Uric acid oxidase secretion signal peptide and application thereof

Abstract

The invention provides a urate oxidase secretion signal peptide and application thereof, and relates to the technical field of gene engineering. Aiming at the problem that urate oxidase is difficult to secrete in pichia pastoris reported in current literatures, endogenous signal peptides of pichia pastoris are screened to obtain signal peptides with relatively strong secretion ability, and the signal peptides are specifically optimized to obtain a signal peptide mutant with relatively strong secretion ability; the urate oxidase can be better secreted out of cells, so that the extracellular enzyme activity of the urate oxidase is improved.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a urate oxidase secretion signal peptide and its application. Background Art

[0002] Hyperuricemia (HUA) is a chronic clinical syndrome in which the blood uric acid content significantly exceeds the normal range due to purine metabolism disorder or abnormal uric acid excretion. It generally refers to the fasting blood uric acid level of males higher than 420μmol / L and females higher than 360μmol / L on two different days under a normal purine diet. Gout (Gout) is a crystalline 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 hypertension, diabetes, coronary heart disease and chronic kidney disease. Gout has become the "fourth high" after diabetes, hypertension and hyperlipidemia, causing serious harm to people's health.

[0003] Clinically, hyperuricemia is generally prevented and treated in three ways. The first way is to reduce the production of uric acid. The drugs used are xanthine oxidase (XOD) inhibitors, such as allopurinol and febuxostat, which 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. The drugs used are URAT1 inhibitors, such as benzbromarone and lesinade, which inhibit the uric acid transporter URAT1 to reduce the reabsorption of uric acid by the renal tubules, thereby increasing the excretion of uric acid and reducing blood uric acid. The third way is to degrade uric acid. The drugs used are urate oxidase (UOX) drugs, such as rasburicase and purikacil, which degrade uric acid into more soluble allantoin and excrete it from the body to reduce 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 class of drugs with great potential.

[0004] At present, research on heterologous expression of urate oxidase in yeast at home and abroad mainly focuses on intracellular expression or the use of α-factor from Saccharomyces cerevisiae as a signal peptide to achieve extracellular secretion of urate oxidase. However, studies have found that the extracellular secretion amount of α-factor as a signal peptide is low, and it is impossible to achieve final industrialization, and the cost of intracellular expression, secretion and purification is high. Some researchers have also achieved extracellular secretion of urate oxidase by screening signal peptides. In 2021, Beijing University of Chemical Technology achieved extracellular secretion of urate oxidase in Pichia pastoris by screening urate oxidase secretion signal peptides, and the extracellular enzyme activity at the shake flask level reached 0.474U / mL (patent number: CN113956989A), but the enzyme activity still has room for improvement, so it is necessary to continue screening and optimizing the secretion signal peptide to improve the extracellular enzyme activity of urate oxidase. Summary of the invention

[0005] In response to the problem that urate oxidase is difficult to secrete in Pichia pastoris as reported in current literature, the present invention screened the endogenous signal peptides of Pichia pastoris, obtained signal peptides with strong secretion ability, and optimized them in a targeted manner, thereby obtaining a signal peptide mutant with strong secretion ability, which can better secrete urate oxidase to the extracellular space, thereby improving the extracellular enzyme activity of urate oxidase.

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

[0007] Preferably, the Fre2 amino acid residue site is at least selected from one or more of position 16, position 17, position 18, and position 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] Wherein, the Fre2 comprises the amino acid sequence 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] Furthermore, it is characterized in that the mutation includes at least one or more of the following A1)-A4):

[0011] A1) a lysine residue (K) and / or an arginine residue (R) is added to the carbon-terminus and / or the nitrogen-terminus of the amino acid residue at position 21; preferably, a lysine residue (K) and an arginine residue (R) are added to the carbon-terminus of the amino acid residue at position 21; more preferably, the signal peptide mutant comprises an amino acid sequence as shown in SEQ ID NO.15 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.15; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as shown in SEQ ID NO.16 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. NO.16 has a nucleotide sequence with 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;

