Urate oxidase secretion signal peptide and use thereof

Abstract

The application provides a uric acid oxidase secretion signal peptide and application thereof, and relates to the technical field of genetic engineering. In view of the problem that uric acid oxidases reported in the current literature are difficult to secrete in Pichia pastoris, the endogenous signal peptide of Pichia pastoris is screened, a signal peptide with strong secretion capacity is obtained, and the signal peptide is optimized in a targeted manner, a signal peptide mutant with strong secretion capacity is obtained, uric acid oxidase can be better secreted to the extracellular, and then the extracellular enzyme activity of uric acid oxidase is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a uric acid oxidase secretion signal peptide and application thereof. BACKGROUND

[0002] Hyperuricemia (HUA) is a chronic clinical syndrome caused by purine metabolism disorder or abnormal uric acid excretion, which leads to a significant increase in blood uric acid content beyond the normal range. Generally, it refers to the fasting blood uric acid level of male being higher than 420 μmol / L and female being higher than 360 μmol / L within 24 hours, which is called hyperuricemia. Gout is a kind of crystal arthritis caused by monosodium urate deposition, which is directly related to hyperuricemia caused by purine metabolism disorder. Hyperuricemia is not only the early stage of gout, but also an independent risk factor for causing hypertension, diabetes, coronary heart disease and chronic kidney disease. Gout has become the "fourth high" after diabetes, hypertension and hyperlipidemia, which causes serious harm to people's health.

[0003] In clinical practice, hyperuricemia is generally prevented and treated by three ways. The first way is to reduce the generation of uric acid by using drugs such as xanthine oxidase (XOD) inhibitors, for example, allopurinol, febuxostat, etc., which can inhibit the activity of xanthine oxidase to reduce the conversion of xanthine to uric acid. The second way is to increase the excretion of uric acid by using drugs such as URAT1 inhibitors, for example, benzbromarone, lesinurad, etc., which can inhibit the uric acid transporter URAT1 to reduce the reabsorption of uric acid in the renal tubule, thereby increasing the excretion of uric acid and reducing blood uric acid. The third way is the degradation of uric acid by using drugs such as uric acid oxidase (UOX) drugs, for example, rasburicase, pegzilgotucase, etc., which can degrade uric acid into allantoin that is more easily dissolved, thereby reducing blood uric acid. Studies have shown that UOX is a safer and more effective alternative drug that can quickly control the concentration of uric acid in plasma, and is a kind of extremely potential drug.

[0004] At present, the research on the heterologous expression of uricase in yeast at home and abroad mainly focuses on intracellular expression or using the alpha-factor derived from Saccharomyces cerevisiae as a signal peptide to realize the extracellular secretion of uricase. However, it is found that the extracellular secretion amount is low when using the alpha-factor as a signal peptide, which cannot realize the final industrialization. The intracellular expression and secretion purification cost is also high. Some researchers have realized the extracellular secretion of uricase by screening the signal peptide. In 2021, Beijing University of Chemical Technology realized the extracellular secretion of uricase in Pichia pastoris by screening the secretion signal peptide of uricase. The extracellular enzyme activity reached 0.474 U / mL at the level of shake flask (Patent No.: CN113956989A). However, the enzyme activity still needs to be improved, so it is necessary to continue to screen and optimize the secretion signal peptide to improve the extracellular enzyme activity of uricase. SUMMARY

[0005] In view of the problem that the uricase is difficult to be secreted in Pichia pastoris reported in the literature, the endogenous signal peptide of Pichia pastoris is screened in the present application, a signal peptide with strong secretion ability is obtained and is optimized, a signal peptide mutant with strong secretion ability is obtained, which can better secrete uricase to the extracellular, and then improve the extracellular enzyme activity of uricase.

[0006] In one aspect, the present application provides a signal peptide mutant, wherein the signal peptide comprises a sequence obtained by mutating the Fre2 amino acid residue site;

[0007] Preferably, the Fre2 amino acid residue site is at least selected from one or more of positions 16, 17, 18, and 21;

[0008] More preferably, the Fre2 comprises an amino acid sequence as shown in SEQ ID NO. 3 or an amino acid sequence having 98% or more identity with SEQ ID NO. 3.

