CN112552382A - Signal peptide mutant and application thereof - Google Patents
Signal peptide mutant and application thereof Download PDFInfo
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Abstract
The invention discloses a signal peptide mutant and application thereof, belonging to the technical field of genetic engineering. The signal peptide mutant is obtained by carrying out site-specific mutagenesis on charged amino acids in an N region of a signal peptide derived from a gram-positive bacterium Sec pathway, so that the charge density of the amino acids in the N region is 0.2-0.8; mutating the signal peptide H region to enable the amino acid hydrophobicity of the H region to be 0-70; and carrying out site-directed mutagenesis on the amino acid in the C region of the signal peptide to mutate the last three-position amino acid in the C region into alanine-any amino acid-alanine. The invention can effectively improve the secretion of heterologous protein in a microbial host and effectively promote the high-efficiency expression and industrial production of target protein.
Description
The application is a divisional application of the following patent applications, the application numbers of the original applications: 2020100747867, filing date: 2020-01-22, invention name: a signal peptide mutant for improving the secretion of heterologous protein and a construction method and application thereof.
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a signal peptide mutant and application thereof.
Background
Recombinant proteins such as industrial enzymes and biopharmaceutical proteins have a very broad market of application. Various prokaryotic and eukaryotic expression systems have been developed to produce recombinant proteins. Among them, bacteria have advantages such as easy handling and many genetic manipulation tools, and are used as expression hosts for recombinant proteins. However, the expression of heterologous proteins in a host often results in the formation of insoluble inclusion bodies and is easily degraded by proteases, which makes the production of heterologous proteins difficult on a large scale. Secretion of the recombinant protein into the growth medium of the bacterial host effectively prevents the recombinant protein from forming insoluble inclusion bodies in the cytoplasm. In addition, the secretion of the recombinant protein can reduce the toxic effect of the protein on a production host, simplify the downstream processing steps of the product and obviously reduce the production cost. It is therefore desirable to be able to secrete recombinant proteins into the culture supernatant of bacterial expression hosts with high efficiency during production.
Signal peptide (abbreviated as SP) is a short peptide chain that guides a newly synthesized protein to the secretory pathway, and is mostly located at the N-terminus of the secretory protein, and its length is usually 16 to 30 amino acid residues. The signal peptide contains three regions: region N, region H and region C. The N region consists of 1-5 positively charged amino acid residues, which interact with negatively charged phospholipids. The middle part of the signal peptide is an H region and consists of 7-15 hydrophobic amino acid residues. The C region is usually 3 to 7 amino acids in length, and mainly includes hydrophilic amino acids that can be recognized and cleaved by signal peptidase. During secretion of the heterologous protein, the nature of the signal peptide plays a crucial role in the amount of secretion of the heterologous protein.
Disclosure of Invention
The invention provides a method for improving the secretion amount of heterologous proteins in a bacterial host by modifying a signal peptide by using site-directed mutagenesis.
The technical scheme for solving the technical problems is as follows:
the invention aims to provide a signal peptide mutant for improving the secretion of heterologous proteins, which is characterized in that charged amino acids in an N region of a signal peptide of an Sec pathway of gram-positive bacteria are subjected to site-directed mutagenesis to ensure that the charge density of the amino acids in the N region is 0.2-0.8; mutating the signal peptide H region to enable the amino acid hydrophobicity of the H region to be 0-70; and carrying out site-directed mutagenesis on the amino acid in the C region of the signal peptide to mutate the last three-position amino acid in the C region into alanine-any amino acid-alanine. The invention can effectively improve the secretion amount of heterologous protein in a microbial host.
The method for calculating the charge density of the N region of the signal peptide comprises the following steps: the charge amount of the amino acid R, K is +1, the charge amount of the amino acid D, E is-1, and the charge amounts of the remaining amino acids are 0. The charge density of the N region is obtained by dividing the total number of the charges of the amino acids in the N region by the total number of the amino acids in the N region.
Wherein, Kyte-Doolittle method is adopted to calculate the hydrophobicity of the amino acid in the H region of the signal peptide, and the corresponding hydrophobicity value of the amino acid is as the following table:
TABLE 1 hydrophobicity values corresponding to different amino acids
Preferably, the signal peptide H region is mutated such that the amino acid hydrophobicity of the H region is 0-38.
More preferably, the signal peptide H region is mutated such that the amino acid hydrophobicity of the H region is 20-38.
Preferably, site-directed mutagenesis is performed on the charged amino acids in the N region of the signal peptide such that the charge density of the amino acids in the N region is 0.25-0.45.
Preferably, the signal peptide is derived from bacillus subtilis or bacillus amyloliquefaciens.
Preferably, the original amino acid sequence of the signal peptide is as set forth in SEQ ID NO:2, is derived from bacillus amyloliquefaciens.
Preferably, the original amino acid sequence of the signal peptide is as set forth in SEQ ID NO:6, is derived from bacillus subtilis.
Preferably, the original amino acid sequence of the signal peptide is as set forth in SEQ ID NO:9, derived from bacillus subtilis.
Preferably, the amino acid sequence of the mutant of the signal peptide is as set forth in SEQ ID NO:2, mutating the last three amino acids in the C region from TSA to ASA, wherein the charge density of the N region is 0.42 and the hydrophobicity of the H region is 36 before and after the mutation of the signal peptide, and the amino acid sequence of the signal peptide mutant is shown as SEQ ID NO:3, respectively.
Preferably, the amino acid sequence of the mutant of the signal peptide is as set forth in SEQ ID NO:6, mutating the charge density of the N region of the signal peptide from 0.5 to 0.29, the hydrophobicity of the H region from 72.9 to 35.7, and the last three-position amino acid of the C region from GRA to AHA to obtain the amino acid sequence of the signal peptide mutant as shown in SEQ ID NO: shown at 7.
Preferably, the amino acid sequence of the mutant of the signal peptide is as set forth in SEQ ID NO:9, mutating the charge density of the N region of the signal peptide from 0.1 to 0.67, the hydrophobicity of the H region from 38.5 to 40.5, and the last three-position amino acid of the C region from VFS to ADA to obtain the amino acid sequence of the signal peptide mutant as shown in SEQ ID NO: shown at 10.
Preferably, the amino acid sequence of the mutant of the signal peptide is as set forth in SEQ ID NO:9, mutating the charge density of the N region of the signal peptide from 0.1 to 0.67, the hydrophobicity of the H region from 38.5 to 35.8, and the last three-position amino acid of the C region from VFS to ADA to obtain the amino acid sequence of the signal peptide mutant as shown in SEQ ID NO: shown at 11.
The second purpose of the invention is to provide the use of the signal peptide mutant in improving the secretion expression of heterologous proteins.
Preferably, the heterologous protein of interest is an alkaline protease derived from Bacillus clausii and having an amino acid sequence shown in SEQ ID NO 1.
The coding gene of the alkaline protease is gene aprE, the nucleotide sequence of the gene aprE is shown as SEQ ID NO. 12, and the GenBank of the gene aprE is FJ 940727.1.
Preferably, the target heterologous protein is alkaline xylanase which is derived from Bacillus pumilus and has an amino acid sequence shown as SEQ ID NO. 5.
The nucleotide sequence of the coding gene of the alkaline xylanase is shown as SEQ ID NO. 13, and the GenBank thereof is KU 301789.1.
Preferably, the heterologous protein of interest is cutinase derived from Fusarium solani and has an amino acid sequence shown in SEQ ID NO. 8.
The nucleotide sequence of the coding gene of the cutinase is shown as SEQ ID NO. 14, and the GenBank thereof is AAA 33335.1.
The third purpose of the invention is to provide a recombinant vector containing the coding gene of the signal peptide mutant.
