24(AN02)的内含子序列，去除后翻译成氨基酸序列，结果如下：
GIM 3.24(AN02)LPL的氨基酸序列如下：
Figure PCTCN2022140539-appb-000006
实施例2：AN02-LPL在毕赤酵母中表达及随机突变
选取AN02-LPL的成熟肽，并以该成熟肽前的前10个氨基酸作为前导肽，获得AN02-LPL，氨基酸序列如下：
Figure PCTCN2022140539-appb-000007
Figure PCTCN2022140539-appb-000008
其对应的DNA序列如下：
Figure PCTCN2022140539-appb-000009
在5’端增加Sac-II酶切位点CCGCGG，送生工生物工程有限公司合成基因序列，通过EcoRI酶切位点克隆至pA0815载体(购自invitrogen)中，得到pic-AN02质粒。用BglII线性化后，用电转化法将载体转化至毕赤酵母GS115菌株(购自invitrogen)的感受态细胞中。将转化物接种于MGYS平板上，30℃培养3天，得到毕赤酵母转化子。挑取平板上的单克隆，至BMMY-大豆磷脂培养基筛选平板上，选取白色沉淀圈大的克隆，命名为pic-AN02-LPL。
以pic-AN02-LPL作为模板使用TaKaRa Taq酶和引物对PLPL-1：TCCCCGCGGCGAAACGATGAGATTTCCTTC(SEQ ID NO：7)/PLPL-2：CCGGAATTCTTAAGAACACTCAGAAATG(SEQ ID NO：8)进行易错PCR(在PCR时额外添加0.3mM的MnCl 2)，得到大小为约1000bp的突变扩增子片段集合。通过Sac-II和EcoRI酶切位点将得到的片段克隆至pic-AN02质粒，并将得到的载体转化入大肠杆菌DH5α菌株。
将含pic-AN02突变体的平板用2ml的无菌水进行洗涤，抽提质粒，用SaiI进行线 性化，回收约8.5kb的片段，作为载体。取500ng载体，用电转化法将载体转化至毕赤酵母GS115菌株的感受态细胞中。将转化物接种于BMM-大豆磷脂筛选培养基平板上，30℃培养3天，得到pic-AN02-LPL的毕赤酵母突变体文库。筛选得到一株突变体pic-AN02m1。
将pic-AN02m1菌株接种于3ml YPD液体培养基中，30℃培养过夜，抽提基因组DNA。以pic-AN0m1菌株的基因组DNA为模板，使用
Figure PCTCN2022140539-appb-000010
DNA聚合酶和引物对AOX-5：GACTGGTTCCAATTGACAAGC(SEQ ID NO：9)和3’-AOX1：GGCAAATGGCATTCTGACATCCTC(SEQ ID NO：10)进行PCR扩增，得到pic-AN02m1菌株中AN02突变体的DNA序列。将得到的序列送往上海生工生物工程公司，用引物对AOX-5/3’-AOX1进行测序。pic-AN02m1菌株的突变LPL的DNA序列及其编码的氨基酸如下所示，突变位点为L86I，G187D，E209K，A245D。
氨基酸序列：
Figure PCTCN2022140539-appb-000011
DNA序列：
Figure PCTCN2022140539-appb-000012
Figure PCTCN2022140539-appb-000013
实施例3：pic-AN02m1编码的LPL基因在毕赤酵母中的定点突变
将pic-AN02m1编码的溶血磷脂酶LPL的255位苏氨酸分别突变为色氨酸、苯丙氨酸和酪氨酸，突变后的基因命名为W、F和Y。
突变体W的氨基酸序列
Figure PCTCN2022140539-appb-000014
其中，前十个氨基酸为前导肽。因此，其功能序列如下：
Figure PCTCN2022140539-appb-000015
突变体f的氨基酸序列
Figure PCTCN2022140539-appb-000016
其中，前十个氨基酸为前导肽。因此，其功能序列如下：
Figure PCTCN2022140539-appb-000017
突变体y的氨基酸序列
