CN118086354A

CN118086354A - Bradyyeast system for expressing human lysozyme as well as construction method and application thereof

Info

Publication number: CN118086354A
Application number: CN202410048748.2A
Authority: CN
Inventors: 孙坚; 贺骞; 黄毓茂; 陆菊; 任昊; 廖晓萍; 刘雅红
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-05-28

Abstract

The invention belongs to the technical field of biology and discloses a Bradyyeast system for expressing human lysozyme as well as a construction method and application thereof. The gastric lavage test of mice shows that the engineering yeast can induce and express hLYZ through Cu ²⁺ in the in-situ environment of the intestinal tracts of the mice, and shows a certain functional activity in the intestinal tracts.

Description

Bradyyeast system for expressing human lysozyme as well as construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a Bradyyeast system for expressing human lysozyme, and a construction method and application thereof.

Background

Antibiotics have been used in animal production for many years and are typically added to animal feed at sub-therapeutic levels to increase growth rate and feed efficiency, for disease prevention, and at higher doses for disease treatment. While antibiotics have been used by growers as effective supplements, today, the use of antibiotics as feed additives in animal production is severely limited or banned, and it is necessary to improve biosafety by avoiding the use of traditional antibiotics as much as possible and finding safer alternatives.

Human lysozyme is a natural protein, is nontoxic and drug-resistant, and in 1992, the food additive society of FAO/WTO has recognized that lysozyme is safe for use in foods. It is widely distributed in various tissues (liver, articular cartilage, plasma) and body fluids (saliva, tears, milk), can cause damage to bacterial cell walls, hydrolyzes 1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine groups in cell walls, shows a certain effect in host defense mechanisms, is widely used in animal husbandry, can be added into animal feeds, can be used as a preservative and a bacteriostatic agent, not only balances microbial flora in animal intestinal tracts, prevents invasion of harmful bacteria, but also can reduce production loss to the maximum extent by improving feed efficiency and reducing susceptibility to bacterial infection and diseases, and can be considered as a suitable substitute for antibiotics.

In the breeding industry, the expression systems such as escherichia coli, pichia pastoris or saccharomyces cerevisiae are often utilized to express lysozyme, then the lysozyme is purified and recovered in a certain mode to prepare a lysozyme preparation, and the lysozyme preparation is added into feed, so that the process is complex, the production cost is high, and the application of the lysozyme in the breeding industry is limited. Saccharomyces boulardii (Saccharomyces boulardii, hereinafter referred to as S.b) is a probiotic yeast that has been used to promote intestinal health and prevent diarrhea. Not only does this yeast exhibit a phenotype beneficial to intestinal health, it also has a longer residence time in the gut than the general Saccharomyces cerevisiae and thus can be an ideal enteral delivery vehicle. Meanwhile, copper is found to be a feed additive with pharmacological dosage in animal production, and the copper is low in cost and can be used as an ideal protein expression inducer.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the problems in the prior art and provide a Cu ²⁺ inducible Saccharomyces boulardii expression system.

It is a second object of the present invention to provide a yarrowia system that expresses human lysozyme.

A third object of the present invention is to provide the use of a Bradyyeast system expressing human lysozyme.

The aim of the invention is achieved by the following technical scheme:

a bradyyeast system for expressing human lysozyme, the expression system comprising the following steps:

s1, respectively constructing HyhMX resistant and KanR resistant homologous arms, inserting and replacing alleles of CUP1 on a Saccharomyces boulardii chromosome one by one, and introducing a Cre-LoxP recombinase system to eliminate HyhMX/KanR resistance to obtain S.b delta CUP1;

S2, respectively constructing HyhMX resistant and KanR resistant homologous arms, inserting and replacing alleles of URA3 on S.b delta CUP1 chromosome one by one, and introducing a Cre-LoxP recombinase system to eliminate HyhMX/KanR resistance to obtain S.b delta CUP1 delta URA3;

S3, constructing pCUP Ura3-cf-hlYZ plasmid: amplifying cf-hLYZ sequence, and amplifying a carrier skeleton part except for alpha f-eGFP by using pCUP Ura 3-alpha f-eGFP plasmid as a template; homologous recombination is adopted to the cf-hlyZ sequence and the vector skeleton fragment to obtain pCUP Ura3-cf-hlyZ plasmid;

S4, transferring pCUP Ura3-cf-hlyZ plasmid into S.b delta CUP1 delta URA3 to obtain the Bradyyeast system for expressing human lysozyme.

Firstly, constructing a Cu ²⁺ inducible expression vector, and realizing secretion expression of human lysozyme by constructing chicken lysozyme signal peptide in the expression vector. Secondly, in order to further verify the application of the engineering yeast in oral delivery, the engineering yeast is irrigated into mice, and whether the engineering yeast is successfully applied to the in-situ environment of the intestinal tracts of the mice is judged by measuring the content of human lysozyme in the intestinal tracts and performing 16s analysis on the intestinal flora.