Preferably, the expression vector used by the recombinant vector is pWB980 or PUB 110.
The fifth object of the present invention is to provide a host cell containing the recombinant vector or the gene encoding the mutant signal peptide.
Preferably, the host cell is bacillus subtilis.
The host cell does not have a function of secreting and expressing the alkaline protease.
More preferably, the host cell is bacillus subtilis WB 600. Refer to the non-patent literature (Shishuo, Lidenke, etc.. two different promoters and their combinations on the heterologous expression of alkaline protease AprE [ J ]. J.Biol.Engineer, China 2019,39 (10): 17-23).
The sixth purpose of the invention is to provide a genetically engineered bacterium, which takes bacillus subtilis as a host cell and expresses and connects the coding gene of the signal peptide mutant and the coding gene of heterologous protein through a vector.
The seventh purpose of the invention is to provide a method for constructing the signal peptide mutant, which comprises the following steps:
and carrying out the same enzyme digestion on the gene sequence coded by the signal peptide mutant and the gene sequence fragment coded by the heterologous protein, then connecting to obtain a connection product, purifying the connection product, cloning to an expression vector to obtain a recombinant vector, transferring the recombinant vector to a host cell, and screening to obtain a positive transformant.
The seventh object of the present invention is to provide a method for producing the alkaline protease, comprising the steps of:
the genetically engineered bacteria containing the signal peptide mutant are transferred into a fermentation culture medium with the inoculation amount of 1.5-5% (v/v) after being activated, and are fermented and cultured for 20-50h at 34-40 ℃ and 215-225 r/min.
Preferably, the fermentation medium consists of: 60-70g/L of corn flour, 30-50g/L of bean cake powder, 2-8g/L of disodium hydrogen phosphate, 0.1-0.6g/L of monopotassium phosphate, 0.5-1.0g/L of high-temperature amylase and the balance of water, p H is natural;
or the composition is as follows: 8-12g/L of xylan, 2-8g/L of peptone, 1-1.5g/L of yeast powder and K2HPO4 1-3g/L，MgSO4·7H20.1-0.4g/L of O, 2-8g/L of sodium chloride, 10-20g/L of agar powder and the balance of water;
or the composition is as follows: 6-12g/L of glucose, 8-16g/L of peptone, 20-30g/L of yeast extract, 8-14g/L of disodium hydrogen phosphate, 1-5g/L of potassium dihydrogen phosphate and the balance of water, p H7.0.0-7.5.
Preferably, the activation step of the genetically engineered bacteria is as follows: inoculating the genetic engineering bacteria into a seed culture medium, and carrying out shaking culture at 34-40 ℃ and 215-225r/min for 10-13 h;
the seed culture medium comprises the following components: 45-55mg/L kanamycin, 4-6g/L yeast powder, 8-12g/L peptone, 4-6g/L sodium chloride and the balance of water;
or LB culture medium;
or the composition is as follows: 6-12g/L of glucose, 8-16g/L of peptone, 20-30g/L of yeast extract, 8-14g/L of disodium hydrogen phosphate, 1-5g/L of potassium dihydrogen phosphate and the balance of water, p H7.0.0-7.5.
Has the advantages that:
(1) the length of the C region of the signal peptide is usually 3-7 amino acids, and mainly comprises hydrophilic amino acids which can be recognized and cut by the signal peptidase. During secretion of the heterologous protein, the nature of the signal peptide plays a crucial role in the amount of secretion of the heterologous protein. The last three amino acids in the C region of the signal peptide are mutated into alanine-any amino acid-alanine, so that the secretory expression quantity of the signal peptide to heterologous proteins is obviously improved. In the signal peptide, mutation of the amino acid in the C region plays a dominant role in the secretory expression of the heterologous protein.
(2) The H region of the signal peptide is composed of a stretch of hydrophobic residues, and the signal peptide hypothesis states that: proteins are transported across membranes primarily due to the presence of hydrophobic structures in the signal peptide. The hydrophobicity of the H region of the conventional signal peptide is approximately between-10 and 100. The hydrophobic region in the signal peptide has important influence on the secretion of the protein, the hydrophobicity of the H region of the signal peptide determined by the invention is 0-70, the protein secretion is more facilitated, and the excessive length or the excessive length of the hydrophobic region is not favorable for the protein secretion. According to the invention, through amino acid mutation, the amino acid hydrophobicity of the H region is 0-70, which is more beneficial to the combination of the signal peptide and the protein on the membrane, so that the better secretion expression of the foreign protein is promoted, the expression quantity of the target gene is effectively improved, and meanwhile, the influence of the N region on the signal peptide (namely the effect of the N region on transmembrane transport of the signal peptide) can be weakened through the mutation of the H region. When the amino acid hydrophobicity of the signal peptide H region is less than 0 or greater than 70, the secretory expression of the foreign protein may be reduced, or even the target protein may not be efficiently secreted. The H region is mutated, the amino acid hydrophobicity of the H region is preferably in the range of 0-38, more preferably in the range of 20-38, and in the preferred value range, the combination of the signal peptide and the protein on the membrane is more facilitated, and the secretion expression amount of the foreign protein is more remarkable.
(3) The N-terminal of the mature protein and the N-terminal of the signal peptide are oppositely charged, and electrostatic interaction between the mature protein and the N-terminal of the signal peptide is favorable for forming a hairpin structure, so that the insertion of a hydrophobic region of the signal peptide into a cell membrane is favorable, and the secretion of the protein is further favorable.
The N-terminal region of the signal peptide at least contains one arginine or lysine, the residue with positive charge is the key of the signal peptide for transmembrane, the signal peptide with positive charge is combined with phospholipid with negative charge so as to enable the signal peptide to be transmembrane, the charge density of the N-terminal region of the conventional signal peptide is approximately between-0.2 and 0.9, and after mutation of amino acid in the N-terminal region, the charge density of the amino acid is controlled between 0.2 and 0.8, so that the transmembrane efficiency of the signal peptide is improved, and the secretion expression amount of the foreign protein is effectively improved. When the hydrophobicity of the amino acid in the N region of the signal peptide is less than 0.2 or greater than 0.8, the secretory expression of the foreign protein may be reduced, or even the target protein may not be efficiently secreted.
The N region is mutated, and the amino acid hydrophobicity of the N region is preferably in the range of 0.25-0.45, so that the transmembrane efficiency of the signal peptide is more efficient, and the secretion expression quantity of the foreign protein is more remarkable.
Drawings
FIG. 1: schematic diagram of signal peptide structure.
FIG. 2: map of target heterologous protein and signal peptide recombinant vector
FIG. 3: the alkaline protease coding gene PCR agarose electrophoresis picture of the embodiment 1 of the invention, wherein Lane 1 is marker, Lane 2 is the alkaline protease coding gene PCR product;
FIG. 4: the alkaline xylanase coding gene enzyme digestion agarose electrophoresis picture of embodiment 2 of the invention, wherein a Lane 1 is an alkaline xylanase coding gene PCR product, and a Lane 2 is a marker;
FIG. 5: the agarose electrophoresis picture of the cutinase coding gene enzyme digestion of the embodiment 3 of the invention, wherein Lane 1 is the PCR product of the cutinase coding gene, and Lane 2 is marker.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The following are the media used in the examples of the invention:
b, preparing a culture medium by bacillus subtilis competence:
SP-A salt solution: (NH)4)2SO4 4g/L，K2HPO4·3H2O 28g/L，KH2PO412g/L, 2g/L sodium citrate and the balance of water;
SP-B salt solution: MgSO (MgSO)4·7H2O0.4 g/L, and the balance of water;
100 × CAYE solution: casein hydrolysate 20g/L, yeast powder 100g/L, and water in balance;
SPI (200 mL): 98mL of SP-A salt solution, 98mL of SP-B salt solution, 2mL of 50% glucose and 100 xCAYE 2 mL;
SPII medium (600 mL): SPI 588mL, 50mmol/L CaCl2 6mL，250mmol/L MgCl2 6mL；
100 × EGTA solution: 10mmol/L EGTA solution.