[0009] Preferably, the Fre2 comprises an amino acid sequence as shown in SEQ ID NO. 3 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO. 3.

[0010] Further, the mutation at least includes any one or more of A1) to A4) as follows:

[0011] A1) adding a lysine residue (K) and / or an arginine residue (R) to the carbon terminus and / or the nitrogen terminus of the 21st amino acid residue; preferably, adding a lysine residue (K) and an arginine residue (R) to the carbon terminus of the 21st amino acid residue; more preferably, the signal peptide mutant comprises an amino acid sequence as set forth in SEQ ID NO. 15 or an amino acid sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 15; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as set forth in SEQ ID NO. 16 or a nucleotide sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 16;

[0012] A2) mutating the 18th amino acid residue from an alanine residue (A) to a threonine residue (T); more preferably, the signal peptide mutant comprises an amino acid sequence as set forth in SEQ ID NO. 21 or an amino acid sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 21; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as set forth in SEQ ID NO. 22 or a nucleotide sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 22;

[0013] A3) the 16th amino acid residue is mutated from a threonine residue (T) to a valine residue (V); more preferably, the signal peptide mutant comprises an amino acid sequence as set forth in SEQ ID NO. 17, or an amino acid sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 17; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as set forth in SEQ ID NO. 18, or a nucleotide sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 18;

[0014] A4) the 17th amino acid residue is mutated from a valine residue (V) to a threonine residue (T); more preferably, the signal peptide mutant comprises an amino acid sequence as set forth in SEQ ID NO. 19, or an amino acid sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 19; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as set forth in SEQ ID NO. 20, or a nucleotide sequence that is 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO. 20;

[0015] Preferably, the 16th amino acid residue is mutated from a threonine residue (T) to a valine residue (V) and the 17th amino acid residue is mutated from a valine residue (V) to a threonine residue (T); more preferably, the signal peptide mutant comprises an amino acid sequence as set forth in SEQ ID NO. 23 or an amino acid sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO. 23; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as set forth in SEQ ID NO. 24 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO. 24.

[0016] It is understood by those skilled in the art that the mutation of the signal peptide of the present application can be accomplished by using existing gene editing technologies.

[0017] In another aspect, the present application also provides a biological material, which comprises at least any one or more of the following B1) to B5):

[0018] B1) a nucleic acid molecule comprising a nucleic acid molecule encoding the signal peptide mutant;

[0019] B2) an expression cassette comprising the nucleic acid molecule of B1); preferably, the expression cassette further comprises a urate oxidase-encoding gene; more preferably, the urate oxidase is a urate oxidase (UOX) derived from Aspergillus flavus, NCBI GeneID: X61766.1, with an amino acid sequence as set forth in SEQ ID No. 1 and a nucleotide sequence as set forth in SEQ ID No. 2;

[0020] B3) a recombinant vector comprising the nucleic acid molecule of B1) and / or the expression cassette of B2);

[0021] B4) a recombinant microorganism comprising the nucleic acid molecule of B1), the expression cassette of B2), and / or the recombinant vector of B3);

[0022] B5) a recombinant cell comprising the nucleic acid molecule of B1), the expression cassette of B2), and / or the recombinant vector of B3).

[0023] The skilled person can envisage that other commonly used expression elements can be added to the expression cassette described herein to assist the expression of the gene of interest, such as tags, fluorescent protein markers, resistance selection markers, etc.

[0024] The skilled person can select a known plasmid for the construction of the recombinant vector and perform routine optimization and modification of the constructed recombinant vector according to the actual situation, and the type of plasmid is not compulsorily limited in the present application. In a preferred embodiment, the vector can be pPICZ alpha A and / or BB3 ck-sgRNA.

[0025] In an alternative embodiment, the expression cassette of the gene of interest is located on the recombinant vector or is introduced into the recombinant microorganism or recombinant cell using the recombinant vector.