The invention also provides application of the Bradyyeast system in preparing lysozyme.

The invention also provides application of the Bradyyeast system in preparing feed additives.

The invention also provides a feed additive, which contains the Bradyyeast system.

The invention also provides application of the Bradyyeast system in improving the abundance of microorganisms in animal intestinal tracts.

Compared with the prior art, the invention has the following beneficial effects:

The invention firstly transfers hlyZ genes fused with chicken lysozyme signal peptide into probiotics S.b delta CUP1 delta URA3 to construct engineering yeast S.b delta CUP1 delta URA3/pCUP1Ura3-cf-hlyZ, and the engineering yeast can secrete and express hlyZ with antibacterial activity. The gastric lavage test of mice shows that the engineering yeast can induce and express hLYZ through Cu ²⁺ in the in-situ environment of the intestinal tracts of the mice, and shows a certain functional activity in the intestinal tracts.

Drawings

FIG. 1 is a schematic diagram of the construction of a Cu ²⁺ hypersensitive yeast strain S.b DeltaCUP 1;

FIG. 2 is a gel electrophoresis diagram for genotyping the WT S.b, S.b #1, S.b #2 transformants; m is DL10000 maker; a is the PCR amplification product of primer pair S.b CUP1-F/S.b CUP1-R, B is the PCR amplification product of primer pair S.b CUP1-F/Hyg-R, C is the PCR amplification product of primer pair S.b CUP 1-F/Kan-R;

FIG. 3 is a diagram of S.b #2-Cre-ble transformant-depleted HyhMX/KanR resistance genotype verification gel electrophoresis; m is DL5000maker; AB is HyhMX/KanR amplification results before elimination; CD is HyhMX/KanR amplification result after elimination;

FIG. 4 is HyhMX/KanR resistance insert and elimination phenotype verification results;

FIG. 5 is a photograph showing the result of a lost replica of pPL5071-TEF1-Cre-ble plasmid in S.b #2-Cre-ble transformant;

FIG. 6 is a genotyping gel electrophoresis chart of pPL5071-TEF1-Cre-ble plasmid in S.b #2-Cre-ble transformant; m is DL1000 maker; a is S.b #2-Cre-ble; b is S.b ΔCUP1;

FIG. 7 is a WT S.b, S.b ΔCUP1 phenotype verification;

FIG. 8 is a schematic representation of the construction of plasmid pCUP Hyg αf-eGFP;

FIG. 9 shows Western Blot results of secretory expression of eGFP at S.b ΔCUP1;

FIG. 10 shows Western Blot results of intracellular expression of eGFP in S.b ΔCUP1;

FIG. 11 is the results of flow cytometry analysis (positive cell fraction) of eGFP expression in S.b ΔCUP1 cells;

FIG. 12 shows the results of fluorescence microscopy of eGFP expression in S.b ΔCUP1 cells;

FIG. 13 is the results of flow cytometry analysis (fluorescence intensity) of eGFP expression levels comparison at WT S.b and S.b ΔCUP1;

FIG. 14WT S.b, S.b ΔCUP1ΔURA3 phenotype verification;

FIG. 15 is a schematic representation of the construction of plasmid pCUP Ura3- αf-eGFP;

FIG. 16 is a Western Blot results of the secretory expression of eGFP at S.b ΔCUP1ΔURA3;

FIG. 17 shows Western Blot results of intracellular expression of eGFP at S.b ΔCUP1ΔURA3;

FIG. 18 is the results of flow cytometry analysis (positive cell fraction) of eGFP expression in S.b ΔCUP1ΔURA3 cells;

FIG. 19 shows the results of fluorescence microscopy of eGFP expression in S.b. DELTA.CUP1. DELTA.URA3 cells;

FIG. 20 is a flow cytometry analysis result (fluorescence intensity) of comparison of eGFP expression levels at S.b ΔCUP1 and S.b ΔCUP1ΔURA3;

FIG. 21 is a schematic representation of the construction of plasmid pCUP Ura 3-cf-hLYZ;

FIG. 22 shows Western Blot results of secretory expression of hLYZ at S.b ΔCUP1ΔURA3;

FIG. 23 is a graph showing the results of an experiment of the antibacterial effect of S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ expression product on Staphylococcus aureus ATCC 29213;

FIG. 24 is a result of an experiment of the antibacterial effect of S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ expression product on E.coli ATCC 25922;

FIG. 25 is a flow chart of a S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ mouse lavage test;

FIG. 26 is a graph showing the results of weight gain in mice after lavage S.b ΔCUP1ΔURA3/pCUP1Ura 3-cf-hLYZ;

FIG. 27 shows the results of the measurement of the content of hLYZ in cecum and colon contents of mice;

FIG. 28 shows the results of a diversity analysis of intestinal microorganisms in mice;