Example 1: preparation of signal peptide mutant and induction of foreign protein expression
The bacillus clausii-derived alkaline protease is taken as a reporter gene, a bacillus amyloliquefaciens-derived signal peptide is modified to obtain a signal peptide mutant, and the secretion amount of the alkaline protease in a bacillus subtilis host is improved, wherein the structural schematic diagram of the signal peptide refers to the attached figure 1.
(1) Acquisition of target Gene
The target gene is alkaline protease gene aprE (the amino acid sequence of the alkaline protease is shown as SEQ ID NO:1, the nucleotide sequence of the protease gene aprE is shown as SEQ ID NO:12, GenBank is FJ940727.1, the construction reference patent document of the target gene is 201910332253.1 patent of invention of gene engineering bacteria for high-efficiency heterologous expression of alkaline protease and construction method thereof, wherein the acquisition of the alkaline protease gene is from the embodiment 1 of the above document, the construction of the recombinant alkaline protease gene engineering bacteria is from the embodiment 2 of the above document, and the PCR agarose electrophoresis chart of the gene coding for the alkaline protease refers to the attached figure 3.
(2) Preparation of signal peptides and mutants thereof:
and (3) according to the sequencing result of the bacillus amyloliquefaciens genome, obtaining a signal peptide through online signal peptide prediction software SignalP 5.0Server prediction. In the embodiment, the signal peptide is named as signal peptide A, is derived from the signal peptide of Sec pathway in Bacillus amyloliquefaciens genome CGMCC No.11218, and has an amino acid sequence shown as SEQ ID NO. 2, and the mutated signal peptide is named as signal peptide B, and has an amino acid sequence shown as SEQ ID NO. 3. The charge density of the N region of the signal peptide before mutation is 0.42, the hydrophobicity of the H region is 36, and the amino acid at the tail end of the C region is TSA. Because the charge density of the N region of the signal peptide is in accordance with 0.2-0.8, and the hydrophobicity of the H region is in accordance with 0-70, the N region and the H region of the signal peptide are not mutated, only the last three amino acids of the C region of the signal peptide are mutated, the charge density of the N region of the mutated signal peptide is still kept at 0.42, the hydrophobicity of the H region is still kept at 36, and the last three amino acids of the C region are mutated to ASA. The mutated signal peptide meets the requirement of the invention that the last amino acid of the C region of the signal peptide is alanine-any amino acid-alanine.
TABLE 2 Signal peptide mutations
The non-mutated signal peptide and the mutated signal peptide are constructed through primer design, and a target fragment is constructed through an overlapping PCR method with a promoter Ply-2 (the nucleotide sequence is shown in SEQ ID NO: 4 (the promoter is from bacillus subtilis amylase, and the construction method is from invention patent of 201910332240.4, a gene engineering bacterium for producing recombinant alkaline protease and the construction method thereof) and the amplification of the signal peptide is carried out by taking the existing bacillus amyloliquefaciens genome in a laboratory as a template.
Taking the genome of bacillus amyloliquefaciens CGMCC No.11218 as a template, A-F as an upstream primer and A-R as a downstream primer, and carrying out PCR amplification to obtain a signal peptide A, wherein the amino acid of the signal peptide A is shown in SEQ ID NO:2, on the basis of the signal peptide A, performing PCR by taking A-F as an upstream primer and B-RT as a downstream primer, mutating the last three amino acids TSA at the C end of the signal peptide into ASA, simultaneously, the charge density of the N region of the signal peptide is 0.42, the hydrophobicity of the H region is 36, and as the requirements of the charge density of the N region of the signal peptide of 0.2-0.8 and the hydrophobicity of the H region of 0-70 are met, the mutation is not performed, so that a signal peptide mutant is obtained, wherein the amino acids are shown as SEQ ID NO:3, respectively.
The expression alkaline protease P constructed in the embodiment 2 of 201910332240.4ly-2+SPDacB) The plasmid of (1) is a template (which contains promoter P)ly-2) With Ply-2F is an upstream primer and P isly-2R is a downstream primer for PCR amplification of Ply-2-a gene fragment.
Method using overlapping PCR with Ply-2-A and signal peptide A gene as template, Ply-2Taking the-F as an upstream primer and the A-R as a downstream primer to carry out PCR amplification to obtain a promoter Ply-2And signal peptide a. Simultaneously with Ply-2-A and mutated signal peptide B as template, with Ply-2taking-F as an upstream primer and B-RT as a downstream primer, and carrying out PCR amplification to obtain a promoter Ply-2And the mutated signal peptide B. Promoter Ply-2And signal peptide A, B are shown in Table 3 below. In Table 3, the restriction sites are underlined, the lower case letters are homologous fragments, and RT is the downstream primer of the mutant.
TABLE 3 promoter Ply-2Primer sequence and enzyme cutting site for overlapping PCR with signal peptide A
Promoter Ply-2The reaction system used for overlap PCR with signal peptide A was 50. mu.L, as follows:
promoter Ply-2The overlap PCR annealing temperature with signal peptide A was 60 ℃ and the extension time corresponded to the gene length by the following reaction procedure:
promoter fragment Ply-2And the signal peptide gene is amplified and purified by overlapping PCR, and then a product enzyme cutting system is recovered by double enzyme cutting (EcoRI-HindIII), and the product enzyme cutting system is constructed on a pWB980 expression vector containing the alkaline protease gene aprE by double enzyme cutting (EcoRI-HindII) (as shown in figure 2). Transformed Bacillus subtilis WB 600. The obtained recombinant strains are respectively named AY (containing a signal peptide A) and BT (containing a signal peptide mutant B)
(3) Expression of recombinant genetically engineered bacteria:
the Bacillus subtilis WB600 transformation method comprises the following steps:
(1) selecting a newly activated Bacillus subtilis WB600 single colony to be cultured in 5mL LB liquid medium at 37 ℃ and 220r/min overnight;
(2) transferring 100 μ L of culture solution into 5mL SPI culture medium, culturing at 37 deg.C and 220r/min to OD of late logarithmic growth phase6001.2 (about 3-4 h);
(3) putting 200 μ L of culture solution growing to the end of logarithmic phase into 2mL of SPII culture medium, culturing at 37 deg.C and 100r/min for 1.5 h;
(4) adding 20 μ L10 mmol/L EGTA into thallus of the SPII culture medium, culturing at 37 deg.C and 100r/min for 10 min;
(5) adding the ligation product, and culturing at 37 ℃ at 100r/min for 30 min;
(6) regulating the rotating speed to 220r/min, continuously culturing for 1.5h, taking bacterial liquid, coating the bacterial liquid on an LB screening plate containing 100 mu g/mL kanamycin, culturing for 12h at 37 ℃, and screening positive transformants for verification.
Inoculating single colonies of recombinant genetic engineering bacteria AY and BT on a fresh plate into a kanamycin-resistant seed culture medium with a final concentration of 50mg/L respectively, carrying out shake culture at 37 ℃ and 220r/min for 12h, transferring the single colonies into a fermentation culture medium with an inoculation amount of 2%, and carrying out fermentation culture at 37 ℃ and 220r/min for 48 h.
