CN113736818A

CN113736818A - Method for improving secretion efficiency and enzyme activity of human lysozyme in pichia pastoris

Info

Publication number: CN113736818A
Application number: CN202111179096.9A
Authority: CN
Inventors: 高晓冬; 李子杰; 王亚森; 许向阳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-10-08
Filing date: 2021-10-08
Publication date: 2021-12-03

Abstract

The invention discloses a method for improving the secretion efficiency and enzymatic activity of human lysozyme in Pichia pastoris. Gene knockout, chaperone co-expression, optimization of fermentation conditions, etc. The technical solution provided by the present invention has a very high effect of improving the secretion efficiency and enzymatic activity of human lysozyme.

Description

Method for improving secretion efficiency and enzyme activity of human lysozyme in pichia pastoris

Technical Field

The invention relates to the technical field of protein engineering or gene engineering, in particular to a method for improving secretion efficiency and enzyme activity of human lysozyme in pichia pastoris.

Background

Lysozyme (lysozyme) has attracted much attention as a novel antimicrobial peptide due to its characteristics of antimicrobial property, antiviral property, immunity enhancement and the like. Unlike traditional antibiotics, lysozyme can hydrolyze beta-1, 4 glycosidic bonds of bacterial cell walls, destroy the structure of the cell walls to dissolve and kill the bacteria, so that the drug resistance problem is not worried about. The lysozyme has good effect of killing microorganisms, is widely applied to the aspects of animal husbandry, food industry, clinical medicine and the like, and also draws the attention of world health organization and food and agricultural organization.

Lysozyme, also known as muramidase (muramidase) or N-acetylmuramidase (N-acetylmuramidase glycohydrolase), is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria. The lysozyme can be divided into animal lysozyme, plant lysozyme and microbial lysozyme, wherein the animal lysozyme is divided into chicken type lysozyme (c type lysozyme), goose type lysozyme (g type lysozyme) and invertebrate type lysozyme. The human lysozyme belongs to the type c lysozyme, consists of 130 amino acids, and has the relative molecular weight of 14700 Da. Compared with other types of lysozyme, the human lysozyme has the advantages of high enzyme activity, high thermal stability and the like, and is human endogenous protein. And due to the reasons of raw material sources, purification cost and the like, the preparation amount of the human lysozyme can not meet the requirements of various fields. At present, various modes for producing the human lysozyme by using a eukaryotic host or a prokaryotic host exist, but the enzyme activity and the yield are in an unsatisfactory state.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The invention is provided in view of the above and/or the problems existing in the existing method for improving the secretion efficiency and enzyme activity of the human lysozyme.

Therefore, one of the purposes of the invention is to overcome the defects of the existing method for improving the secretion efficiency and the enzyme activity of the human lysozyme and provide a method for improving the secretion efficiency and the enzyme activity of the human lysozyme in pichia pastoris.

To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a method for improving secretion efficiency and enzyme activity of human lysozyme in pichia pastoris comprises the following steps:

optimizing a base sequence: artificially synthesizing and constructing a base sequence of a codon preference optimization code of pichia pastoris into a plasmid to obtain a recombinant plasmid;

optimization of promoters and optimization of fermentation conditions: replacing an inducible promoter in the recombinant plasmid with a constitutive promoter, and performing production intensity comparison and fermentation condition optimization on the two promoters;

optimizing a signal peptide: optimizing a secretion signal peptide of hLYZ, and verifying the optimized result of the signal peptide;

knocking out genes: knocking out one or two genes of VPS10-1 and VPS10-2 to form a single-knock-out strain or a double-knock-out strain;

co-expression of chaperonin: the bacterial strain with the gene knocked out is subjected to chaperonin co-expression, and whether the bacterial strain can be stable or not is verified, and the enzyme activity is improved;