FIG. 29 is a graph showing the results of analysis of the composition of the intestinal microbiota level of mice;

FIG. 30 is the results of the ratio of microorganisms of the phylum Zymobacter enterica and the phylum Bacteroides (F/B);

FIG. 31 is a thermal diagram of a species abundance analysis at the mouse intestinal microbiota level;

FIG. 32 is a graph showing the difference in abundance of intestinal microorganisms LEfSe in mice.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

EXAMPLE 1 construction of Cu ²⁺ highly sensitive Yeast Strain S.b DeltaCUP 1

By constructing homologous arms with different resistances, inserting and replacing the homologous arms one by one on the alleles of CUP1 on S.b chromosome (see figure 1), and removing the double antibodies by using a Cre-LoxP recombinase system, thereby realizing the construction of S.b delta CUP 1.

(1) Acquisition of Loxp-HyhMX/KanR-Loxp insert

To obtain homology arms with Hyg resistance (HyhMX) and G418 resistance (KanR), the plasmids pCEV-G4-Hyg and pCEV-G4-Km were PCR amplified using the primers Loxp-F1/Loxp-R1, respectively, with the following reaction system and conditions:

Loxp-F1(5’-3’)：

GGAGTATAATTATTGACAAGGATTTGGAATCTGATAATCTGGGTATTACTTATTGGAT CATGGTAGACAACCC

Loxp-R1(5’-3’)：

TCCAAGAGGTACCACGTTGAATGTCCATTTGGGTAATTTAGAATGGTTAATCGCTCC TAGTGGATCTGATATC

the underlined parts are homologous parts at the left and right ends of the S.b chromosome group CUP1 gene.

The PCR amplification system (50. Mu.L) was: 2X Phanta Max Master Mix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 30s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.2% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

(2) Insertion of Loxp-HyhMX/KanR-Loxp fragment

YPD medium resuscitator laboratory stored S.b (ATCC MYA-796), reference Coolabe Super yeast competent preparation and transformation kit Plus instructions S.b competent, and add inserts to the transformation solution to prepare a premix.

360. Mu.L of the premix was added to 150. Mu.L S.b of competent cells, and the mixture was repeatedly blown and mixed to thoroughly suspend the yeast cells in the premix. Incubating in a water bath at 30 ℃ for 60min, and mixing the materials uniformly after reversing every 10 min. Centrifuging at 8000rpm for 2min, and discarding the supernatant. The bacterial pellet was resuspended in 500. Mu. LYPD Liquid Medium and shake-cultured at 180rpm on a shaker at 30℃for 1h. Centrifuging at 8000rpm for 1min, and discarding the supernatant. The pellet was resuspended in 500. Mu.L of sterile deionized water, 100. Mu.L of the transformation solution was pipetted into YPD medium containing 0.1mg/mL Hyg and incubated at 30℃for 3 days. Three pairs of primer amplification verification was performed by S.b CUP1-F/S.b CUP1-R, S.b CUP1-F/Hyg-R, S.bCUP1-F/Kan-R as primer pairs, with wild type (WTS.b) and transformant (S.b #1) as templates. The reaction system and conditions were as follows:

S.b CUP1-F(5’-3’):TGTTACAGATTATAGTCAGTCGG

S.b CUP1-R(5’-3’):TTATTCCTTTGCCCTCGGA

Hyg-R(5’-3’):CCTATATCAATTAGAGCACATGC

Kan-R(5’-3’):TTAGAAAAACTCATCGAGCATC

The PCR amplification system (50. Mu.L) was: green Taq Mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, template 1. Mu.L, double pure water 21. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 2min for 30s, 35 cycles total; final extension at 68℃for 5min; preserving at 4 ℃. After the PCR product is separated by 1% agarose gel electrophoresis, the wild type can only amplify a 5214bp band, S.b #1 transformant can respectively amplify a 5214bp band and a 2046bp band (see figure 2), and the result shows that Loxp-HyhMX-Loxp is successfully inserted into one of the allele regions of CUP1 on a chromosome.

The S.b #1 transformant was picked up to prepare competent cells and Loxp-KanR-Loxp was transformed into the prepared competent cells in the same manner, and screening was performed on the S.b #2 transformant on the double plate by gene screening using YPD medium containing 0.1mg/mLHyg and 0.2mg/mL G418. After the PCR product is separated by 1.2% agarose gel electrophoresis, S.b #2 transformant is respectively amplified into 2046bp and 1828bp bands, and the amplified bands of 5214bp are not amplified (see figure 2), so that the Loxp-KanR-Loxp is proved to be successfully inserted into another allele region of CUP1 on the chromosome.