[0026] Further, the nucleic acid molecule of B1) comprises any one of the following b11) to b16):

[0027] b11) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO. 16 or a DNA molecule having 98% or more identity to SEQ ID NO. 16 and encoding the signal peptide mutant;

[0028] b12) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO. 18 or a DNA molecule having 98% or more identity to SEQ ID NO. 18 and encoding the signal peptide mutant;

[0029] b13) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO. 20 or a DNA molecule having 98% or more identity to SEQ ID NO. 20 and encoding the signal peptide mutant;

[0030] b14) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO. 22 or a DNA molecule having 98% or more identity to SEQ ID NO. 22 and encoding the signal peptide mutant;

[0031] b15) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO. 24 or a DNA molecule having 98% or more identity to SEQ ID NO. 24 and encoding the signal peptide mutant;

[0032] b16) the DNA molecule hybridizes to any of the nucleotide sequences defined in b11) to b15) under stringent conditions and encodes the signal peptide mutant.

[0033] In another aspect, the application also provides an engineered bacterium for producing uricase, wherein the engineered bacterium comprises a gene expressing the signal peptide mutant and a gene expressing uricase.

[0034] Further, the engineered bacterium is one or more of Pichia pastoris, Saccharomyces cerevisiae and Candida.

[0035] Preferably, the Pichia pastoris is more preferably Pichia pastoris X-33.

[0036] In another aspect, the application also provides a method for producing uricase, comprising fermenting the engineered bacterium to obtain uricase.

[0037] Preferably, the uricase is an extracellular enzyme.

[0038] More preferably, the extracellular enzyme activity of the uricase is >0.6 U / mL.

[0039] More preferably, the extracellular enzyme activity of the uricase is 0.6 U / mL-0.9 U / mL.

[0040] More preferably, the extracellular enzyme activity of the uricase is 0.708 U / mL-0.896 U / mL.

[0041] It is understood by those skilled in the art that the Pichia pastoris can be activated and fermented to obtain a crude uricase solution, and the crude uricase solution can be purified by conventional purification methods to obtain high-purity uricase.

[0042] In a preferred embodiment, the method comprises activating the engineered bacterium, inoculating the bacterium into a fermentation medium at an inoculation amount of 1%-10% and culturing at 25-40°C and 200-300 rpm for 12-48 h, collecting the bacterium, resuspending the bacterium in the fermentation medium at 25-40°C and 200-300 rpm, and inducing the bacterium with 1%-5% methanol for 12-48 h, and collecting the supernatant to obtain a crude uricase solution.

[0043] Preferably, the fermentation medium is BMGY medium.

[0044] In the examples of the application, the extracellular enzyme activity of the obtained uricase can be as high as 0.896 U / mL, which is 89% higher than the extracellular enzyme activity reported in the prior art, proving that the signal peptide mutant described in the application can effectively promote the extracellular secretion of uricase and thus increase the extracellular enzyme activity.

[0045] In another aspect, the present application also provides an application of a signal peptide containing an amino acid sequence shown in SEQ ID No. 7 in promoting the exocytosis of urate oxidase and / or increasing the exocytosis amount of urate oxidase and / or increasing the exocytosis activity of urate oxidase and / or in preparing urate oxidase.

[0046] In the present application, it is found for the first time that signal peptide 0030 can effectively promote the exocytosis of urate oxidase and increase the exocytosis activity of urate oxidase, wherein the amino acid sequence of signal peptide 0030 is shown in SEQ ID No. 7, and the nucleotide sequence encoding signal peptide 0030 is shown in SEQ ID No. 8 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID No. 8.

[0047] In another aspect, the present application also provides an engineered bacterium for producing urate oxidase, wherein the engineered bacterium contains a signal peptide gene expressing the amino acid sequence shown in SEQ ID No. 7 and a gene expressing urate oxidase.

[0048] Further, the engineered bacterium is one or more of Pichia pastoris, Saccharomyces cerevisiae and Candida.

[0049] Preferably, the engineered bacterium is Pichia pastoris, and more preferably, the engineered bacterium is Pichia pastoris X-33.

[0050] In another aspect, the present application also provides a method for producing urate oxidase, which comprises fermenting the engineered bacterium to obtain urate oxidase.

[0051] Preferably, the exocytosis activity of urate oxidase is 0.6-0.7 U / mL.

[0052] More preferably, the exocytosis activity of urate oxidase is 0.653 U / mL.

[0053] In another aspect, the present application also provides an application of the signal peptide mutant, the biomaterial or the engineered bacterium in promoting the exocytosis of urate oxidase and / or increasing the exocytosis amount of urate oxidase and / or increasing the exocytosis activity of urate oxidase.