and (3) horizontally optimizing a fermentation tank: the optimized strain is subjected to 5L fermentation tank fermentation condition discussion, and the optimal fermentation condition is explored.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, in the optimized base sequence, the base sequence of the codon preference optimized code of the pichia pastoris is hLYZ.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, in the optimized base sequence, the plasmid is pPIC 9K.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, the optimized base sequence comprises one or more of an inducible promoter and a constitutive promoter, and the promoter comprises P_AOX1、P_GAPOne or two of them.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, restriction endonucleases used in the optimized base sequence are EcoRI and Not I.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, the invention optimizes a promoter and optimizes an inducible promoter P in fermentation conditions_AOX1The optimal temperature for the fermentation production of hLYZ is 26 ℃, and the optimal pH is 6.0; constitutive promoter P_GAPThe optimum temperature for fermentation production of hLYZ is 28 deg.C, and the optimum pH is 6.0.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, in the optimization of the signal peptide, the replaced signal peptide is alpha signal peptide, and the types of the signal peptide for replacement comprise one of HFBI signal sequences (Hss), INUIA signal sequences (Iss), W1 signal sequences (Wss), Killer signal sequences (Ks), Invertase signal sequences (Inss), hLYZ signal sequences (Hss), Albumin signal sequences (Alss), Lipase signal sequences (Lss), Apre signal sequences (Apss) and Glucoamyase signal sequences (Gs).

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, a primer used for replacing a signal peptide is F, R in signal peptide optimization, and F1 and R1 comprise one or more than one of Iss-F, Iss-R, Hss-F, Hss-R, Inss-F, Inss-R, Kss-F, Kss-R, Lss-F, Lss-R, Alss-F, Alss-R, Gss-F, Gss-R, hss-F, hss-R, Wss-F, Wss-R, Apss-F, Apss-R.

As a preferable scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, in the co-expression chaperone protein, the co-expressed chaperone protein is one or more of Sso2, Ero1, Pdi1, Kex2, Aft1, Hac1, Hrd1, Bmh2, Ydj1 and Cne 1.

As a preferred scheme in the method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris, the restriction endonucleases used in the coexpression chaperonin are EcoR I and Sal I or Xho I and Not I.

The invention provides a human lysozyme expression and secretion strain which is inserted with a plasmid containing gene preference codes and is combined with the optimization of a promoter, a signal peptide and chaperonin to obtain a good effect on secretion efficiency and enzyme activity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 shows that the promoter in example 1 is P_AOX1SDS-PAGE and Western blot patterns of hLYZ expression;

FIG. 2 example 1 where the promoter is P_AOX1Expression levels of the strains at different temperatures, pH, and fermentation times;

FIG. 3 shows the case of example 2 where P is used as the promoter_GAPAn SDS-PAGE picture and a Western blot picture are obtained;

FIG. 4 shows the case of example 2 where P is used as the promoter_GAPThe expression levels of the strains at different temperatures, pH and fermentation times thereafter;

FIG. 5 is a SDS-PAGE pattern of the strains after insertion of different signal peptides in example 3;

FIG. 6 shows the level of enzyme activity of the strain obtained in example 3 after insertion of different signal peptides;

FIG. 7 is an SDS-PAGE pattern of strains after single-or double-knock-out in example 4, wherein the four lanes contain no-knock-out, VPS10-1 knock-out, VPS10-2 knock-out, and VPS10-1/2 double-knock-out;

FIG. 8 shows the level of enzyme activity of single-or double-knockout strains in example 4;

FIG. 9 is an SDS-PAGE pattern of the strains after co-expression of different chaperones in example 5;

FIG. 10 shows the enzyme activity levels of the strains after co-expression of different chaperones in example 5;

FIG. 11 is the data relating to the fermentation process in example 6, wherein a is a schematic representation of the fermentation process and b is at OD₆₀₀Feeding methanol at 200, 300, 400 and at OD₆₀₀Co-adding sorbitol and methanol to induce expression when the value is 300, and c is different fermentation processes or different OD₆₀₀The value is the protein expression, d is OD₆₀₀300 f fermentation profile.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1