(3) Elimination of Loxp-HyhMX/KanR-Loxp insert

After the S.b #2 transformant was prepared to be competent, plasmid pPL5071-TEF1-Cre-ble was transformed into competent cells in the same manner, and the Cre enzyme was introduced to eliminate HyhMX/KanR resistance. The transformant was selected from the transformant S.b #2-Cre-ble in YPD medium of 0.05mg/mL Zeocin. Recombinant strain S.b #2-Cre-ble was cultured in YPD Zeocin (0.1 mg/mL) medium for 3 days, cre enzyme was expressed, hyhMX/KanR resistance was eliminated, PCR amplification was performed with S.b CUP1-F/Hyg-R and S.bCUP1-F/Kan-R as primer pairs, S.b #2 as positive control, and whether HyhMX/KanR resistance was eliminated was verified, with the following reaction system and conditions:

S.b CUP1-F(5’-3’):TGTTACAGATTATAGTCAGTCGG

Hyg-R(5’-3’):CCTATATCAATTAGAGCACATGC

Kan-R(5’-3’):TTAGAAAAACTCATCGAGCATC

The PCR amplification system (50. Mu.L) was: green Taq Mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, template 1. Mu.L, double pure water 21. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 1min for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. After the PCR products are separated by 1.2% agarose gel electrophoresis, S.b #2 is respectively amplified into bands of 2046bp and 1828bp, but S.b #2-Cre-ble is not amplified into the corresponding target band (see figure 3), which proves that HyhMX/KanR resistance is successfully eliminated.

WT S.b, S.b #1, S.b #2 and S.b #2-Cre-ble were cultured at 37℃overnight at 250rpm, the concentration of the bacterial solution was adjusted and the bacterial solution was subjected to gradient dilution, and bacterial solutions at different dilutions (10 ^-1、10^-2、10-³、10^-4) were dropped on YPD Hyg (0.2 mg/mL), YPD G418 (0.1 mg/mL), YPD Hyg (0.2 mg/mL) +G418 (0.1 mg/mL) medium and cultured at 37℃for 3 days to confirm the phenotype of HyhMX/KanR resistance gene insertion and elimination. As a result, it was confirmed that all strains were able to grow on Hyg (0.2 mg/mL) resistant plates, as S.b #1 had inserted the R1-HyhMX6-R2 fragment, and S.b #2 had inserted the R1-HyhMX6-R2 and R1-KanR-R2 fragments, and thus were able to grow normally on double resistant plates, S.b #2-Cre-ble had eliminated the HyhMX6/KanR resistance gene, and were unable to grow on YPD Hyg (0.2 mg/mL) and YPD G418 (0.1 mg/mL) resistant plates. Verification from both genotype and phenotype demonstrated successful insertion of the knockout fragments R1-HyhMX-R2 and R1-KanR-R2 into the CUP1 region on the chromosome and successful elimination of the HyhMX/KanR gene by Cre enzyme.

(4) Passage loss of pPL5071 plasmid

Strains successfully eliminated HyhMX/KanR resistance markers were subcultured in YPD blank medium, and screened for loss of plasmid pPL5071-TEF1-Cre-ble by photolithography (see FIG. 5), and a monoclonal strain which grew on blank YPD plates but could not normally grow on YPD Zeocin (0.05 mg/mL) plates was selected, and PCR amplification was performed with Z-F/Z-R as a primer and S.b #2-Cre-ble as a positive control, with the following reaction system and conditions:

Z-F(5’-3’)：GGCGAGCTCGAATTCGAAATGAC

Z-R(5’-3’)：TCATGAGATGCCTGCAAGCAAT

the PCR amplification system (50. Mu.L) was: green Taq Mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, template 1. Mu.L, double pure water 21. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 1min for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. After the PCR product was separated by 1.2% agarose gel electrophoresis, the strain after the loss of the plasmid could not be amplified to a 387bp ble gene band (see FIG. 6), indicating that the pPL5071 plasmid was successfully lost and designated S.b ΔCUP1.

(5) Phenotypic verification of CUP1 knockout strain

WT S.b and S.b DeltaCUP 1 were grown to logarithmic growth phase, the bacterial solution concentration was adjusted and subjected to gradient dilution, bacterial droplets at 5. Mu.L of different dilutions (10 ^-1、10^-2、10^-3、10^-4) were pipetted onto SD/-Cu ²⁺ plates containing different copper ion concentrations, incubated at 37℃for 2 days, and the phenotypic differences of CUP1 knockout strain compared to the original strain (see FIG. 7). Because the metallothionein coded by the CUP1 gene can chelate a plurality of Cu ²⁺, the tolerance of the strain to Cu ²⁺ is increased, the minimum inhibitory concentration of WTS.b is 0.10mM, and the strain S.b delta CUP1 with the CUP1 gene knocked out is obviously inhibited at the concentration of Cu ²⁺ of 0.05mM, compared with the WT, the minimum inhibitory concentration of the CUP1 knocked-out bacteria is reduced by 2 times. The phenotype verification result accords with the experimental expectation, and shows that the Cu ²⁺ high-sensitivity yeast strain S.b delta CUP1 is successfully constructed.