[0054] In another aspect, the present application also provides an application of the signal peptide mutant, the biomaterial or the engineered bacterium in preparing urate oxidase.

[0055] The present application has the following beneficial effects:

[0056] In view of the problem that the uric acid oxidase reported in the prior art is difficult to be secreted in Pichia pastoris, the application obtains signal peptides Fre2, Dse4 and 0030 with strong secretion ability by screening endogenous signal peptides of Pichia pastoris, and it is found for the first time that 0030 can be used as an extracellular secretion signal peptide of uric acid oxidase, thereby providing a new biological material for engineering modification of uric acid oxidase.

[0057] In addition, in the application, the signal peptide Fre2 is subjected to targeted sequence optimization, directional mutation or modification and mutation combination are performed on the 21st, 16th, 17th and 18th amino acid residues, thereby obtaining signal peptide mutants Fre2(T16V) and Fre2(T16V, V17T) with strong secretion ability, wherein the extracellular enzyme activity of Fre2(T16V, V17T) can reach 0.896 U / mL, which can better secrete the uric acid oxidase to the extracellular, thereby improving the extracellular enzyme activity of the uric acid oxidase and increasing the yield of the extracellular uric acid oxidase. BRIEF DESCRIPTION OF DRAWINGS

[0058] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0059] Figure 1 It is a fermentation result diagram of different signal peptide strains in a flask;

[0060] Figure 2 It is a fermentation result diagram of signal peptide Fre2 strains with different optimization modes in a flask;

[0061] Figure 3 It is a structure diagram of plasmid BB3ck-sgRNA. DETAILED DESCRIPTION

[0062] Technical terms:

[0063] Signal peptide: a peptide chain that guides the newly synthesized protein to the secretion pathway.

[0064] Identity: refers to the similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution research.

[0065] Recombination: in a broad sense, any gene exchange process that causes genotypic changes is called recombination.

[0066] Expression cassette: the expression cassette refers to a group of DNA sequences composed of a promoter, a target gene and a reporter gene, which can be expressed in specific tissues and easily detected.

[0067] Recombinant vector: The recombinant vector is the vector which is based on the basic skeleton of the cloning vector and the target gene is introduced into the vector, so that the target gene can be expressed.

[0068] Recombinant microorganism: The recombinant microorganism is the strain of the bacterial cell which is obtained by the genetic engineering method and the foreign gene is highly expressed.

[0069] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present application. The term "recombinant cell" encompasses any progeny of the parent cell that results from the replication of the parent cell that has a mutation arising during replication.

[0070] In order to more clearly illustrate the overall concept of the present application, the following will be described in detail with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without one or more of these specific details. In other instances, well-known features have not been described in detail in order to avoid obscuring the present application.

[0071] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0072] It should be noted that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise, and it will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components and / or combinations thereof.

[0073] In the following embodiments, unless otherwise specified, the reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained by commercial purchase. The plasmids, endonucleases, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed according to the kit instructions.

[0074] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application. Unless otherwise specified, the experimental methods, detection methods, preparation methods, etc. disclosed in the present application are preferred experimental methods, detection methods, preparation methods, etc. known to those skilled in the art at the filing date of the present application.

[0075] The methods all employ, unless otherwise indicated, conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques and related arts, as is routine in the art. See, e.g., Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0076] In the present specification, the amino acid at the corresponding site is represented by the generally accepted IUPAC one-letter abbreviation, in which each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0077] In the present specification, regarding the mutation of an amino acid, it is expressed as "original amino acid, site, substituted amino acid". For example, the mutation of threonine T at site 16 to valine V is expressed as T16V.

[0078] BB3cK_pGAP_23*_pPFK300_Cas9 plasmid addgene: #1000000136.

[0079] The culture media involved in the following examples include:

[0080] LB medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0081] YPD medium: yeast extract 10 g / L, tryptone 20 g / L, glucose 20 g / L.

[0082] BMGY medium: yeast extract 10 g / L, tryptone 20 g / L, glycerol 10 g / L, YNB 13.4 g / L (sterilized by filtration), 1 M potassium phosphate buffer pH 6.4 100 mL / L, 0.02% biotin 2 mL / L (sterilized by filtration).