Designing a base sequence of a pichia pastoris codon preference optimization code (hLYZ), artificially synthesizing the base sequence, inserting the base sequence into a pichia pastoris methanol induced secretion type plasmid pPIC9K, inserting the hLYZ into the pichia pastoris methanol induced secretion type plasmid pPIC9K by using a double enzyme digestion technology, wherein endonucleases used in double enzyme digestion are EcoRI and Not I, and constructing the plasmid pPIC9K-hLYZ after the double enzyme digestion is finished.

The constructed plasmid pPIC9K-hLYZ was subjected to promoter substitution using the endonucleases Sac I and BamH I, and constitutive promoter P was used_GAPSubstitution of the inducible promoter P in pPIC9K-hLYZ_AOX1In which P is_AOX1The sequence of (A) is shown in Seq ID No.1，P_GAPThe sequence of (a) is shown as Seq ID No.2, the sequence of human lysozyme is shown as Seq ID No.3, and the recombinant plasmid pPIC9K-P is obtained_GAP-hLYZ。

The plasmid pPIC9K-P was isolated using Sal I enzyme_GAPAfter linearization of hLYZ, the hLYZ gene was inserted into the genome of Pichia pastoris GS115 by electrotransformation with a set voltage of 1500V and a resistance of 200 Ω, and 1ml of 1mol L was added rapidly^-1The D-sorbitol is pre-cultured in a shaker at 30 ℃ for 2h at 220rpm, and then spread on MD plates, and YPD plates with high concentration G418 of 3mg/ml are used for screening out high copy strains as the starting strain GS 115/hLYZ.

The specific operation of introducing the plasmid into the strain to realize the inclusion of the target plasmid in the strain comprises the following steps: linearizing pGAPZA type plasmid by using restriction endonuclease BlnI, uniformly mixing the purified linear plasmid and competent Pichia pastoris in an electric rotating cup, standing on ice for 15min, inserting the linear plasmid into Pichia pastoris genome through electric transformation (1500V and 250 omega), and quickly adding 1 mol. L^-1D-sorbitol (Pre-cooled), pre-incubated at 30 ℃ for 2h, applied to a medium containing 100. mu.g.mL^-1Bleomycin was grown on YPD plates for 3 days.

The products cultured at the shake flask level were verified using SDS-PAGE and Western blot, and the specific conditions for shake flask culture were as follows: culturing the knockout strain and the original strain, culturing in YPD medium for 12h, transferring to BMGY medium, and culturing for 24h to OD ₆₀₀15, resuspend to BMGY and add 0.5% methanol every 12 h. The results of SDS-PAGE and Western blot are shown in FIGS. 1 and 3.

As can be seen from FIGS. 1 and 3, the strain GS115/hLYZ constructed in my invention carries the target gene, i.e., the plasmid can be normally introduced into the target strain, and the target gene can stably exist, and the optimal expression conditions of the strain are determined according to the method of example 2.

Example 2

The activity of the shake flask-cultured products was tested using Micrococcus muralis as indicator.

The protein expression level under different temperature and pH conditions is measured by adjusting the temperature and pH, and the fermentation temperature, pH and time are adjusted to obtain the expression condition of the strain under the optimal expression level.

FIG. 2 shows that the inducible promoter P_AOX1The optimum conditions for producing hLYZ by fermentation are 26 deg.C, pH 6.0, enzyme activity reaches the highest after methanol induction for 96h, and the enzyme activity can reach 16,400 + -700 U.mL^-1(ii) a Constitutive promoter P_GAPThe optimum condition for fermentation production of hLYZ is 28 deg.C, pH 6.0, and the enzyme activity reaches the highest 48h after using glucose as carbon source, but only reaches 310 + -17.3 U.mL^-1Therefore, inducible promoter P was selected for this study_AOX1Fermenting to produce hLYZ.