[0012] A2) the amino acid residue at position 18 is mutated from an alanine residue (A) to a threonine residue (T); more preferably, the signal peptide mutant comprises an amino acid sequence as shown in SEQ ID NO.21 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.21; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as shown in SEQ ID NO.22 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. ID NO. 22 has a nucleotide sequence with 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;

[0013] A3) the amino acid residue at position 16 is mutated from a threonine residue (T) to a valine residue (V); more preferably, the signal peptide mutant comprises an amino acid sequence as shown in SEQ ID NO.17 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.17; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as shown in SEQ ID NO.18 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.18 ID NO. 18 has a nucleotide sequence with 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;

[0014] A4) the amino acid residue at position 17 is mutated from a valine residue (V) to a threonine residue (T); more preferably, the signal peptide mutant comprises an amino acid sequence as shown in SEQ ID NO.19 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.19; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as shown in SEQ ID NO.20 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. ID NO. 20 has a nucleotide sequence with 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;

[0015] Preferably, the amino acid residue at position 16 is mutated from a threonine residue (T) to a valine residue (V) and the amino acid residue at position 17 is mutated from a valine residue (V) to a threonine residue (T); more preferably, the signal peptide mutant comprises an amino acid sequence as shown 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 with SEQ ID NO.23; more preferably, the nucleic acid molecule encoding the signal peptide mutant comprises a nucleotide sequence as shown in SEQ ID NO.24 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. NO.24 has a nucleotide sequence with 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%, and 99.9% identity.

[0016] It is understandable to those skilled in the art that the mutation of the signal peptide of the present application can be completed by using existing gene editing technology.

[0017] On the other hand, the present application also provides a biomaterial, which comprises at least one or more of the following B1)-B5):

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

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

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

[0021] B4) a recombinant microorganism, which contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3);

[0022] B5) A recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3).

[0023] Those skilled in the art will appreciate that other commonly used expression elements can be adaptively added to the expression cassette described in the present application to assist in the expression of the target gene, such as adding tags, fluorescent protein markers, resistance screening markers, etc.

[0024] A person skilled in the art can select a known plasmid to construct a recombinant vector according to actual conditions and perform conventional optimization and modification on the constructed recombinant vector. The present application does not impose mandatory restrictions on the type of plasmid. In a preferred embodiment, the vector may be pPICZαA and / or BB3ck-sgRNA.

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

[0026] Furthermore, the nucleic acid molecule described in B1) comprises any one of the following b11)-b16):

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

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

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

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

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

[0032] b16) A DNA molecule which hybridizes with the nucleotide sequence specified in any one of b11) to b15) under stringent conditions and encodes the signal peptide mutant.

[0033] On the other hand, the present application also provides an engineered bacterium for producing uricase, wherein the engineered bacterium contains a gene expressing the signal peptide mutant and a gene expressing uricase.

[0034] Furthermore, the engineered bacteria are one or more of Pichia pastoris, Saccharomyces cerevisiae, and Candida.

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

[0036] On the other hand, the present application also provides a method for producing urate oxidase, the method comprising: obtaining urate oxidase by fermentation using the engineered bacteria.

[0037] Preferably, the urate oxidase is an extracellular enzyme.

[0038] More preferably, the extracellular enzymatic activity of urate oxidase is >0.6 U / mL.

[0039] More preferably, the extracellular enzymatic activity of the urate oxidase is 0.6 U / mL-0.9 U / mL.

[0040] More preferably, the extracellular enzymatic activity of the urate oxidase is 0.708 U / mL-0.896 U / mL.

[0041] It is understood by those skilled in the art that a general method can be used to activate and ferment the Pichia pastoris to obtain a crude urate oxidase enzyme solution, and a conventional urate oxidase purification method can be used to purify and extract the crude enzyme solution to obtain high-purity urate oxidase.