Figure PCTCN2022140539-appb-000018
Figure PCTCN2022140539-appb-000019
其中，前十个氨基酸为前导肽。因此，其功能序列如下：
Figure PCTCN2022140539-appb-000020
送生工生物工程有限公司合成基因序列，克隆至pic-AN02-LPL载体中，得到突变体W、F和Y质粒。用SalI线性化后，用电转化法将载体转化至毕赤酵母GS115菌株的感受态细胞中。将转化物接种于MGYS平板上，30℃培养3天，得到突变体W，F和Y的毕赤酵母转化子。挑取平板上的单克隆，至BMM-大豆磷脂培养基筛选平板上，选取白色沉淀圈大的克隆，分别命名为突变体W、F和Y。
取pic-AN02m1、pic-AN02-LPL毕赤酵母表达菌株和突变体W、F和Y毕赤酵母表达菌株。先在液体YPD中活化，然后接种于BMGY培养基中，在30℃下，220rpm振荡培养过夜。将培养物转至BMMY培养基中，初始OD600为6。
首先，用2％甲醇进行诱导，在24h和32h后各补加1％甲醇，48h和56h后各补加1％甲醇，72h取样。
将获得的发酵也用截留分子量为10kDa的超滤管进行超滤脱盐浓缩30倍。将处理后的样品加入缓冲液中(20mM柠檬酸-柠檬酸钠缓冲液(pH4.0))。
将浓缩的酶液按如下方法测定溶血磷脂酶活力：
9ml底物：5ml 1％溶血磷脂，1ml 20％Triton X-100，2.5ml 0.1M柠檬酸-柠檬酸钠缓冲液。
10ul稀释的酶液+90ul底物50℃反应10min，95℃5min灭活，7000rpm离心5min，取1ul上清加NEFA试剂盒中的试剂A 80ul，37℃反应10min，加试剂B 160ul反应10min。测定550nm吸光值，从而计算得到酶活。
蛋白浓度利用Bradford试剂盒测定。通过酶活和蛋白浓度计算得到picAN02m1编码的LPL、pic-AN02-LPL编码的LPL、以及突变体W，Y，F的溶血磷脂酶的比酶活。如图1所示，突变体W、F的溶血磷脂酶比酶活为30224U/mg和18973U/mg；picAN02m1(M1) 和pic-AN02-LPL(WT)的溶血磷脂酶比酶活为13473U/mg和12746U/mg。相比于起始序列M1即picAN02m1，比酶活为13473U/mg，比酶活分别提高了124％和40％。
二.本发明的突变的黑曲霉菌株的制备和功能验证。
初始菌株黑曲霉购自中国工业微生物菌种保藏管理中心(简称CICC)，菌株保藏编号CICC2243。初始菌株先涂布在MM固体培养基平板上培养孢子，洗脱孢子后，再经过紫外诱变后，最后通过在筛选平板上添加5-氟乳清酸和尿嘧啶来进行筛选，获得了乳清酸磷酸核糖基转移酶营养缺陷型菌株AN19E。
再利用ARTP诱变的方式，筛选到了一株菌株AN19E-13。其在添加了尿嘧啶的溶血磷脂酶筛选平板上，与诱变出发菌株AN19E相比，AN19E-13的沉淀圈明显变大。检测发现AN19E-13其自身溶血磷脂酶(LPL)酶活比出发菌株AN19E提高了10.8倍。
尝试在黑曲霉菌株AN19E-13中进行溶血磷脂酶(LPL)的异源重组表达，发现该菌株表达LPL的能力提高了112％。从而证明其作为蛋白表达系统，尤其是作为食品用溶血磷脂酶的表达系统，具有明显的优势。
在本发明中，术语“ARTP”是常压室温等离子体(Atmospheric and Room Temperature Plasma)的简称，具体指，能够在大气压下产生温度在25-40℃之间的、具有高活性粒子(包括处于激发态的氦原子、氧原子、氮原子、OH自由基等)浓度的等离子体射流。术语“ARTP诱变”，即利用常压室温等离子体技术进行菌株诱变，具体而言，采用氦气为工作气体的常压室温等离子体源中含有多种化学活性粒子成分，如OH、氮分子二正系统、氮分子一负系统、激发态氦原子、氢原子和氧原子等。ARTP富含的活性能量粒子对菌株/植株/细胞等的遗传物质造成损伤，并诱发生物细胞启动SOS修复机制。SOS修复过程为一种高容错率修复，因此修复过程中会产生种类丰富的错配位点，并最终稳定遗传进而形成突变株。SOS修复强度，和DNA受损伤的程度有很大关联。