The expression level of enzyme activity can be obtained, the activity of the enzyme is increased greatly when the methanol is used for inducing the promoter, and the activity of the enzyme is lower when a constitutive promoter is used, but an external inducer is not needed for inducing, so that the promoter has more stable and safer effect compared with an induced promoter.

Example 3

Optimizing signal peptides of the constructed GS115/hLYZ strain, respectively using ten kinds of signal peptides to replace alpha signal peptides in pPIC9K-hLYZ, respectively, optimizing gene sequences of signal peptides HFBI signal sequences (Hss), INUIA signal sequences (Iss), W1 signal sequences (Wss), Killer signal sequences (Kss), Invertase signal sequences (Inss), hLYZ signal sequences (Hss), Albumin signal sequences (Alss), Lipase signal sequences (Lss), Apre signal sequences (Apss), Glucomylase signal sequences (Gs), using primers Hss-F/R, IssF/R, Wss-F/R, Kss-F/R, Inss-F/R, Hss-F/465/R, Apss-F/48325/F-R, Gss/F, the gene sequences of signal peptides Hss, Iss, Wss, Kss, Inss, Hss, Alss, Lss, Apss and Gs are amplified by taking the synthesized signal peptide gene sequence as a template, and are connected to pPIC9K-hLYZ after being cut by BamH I and EcoR I to replace alpha signal peptide in the plasmid, so as to construct plasmids pPIC9K-Hss-hLYZ, pPIC9K-Iss-hLYZ, pPIC9K-Wss-hLYZ, pPIC9K-Kss-hLYZ, pPIC9K-Inss-hLYZ, pPIC9K-Hss-hLYZ, pPIC9K-Alss-hLYZ, pPIC9K-Lss-hLYZ, pPIC9K-Apss-hLYZ, pPIC9K-hLYZ, and the corresponding sequences of the signal peptides and the signal peptides are shown in Table 1.

TABLE 1 Signal peptides and their corresponding sequences

The optimization of the signal peptide is signal peptide insertion, and the specific operation method is as follows: design of primers F1 and R1 for inserting codon-optimized signal peptide sequence into pichia pastoris methanol-induced secretion type plasmid pPIC9KEcoRI andNotand (3) realizing the insertion of a signal peptide in the enzyme cutting site I, wherein a signal peptide primer and a corresponding sequence thereof are shown in a table 2.

TABLE 2 Signal peptides and their corresponding sequences

Iss-F	GCGCGGATCCATGAAGTTGGCTTACTCCTT
		Iss-R	GCGCGAATTCGTAGTTAATAACGGAAGCA
Hss-F	GCGCGGATCCATGAAGTTCTTTGCTATTGCT
		Hss-R	GCGCGAATTCAGCAACAGCAGCAGCAGCAA
Inss-F	GCGCGGATCCATGTTGTTGCAAGCTTTTTT
		Inss-R	GCGCGGATCCAGCACTAATCTTAGCAGCAA
Kss-F	GCGCGGATCCATGAGTTTGATGGTTGTTAGT
		Kss-R	GCGCGGATCCTGGCCAAGCACCTTGCAAC
Lss-F	GCGCGGATCCATGAAGTTGTTGAGTTTGACT
		Lss-R	GCGCGGATCCAGCAACACAAGTAGCCAAAA
Alss-F	GCGCGGATCCATGAAGTGGACGACTTTTAT
		Alss-R	GCGCGGATCCACTGTAAGCACTACTAAACAA
Gss-F	GCGCGGATCCATGAGTTTTAGATCTTTGTTGG
		Gss-R	GCGCGGATCCGGTTTGTACTGGTTTGGCT
hss-F	GCGCGGATCCATGAAGGCTTTGATTGTTTTG
		hss-R	GCGCGGATCCACCTTGAACAGTAACAGACAA
Wss-F	GCGCGGATCCATGAGAAGAAGAGCTATTCC
		Wss-R	GCGCGAATTCAGCCAAAGCAGATGAACCCA
Apss-F	GCGCGGATCCATGAGATTTCCTTCTATTTT
		Apss-R	GCGCGAATTCAGCCAAAGCGGATGAAGCAGC

The strain with different signal peptides replaced was subjected to shake flask fermentation, and the secretion expression of hLYZ was detected by SDS-PAGE, the results are shown in FIG. 5.