[0083] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, YNB 13.4 g / L (sterilized by filtration), 1M potassium phosphate buffer pH 6.8 100 mL / L, 0.02% biotin 2 mL / L (sterilized by filtration).

[0084] In addition, the "water" in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high-purity water, purified water, and any feasible water that can be used in the field.

[0085] In the following examples, % means wt%, i.e. weight percentage, if no other specific description.

[0086] Example 1 Screening of signal peptides

[0087] 1. Construction of different signal peptide plasmids

[0088] The aspergillus flavus-derived uric acid oxidase (UOX) gene (NCBI Gene ID: X61766.1, the amino acid sequence is shown as SEQ ID No. 1, and the nucleotide sequence is shown as SEQ ID No. 2) was synthesized by Beijing Qikexin Biotechnology Co., Ltd., the vector was pPICZαA, and the pPICZαA-UOX plasmid was obtained. In this embodiment, the signal peptide fragments of UOX were screened, which specifically involved fragments Fre2, Dse4, 0030, UTH1, SCW10, and 0337. Among them, the amino acid sequence of Fre2 is shown as SEQ ID No. 3, and the nucleotide sequence is shown as SEQ ID No. 4; the amino acid sequence of Dse4 is shown as SEQ ID No. 5, and the nucleotide sequence is shown as SEQ ID No. 6; the amino acid sequence of 0030 is shown as SEQ ID No. 7, and the nucleotide sequence is shown as SEQ ID No. 8; the amino acid sequence of UTH1 is shown as SEQ ID No. 9, and the nucleotide sequence is shown as SEQ ID No. 10; the amino acid sequence of SCW10 is shown as SEQ ID No. 11, and the nucleotide sequence is shown as SEQ ID No. 12; the amino acid sequence of 0337 is shown as SEQ ID No. 13, and the nucleotide sequence is shown as SEQ ID No. 14.

[0089] Take the construction of plasmid pPICZ alpha A-Fre2-UOX as an example, pPICZ alpha A-UOX plasmid as a template, using primer Fre2-F / R for PCR amplification, 5 μL of DNA gel electrophoresis verification after amplification, the remaining reaction liquid using DpnI enzyme to remove template, correct band size then purify the DNA fragment, using T4 ligase blunt end connection after transformation into E. coli DH5 alpha competent cells, incubate 1 h after coating LB (plate) medium, 37°C overnight culture, pick single colony in LB (liquid) medium activation, using primer seq-F sequencing, sequencing work is completed by Beijing Qikexi Biotechnology Co., Ltd., sequencing results correct then plasmid pPICZ alpha A-Fre2-UOX construction is completed. Using the primer 0337-F / R, 0030-F / R, SCW10-F / R, UTH1-F / R, Dse4-F / R in table 1 according to the above method respectively constructs plasmid pPICZ alpha A-0337-UOX, pPICZ alpha A-0030-UOX, pPICZ alpha A-SCW10-UOX, pPICZ alpha A-UTH1-UOX, pPICZ alpha A-Dse4-UOX.

[0090] 2. Construction of sgRNA plasmid and assembly of integration fragment

[0091] Plasmid BB3ck-sgRNA plasmid structure diagram is shown in Figure 3 (constructing according to the method described in“CRISPR / Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris” with plasmid BB3cK_pGAP_23*_pPFK300_Cas9 as a vector), using primer sgRNA-F, sgRNA-PNS1-R to amplify, 5 μL of DNA gel electrophoresis verification after amplification, the remaining reaction liquid using DpnI enzyme to remove template, correct band size then purify the DNA fragment, 2 μL of purified DNA fragment is transformed into E. coli DH5 alpha competent cells, incubate 1 h after coating LB plate, 37°C overnight culture, pick single colony in LB liquid medium activation, using primer sgRNA-seq-F sequencing, sequencing work is completed by Beijing Qikexi Biotechnology Co., Ltd., sequencing results correct then plasmid BB3ck-sgRNA-PNS1 is constructed, BB3ck-sgRNA-PNS1 plasmid contains Cas9, sgRNA expression cassette, used for targeting the target gene for cutting.