EXAMPLE 2 use of Cu ²⁺ -induced Bradyyeast expression System

(1) Construction of pCUP Hyg alpha f-eGFP, pCUP1Hyg-eGFP plasmid

The TEF promoter was replaced with the CUP1 promoter using the pCEVHyg αf-eGFP, pCEVHyg-eGFP and empty vector plasmids stored in the laboratory as base plasmids. The pCUP Hygαf-eGFP construct was used as an example (see FIG. 8).

The CUP1 promoter is amplified by taking CUP1-F/CUP1-R as a primer and WTS.b as a template, and the reaction system and the conditions are as follows:

CUP1-F(5’-3’):

ACAAATTCCTGCATACCCCTCATTTCTAGTTAGAAAAAGACATTTTTGC

CUP1-R(5’-3’):

GGATCCTTGTAATCTATTTCGATGACTTCTATATGATATTGC

The PCR amplification system (50. Mu.L) was: 2X PhantaMax MasterMix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 15s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.5% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

The P-F/P-R is used as a primer, pCUP Hyg alpha F-eGFP, pCUP1Hyg-eGFP plasmid and empty load template thereof are used for PCR amplification of a carrier framework part, and the reaction system and conditions are as follows:

P-F(5’-3’):AAATGAGGGGTATGCAGGAATTTG

P-R(5’-3’):CGAAATAGATTACAAGGATCC

The PCR amplification system (50. Mu.L) was: 2X PhantaMax MasterMix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 30s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.5% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

According to the Invirginiane homologous recombination enzyme Clon Express II instruction, the vector and fragment usage amounts are calculated, pCUP1 recovery fragment, vector frame recovery fragment, 4. Mu.L of 5 XCE II Buffer and 2. Mu. LExnase II are respectively added to ice, double distilled water is added to the total volume of 20. Mu.L, the reaction is carried out at 37 ℃ for 37min, and the homologous recombination products are immediately placed on ice for cooling.

10. Mu.L of the homologous recombination product was pipetted into DH 5. Alpha. Competence, ice-cooled for 30min, heat-shocked at 42℃for 90s, and immediately after the heat-shock was completed, ice-cooled for 3min. 400 μLLB broth was then added and resuscitated at 37℃at 180rpm for 1h.

Centrifuging at 8000rpm for 2min at room temperature, discarding 400 μl of culture medium, re-suspending the rest bacterial liquid, uniformly coating 100 μl of bacterial liquid on LB plate containing 100 μg/mLAmp, incubating at 37deg.C for 15min, culturing for 24 hr, and picking single colony for PCR verification. The reaction system and conditions were as follows:

CUP1-F1(5’-3’):ACGCATCGCTCTTTTTGCTTCTA

V-R(5’-3’):CAAGTTGAGCTTGTTTGTTCTTGA

The PCR amplification system (50. Mu.L) was: green Taq Mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, template 1. Mu.L, double pure water 21. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 1min for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR products are identified by 1% agarose gel electrophoresis, single colony which is successfully identified is amplified and cultured at 37 ℃ and 180rpm, plasmid extraction is carried out by referring to the specification of an OMEGA plasmid extraction kit, the extracted plasmid is sent to the Shanghai biological engineering Limited company for sequencing and identification, and sequencing results show that the constructed pCUP Hyg alpha f-eGFP, pCUP1Hyg-eGFP plasmids and empty mutation-free sites thereof exist.

(2) PCUP1Hyg alpha f-eGFP plasmid and its empty transformation and secretory expression.

The S.b. DELTA.CUP1 strain in example 1 was recovered by YPD, competent preparation of the yeast with reference to Coolabe Super and preparation of the transformation kit Plus instruction, and the constructed pCUP Hyg. Alpha.f-eGFP plasmid and its empty vector were transferred into the prepared S.b. DELTA.CUP1 competent, and the transformation solution was spread onto YPD Hyg (0.2 mg/mL) medium and incubated at 37℃for 4d at constant temperature to obtain S.b. DELTA.CUP1/pCUP 1Ura 3. Alpha.f-eGFP and its empty vector.

Single colonies on YPD Hyg (0.2 mg/mL) medium were picked and inoculated into 10mLYPD Hyg (0.2 mg/mL) liquid medium, respectively, and cultured overnight at 37℃and 1% of the inoculum size was inoculated into a new 100mL liquid medium the next day, cu ²⁺ with a final concentration of 0.1mM was added as inducer, no-load strain was used as negative control, and cultured at 180rpm at 37℃for 3 days.

The fermentation supernatant was collected by centrifugation at 10000rpm for 10min, filtered with a 0.22 μm filter membrane, and the fermentation broth was concentrated to about 1mL by ultrafiltration using a 10kDa ultrafiltration tube, and verified by Western Blot analysis.