The protein expression was measured by referring to the optimal protein expression conditions in example 3, i.e., using 26 ℃, pH 6.0, 96 hours after methanol induction.

The hLYZ enzyme activity levels of the strains after the replacement of the different signal peptides are shown in FIG. 6.

From FIG. 5, it can be seen that my invention can achieve efficient and stable insertion of signal peptides.

As shown in FIG. 6, the enzyme activity of the protein was the highest after insertion of the HFBI signal peptide, and the secretion activity of hLYZ was increased to 24,880 + -1034.6 U.mL^-1It is 1.51 times of the original strain.

It can be further found that the signal peptide designed in this example can achieve a certain degree of promotion effect on the expression level of the protein, i.e., a certain degree of promotion effect on the expression of the protein is achieved.

Example 4

GS115/Hss-hLYZ is used as an initial strain, and 1ng of plasmid and 5ng of plasmid are addedThe donner DNA and 80. mu.l of competence were placed in an electric rotor and left to stand for 5min, and then transferred into competence by means of electric conversion with a set parameter of 1500V and a resistance of 200 ohms. 1ml of 1 mol. L was added rapidly^-1The D-sorbitol was pre-cultured in a shaker at 30 ℃ for 2h at 220rpm and spread on YPD plates containing hygromycin for 3 days.

Designing primers VPS10-1-1300-F/R at 1300bp upstream and downstream of a target gene, and primarily screening positive clones by using a colony PCR method, wherein the screening standard is that 1300bp upstream and downstream of the target gene is used as a primer, if 2600 bands are obtained by PCR, the positive transformants are obtained, and if 2600 and 5400 target genes are obtained, the cases are failed. The genome of the single clone was then extracted, PCR was performed again using the above primers, and the PCR product was sent to the sequencing company for sequencing. Finally, the VPS10-1 delta single knockout strain is obtained.

F/R, VPS 10-2-F/R on the primer VPS10-2 is designed, and the upstream and downstream 1000bp of the VPS10 gene are fused together to form 2000bp to be used as gene knocked-out donner DNA. GS115/Hss-hLYZ is used as an initial strain, 1ng of plasmid, 5ng of donner DNA and 80 mu l of competence are placed in an electric rotating cup to stand for 5min, and then the cells are transferred into competence in an electric conversion mode with the set parameters of voltage 1500V and resistance 200 omega. 1ml of 1 mol. L was added rapidly^-1The D-sorbitol was pre-cultured in a shaker at 30 ℃ for 2h at 220rpm and spread on YPD plates containing hygromycin for 2-3 days.

Designing primers VPS10-2-1300-F/R at 1300bp positions of the upstream and downstream of a target gene, and primarily screening positive clones by using a colony PCR method, wherein the screening standard is that the 1300bp positions of the upstream and downstream of the target gene are used as primers, if 2600bp bands are obtained by PCR, a positive transformant is obtained, and if the 2600bp bands are added with the 5400bp target genes, a failure case is obtained. The genome of the single clone was then extracted, PCR was performed again using the above primers, and the PCR product was sent to the sequencing company for sequencing. Finally obtaining the VPS10-1 delta VPS10-2 delta double knockout strain.

The obtained knockout strain is subjected to shake flask fermentation, and the fermentation supernatant is subjected to SDS-PAGE detection, and the result is shown in FIG. 7.