[0092] As an example of the assembly of Donor-Fre2-UOX, the Fre2-UOX expression cassette is amplified from the plasmid pPICZ alpha A-Fre2-UOX using primers PAOX1-F and TAOX1-R, and the upstream and downstream homologous arm fragments are amplified from the Pichia pastoris X-33 genome using primers PNS1-up-F / R and PNS1-down-F / R. The upstream and downstream fragments are fused with the Fre2-UOX expression cassette by overlap PCR to assemble Donor-Fre2-UOX. Donor-0337-UOX, Donor-0030-UOX, Donor-SCW10-UOX, Donor-UTH1-UOX, and Donor-Dse4-UOX are assembled according to the above method.

[0093] 3. Screening of signal peptide

[0094] As an example of the construction of the X33-Fre2-UOX recombinant strain, first, the Pichia pastoris X-33 competent cells are prepared according to the method in the Pichia pastoris expression manual. 1 μg of Donor-Fre2-UOX and 0.5 μg of plasmid sgRNA-PNS1 are mixed with the Pichia pastoris X-33 competent cells and placed on ice for 2 min. The mixture is transferred to a pre-cooled electroporation cup, and an electroporator is used for electroporation. The electroporation conditions are 1.5 kV / cm, capacitance 25 μF, and resistance 200 Ω. After electroporation, 1 mL of YPD medium is immediately added to the electroporation cup, and the mixture is gently mixed and incubated at 30°C for 1-2 h. The recovered cells are spread on a YPD selection plate and incubated at 30°C for 2-3 days until visible colonies are formed. A single colony is picked and subjected to colony PCR using primers PNS1-up-F and PNS1-down-R. The strain with the correct band size is X33-Fre2-UOX. Recombinant strains X33-0337-UOX, X33-0030-UOX, X33-SCW10-UOX, X33-UTH1-UOX, and X33-Dse4-UOX are constructed according to the above method.

[0095] The recombinant strains constructed above are streaked on YPD (plate) medium, a single colony is inoculated into YPD (liquid) medium and cultured for 14-16 h, then inoculated into BMGY medium at a concentration of 1%, and cultured at 30°C, 220 rpm for 24 h. The cells are collected by centrifugation, resuspended in BMMY medium, and cultured at 25°C, 220 rpm. Every 24 h, 1% of methanol is added for induction. After 48 h of induction, 1 mL of fermentation broth is centrifuged to collect the supernatant, and the uric acid oxidase enzyme activity is detected.

[0096] In this example, the method for detecting the uric acid oxidase enzyme activity is as follows:

[0097] Uricase can catalyze the oxidation of uric acid to allantoin, and their maximum absorbance wavelengths are different, uric acid is 293 nm, while allantoin is 224 nm, and within a certain range, the concentration is proportional to the absorbance. Thus, the corresponding change in uric acid concentration can be calculated by measuring the change in absorbance of the solution at 293 nm, and then the enzyme activity can be calculated.

[0098] Take 1 mL of fermentation broth, centrifuge at 4°C, 12000 rmp for 10 min, take the supernatant and dilute it to the corresponding multiple as the crude enzyme solution, add 2.8 mL of 0.05M Tris-HCl, 0.6 mL of 0.6 mM uric acid, and 0.4 mL of crude enzyme solution in a 5 mL centrifuge tube, react in a 30°C water bath for 5 min, terminate the reaction with 5M HCl, measure the absorbance of the reaction solution at 293 nm, and then calculate the concentration of uric acid and enzyme activity. The definition of uricase enzyme activity is: the amount of enzyme required to catalyze 1 μM of uric acid per minute at 30°C is one unit. The fermentation results are shown in Figure 1

[0099] Table 1 Primer sequences involved in Example 1

[0100]

[0101]

[0102] As Figure 1 shown, when the signal peptide Fre2 is used as the secretion signal peptide of uricase, the extracellular enzyme activity is the highest, reaching 0.708 U / mL, followed by the Dse4 signal peptide, with an extracellular enzyme activity of 0.687 U / mL, and the signal peptide 0030 also has good secretion effect, with an extracellular enzyme activity of 0.653 U / mL. In order to achieve better secretion effect, the signal peptide Fre2 will be optimized and improved.