Western Blot shows (see FIG. 9), that S.b ΔCUP1/pCUP Hygαf-eGFP fermentation broth has a specific band at about 26.9kDa, and the specific band is consistent with the expected protein size, which shows that eGFP is successfully secreted and expressed, and proves that the constructed Bradyyeast Cu ²⁺ inducible expression system can be successfully applied to secretory and expression of exogenous proteins.

(3) PCUP1Hyg-eGFP plasmid transformation and intracellular expression.

The S.b. DELTA.CUP1 strain in example 1 was recovered by YPD, competent preparation of yeast with reference to Coolabe Super and preparation of a transformation kit Plus instruction, and the pCUP Hyg-eGFP plasmid constructed in example 3 was transferred to the prepared S.b. DELTA.CUP1 competent, and the transformation solution was spread on YPD Hyg (0.2 mg/mL) medium and incubated at 37℃for 4 days to obtain S.b. DELTA.CUP1DELTA.URA 3/pCUP1Ura3-eGFP.

Single colonies on YPD Hyg (0.2 mg/mL) medium were picked and inoculated into 10mL of liquid medium, respectively, and cultured overnight at 37℃and 1% of the inoculum size was inoculated into a new 100mL of liquid medium the next day, cu ²⁺ as an inducer was added at a final concentration of 0.1mM, and the recombinant strain without the inducer was used as a negative control, and cultured at 180rpm at 37℃for 3 days.

The bacterial precipitate was collected by centrifugation at 10000rpm for 10min, and a part of the bacterial precipitate was lysed with a yeast lysate, followed by WesternBlot analysis.

WesternBlot shows (see FIG. 10), S.b ΔCUP1/pCUP Hyg-eGFP lysate showed a specific band around 26.9kDa, consistent with the expected protein size, indicating successful intracellular expression of eGFP.

Another portion of the cell pellet was resuspended in 0.01M PBS and analyzed for eGFP fluorescence intensity by flow cytometry and fluorescence microscopy. The flow cytometry analysis results show that the recombinant strain has obvious fluorescence signals after 3 days of expression at the induction dose of 0.1mM Cu ²⁺ (see FIG. 11), the positive cell ratio of the recombinant strain is about 27.9%, and green fluorescence emitted by the thalli can be observed by a fluorescence microscope (see FIG. 12).

In conclusion, the constructed Bradyyeast Cu ²⁺ inducible expression system is proved to be successfully applied to intracellular expression of exogenous proteins.

(4) Comparison of eGFP expression levels at WTS.b and S.b ΔCUP1

Resuscitates WTS.b by YPD, prepares competence by referring to Coolabe Super yeast competence and prepares a transformation kit Plus instruction, transfers the constructed pCUP Hyg-eGFP plasmid into the prepared WTS.b competence, and coats the transformation solution onto YPD Hyg (0.2 mg/mL) culture medium for 4d at a constant temperature of 37 ℃ to obtain S.b/pCUP Hyg-eGFP.

Single colonies of S.b/pCUP Hyg-eGFP and S.b DeltaCUP 1/pCUP Hyg-eGFP on the culture medium were picked and inoculated into 10mL of liquid culture medium respectively, cultured overnight at 37℃and inoculated into a new 100mL of liquid culture medium the next day in an inoculum size of 1%, and then 0.01, 0.1 and 0.3mM Cu ²⁺ were added as inducers, respectively, and cultured at 180rpm at 37℃for 3 days.

The bacterial pellet was collected by centrifugation at 10000rpm for 10min, resuspended in 0.01M PBS, and semi-quantitatively analyzed by flow cytometry for protein expression levels of WT S.b/pCUP Hyg-eGFP and S.b DeltaCUP 1/pCUP Hyg-eGFP.

The flow cytometry analysis results show (see fig. 13), that the expression level of eGFP at S.b Δcup1 is significantly higher than that at WT S.b (P < 0.05) at low induction dose (0.01 mM Cu ²⁺), medium induction dose (0.10 mM Cu ²⁺) and high induction dose (0.30 mM Cu ²⁺), indicating that the constructed buddyyeast Cu ²⁺ inducible expression system has higher Cu ²⁺ responsiveness and stronger exogenous protein expression capacity than the wild type.

EXAMPLE 3 construction of Cu ²⁺ highly susceptible and uracil auxotrophic Yeast Strain S.b DeltaCUP1DeltaURA 3

In example 2, it was found that, although the expression level of eGFP in S.b Δcup1 was significantly higher than that in wts.b, the positive cell ratio was only about 28%, so that in order to increase the expression level of the foreign protein in the system, S.b Δcup1 was further modified to be uracil auxotroph, so as to increase the stability of the expression plasmid in the strain, and thus to increase the protein expression level.