The enzyme activities of the strains after the single-knockout and the double-knockout were measured, the measured enzyme activity levels were the temperature and pH expressed in example 3, methanol was used as a carbon source, the measurement time was 96 hours after the cultivation, and the measured enzyme activity data are recorded in fig. 8.

From FIG. 7, it can be seen that the methods provided in my invention achieve effective single or double knockout in strains.

From FIG. 8, the data of the enzyme activity of the single-knockout and double-knockout can be obtained as follows: the enzyme activity of the VPS10-1 delta single knockout strain is 56,100 +/-1876.67U/mL, the enzyme activity of the VPS10-2 delta single knockout strain is 59,080 +/-3040U/mL, and the enzyme activity of the VPS10-1 delta VPS10-2 delta double knockout strain is 6,050 +/-630 U.mL^-1The VPS10-2 delta single knockout strain can be obtained, and the enzyme activity is higher.

Example 5

The strain subjected to the knockout in the example 4 is optimized for chaperonin, ten genes of chaperonin are respectively added to the genome of the strain to realize the expression of the chaperonin by the strain, the ten chaperonin are respectively subjected to PCR amplification by taking SSO2-F/R, ERO1-F/R, PDI1-F/R, KEX2-F/R, AFT1-F/R, HAC1-F/R, HRD1-F/R, BMH2-F/R, YDJ1-F/R, CNE1-F/R as primers to respectively obtain SSO2, ERO1, PDI1, KEX2, AFT1, HAC1, HRD1, BMH2, YDJ1 and CNE1 gene fragments, and the specific insertion method of the chaperonin is as follows: the gene fragment of chaperonin is cut by restriction endonucleases EcoR I and Sal I (the gene sequence containing cutting sites of EcoR I and Sal I inside uses restriction endonucleases Xho I and Not I) by taking a Pichia pastoris GS115 genome as a template, and then is connected to a pGAPZA vector to construct corresponding plasmids pGAPZA-SSO2, pGAPZA-ERO1, pGAPZA-PDI1, pGAPZA-KEX2, pGAPZA-AFT1, pGAPZA-HAC1, pGAPZA-HRD1, pGAPZA-BMH2, pGAPZA-YDJ1 and pGAPZA-CNE 1. Then linearizing the constructed plasmid with pGAPZA type plasmid through restriction endonuclease BlnI, mixing the purified linear plasmid and pichia in an electric rotating cup uniformly, standing on ice for 15min, inserting the linear plasmid into pichia genome through electric transformation (1500V, 250 omega), and rapidly adding 1 mol. L^-1D-sorbitol (Pre-cooled), pre-incubated at 30 ℃ for 2h, applied to a medium containing 100. mu.g.mL^-1Growing bleomycin on YPD plate for 3 days, and collecting the obtained strainThe strains were subjected to shake flask fermentation and the fermentation supernatants were examined by SDS-PAGE and the results are shown in FIG. 9.

The expression level after insertion of the signal peptide was measured according to the optimum temperature and pH measured in example 3 using methanol as a carbon source, and the enzyme activity level was measured after 96 hours of culture, and the measurement results are shown in FIG. 10.

As can be seen from fig. 9, stable expression of chaperonin was achieved in my invention.

From fig. 10, the strain after signal peptide optimization contains the inserted chaperone gene, and different chaperone proteins have different degrees of effects on the production of target proteins, wherein two chaperone proteins of Ero1 and Pdi1 increase the secretase activity of hLYZ, and finally the highest secretase activity of hLYZ can be increased to 81600 ± 5230U · mL at the level of a shake flask^-1The strain is 5.41 times of the original strain, and is the highest reported at the shake flask level.