[0042] In a preferred embodiment, the method comprises: activating the engineered bacteria, inoculating into a fermentation medium at an inoculation rate of 1%-10%, culturing at 25°C-40°C, 200-300rpm for 12-48h, collecting the bacteria, resuspending in a fermentation medium at 25°C-40°C, 200-300rpm, 1%-5% methanol induction culture for 12-48h, and collecting the supernatant, which is the crude urate oxidase enzyme solution.

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

[0044] In the examples of the present application, the extracellular enzyme activity of the obtained urate oxidase can reach up to 0.896U / mL, which is 89% higher than the extracellular enzyme activity recorded in the prior art, proving that the signal peptide mutant described in the present application can effectively promote the extracellular secretion of urate oxidase and thus increase its extracellular enzyme activity.

[0045] On the other hand, the present application also provides the use of a signal peptide containing the amino acid sequence shown in SEQ ID No.7 in promoting the extracellular secretion of uricase oxidase and / or increasing the extracellular secretion amount of uricase oxidase and / or increasing the extracellular enzyme activity of uricase oxidase and / or in the preparation of uricase oxidase.

[0046] In the present application, it is discovered for the first time that signal peptide 0030 can effectively promote the extracellular secretion of urate oxidase and thus improve its extracellular enzyme activity, wherein the amino acid sequence of signal peptide 0030 is as shown in SEQ ID No.7, and the nucleotide sequence encoding the signal peptide 0030 is as 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] On the other hand, the present application also provides an engineered bacterium for producing uricase, wherein the engineered bacterium contains a signal peptide gene expressing the amino acid sequence shown in SEQ ID No.7 and a gene expressing uricase.

[0048] Furthermore, the engineered bacteria are one or more of Pichia pastoris, Saccharomyces cerevisiae, and Candida.

[0049] Preferably, it is Pichia pastoris, and more preferably, it is Pichia pastoris X-33.

[0050] On the other hand, the present application also provides a method for producing urate oxidase, the method comprising: obtaining urate oxidase by fermentation using the engineered bacteria.

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

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

[0053] On the other hand, the present application also provides the use of the signal peptide mutant or the biomaterial or the engineered bacteria in promoting the extracellular secretion of urate oxidase and / or increasing the extracellular secretion amount of urate oxidase and / or increasing the extracellular enzyme activity of urate oxidase.

[0054] On the other hand, the present application also provides the use of the signal peptide mutant or the biomaterial or the engineered bacteria in the preparation of urate oxidase.

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

[0056] In response to the problem that urate oxidase is difficult to secrete in Pichia pastoris as reported in current literature, the present invention screened endogenous signal peptides of Pichia pastoris and obtained signal peptides Fre2, Dse4 and 0030 with strong secretion ability. It was found for the first time that 0030 can be used as an extracellular secretion signal peptide of urate oxidase, providing a new biomaterial for the engineering modification of urate oxidase.

[0057] In addition, in the present application, targeted sequence optimization is performed on the signal peptide Fre2, and directed mutation or modification and mutation combination are performed on the 21st amino acid residue, the 16th amino acid residue, the 17th amino acid residue, and the 18th amino acid residue, to obtain a signal peptide mutant Fre2 (T16V) and Fre2 (T16V, V17T) with strong secretion ability, among which the extracellular enzyme activity of Fre2 (T16V, V17T) can reach 0.896U / mL, which can better secrete urate oxidase to the extracellular space, thereby increasing the extracellular enzyme activity of urate oxidase and increasing the extracellular urate oxidase production. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0059] Figure 1 The figure shows the results of shake flask fermentation of different signal peptide strains;

[0060] Figure 2 This is a diagram showing the shake flask fermentation results of strains with different optimization methods for signal peptide Fre2;

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

[0062] Technical terms:

[0063] Signal peptide: a peptide chain that directs newly synthesized proteins to the secretory pathway.