[0103] Example 2 Optimization of signal peptide

[0104] ​The signal peptide mutants were constructed by mutating different amino acid sites of the signal peptide Fre2 using the primers Fre2(+KR)-F / R, Fre2(A18T)-F / R, Fre2(T16V)-F / R, Fre2(V17T)-F / R, Fre2(T16V, V17T)-F / R in Table 2. Specifically, the amino acid sequence of Fre2(+KR) is shown as SEQ ID No. 15, and the nucleotide sequence is shown as SEQ ID No. 16, the amino acid sequence of Fre2(T16V) is shown as SEQ ID No. 17, and the nucleotide sequence is shown as SEQ ID No. 18, the amino acid sequence of Fre2(V17T) is shown as SEQ ID No. 19, and the nucleotide sequence is shown as SEQ ID No. 20, the amino acid sequence of Fre2(A18T) is shown as SEQ ID No. 21, and the nucleotide sequence is shown as SEQ ID No. 22, the amino acid sequence of Fre2(T16V, V17T) is shown as SEQ ID No. 23, and the nucleotide sequence is shown as SEQ ID No. 24.

[0105] The plasmids pPICZαA-Fre2(+KR)-UOX, pPICZαA-Fre2(A18T)-UOX, pPICZαA-Fre2(T16V)-UOX, pPICZαA-Fre2(V17T)-UOX, pPICZαA-Fre2(T16V, V17T)-UOX were constructed according to the method of Example 1. The Donor-Fre2(+KR)-UOX, Donor-Fre2(A18T)-UOX, Donor-Fre2(T16V)-UOX, Donor-Fre2(V17T)-UOX, Donor-Fre2(T16V, V17T)-UOX were assembled according to the method of Example 1. The strains X33-Fre2(+KR)-UOX, X33-Fre2(A18T)-UOX, X33-Fre2(T16V)-UOX, X33-Fre2(V17T)-UOX, X33-Fre2(T16V, V17T)-UOX were constructed according to the method of Example 1. After 48h of shake flask fermentation induction, the supernatant was centrifuged to detect enzyme activity. The fermentation results are shown in Table 3. Figure 2

[0106] Table 2 Primer sequences involved in Example 2

[0107]

[0108]

[0109] As Figure 2 ​As shown, after mutating different amino acid sites of the signal peptide Fre2, it was found that the secretion effect of the signal peptide Fre2(T16V, V17T) was the most obvious, the extracellular enzyme activity was 0.896 U / mL, which was increased by 26.5% compared with the control group (Fre2), and the second was the signal peptide Fre2(T16V), the extracellular enzyme activity was 0.782 U / mL, which was increased by 10.4% compared with the control group.

[0110] The above only describes the embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of claims of the present application.

Claims

1. A signal peptide mutant, characterized in that, The signal peptide is a sequence obtained by mutating the Fre2 amino acid residue site; The Fre2 amino acid sequence is shown in SEQ ID NO.3; The mutation is: A1) The 16th amino acid residue is mutated from a threonine residue to a valine residue; A2) The 16th amino acid residue is mutated from a threonine residue to a valine residue and the 17th amino acid residue is mutated from a valine residue to a threonine residue.

2. A biomaterial, characterized in that, The biomaterial comprises at least one or more of the following B1)-B5): B1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding the signal peptide mutant of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B5) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that, The expression cassette also contains a gene encoding urate oxidase.

4. The biomaterial according to claim 2, characterized in that, B1) The nucleic acid molecule mentioned is any one of the following b11)-b12): b11) The nucleotide sequence is shown in SEQ ID NO.18; b12) The nucleotide sequence is shown in SEQ ID NO.

24.

5. An engineered bacterium for producing uricase, characterized in that, The engineered bacteria contain a gene expressing the signal peptide mutant of claim 1 and a gene expressing uricase.

6. The engineered bacteria according to claim 5, characterized in that, The engineered bacteria are one or more of Pichia pastoris, Saccharomyces cerevisiae, and Candida albicans.

7. A method for producing uricase, characterized in that, The method includes: obtaining urate oxidase by fermentation using engineered bacteria as described in claim 5 or 6.

8. The use of the signal peptide mutant as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the engineered bacteria as described in claim 5 or 6 in promoting the extracellular secretion of uricase and / or increasing the extracellular secretion amount of uricase and / or increasing the extracellular enzyme activity of uricase.

9. The use of the signal peptide mutant as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the engineered bacteria as described in claim 5 or 6 in the preparation of uricase.

Images