Example 6

Detecting the expression condition of hLYZ by the prepared strain: selecting single strain with good growth condition, inoculating to 5mL YPD culture medium, culturing at 30 deg.C for 16 hr to obtain first-stage seed solution, transferring the first-stage seed solution to shake flask containing 50mL BMGY culture medium, and culturing at 30 deg.C for 24 hr to obtain second-stage seed solution. Inoculating the secondary seed liquid into a fermentation tank according to the inoculation amount of 10%, wherein the culture liquid in the fermentation tank is RDMY culture liquid and comprises the following raw materials as shown in Table 1:

TABLE 1 raw materials contained in RDMY Medium

The initial liquid loading amount in the fermentation process is 2L, the temperature is controlled to be 30 ℃, the pH value is 6.0, the ventilation ratio is 1.3vvm, the pH value of the fermentation liquid is adjusted by using 40% ammonia water, the stirring is gradually increased from 300 to 800rpm in the fermentation process, the Dissolved Oxygen (DO) in the fermentation tank is kept above 10%, and a proper amount of antifoaming agent is added to control the fermentation foam when necessary. The fermentation process of methanol type Pichia pastoris includes batch fermentation stage, fed-batch fermentation stage and methanol induction stage. In the batch fermentation stage, the thalli consume the carbon source in the initial culture medium, when DO in the fermentation tank rises and proves that the carbon source in the initial culture medium is exhausted, the fed-batch fermentation stage is started, and glycerol is fed in a DO-Star mode in a flowing mode, so that the glycerol in the culture medium is in an extremely low state, and the growth of the thalli is facilitated. When the bacterial concentration in the culture medium reaches an expected value, stopping the flow addition of glycerol, starving the cells for 2h, maintaining the DO in the fermentation tank to be above 60%, finally entering a methanol induction stage, adjusting the pH and the temperature of the fermentation liquid to the optimal fermentation conditions determined in the embodiment 3, slowly flowing and adding methanol into the culture liquid, detecting the content of the methanol in the fermentation liquid through a methanol detector, and regularly sampling to detect the expression condition of hLYZ.

Adopting pH and temperature optimized in shake flask level, fermenting with 5L fermentation tank to produce hLYZ, and monitoring methanol content in the fermentation broth to 5 g.L in real time with methanol detector^-1Fermenting with RDMY culture medium to produce hLYZ, and respectively fermenting in glycerol growth phase with thallus OD in the fermentation tank₆₀₀Methanol-induced secretory expression of hLYZ was performed at 200, 300, and 400. The secretion expression condition of hLYZ protein is detected by SDS-PAGE, and the activity is determined, the result is shown as a, b, c and d in figure 11, the secretion expression of hLYZ in the fermentation tank is different from the shake flask level, and the enzyme activity reaches the highest 72h after methanol induction. At OD₆₀₀The hLYZ is induced and expressed by methanol when the hLYZ is 200, 300 and 400, and the highest enzyme activity is 225,600 +/-13665.4 U.mL respectively^-1、352,000±16696.5U·mL^-1、267,000±10405.8U·mL^-1. Wherein, when the thallus OD in the fermentation liquor₆₀₀When the enzyme activity of hLYZ obtained by adding methanol to induce reaches 300 hours, the total protein concentration reaches 3.18 mg/mL^-1. The resulting data are recorded in FIG. 11

As shown in FIG. 11, the increase of hLYZ secretion expression with time during fermentation is not linear, which may be due to the fact that the dissolved oxygen in the fermentation broth is controlled to be above 10% before the methanol induction, but the dissolved oxygen in the fermentation broth is too low and 0% for a long time at the middle and later stages of the induction, which affects hLYZ secretion expression and generates some toxic byproducts.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

<110> university of south of the Yangtze river

<120> method for improving secretion efficiency and enzyme activity of human lysozyme in pichia pastoris

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tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat tattagctta 840

ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac ttttaacgac 900

aacttgagaa gatcaaaaaa caactaatta ttcgaa 936

<210> 2

<211> 477

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tttttgtaga aatgtcttgg tgtcctcgtc caatcaggta gccatctctg aaatatctgg 60

ctccgttgca actccgaacg acctgctggc aacgtaaaat tctccggggt aaaacttaaa 120

tgtggagtaa tggaaccaga aacgtctctt cccttctctc tccttccacc gcccgttacc 180

gtccctagga aattttactc tgctggagag cttcttctac ggcccccttg cagcaatgct 240

cttcccagca ttacgttgcg ggtaaaacgg aggtcgtgta cccgacctag cagcccaggg 300

atggaaaagt cccggccgtc gctggcaata atagcgggcg gacgcatgtc atgagattat 360

tggaaaccac cagaatcgaa tataaaaggc gaacaccttt cccaattttg gtttctcctg 420

acccaaagac tttaaattta atttatttgt ccctatttca atcaattgaa caactat 477

<210> 3

<211> 393

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaggtattcg aaagatgtga acttgcaaga actctaaagc gtttgggaat ggacggatac 60

cgtggtatca gtttggcaaa ttggatgtgt cttgccaagt gggagtccgg ttacaatacg 120

agggctacga actataacgc tggtgataga tcaactgact atggcatctt ccagattaac 180

agtaggtatt ggtgcaacga cggcaagacg cctggagcag taaacgcttg tcatctatct 240

tgttctgcac tgcttcaaga caacatcgca gacgctgtag cctgcgctaa aagagtggtc 300

cgtgatccac aaggcataag agcttgggtg gcctggagga acaggtgcca aaatagagat 360

gtgagacagt acgttcaagg ctgtggcgtt taa 393

Claims

1. A method for improving the secretion efficiency and the enzyme activity of human lysozyme in pichia pastoris is characterized in that: the method comprises the following steps:

2. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: in the optimized base sequence, the base sequence of the codon preference optimized code of the pichia pastoris is hLYZ.

3. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: in the optimized base sequence, the plasmid is pPIC 9K.

4. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: in the optimized base sequence, the type of the promoter comprises one or more of an inducible promoter and a constitutive promoter, and the promoter comprises P_AOX1、P_GAPOne or two of them.

5. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: in the optimized base sequence, the restriction endonucleases used are EcoRI and Not I.

6. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: said optimized promoter and optimized fermentation conditions, inducible promoter P_AOX1The optimal temperature for the fermentation production of hLYZ is 26 ℃, and the optimal pH is 6.0; constitutive promoter P_GAPThe optimum temperature for fermentation production of hLYZ is 28 deg.C, and the optimum pH is 6.0.

7. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: in the signal peptide optimization, the replaced signal peptide is alpha signal peptide, and the signal peptide to be replaced comprises one of HFBI signal sequence (Hss), INUIA signal sequence (Iss), W1 signal sequence (Wss), Killer signal sequence (Ks), inverse signal sequence (ins), hLYZ signal sequence (Hss), Albumin signal sequence (Alss), Lipase signal sequence (Lss), Apre signal sequence (Apss) and Glucomylase signal sequence (Gs).

8. The method for improving the secretion efficiency and the enzyme activity of the human yeast in the pichia pastoris according to claim 1 or 7, wherein the method comprises the following steps: in the signal peptide optimization, a primer used for replacing a signal peptide is F, R, and the F1 and R1 comprise one or more of Iss-F, Iss-R, Hss-F, Hss-R, Inss-F, Inss-R, Kss-F, Kss-R, Lss-F, Lss-R, Alss-F, Alss-R, Gss-F, Gss-R, hss-F, hss-R, Wss-F, Wss-R, Apss-F, Apss-R.

9. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1, wherein the method comprises the following steps: in the co-expression chaperonin, co-expressed chaperonin is one or more of Sso2, Ero1, Pdi1, Kex2, Aft1, Hac1, Hrd1, Bmh2, Ydj1 and Cne 1.

10. The method for improving the secretion efficiency and the enzyme activity of the human lysozyme in the pichia pastoris according to claim 1 or 9, wherein the method comprises the following steps: in the co-expression chaperonin, the restriction endonucleases used are EcoR I and Sal I or Xho I and Not I.