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

[0065] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

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

[0067] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0068] Recombinant microorganism: A fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[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 invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0070] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0071] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0072] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0073] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. The plasmids, endonucleases, PCR enzymes, column-based 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 invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the protection scope of the present invention. Unless otherwise specified, the experimental methods, detection methods, preparation methods disclosed in the present invention are not intended to limit the protection scope of the present invention.

[0075] The methods all adopt conventional techniques in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0076] In the present specification, the amino acids at the corresponding positions are represented by the recognized IUPAC single-letter abbreviations, wherein each amino acid and its abbreviation are: 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 this specification, amino acid mutations are represented by "original amino acid, position, substituted amino acid." For example, the mutation of threonine T at position 16 to valine V is represented by T16V.

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

[0079] The culture medium involved in the following embodiments includes:

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

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

[0082] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L, YNB 13.4 g / L (filter sterilized), 100 mL / L 1 M potassium phosphate buffer with a pH of 6.4, and 2 mL / L 0.02% biotin (filter sterilized).

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

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

[0085] In the following examples, unless otherwise specified, % means wt %, i.e. weight percentage.

[0086] Example 1 Screening of signal peptides

[0087] 1. Construction of different signal peptide plasmids

[0088] The urate oxidase (UOX) gene from Aspergillus flavus (NCBI Gene ID: X61766.1, the amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2) was synthesized by Beijing Qingke 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, specifically involving fragments Fre2, Dse4, 0030, UTH1, SCW10, and 0337, wherein the amino acid sequence of Fre2 is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.4, the amino acid sequence of Dse4 is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6, the amino acid sequence of 0030 is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.8, the amino acid sequence of UTH1 is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.10, the amino acid sequence of SCW10 is shown in SEQ ID No.11, and the nucleotide sequence is shown in SEQ ID No.12, and the amino acid sequence of 0337 is shown in SEQ ID No.13, and the nucleotide sequence is shown in SEQ ID No.14.

[0089] Taking the construction of plasmid pPICZαA-Fre2-UOX as an example, pPICZαA-UOX plasmid was used as a template, and primers Fre2-F / R were used for PCR amplification. After the amplification was completed, 5 μL was taken for DNA gel electrophoresis verification, and the remaining reaction solution was digested with DpnI enzyme. If the band size was correct, the DNA fragment was purified, and after blunt-end ligation using T4 ligase, it was transformed into Escherichia coli DH5α competent cells. After incubation for 1 hour, LB (plate) medium was applied, cultured overnight at 37°C, and a single colony was picked to activate in LB (liquid) medium, and sequencing was performed using primer seq-F. The sequencing work was completed by Beijing Qingke Biotechnology Co., Ltd. If the sequencing results were correct, the construction of plasmid pPICZαA-Fre2-UOX was completed. Using primers 0337-F / R, 0030-F / R, SCW10-F / R, UTH1-F / R, and Dse4-F / R in Table 1, plasmids pPICZαA-0337-UOX, pPICZαA-0030-UOX, pPICZαA-SCW10-UOX, pPICZαA-UTH1-UOX, and pPICZαA-Dse4-UOX were constructed according to the above method.

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

[0091] Plasmid BB3ck-sgRNA plasmid structure diagram is shown in Figure 3 (Use plasmid BB3cK_pGAP_23*_pPFK300_Cas9 as a vector according to the “CRISPR / Cas9-Mediated Homology-Directed Genome Editing in Pichiapastoris”), using BB3ck-sgRNA plasmid as template, using primers sgRNA-F and sgRNA-PNS1-R for amplification, after amplification, 5 μL was taken for DNA gel electrophoresis verification, and the remaining reaction solution was digested with DpnI enzyme template, and the DNA fragment was purified if the band size was correct, and 2 μL of the purified DNA fragment was transformed into Escherichia coli DH5α competent cells, incubated for 1 hour, coated on LB plates, cultured at 37°C overnight, picked single colonies and activated in LB liquid culture medium, and sequenced using primer sgRNA-seq-F. The sequencing work was completed by Beijing Qingke Biotechnology Co., Ltd., and the construction of plasmid BB3ck-sgRNA-PNS1 was completed if the sequencing result was correct. The BB3ck-sgRNA-PNS1 plasmid contained Cas9 and sgRNA expression cassettes for targeting the target gene for cutting.