By constructing homologous arms with different resistances, inserting and replacing the homologous arms one by one on alleles of URA3 on S.b DeltaCUP 1 chromosome (refer to knockout of CUP1 gene in figure 1), and then eliminating double antibodies by using a Cre-LoxP recombinase system, thereby achieving the construction of S.b DeltaCUP1DeltaURA 3.

(1) Knock-out of URA3 Gene

To obtain homology arms with Hyg resistance (HyhMX) and G418 resistance (KanR), plasmids pCEV-G4-HyhMX6 and pCEV-G4-KanR were PCR amplified using primers Loxp-F2/Loxp-R2, respectively, with the following reaction system and conditions:

Loxp-F2(5’-3’)：

ACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCTTGGATCATG GTAGACAACCC

Loxp-R2(5’-3’)：

AATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTCGCTCCTAGTGGA TCTGATATC

The underlined parts are homologous parts at the left and right ends of the S.b ΔCUP1 genome URA3 gene.

The PCR amplification system (50. Mu.L) was: 2X PhantaMax Master Mix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 30s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.2% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

The recovered Loxp-HyhMX/KanR-Loxp fragment was inserted on both sides of the S.b DeltaCUP 1 chromosome set URA3 allele in the same manner as in example 1 to obtain S.b DeltaCUP 1#1 and S.b DeltaCUP 1#2, respectively, and the pPL5071-TEF1-Cre-ble plasmid was transferred to S.b DeltaCUP 1#2 in the manner as in example 1 to obtain S.b DeltaCUP 1#2-Cre-ble, the resistance was eliminated by constitutive expression of Cre enzyme, hyhMX/KanR-resistant strain S.b DeltaCUP 1#2-Cre-ble was serially subcultured in YPD medium to obtain the lost pPL5071-TEF1-Cre-ble plasmid, which was designated S.b DeltaCUP 1 DeltaURA 3 by replica plating and PCR screening.

(2) Phenotype verification of Gene knockout strains

WTS. B and S.b ΔCUP1ΔURA3 were cultured to logarithmic phase, the bacterial liquid concentration was adjusted and subjected to gradient dilution, 5. Mu.L of bacterial liquid at different dilutions (10 ^-1、10^-2、10^-3、10^-4) was aspirated and the bacterial liquid was respectively dropped onto YPD, SD/-Ura, 5FOA plate medium and cultured at 37℃for 2 days to verify the URA3 gene deletion phenotype.

All strain phenotypes were confirmed to be in agreement with the experimental expectation (see FIG. 14), S.b. DELTA. CUP 1. DELTA. URA3 was unable to grow in auxotrophic media (SD/-Ura media) lacking uracil (Uracil) due to deletion of the URA3 gene, but could grow on media supplemented with Uracil and 5-FOA (5 FOA media), demonstrating that the strain URA3 gene was knocked out. Whereas WT S.b contains the URA3 gene, it can convert 5-fluoroorotic acid (5-FOA) into toxic substances, and thus cannot grow in 5FOA medium. Taken together, it was shown that the Cu ²⁺ hypersensitive and uracil auxotrophic yeast strain S.b DeltaCUP1DeltaURA 3 was successfully constructed.

EXAMPLE 4 use of Cu ²⁺ -induced uracil auxotroph Brettanomyces expression System

(1) Construction of pCUP1Ura 3. Alpha.f-eGFP, pCUP1Ura3-eGFP plasmid

The HyhMX6 resistance gene fragment was replaced with the URA3 gene fragment using pCUP. Alpha.f-eGFP of Hyg, pCUP1Hyg-eGFP plasmid and empty vector constructed in example 2 as templates, respectively, and the pCUP Ura 3. Alpha.f-eGFP construction procedure was exemplified (see FIG. 15).

The URA3 gene and its promoter were amplified by PCR using the laboratory stored plasmid pPL5071-TEF1-Cre-URA3 as a template. The reaction system and conditions were as follows:

URA3-F(5’-3’)：

CATGGTAGACAACCCTTAATTCACAGCTTGTCTGTAAGCGG

URA3-R(5’-3’)：

CCTAGTGGATCTGATATCACCTACCTGATGCGGTATTTTCTCC

The PCR amplification system (50. Mu.L) was: 2X PhantaMax Master Mix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water.

The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 15s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃.