Wherein the seed culture medium comprises the following components: 50mg/L kanamycin, 5g/L yeast powder, 10g/L peptone, 5g/L sodium chloride and the balance of water;
the fermentation medium comprises the following components: 64g/L of corn flour, 40g/L of bean cake powder, 4g/L of disodium hydrogen phosphate, 0.3g/L of monopotassium phosphate, 0.7g/L of high-temperature amylase and the balance of water, and p H is natural.
And (3) determining the enzyme activity of the alkaline protease in the fermentation supernatant of the recombinant genetic engineering bacteria according to a national standard GB/T23527-2009 appendix B Folin phenol method. Through determination, the activity of the recombinant alkaline protease in the fermentation supernatant of the two recombinant bacteria within 48 hours reaches the highest. The enzyme activity of the recombinant alkaline protease is measured, and compared with the enzyme activity of the non-mutated recombinant strain, the activity of the recombinant strain of the signal peptide mutant constructed by the invention for expressing the alkaline protease is improved. The enzyme activity is shown in Table 4:
TABLE 4 comparison of enzyme activities of recombinant bacteria before and after signal peptide mutation
According to the signal peptide mutant constructed by the invention, the tail amino acid of the C region of the signal peptide is mutated into alanine-any amino acid-alanine, so that the secretion amount of heterologous protein can be effectively improved.
Example 2: preparation of signal peptide mutant and induction of foreign protein expression
Alkaline xylanase from bacillus pumilus is taken as a reporter gene, a signal peptide YwjE from bacillus subtilis is modified to obtain a signal peptide mutant, and the secretion of the alkaline xylanase in a bacillus subtilis host is improved, wherein the structural schematic diagram of the signal peptide refers to the attached drawing 1.
(1) Obtaining the target gene alkaline xylanase gene:
the target gene alkaline xylanase gene (alkaline xylanase amino acid sequence shown as SEQ ID NO:5, nucleotide sequence of alkaline xylanase gene shown as SEQ ID NO:13, 687bp, GenBank: KU301789.1) was synthesized by Suzhou Jinweizhi Co., Ltd.
Constructing recombinant alkaline xylanase gene engineering bacteria:
plasmid containing alkaline xylanase gene is subjected to double enzyme digestion by BamHI-SphI, a 687bp band is recovered by cutting glue, and the plasmid is connected to a pWB980 expression vector subjected to double enzyme digestion by Solution I ligase (shown in figure 2) to transform the Bacillus subtilis WB 600. Expression of hosts for subsequent construction of signal peptides reference is made to the following patent documents: the invention of the invention patent with application number 201910332253.1, genetically engineered bacterium for high efficiency heterologous expression of alkaline protease and construction method thereof, wherein the agarose electrophoresis pattern of alkaline xylanase coding gene PCR product refers to FIG. 4.
(2) Preparation of signal peptide and its mutant:
the signal peptide is signal peptide of Bacillus subtilis YwjE gene (GenBank: JQ 302237.1). The amino acid sequence before mutation is shown as SEQ ID NO. 6, the charge density of the N region of the signal peptide is 0.5, the hydrophobicity of the H region of the signal peptide is 72.9, and the tail amino acid of the C region of the signal peptide is GRA. Wherein the hydrophobicity of the H region does not meet the requirement of the invention that the amino acid hydrophobicity of the H region of the signal peptide is between 0 and 70. The requirement of mutating the last amino acid of the C region of the signal peptide into alanine-any amino acid-alanine is not met by the last amino acid of the C region of the signal peptide. The amino acid sequence of the mutated signal peptide is shown as SEQ ID NO. 7, the charge density of the N region of the mutated signal peptide is 0.29, so that the charge density of the amino acid of the N region of the signal peptide is in a preferred range of 0.25-0.45, the hydrophobicity of the H region is 35.7, and the requirement of the invention on the amino acid hydrophobicity of the H region of the signal peptide is between 0-70 is met. The tail amino acid of the C region of the signal peptide is AHA after mutation, which meets the requirement that the tail amino acid of the C region of the signal peptide is alanine-any amino acid-alanine.
TABLE 5 Signal peptide mutations
Signal peptide and P before and after mutationly-2The promoter fragment is synthesized by Suzhou Jinweizhi GmbH, the synthesized band is subjected to double enzyme digestion (EcoRI-HindII), and the band is cut and recovered. The bacillus subtilis WB600 is transformed by connecting Solution I ligase to a pWB980 expression vector containing alkaline xylanase gene which is subjected to double enzyme digestion (EcoRI-HindII). The transformation procedure was as in example 1. The obtained recombinant strains were named YE (containing a signal peptide) and YE-T (containing a signal peptide mutant)
(3) Expression of recombinant genetically engineered bacteria:
single colonies of recombinant genetic engineering bacteria YE and YE-T on a fresh plate are picked and inoculated into an LB culture medium, shaking culture is carried out for 16h at 37 ℃ and 220rpm, seed liquid is obtained, and the seed liquid (OD600 is about 1.0) is prepared by overnight culture. Respectively inoculating the seed liquid into 30mL of liquid xylan fermentation medium in a 250mL triangular flask according to the inoculation amount of 1.0%, performing shake culture at 37 ℃ for 48h, taking the fermentation liquid at 4 ℃ and 12000g, centrifuging for 10min, and taking the supernatant as crude enzyme liquid to perform enzyme activity determination.
Xylan fermentation medium:
10.0g/L of xylan, 5.0g/L of peptone, 0.5g/L of yeast powder and K2HPO4 1.5g/L，MgSO4·7H20.2 g/L of O, 5.0g/L of sodium chloride, 15.0g/L of agar powder and the balance of water, p H is natural.
The enzyme activity determination method comprises the following steps:
and (3) enzyme activity determination: sucking 0.6mL of xylan substrate solution as substrate, placing into 25mL graduated tube with plug, adding appropriate dilution of crude enzyme solution 0.4mL, water bathing at constant temperature for 10min, adding 1.5mL of DNS, and boiling for 5 min. And simultaneously preparing a blank control, namely adding 0.4mL of crude enzyme solution diluted in a proper proportion into a 25mL colorimetric tube, performing inactivation for 10min in a boiling water bath, then respectively adding 0.6mL of 1% sodium carboxymethylcellulose and 1.5mL of 3, 5-dinitrosalicylic acid solution, and uniformly mixing to obtain the blank control. And (5) after the mixed solution is cooled, carrying out color comparison at 540nm, and calculating the enzyme activity according to the absorbance value at A540 nm.
Wherein, the preparation of the xylan substrate solution: weighing 1.0g birchwood xylan, dissolving in 80mL acidic buffer solution (in acetic acid-sodium acetate buffer solution) or alkaline buffer solution (Tris-HCl and glycine-NaOH buffer solution), heating to dissolve, diluting to 100mL, and storing at 4 deg.C for use.
3, 5-dinitrosalicylic acid (DNS) solution composition: 6.3g of 3, 5-dinitrosalicylic acid is dissolved in a small amount of water, 21.0g of NaOH, 182.0g of potassium sodium tartrate, 5.0g of redistilled phenol and 5.0g of sodium sulfite are sequentially added, the volume is kept to 1000mL, and the mixture is stored in a dark place for later use.
Alkaline xylanase activity is defined as: the amount of enzyme required to degrade the substrate per minute to produce 1. mu. mol of reducing sugar is defined as one unit of enzyme activity, expressed in U/mL.
And (3) measuring the enzyme activity of the recombinant alkaline xylanase, wherein the activity of the recombinant strain of the signal peptide mutant constructed by the invention for expressing the alkaline xylanase is improved compared with that of the non-mutant recombinant strain. The enzyme activity is shown in the following table 6:
TABLE 6 comparison of enzyme activities of recombinant bacteria before and after signal peptide mutation
According to the signal peptide mutant constructed by the invention, the H region of the signal peptide is mutated to ensure that the amino acid hydrophobicity of the H region is between 0 and 70, and the amino acid of the C region of the signal peptide is subjected to site-directed mutation to ensure that the tail amino acid of the C region is mutated into alanine-any amino acid-alanine, so that the secretion amount of heterologous protein can be effectively improved.