[0092] Taking the assembly of Donor-Fre2-UOX as an example, plasmid pPICZαA-Fre2-UOX was used as a template, primers PAOX1-F and TAOX1-R were used to amplify the Fre2-UOX expression cassette, and the Pichia pastoris X-33 genome was used as a template, and primers PNS1-up-F / R and PNS1-down-F / R were used to amplify the upstream and downstream homology arm fragments. The upstream and downstream fragments were 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 were assembled according to the above method.

[0093] 3. Screening of signal peptides

[0094] Taking the construction of X33-Fre2-UOX recombinant strain as an example, first use the method in the Pichia pastoris expression operation manual to make Pichia pastoris X-33 competent cells, take 1μg Donor-Fre2-UOX, 0.5μg plasmid sgRNA-PNS1 and Pichia pastoris X-33 competent cells, mix them evenly and place them on ice for 2min, transfer the mixture to a pre-cooled electroporation cup, and use an electroporator for electroporation. The electroporation conditions are 1.5kV / cm, capacitance 25μF, and resistance 200Ω. After the electroporation, immediately add 1mL of YPD medium to the electroporation cup, mix gently, and resuscitate at 30℃ for 1-2h. The revived cells are spread on the YPD screening plate and cultured at 30℃ for 2-3 days until visible colonies are formed. Pick a single clone and use primers PNS1-up-F and PNS1-down-R for colony PCR verification. 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 were constructed according to the above method.

[0095] The recombinant strain constructed above was streaked and activated on YPD (plate) medium, a single clone was picked and inoculated into YPD (liquid) medium and cultured for 14-16 h, and then inoculated into BMGY medium at a 1% inoculum size, cultured at 30°C, 220 rpm for 24 h, centrifuged to collect the bacteria, resuspended in BMMY medium, and cultured in a shaker at 25°C, 220 rpm, induced by adding 1% methanol every 24 h, and 1 mL of the fermentation broth was centrifuged after 48 h of induction to collect the supernatant for detection of urate oxidase activity.

[0096] The method for determining the activity of urate oxidase in this embodiment is as follows:

[0097] Urate oxidase can catalyze the oxidation of uric acid to produce allantoin. The wavelengths of their maximum absorbance are different, uric acid is 293nm, and allantoin is 224nm. Within a certain range, their concentration and absorbance are proportional. Therefore, the corresponding change in uric acid concentration can be calculated by measuring the change in the absorbance value of the solution at 293nm, and then the enzyme activity can be calculated.

[0098] Take 1mL of fermentation liquid and centrifuge at 4℃ and 12000rmp for 10min. Take out the supernatant and dilute it accordingly as the crude enzyme solution. Add 2.8mL of 0.05M Tris-HCl, 0.6mL of 0.6mM uric acid, and 0.4mL of crude enzyme solution to a 5mL centrifuge tube. React in a 30℃ water bath for 5min. Terminate the reaction with 5M HCl and measure the absorbance of the reaction solution at 293nm to calculate the uric acid concentration and enzyme activity. The definition of urate oxidase activity is: at 30℃, the amount of enzyme required to catalyze 1μM uric acid per minute is one unit. The fermentation results are as follows: Figure 1 shown.

[0099] Table 1 Primer sequences involved in Example 1

[0100]

[0101]

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

[0103] Example 2 Optimization of signal peptide

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

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

[0106] Table 2 Primer sequences involved in Example 2

[0107]

[0108]

[0109] like Figure 2As 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 most significantly improved, with an extracellular enzyme activity of 0.896 U / mL, an increase of 26.5% relative to the control group (Fre2), followed by the signal peptide Fre2 (T16V), with an extracellular enzyme activity of 0.782 U / mL, an increase of 10.4% relative to the control group.