实施例4：乳清酸磷酸核糖基转移酶营养缺陷型黑曲霉菌株的获得
将黑曲霉CICC2243菌株孢子接种涂布至MM固体培养基(1％葡萄糖，0.15％KH 2PO 4，0.6％NaNO 3，0.05％KCl，0.05％MgSO 4，2％琼脂粉)上，28℃，静置培养5天，获得黑曲霉孢子。用孢子洗液(0.9％NaCl，0.05％吐温80)洗脱新鲜的黑曲霉CICC2243孢子，通过Miracloth(Calbiochem，Cat#475885)过滤制备成孢子悬液，用无菌水洗涤菌体2次并调整至1×10 7个/mL。取2mL孢子悬浮液均匀分散在培养皿表层，置于超净工作台紫外灯下照射90s，取100μL涂布于添加了0.3％尿嘧啶(Uracil)和1mg/mL 5-氟乳清酸(5-FOA)的MM固体培养基上，28℃，避光培养(全过程在红光下操作，防止回复突变)7天。将上一步在MM固体培养基上长出来的单菌落，转接到MM固体培养基、MM-Uracil固体培养基上，挑取只能在MM-Uracil固体培养基上生长的菌株，从而获得 乳清酸磷酸核糖基转移酶(pyrE)营养缺陷型黑曲霉AN19E菌株。通过对黑曲霉AN19E菌株的pyrE基因测序发现，其第64和65位核苷酸由TT发生了缺失，从而导致pyrE基因失活。
实施例5：LPL表达载体的构建
外源的黑曲霉溶血磷脂酶(LPL)基因序列如下：
核酸序列：
Figure PCTCN2022140539-appb-000021
氨基酸序列：
Figure PCTCN2022140539-appb-000022
具体操作参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)的方法，构建LPL基因表达载体pANE-LPL，如图2所示，过程如下：
用SphI和HindIII酶切位点将从生工生物工程(上海)股份有限公司全基因合成获得的LPL基因(SEQ ID NO：19，带米曲霉α-淀粉酶信号肽(NCBI序列号：XM_001821384.2，1-63bp序列))插入到含米曲霉烯醇化酶启动子(NCBI序列号：D63941.1，215-734bp；含12拷贝增强子序列(gtcgtgtcgggcatttatcgggggatggaccaatcagcgtagg，SEQ ID NO：21)和黑曲霉糖化酶终止子(NCBI序列号：AF214480.1，其中终止子序列 部分)的表达框中，将整个表达框以BglII和XhoI插入到克隆载体pSP72的多克隆位点，最后将黑曲霉来源的PyrE表达基因(NCBI序列号：AY840014.1)以XhoI酶切位点插入到载体上，从而构建好LPL基因表达载体pANE-LPL。见图2。
实施例6：黑曲霉AN19E回补实验
用孢子洗液洗脱新鲜的黑曲霉AN19E孢子，通过Miracloth过滤制备成孢子悬液，并调整至1×10 7个/mL。接种1mL孢子悬液至菌丝培养基(2％胰蛋白胨，1％酵母抽提物，2％葡萄糖，0.3％尿嘧啶)中，于28℃，180rpm，培养40小时，用灭菌Miracloth过滤收集长出的菌丝。
收集的菌丝用灭菌的渗透压稳定剂(0.6M MgSO 4，10mM NaH 2PO 4，pH＝5.8)冲洗三次，压干。菌丝转移至100mL三角瓶中，每0.8g菌丝重悬在20mL的酶解液(用渗透压稳定剂配制1％裂解酶，1％纤维素酶，0.1％蜗牛酶的酶解液，用0.22 μm的微孔滤膜过滤除菌)中，于30℃，90rpm，酶解60-90min。酶解好的原生质体混合液用Miracloth过滤，收集滤液，4℃，1000g离心10min，用5mL预冷的1.0mol/L山梨醇溶液重悬原生质体沉淀，于800g，4℃离心10min，弃上清。再用预冷的STC溶液(1.0M山梨醇，50mM CaCl 2，50mM Tris-HCl， pH＝7.5)将原生质体调整至1×10 7个/mL，冰浴待用。
向200μL原生质体悬液中，加入10μL浓度为1μg/μL的LPL表达载体pANE-LPL，再添加50μL PTC溶液(40％PEG4000，50mM CaCl 2，50mM Tris-HCl，pH＝7.5)，混匀后冰浴30min。加入0.2mL PTC溶液，混匀后再加入0.8mL PTC溶液，混匀，室温保持30min。