Comparative example 1: a signal peptide YpuA (GenBank: NC-000964.3) derived from Bacillus subtilis 168 and having an amino acid sequence as shown in SEQ ID NO: as shown at 20, the flow of the gas, the amino acid hydrophobicity of the H region is 40.5, which is between 0 and 70, the last three-position amino acid sequence of the C region is ADA, which is satisfied with alanine-arbitrary amino acid-alanine, but the charge density of the amino acid in the N region is 1, and does not satisfy the range of 0.2-0.8, the signal peptide is expressed by the recombinant gene engineering bacteria of the embodiment 2 of the invention to construct a recombinant strain YY, the alkaline xylanase is used as a target protein for expression, and the enzyme activity is measured, the alkaline xylanase activity is 0U/mL, the signal peptide YpuA can not effectively secrete the alkaline xylanase, therefore, when the H region of the signal peptide does not satisfy the range of 0.2-0.8, the signal peptide cannot effectively secrete the target protein or the expression level is extremely low.
Example 3:
a signal peptide mutant is obtained by modifying a bacillus subtilis-derived signal peptide YjiA by using fusarium solani-derived cutinase as a reporter gene, and the secretion amount of the cutinase in a bacillus subtilis host is improved, wherein the structural schematic diagram of the signal peptide refers to the attached figure 1.
(1) Obtaining the target gene:
the target gene cutinase gene (the amino acid sequence of cutinase is shown as SEQ ID NO:8, the nucleotide sequence of cutinase gene is shown as SEQ ID NO:14, GenBank: M29759.1) was synthesized by Jinweizhi, Suzhou, Inc.
Construction of recombinant cutinase gene engineering bacteria:
the plasmid containing cutinase gene was digested with BamHI-SphI, cut to remove 2770bp band, ligated to the digested pWB980 expression vector with BamHI-SphI by Solution I ligase (see FIG. 2), and transformed into Bacillus subtilis WB 600. Expression of hosts for subsequent construction of signal peptides reference patents: the gene engineering bacterium for efficiently and heterologously expressing alkaline protease and the method for constructing the same, having application No. 201910332253.1, are described in example 2, wherein the agarose electrophoresis image of the gene encoding cutinase is shown in FIG. 5.
(2) Preparation of signal peptide and its mutant:
the signal peptide is signal peptide of Bacillus subtilis YjiA gene (GenBank: JQ 302291.1). The amino acid sequence before the mutation of the signal peptide is shown as SEQ ID NO. 9, the charge density of the N region of the signal peptide is 0.1, the hydrophobicity of the H region of the signal peptide is 38.5, and the tail amino acid of the C region of the signal peptide is VFS. The charge density of the N region of the signal peptide does not meet the requirement of the invention on the charge density of 0.2-0.8 of the amino acid in the N region of the signal peptide, and the tail amino acid in the C region does not meet the requirement of the invention on the mutation of the tail amino acid in the C region of the signal peptide into alanine-any amino acid-alanine. The amino acid sequence of the mutated signal peptide is shown as SEQ ID NO. 10, the charge density of the N region of the mutated signal peptide is 0.67, and the requirement of the invention on the charge density of the amino acid of the N region of the signal peptide is between 0.2 and 0.8 is met. The hydrophobicity of the H region of the signal peptide after mutation is 40.5, the requirement of the invention on the amino acid hydrophobicity of the H region of the signal peptide to be 0-70 is met, and the H region is used as comparison of the amino acid hydrophobicity value of the H region outside the preferred range. The last amino acid in the C region of the signal peptide is ADA after mutation, which meets the requirement of the invention that the last amino acid in the C region of the signal peptide is alanine-any amino acid-alanine. YA-T2 is based on the unchanged YA-T1 signal peptide N area and C area, the hydrophobicity of H area is mutated, the amino acid hydrophobicity value of H area is mutated from 40.5 to 35.8, the amino acid hydrophobicity of H area is in the preferred range 0-38, the amino acid sequence after signal peptide mutation is shown in SEQ ID NO. 11.
TABLE 7 Signal peptide mutations
Signal peptide and P before and after mutationly-2The promoter fragment is synthesized by Suzhou Jinweizhi GmbH, the synthesized band is subjected to double enzyme digestion (EcoRI-HindIII), and the gel is cut and recovered. The bacillus subtilis WB600 is transformed by connecting Solution I ligase to a pWB980 expression vector containing cutinase gene which is subjected to double enzyme digestion (EcoRI-HindIII). The transformation procedure was as in example 1. The obtained recombinant strains are respectively named as YA (containing a proto-signal peptide) and YA-T (containing a signal peptide mutant)
(3) Expression of recombinant genetically engineered bacteria:
the single colonies of the recombinant gene engineering bacteria YjiA and YjiA-T on a fresh plate are respectively inoculated into a seed culture medium, 50mL of the culture medium is bottled in 500mL of triangular bottles, 200r/min and 37 ℃ for 12 h. Then inoculating the seeds into a fermentation culture medium at an inoculation amount of 5% (v/v), and culturing for 22h at the temperature of 37 ℃ at 200 r/min. The liquid loading amount is 25mL in a 250mL triangular flask, unless otherwise specified.
The seed culture medium comprises the following components: 10g/L of glucose, 12g/L of peptone, 24g/L of yeast extract, 12.54g/L of disodium hydrogen phosphate, 2.31g/L of potassium dihydrogen phosphate and the balance of water, p H7.5.5.
The fermentation medium comprises the following components: 10g/L of glucose, 12g/L of peptone, 24g/L of yeast extract, 12.54g/L of disodium hydrogen phosphate, 2.31g/L of potassium dihydrogen phosphate and the balance of water, p H7.5.5.
Preparing a crude cutinase enzyme solution:
centrifuging a certain amount of fermentation liquor at 10000r/min for 5min, and taking supernate as cutinase enzyme activity determination liquid.
Measurement of the cutinase Activity:
spectrophotometry is used, and reference is made to non-patent literature: MaartenR, Egmond JDV Fusarium solanipisciutanase [ J ]. Biochimie,2000,82: 67-69. The volume of the reaction solution was 1mL, and the reaction solution contained 20. mu.L of the enzyme solution and 980. mu.L of 50mmol/L sodium deoxycholate buffer solution (pH 8.0, containing 50mmol/L p-nitrobenzyl butyrate). The rate of para-nitrophenol formation was recorded at 405nm at 20 ℃.
Definition of enzyme activity:
at 20 ℃, the enzyme amount for catalyzing the hydrolysis of the p-nitrobenzoate to generate 1 mu mol of p-nitrophenol per minute is an enzyme activity unit.
The enzyme activity of the recombinant cutinase is measured, and compared with the enzyme activity of the non-mutated recombinant strain, the activity of the recombinant strain of the signal peptide mutant constructed by the invention for expressing cutinase is improved compared with the activity of the non-mutated recombinant strain. The enzyme activity is shown in the following table 6:
TABLE 8 comparison of enzyme activities of recombinant bacteria before and after signal peptide mutation
According to the signal peptide mutant constructed by the invention, the N region of the signal peptide is mutated to ensure that the charge density of the amino acid in the N region is 0.2-0.8, and the amino acid in the C region of the signal peptide is subjected to site-directed mutation to ensure that the tail amino acid in the C region is mutated into alanine-any amino acid-alanine, so that the secretion amount of heterologous protein can be effectively improved.