[0110] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A signal peptide mutant, characterized in that The signal peptide comprises a sequence obtained by mutation of the Fre2 amino acid residue site; Preferably, the Fre2 amino acid residue site is at least selected from one or more of position 16, position 17, position 18, and position 21; 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.

2. The signal peptide mutant according to claim 1, characterized in that The mutations include at least one or more of the following A1)-A4): A1) a lysine residue and / or an arginine residue is added to the carbon-terminus and / or nitrogen-terminus of the 21st amino acid residue; preferably, a lysine residue and an arginine residue are added to the carbon-terminus of the 21st amino acid residue; A2) the amino acid residue at position 18 is mutated from an alanine residue to a threonine residue; A3) the amino acid residue at position 16 is mutated from a threonine residue to a valine residue; A4) the amino acid residue at position 17 is mutated from a valine residue to a threonine residue; Preferably, the amino acid residue at position 16 is mutated from a threonine residue to a valine residue and the amino acid residue at position 17 is mutated from a valine residue to a threonine residue.

3. Biomaterial, characterized in that The biological material comprises at least one or more of the following B1)-B5): B1) a nucleic acid molecule comprising a nucleic acid molecule encoding the signal peptide mutant according to any one of claims 1 or 2; B2) an expression cassette, the expression cassette comprising the nucleic acid molecule of B1); preferably, the expression cassette further comprises a gene encoding urate oxidase; B3) a recombinant vector, the recombinant vector comprising the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) a recombinant microorganism, which contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3); B5) A recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecule described in B1) comprises any one of the following b11) to b16): b11) a nucleic acid molecule having a nucleotide sequence containing SEQ ID NO.16 or a DNA molecule having 98% or more identity with SEQ ID NO.16 and encoding the signal peptide mutant according to any one of claims 1 or 2; b12) a DNA molecule having a nucleotide sequence containing a nucleic acid molecule as shown in SEQ ID NO.18 or having 98% or more identity with SEQ ID NO.18 and encoding the signal peptide mutant according to any one of claims 1 or 2; b13) a nucleic acid molecule having a nucleotide sequence containing SEQ ID NO. 20 or a DNA molecule having 98% or more identity with SEQ ID NO. 20 and encoding the signal peptide mutant according to any one of claims 1 or 2; b14) a nucleic acid molecule having a nucleotide sequence containing SEQ ID NO.22 or a DNA molecule having 98% or more identity with SEQ ID NO.22 and encoding the signal peptide mutant according to any one of claims 1 or 2; b15) a DNA molecule having a nucleotide sequence containing a nucleic acid molecule as shown in SEQ ID NO. 24 or having 98% or more identity with SEQ ID NO. 24 and encoding the signal peptide mutant according to any one of claims 1 or 2; b16) A DNA molecule which hybridizes with the nucleotide sequence specified in any one of b11) to b15) under stringent conditions and encodes the signal peptide mutant according to any one of claims 1 or 2.

5. An engineered bacterium for producing urate oxidase, characterized in that: The engineered bacteria contains a gene for expressing the signal peptide mutant according to claim 1 or 2 and a gene for expressing urate oxidase.

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.

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

8. Use of a signal peptide containing the amino acid sequence shown in SEQ ID No. 7 in promoting the extracellular secretion of uricase oxidase and / or increasing the extracellular secretion amount of uricase oxidase and / or increasing the extracellular enzyme activity of uricase oxidase and / or in the preparation of uricase oxidase.

9. Use of the signal peptide mutant according to claim 1 or 2, or the biomaterial according to claim 3 or 4, or the engineered bacteria according to claim 5 or 6 in promoting the extracellular secretion of urate oxidase and / or increasing the extracellular secretion amount of urate oxidase and / or increasing the extracellular enzyme activity of urate oxidase.

10. Use of the signal peptide mutant according to claim 1 or 2, or the biomaterial according to claim 3 or 4, or the engineered bacteria according to claim 5 or 6 in the preparation of urate oxidase.

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