将上述混合液铺于再生培养基(1％葡萄糖，0.6％NaNO 3，0.15％KH 2PO 4，0.05％KCl，0.05％MgSO 4，0.001％FeSO 4，1M蔗糖，2％琼脂粉)上，28℃，培养7天，待菌落长出。
将在平板上长出的菌落，转接至溶血磷脂酶LPL筛选培养基上，28℃，培养3天。
LPL筛选培养基成分如下：
A液：2％麦芽糖，1.34％YNB，柠檬酸6.88g/500mL，柠檬酸钠5.07g/500mL，5mM CaCl2加水至200mL。
B液：1％卵磷脂加水200ml均质机乳化2％琼脂糖、0.02％Triton-x-100加水至300ml
灭菌后，混合A、B液，倒平板。
转化发现黑曲霉AN19E可以很好的实现pyrE基因回补实验，且转化子在溶血磷脂酶LPL筛选培养基表现出溶血磷脂酶LPL的活性，见图3和图4，进一步证明成功的建 立了以乳清酸磷酸核糖基转移酶营养缺陷型黑曲霉AN19E为宿主的黑曲霉表达系统。
实施例7：黑曲霉AN19E ARTP诱变实验
用孢子洗液洗脱新鲜的黑曲霉AN19E孢子，通过Miracloth过滤制备成孢子悬液，并调整至2×10 7个/mL。将孢子悬液与10％甘油1 ∶1混合后，取10μL混合液至铁片上用ARTP仪器(无锡源清天木生物科技有限公司，仪器型号：ARTP-M)处理100s，仪器参数设置：射频功率量程120W、氦气量10SLM(99.999％的高纯氦气)、照射距离2mm。
处理结束后，将铁片取下放入已装有1mL无菌水的离心管中，后用枪头反复吸取，使铁片上的菌体洗下来，稀释到大约100菌落/筛选平板，最后再涂布至添加了尿嘧啶的溶血磷脂酶筛选平板上，放置在30℃培养箱中培养3d。
实施例8：诱变菌株的筛选
将实施例7中诱变完成的孢子，涂布在添加了尿嘧啶的溶血磷脂酶筛选平板上进行筛选，发现了一株菌株沉淀圈明显变大的菌株，见图5，命名为AN19E-13。
将沉淀圈明显变大的菌株AN19E-13和出发菌株AN19E，进行摇瓶发酵，发酵培养基(2％麦芽糖，1.34％YNB，1.38％柠檬酸，1％柠檬酸钠，5mM CaCl 2，1％卵磷脂)，115℃，高压灭菌15min，发酵条件为28℃，200rpm，5d，接种量1×10 7个孢子/50mL，测定溶血磷脂酶LPL活性。
溶血磷脂酶LPL活性测定法如下：
9mL底物：5mL 1％大豆磷脂，1mL 20％Triton X-100，2.5mL 0.1M pH4.0柠檬酸-柠檬酸钠缓冲液。
10uL合适稀释的酶液+90uL底物50℃反应10min，95℃5min灭活，7000rpm离心5min，取1uL上清加NEFA试剂盒(Wako：294-63601)中的试剂A 80uL，37℃反应10min，加试剂B 160uL反应10min。测定550nm吸光值。
酶活测定结果如图6所示。
结果显示，沉淀圈明显变大的菌株AN19E-13其自身表达的溶血磷脂酶LPL活性为1058U/mL比出发菌株AN19E(98U/mL)提高了10.8倍。
将菌株AN19E-13于2021年12月20日保藏于中国普通微生物菌种保藏管理中心(CGMCC)，北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏号CGMCC No.40011，分类命名为黑曲霉(Aspergillus niger)。
实施例9：黑曲霉AN19E-13的表达能力的考察
黑曲霉AN19E-13的转化操作同实施例6，将LPL表达载体pANE-LPL转化入菌株中进行重组表达。
将实施例6中黑曲霉AN19E转化子作为对照，另外将pANE-LPL转化入黑曲霉CICC2243中，由于pANE-LPL无法使用pyrE为筛选标记，所以使用p3SR2(BCCM/LMBP：Accession number：2363)，含乙酰胺酶(amdS)基因，来进行转化筛选。再生培养基需要去除硝酸钠，另外需要补加15mM的乙酰胺和20mM的氯化铯。