Compared with the initial signal peptide, the cutinase enzyme activity produced by the recombinant strain of the mutant signal peptide 1 and the mutant signal peptide 2 is obviously improved.
Compared with the mutated signal peptide 1, on the basis that the N region (the charge density is 0.67) and the C region are not changed (ADA) of the mutated signal peptide 2, the hydrophobicity value of the H region is mutated from 40.5 to 35.8 after mutation, so that the amino acid hydrophobicity of the H region is in the preferred range of 0-38, and the enzyme activity of the cutinase is improved after mutation.
It should be noted that the signal peptide YjiA has a hydrophobicity in the H region of 38.5, and differs from 38 in the range of values from 0 to 38 in the preferred range of the hydrophobicity of the signal peptide H region by only 0.5, but the signal peptide YjiA has a significant difference in both the amino acid type and the amino acid number between 38.5 in the hydrophobicity of the H region of the signal peptide YjiA and 38 in the preferred range because the hydrophobicity of the H region is obtained by adding the hydrophobicity values of a plurality of amino acids and the number of amino acids in the hydrophobic region of the signal peptide YjiA (i.e., the amino acid sequence is VVGILLSLAFVLF) is large.
Comparative example 2: a signal peptide YwqC (GenBank: Z92952.1) derived from Bacillus subtilis 168 and having an amino acid sequence as shown in SEQ ID NO: as shown at 21, the first and second side walls of the chamber, the amino acid hydrophobicity of the H region is 31.6, which is between 0 and 70, the last three-position amino acid sequence of the C region is ATA, which is satisfied alanine-arbitrary amino acid-alanine, but the charge density of the amino acid in the N region is-0.1, but does not satisfy the range of 0.2-0.8, the signal peptide is expressed by the recombinant gene engineering bacteria of the embodiment 3 of the invention to construct a recombinant strain YC, the cutinase enzyme activity is taken as the target protein for expression, and the enzyme activity is measured, the enzyme activity of the cutinase is 0U/mL, the signal peptide YwqC can not effectively secrete the cutinase, therefore, when the H region of the signal peptide does not satisfy the range of 0.2-0.8, the signal peptide cannot effectively secrete the target protein or the expression level is extremely low.
SEQUENCE LISTING
<110> Tianjin science and technology university
SHANDONG LONCT ENZYMES Co.,Ltd.
<120> a signal peptide mutant and use thereof
<130> 1
<160> 21
<170> PatentIn version 3.5
<210> 1
<211> 380
<212> PRT
<213> Bacillus clausii
<400> 1
Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile
1 5 10 15
Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Glu Glu Ala Lys
20 25 30
Glu Lys Tyr Leu Ile Gly Phe Asn Glu Gln Glu Ala Val Ser Glu Phe
35 40 45
Val Glu Gln Val Glu Ala Asn Asp Glu Val Ala Ile Leu Ser Glu Glu
50 55 60
Glu Glu Val Glu Ile Glu Leu Leu His Glu Phe Glu Thr Ile Pro Val
65 70 75 80
Leu Ser Val Glu Leu Ser Pro Glu Asp Val Asp Ala Leu Glu Leu Asp
85 90 95
Pro Ala Ile Ser Tyr Ile Glu Glu Asp Ala Glu Val Thr Thr Met Ala
100 105 110
Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His
115 120 125
Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp Thr
130 135 140
Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe
145 150 155 160
Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr His
165 170 175
Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly
180 185 190
Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala Ser
195 200 205
Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly
210 215 220
Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro
225 230 235 240
Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly Val
245 250 255
Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser Tyr
260 265 270
Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln Asn
275 280 285
Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val
290 295 300
Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala
305 310 315 320
Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala Ala
325 330 335
Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg
340 345 350
Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr
355 360 365
Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
370 375 380
<210> 2
<211> 31
<212> PRT
<213> Bacillus amyloliquefaciens
<400> 2
Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met
1 5 10 15
Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala
20 25 30
<210> 3
<211> 31
<212> PRT
<213> Artificial sequence
<400> 3
Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met
1 5 10 15
Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Ala Ser Ala
20 25 30
<210> 4
<211> 596
<212> DNA
<213> Bacillus subtilis
<400> 4
cattatgttt gaatttccgt ttaaagaatg ggctgcaagc cttgtgtttt tgttcatcat 60
tatcttatat tactgcatca gggctgcggc atccggaatg ctcatgccga gaatagacac 120
caaagaagaa ctgcaaaaac gggtgaagca gcagcgaata gaatcaattg cggtcgcctt 180
tgcggtagtg gtgcttacga tgtacgacag ggggattccc catacattct tcgcttggct 240
gaaaatgatt cttcttttta tcgtctgcgg cggcgttctg tttctgcttc ggtatgtgat 300
tgtgaagctg gcttacagaa gagcggtaaa agaagaaata aaaaagaaat catctttttt 360
gtttggaaag cgagggaagc gttcacagtt tcgggcagct ttttttatag gaacattgat 420
ttgtattcac tctgccaagt tgttttgata gagtgattgt gataatttta aatgtaagcg 480
ttaacaaaat tctccagtct tcacatcggt ttgaaaggag gaagcggaag aatgaagtaa 540
gagggatttt tgactccgaa gtaagtcttc aaaaaatcaa ataaggagtg tcaaga 596
<210> 5
<211> 228
<212> PRT
<213> Bacillus pumilus
<400> 5
Met Asn Leu Arg Lys Leu Arg Leu Leu Phe Val Met Cys Ile Gly Leu
1 5 10 15
Thr Leu Ile Leu Thr Ala Val Pro Ala His Ala Arg Thr Ile Thr Asn
20 25 30
Asn Glu Met Gly Asn His Ser Gly Tyr Asp Tyr Glu Leu Trp Lys Asp
35 40 45
Tyr Gly Asn Thr Ser Met Thr Leu Asn Asn Gly Gly Ala Phe Ser Ala
50 55 60
Gly Trp Asn Asn Ile Gly Asn Ala Leu Phe Arg Lys Gly Lys Lys Phe
65 70 75 80
Asp Ser Thr Arg Thr His His Gln Leu Gly Asn Ile Ser Ile Asn Tyr
85 90 95
Asn Ala Ser Phe Asn Pro Gly Gly Asn Ser Tyr Leu Cys Val Tyr Gly
100 105 110
Trp Thr Gln Ser Pro Leu Ala Glu Tyr Tyr Ile Val Asp Ser Trp Gly
115 120 125
Thr Tyr Arg Pro Thr Gly Ala Tyr Lys Gly Ser Phe Tyr Ala Asp Gly
130 135 140
Gly Thr Tyr Asp Ile Tyr Glu Thr Thr Arg Val Asn Gln Pro Ser Ile
145 150 155 160
Ile Gly Ile Ala Thr Phe Lys Gln Tyr Trp Ser Val Arg Gln Thr Lys
165 170 175
Arg Thr Ser Gly Thr Val Ser Val Ser Ala His Phe Arg Lys Trp Glu
180 185 190
Ser Leu Gly Met Pro Ile Gly Lys Met Tyr Glu Thr Ala Phe Thr Val
195 200 205
Glu Gly Tyr Gln Ser Ser Gly Ser Ala Asn Val Met Thr Asn Gln Leu
210 215 220
Phe Ile Gly Asn
225
<210> 6
<211> 27
<212> PRT
<213> Bacillus subtilis
<400> 6
Met Lys Val Phe Ile Val Ile Met Ile Ile Val Val Ile Phe Phe Ala
1 5 10 15
Leu Ile Leu Leu Asp Ile Phe Met Gly Arg Ala
20 25
<210> 7
<211> 27
<212> PRT
<213> Artificial sequence
<400> 7
Met Lys Asn Met Ser Cys Lys Leu Val Val Ser Val Thr Leu Phe Phe
1 5 10 15
Ser Phe Leu Thr Ile Gly Pro Leu Ala His Ala
20 25
<210> 8
<211> 230
<212> PRT
<213> Fusarium solani
<400> 8
Met Lys Phe Phe Ala Leu Thr Thr Leu Leu Ala Ala Thr Ala Ser Ala
1 5 10 15
Leu Pro Thr Ser Asn Pro Ala Gln Glu Leu Glu Ala Arg Gln Leu Gly
20 25 30
Arg Thr Thr Arg Asp Asp Leu Ile Asn Gly Asn Ser Ala Ser Cys Arg
35 40 45
Asp Val Ile Phe Ile Tyr Ala Arg Gly Ser Thr Glu Thr Gly Asn Leu
50 55 60
Gly Thr Leu Gly Pro Ser Ile Ala Ser Asn Leu Glu Ser Ala Phe Gly
65 70 75 80
Lys Asp Gly Val Trp Ile Gln Gly Val Gly Gly Ala Tyr Arg Ala Thr
85 90 95
Leu Gly Asp Asn Ala Leu Pro Arg Gly Thr Ser Ser Ala Ala Ile Arg
100 105 110
Glu Met Leu Gly Leu Phe Gln Gln Ala Asn Thr Lys Cys Pro Asp Ala
115 120 125
Thr Leu Ile Ala Gly Gly Tyr Ser Gln Gly Ala Ala Leu Ala Ala Ala
130 135 140
Ser Ile Glu Asp Leu Asp Ser Ala Ile Arg Asp Lys Ile Ala Gly Thr
145 150 155 160
Val Leu Phe Gly Tyr Thr Lys Asn Leu Gln Asn Arg Gly Arg Ile Pro
165 170 175
Asn Tyr Pro Ala Asp Arg Thr Lys Val Phe Cys Asn Thr Gly Asp Leu
180 185 190
Val Cys Thr Gly Ser Leu Ile Val Ala Ala Pro His Leu Ala Tyr Gly
195 200 205
Pro Asp Ala Arg Gly Pro Ala Pro Glu Phe Leu Ile Glu Lys Val Arg
210 215 220
Ala Val Arg Gly Ser Ala
225 230
<210> 9
<211> 26
<212> PRT
<213> Bacillus subtilis
<400> 9
Met Ala Ala Gln Thr Asp Tyr Lys Lys Gln Val Val Gly Ile Leu Leu
1 5 10 15
Ser Leu Ala Phe Val Leu Phe Val Phe Ser
20 25
<210> 10
<211> 26
<212> PRT
<213> Artificial sequence
<400> 10
Met Lys Lys Ile Trp Ile Gly Met Leu Ala Ala Ala Val Leu Leu Leu
1 5 10 15
Met Val Pro Lys Val Ser Leu Ala Asp Ala
20 25
<210> 11
<211> 26
<212> PRT
<213> Artificial sequence
<400> 11
Met Lys Lys Ala Phe Ile Leu Ser Ala Ala Ala Ala Val Gly Leu Phe
1 5 10 15
Thr Phe Gly Gly Val Gln Gln Ala Asp Ala
20 25
<210> 12
<211> 1171
<212> DNA
<213> Bacillus clausii
<400> 12
atgaggaggg aaccgaatga agaaaccgtt ggggaaaatt gtcgcaagca ccgcactact 60
catttctgtt gcttttagtt catcgatcgc atcggctgct gaagaagcaa aagaaaaata 120
tttaattggc tttaatgagc aggaagctgt cagtgagttt gtagaacaag tagaggcaaa 180
tgacgaggtc gccattctct ctgaggaaga ggaagtcgaa attgaattgc ttcatgaatt 240
tgaaacgatt cctgttttat ccgttgagtt aagcccagaa gatgtggacg cgcttgaact 300
cgatccagcg atttcttata ttgaagagga tgcagaagta acgacaatgg cgcaatcagt 360
gccatgggga attagccgtg tgcaagcccc agctgcccat aaccgtggat tgacaggttc 420
tggtgtaaaa gttgctgtcc tcgatacagg tatttccact catccagact taaatattcg 480
tggtggcgct agctttgtac caggggaacc atccactcaa gatgggaatg ggcatggcac 540
acatgtggcc gggacgattg ctgctttaaa caattcgatt ggcgttcttg gcgtagcgcc 600
gagcgcggaa ctatacgctg ttaaagtatt aggggcgagc ggttcaggtt cggtcagctc 660
gattgcccaa ggattggaat gggcagggaa caatggcatg cacgttgcta atttgagttt 720
aggaagccct tcgccaagtg ccacacttga gcaagctgtt aatagcgcga cttctagagg 780
cgttcttgtt gtagcggcat ctgggaattc aggtgcaggc tcaatcagct atccggcccg 840
ttatgcgaac gcaatggcag tcggagctac tgaccaaaac aacaaccgcg ccagcttttc 900
acagtatggc gcagggcttg acattgtcgc accaggtgta aacgtgcaga gcacataccc 960
aggttcaacg tatgccagct taaacggtac atcgatggct actcctcatg ttgcaggtgc 1020
agcagccctt gttaaacaaa agaacccatc ttggtccaat gtacaaatcc gcaatcatct 1080
aaagaatacg gcaacgagct taggaagcac gaacttgtat ggaagcggac ttgtcaatgc 1140
agaagcggca acacgctaat caataataaa a 1171
<210> 13
<211> 687
<212> DNA
<213> Bacillus pumilus
<400> 13
atgaatttga gaaaattaag actgttgttt gtgatgtgta ttggactgac gcttatactg 60
acggctgtac cagcccatgc gagaaccatt acgaataatg aaatgggtaa ccatagcggg 120
tacgattatg aattatggaa ggattatgga aatacctcga tgacactcaa taacggcggg 180
gcatttagtg caggctggaa caatatcgga aatgctttat ttagaaaagg gaaaaagttt 240
gattccacta gaactcacca tcagcttggc aacatctcca tcaattacaa cgcaagtttt 300
aacccaggcg ggaattccta tctatgtgtc tatggctgga cacaatctcc attagcagaa 360
tactacattg ttgattcatg gggcacgtat cgtccaacag gagcgtataa aggatcattt 420
tatgctgatg gaggcacata tgacatttat gaaacaaccc gtgtcaatca gccttccatt 480
atcgggatcg caaccttcaa gcaatattgg agtgtacgtc aaacgaaacg tacaagcgga 540
acggtctccg tcagtgcgca ttttagaaaa tgggaaagct tagggatgcc aatagggaaa 600
atgtatgaaa cggcatttac tgtagaaggc taccaaagca gcggaagtgc aaatgtgatg 660
accaatcagc tgtttattgg caactaa 687
<210> 14
<211> 693
<212> DNA
<213> Fusarium solani
<400> 14
atgaaattct tcgctctcac cacacttctc gccgccacgg cttcggctct gcctacttct 60
aaccctgccc aggagcttga ggcgcgccag cttggtagaa caactcgcga cgatctgatc 120
aacggcaata gcgcttcctg cgccgatgtc atcttcattt atgcccgagg ttcaacagag 180
acgggcaact tgggaactct cggtcctagc attgcctcca accttgagtc cgccttcggc 240
aaggacggtg tctggattca gggcgttggc ggtgcctacg cagccactct tggagacaat 300
gctctccctc gcggaacctc tagcgccgca atcagggaga tgctcggtct cttccagcag 360
gccaacacca agtgccctga cgcgactttg atcgccggtg gctacagcca gggtgctgca 420
cttgcagccg cctccatcga ggacctcgac tcggccattc gtgacaagat cgccggaact 480
gttctgttcg gctacaccaa gaacctacag aaccgtggcc gaatccccaa ctaccctgcc 540
gacaggacca aggtcttctg caatacaggg gatctcgttt gtactggtag cttgatcgtt 600
gctgcacctc acttggctta tggtcctgat gctcgtggcc ctgcccctga gttcctcatc 660
gagaaggttc gggctgtccg tggttctgct tga 693
<210> 15
<211> 41
<212> DNA
<213> Artificial sequence
<400> 15
ccggaattcc attatgtttg aatttccgtt taaagaatgg g 41
<210> 16
<211> 35
<212> DNA
<213> Artificial sequence
<400> 16
cgctttcgtt tttgaatcat tcttgacact cctta 35
<210> 17
<211> 35
<212> DNA
<213> Artificial sequence
<400> 17
taaggagtgt caagaatgat tcaaaaacga aagcg 35
<210> 18
<211> 33
<212> DNA
<213> Artificial sequence
<400> 18
cgcggatccg gctgatgttt ttgtaatcgg caa 33
<210> 19
<211> 33
<212> DNA
<213> Artificial sequence
<400> 19
cgcggatccg gctgatgctt ttgtaatcgg caa 33
<210> 20
<211> 25
<212> PRT
<213> Bacillus subtilis 168
<400> 20
Lys Lys Ile Trp Ile Gly Met Leu Ala Ala Ala Val Leu Leu Leu Met
1 5 10 15
Val Pro Lys Val Ser Leu Ala Asp Ala
20 25
<210> 21
<211> 30
<212> PRT
<213> Bacillus subtilis 168
<400> 21
Met Gly Glu Ser Thr Ser Leu Lys Glu Ile Leu Ser Thr Leu Thr Lys
1 5 10 15
Arg Ile Leu Leu Ile Met Ile Val Thr Ala Ala Ala Thr Ala
20 25 30
Claims (8)
1. A mutant signal peptide for increasing the secretion of a heterologous protein, which comprises: the amino acid sequence of the signal peptide mutant is shown as SEQ ID NO: shown at 10.