三种转化子每种都挑取40个转化子进行摇瓶发酵，发酵培养基(2％葡萄糖，15％麦芽糖，7％柠檬酸钠，1.5％硫酸铵，4％TSB，0.1％磷酸二氢钠，0.1％硫酸镁，0.07％Tween 80，微量元素)，115℃，高压灭菌15min，发酵条件为28℃，200rpm，8d，接种量1×10 7个孢子/50mL，测定溶血磷脂酶LPL活性。
溶血磷脂酶LPL活性测定法见实施例8。
酶活测定结果如图7所示。
结果显示，AN19E-13重组表达的溶血磷脂酶LPL酶活为25920U/mL，与出发菌株CICC2243(12230U/mL)和AN19E(12990U/mL)相比，分别提高了112％和100％。
聚丙烯酰胺凝胶电泳分析：使用0.22μm滤膜过滤处理上清液，将等量上清液使用Milipore 10KDa超滤浓缩罐浓缩到相同体积后，取相同体积的浓缩酶液进行聚丙烯酰胺凝胶电泳分析。电泳结果如图8所示。
蛋白电泳图的结果显示，AN19E-13转化子的LPL蛋白条带浓度明显高于对照。

Claims (20)

  1. 溶血磷脂酶，其包含SEQ ID NO：14或16氨基酸序列。
  2. 生产溶血磷脂酶的方法，包括在宿主细胞中表达编码权利要求1的溶血磷脂酶的核酸分子，并且回收得到的多肽。
  3. 可用于表达食品用溶血磷脂酶的突变的黑曲霉(Aspergillus niger)菌株，其是乳清酸磷酸核糖基转移酶营养缺陷型菌株，并且其相对于未突变的菌株，内源酶，优选溶血磷脂酶的生产能力提高。
  4. 权利要求3的突变的黑曲霉(Aspergillus niger)菌株，其乳清酸磷酸核糖基转移酶pyrE基因第64和65位的核苷酸TT缺失。
  5. 突变的黑曲霉菌株，其保藏号为CGMCC NO.40011。
  6. 核酸分子，其包含：
    (a)编码权利要求1所述的溶血磷脂酶的核苷酸序列；和
    (b)与(a)所述的核苷酸序列互补的核苷酸序列。
  7. 载体，其包含权利要求6的核酸分子。
  8. 宿主细胞，其包含权利要求6的核酸分子，或权利要求7的载体。
  9. 权利要求8的宿主细胞，其中所述宿主细胞选自细菌细胞、真菌细胞、哺乳动物细胞、昆虫细胞和植物细胞。
  10. 权利要求9的宿主细胞，其中所述宿主细胞是真菌细胞，优选毕赤酵母细胞或黑曲霉细胞，优选保藏号为CGMCC NO.40011的黑曲霉细胞。
  11. 组合物，其包含权利要求1所述的溶血磷脂酶或权利要求8-10的任一项所述的宿主细胞的发酵液、发酵上清和/或发酵浓缩液。
  12. 权利要求8-10的任一项所述的宿主细胞的发酵液、发酵上清或发酵浓缩液。
  13. 权利要求1所述的溶血磷脂酶或权利要求11所述的组合物或权利要求8-10的任一项所述的宿主细胞的发酵液、发酵上清和/或发酵浓缩液在油脂脱胶中的用途。
  14. 重组黑曲霉菌株，其通过在权利要求3-5的任一项的菌株中引入编码外源蛋白的基因而获得。
  15. 权利要求14的重组黑曲霉菌株，其中所述外源蛋白是酶，优选溶血磷脂酶，优选权利要求1所述的溶血磷脂酶。
  16. 生产目标蛋白的方法，包括将编码所述目标蛋白的基因引入权利要求3-5的任一项的菌株，并且培养所述菌株以产生目标蛋白，或者培养权利要求14-15的任一项重组黑曲霉菌株以产生目标蛋白。
  17. 权利要求16的方法，其中所述目标蛋白是酶，优选溶血磷脂酶，优选权利要求1所述的溶血磷脂酶。
  18. 生物催化剂，其包含权利要求3-5的任一项的突变的黑曲霉菌株，所述菌株中引入了编码酶，优选溶血磷脂酶，优选权利要求1所述的溶血磷脂酶的基因。
  19. 由权利要求3-5和14-15的任一项的菌株生产的外源蛋白，所述外源蛋白优选是酶，更优选溶血磷脂酶；和/或所述外源蛋白优选可用于食品，优选是食品用酶，更优选是食品用溶血磷脂酶。
  20. 重组微生物细胞，其中引入了源自权利要求3-5的任一项的菌株的菌内成分。
PCT/CN2022/140539 2021-12-23 2022-12-21 突变的溶血磷脂酶以及用于表达该酶的突变的黑曲霉菌株 WO2023116738A1 (zh)

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