2. A gene encoding the signal peptide mutant according to claim 1.
3. Use of the signal peptide mutant of claim 1 for increasing the secretory expression of a heterologous protein.
4. The use according to claim 3, wherein the heterologous protein is cutinase having the amino acid sequence shown in SEQ ID NO 8.
5. A recombinant vector characterized by: comprising the gene of claim 2.
6. A host cell, characterized in that: a recombinant vector comprising the gene according to claim 2 or the recombinant vector according to claim 5.
7. A method for producing a heterologous protein, comprising: the method comprises the following steps:
activating the gene engineering bacteria containing the coding gene of the signal peptide mutant and the coding gene of the heterologous protein in the claim 1, transferring the activated gene engineering bacteria to a fermentation culture medium in an inoculation amount of 1.5-5%, and performing fermentation culture at 34-40 ℃ and 215-225r/min for 20-50 h; the fermentation medium comprises the following components: 60-70g/L of corn flour, 30-50g/L of bean cake powder, 2-8g/L of disodium hydrogen phosphate, 0.1-0.6g/L of monopotassium phosphate, 0.5-1.0g/L of high-temperature amylase and the balance of water;
or the fermentation medium consists of: 8-12g/L of xylan, 2-8g/L of peptone, 1-1.5g/L of yeast powder and K2HPO41-3g/L，MgSO4·7H20.1-0.4g/L of O, 2-8g/L of sodium chloride, 10-20g/L of agar powder and the balance of water;
or the fermentation medium consists of: 6-12g/L of glucose, 8-16g/L of peptone, 20-30g/L of yeast extract, 8-14g/L of disodium hydrogen phosphate, 1-5g/L of potassium dihydrogen phosphate and the balance of water, p H7.0.0-7.5.
8. The production method according to claim 7, wherein: the heterologous protein is cutinase, and the amino acid sequence of the heterologous protein is shown as SEQ ID NO. 8; the fermentation medium comprises the following components: 10g/L of glucose, 12g/L of peptone, 24g/L of yeast extract, 12.54g/L of disodium hydrogen phosphate, 2.31g/L of potassium dihydrogen phosphate and the balance of water, p H7.5.5.
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| CN202011580810.0A CN112552382A (en) | 2020-01-22 | 2020-01-22 | Signal peptide mutant and application thereof |
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| CN202010074786.7A CN111205354B (en) | 2020-01-22 | 2020-01-22 | Signal peptide mutant for improving secretion of heterologous protein and construction method and application thereof |
| CN202011580810.0A CN112552382A (en) | 2020-01-22 | 2020-01-22 | Signal peptide mutant and application thereof |
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| CN202011580792.6A Active CN112521466B (en) | 2020-01-22 | 2020-01-22 | Signal peptide mutant for improving secretion of heterologous protein and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957071A (en) * | 2021-09-30 | 2022-01-21 | 华南理工大学 | Combined DNA fragment with double promoters and double secretion signal functions and application thereof |
| CN115125248A (en) * | 2022-06-14 | 2022-09-30 | 天津科技大学 | Combined promoter pctsR-alpha 2 and application thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153968B (en) * | 2020-01-22 | 2022-04-08 | 天津科技大学 | Signal peptide mutant for improving expression quantity of exogenous alkaline protease and construction method and application thereof |
| CN111205354B (en) * | 2020-01-22 | 2021-01-22 | 天津科技大学 | Signal peptide mutant for improving secretion of heterologous protein and construction method and application thereof |
| CN114395577A (en) * | 2022-01-06 | 2022-04-26 | 上海应用技术大学 | Preparation method of genetically engineered bacterium, genetically engineered bacterium and recombinant chitosanase |
| CN115125246B (en) * | 2022-06-14 | 2024-02-06 | 天津科技大学 | Promoter mutant P alpha-rapA and application thereof |
| CN115851679A (en) * | 2022-10-09 | 2023-03-28 | 天津科技大学 | Low-temperature high-activity alkaline protease mutant and application thereof |
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| CN107022005B (en) * | 2017-05-18 | 2020-02-14 | 江南大学 | Signal peptide mutant for improving protein secretion efficiency and application thereof |
| CN111205354B (en) * | 2020-01-22 | 2021-01-22 | 天津科技大学 | Signal peptide mutant for improving secretion of heterologous protein and construction method and application thereof |
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- 2020-01-22 CN CN202010074786.7A patent/CN111205354B/en active Active
- 2020-01-22 CN CN202011580792.6A patent/CN112521466B/en active Active
- 2020-01-22 CN CN202011580810.0A patent/CN112552382A/en not_active Withdrawn
- 2020-12-29 WO PCT/CN2020/140530 patent/WO2021147623A1/en active Application Filing
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957071A (en) * | 2021-09-30 | 2022-01-21 | 华南理工大学 | Combined DNA fragment with double promoters and double secretion signal functions and application thereof |
| CN113957071B (en) * | 2021-09-30 | 2023-07-18 | 华南理工大学 | Combined DNA fragment with double promoter and double secretion signal functions and application thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
| CN115125248A (en) * | 2022-06-14 | 2022-09-30 | 天津科技大学 | Combined promoter pctsR-alpha 2 and application thereof |
| CN115125248B (en) * | 2022-06-14 | 2024-02-06 | 天津科技大学 | Combined promoter pctsR-alpha 2 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111205354B (en) | 2021-01-22 |
| CN112521466A (en) | 2021-03-19 |
| CN112521466B (en) | 2022-06-17 |
| WO2021147623A1 (en) | 2021-07-29 |
| CN111205354A (en) | 2020-05-29 |
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