The PCR product was separated by 1.5% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

The carrier frame part was PCR amplified using P-F1/P-R1 as primers, pCUP Hyg. Alpha.f-eGFP, pCUP1Hyg-eGFP plasmid and empty vector as templates, and the reaction system and conditions were as follows:

P-F1(5’-3’):TAGGTGATATCAGATCCACTAGG

P-R1(5’-3’):ATTAAGGGTTGTCTACCATG

The PCR amplification system (50. Mu.L) was: 2X PhantaMax Master Mix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 15s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.5% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

Homologous recombination was performed by reference to the method described in example 2 above, and the ligation fragment was transformed into DH 5. Alpha. Competence, spread on LB plates containing 100. Mu.g/mLAmp, incubated at 37℃for 24h, and single colonies were picked for PCR verification. The reaction system and conditions were as follows:

2μ-F(5’-3’):GTATCGTATGCTTCCTTCAGC

URA3-R1(5’-3’):TTGTCGCTCTTCGCAATGTC

The PCR amplification system (50. Mu.L) was: green Taq Mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, template 1. Mu.L, double pure water 21. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 15s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR products are identified by 1% agarose gel electrophoresis, single colony which is successfully identified is amplified and cultured at 37 ℃ and 180rpm, plasmid extraction is carried out by referring to the specification of an OMEGA plasmid extraction kit, the extracted plasmid is sent to the Shanghai engineering biological engineering Co, and sequencing identification is carried out, and the sequencing result shows that the constructed pCUP Ura3 alpha f-eGFP, pCUP1Ura3-eGFP plasmid and no-load mutation sites thereof exist.

(2) PCUP1Ura 3. Alpha.f-eGFP plasmid and its empty transformation and secretory expression.

The S.b DeltaCUP1DeltaURA 3 strain in example 3 was recovered by YPD, competent preparation of Coolabe Super yeast and preparation of a transformation kit Plus instruction, constructed pCUP Ura 3. Alpha. F-eGFP plasmid and empty vector thereof were transferred into the prepared S.b DeltaCUP1DeltaURA 3 competent, and the transformation solution was spread on SD/-Ura medium for culturing for 4d at 37℃to obtain S.b DeltaCUP1DeltaURA 3/pCUP1Ura 3. Alpha. F-eGFP and empty vector thereof.

Single colonies on SD/-Ura culture medium are selected and respectively inoculated into 10mL of SD/-Ura liquid culture medium for overnight culture at 37 ℃, the next day, 1% of inoculum size is inoculated into new 100mL of SD/-Ura liquid culture medium, cu ²⁺ with the final concentration of 0.1mM is added as an inducer, no-load strain is used as a negative control, and the culture is carried out for 3 days at 180rpm at 37 ℃.

The fermentation supernatant was collected by centrifugation at 10000rpm for 10min, filtered with a 0.22 μm filter membrane, and the fermentation broth was concentrated to about 1mL by ultrafiltration using a 10kDa ultrafiltration tube, and verified by Western Blot analysis. Western Blot shows (see FIG. 16), that S.b ΔCUP1ΔURA3/pCUP1Ura3αf-eGFP fermentation broth has a specific band at about 26.9kDa, and the specific band is consistent with the expected protein size, which shows that eGFP is successfully secreted and expressed, and proves that the constructed uracil nutritional type Bradyyeast Cu ²⁺ inducible expression system can be successfully applied to secretory expression of exogenous proteins.

(3) PCUP1 transformation and intracellular expression of the Ura3-eGFP plasmid.

The S.b DeltaCUP1DeltaURA 3 strain in example 3 was recovered by YPD, competent preparation of yeast with reference to Coolabe Super and preparation of a transformation kit Plus instruction, and the constructed pCUP Ura3-eGFP plasmid was transferred into the prepared S.b DeltaCUP1DeltaURA 3 competent, and the transformation solution was spread on SD/-Ura medium and cultured at 37℃for 4 days to obtain S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-eGFP.

Single colonies on SD/-Ura culture medium are selected and respectively inoculated into 10mL of SD/-Ura liquid culture medium for overnight culture at 37 ℃, the next day, 1% of inoculum size is inoculated into new 100mL of SD/-Ura liquid culture medium, cu ²⁺ with the final concentration of 0.1mM is added as an inducer, a recombinant strain without the inducer is taken as a negative control, and the recombinant strain is cultured for 3 days at 180rpm at 37 ℃.

The bacterial precipitate was collected by centrifugation at 10000rpm for 10min, and a part of the bacterial precipitate was lysed with a yeast lysate, followed by WesternBlot analysis. Western Blot (see FIG. 17) shows that, with the control of the bacterial lysate without inducer, the bacterial lysate with inducer has a specific band at about 26.9kDa, consistent with the expected protein size, indicating successful intracellular expression of eGFP,

Another portion of the cell pellet was resuspended in 0.01M PBS and analyzed for eGFP fluorescence intensity by flow cytometry and fluorescence microscopy. The flow cytometry analysis results show that the recombinant strain has obvious fluorescence signals after 3 days of expression at the induction dose of 0.1mM Cu ²⁺ (see FIG. 18), the positive cell ratio of the recombinant strain is about 58.8%, and green fluorescence emitted by the thalli can be observed by a fluorescence microscope (see FIG. 19).

In conclusion, it is proved that the construction of uracil nutritional type Bradyyeast Cu ²⁺ inducible expression system can be successfully applied to intracellular expression of exogenous proteins.

(4) Comparison of eGFP expression levels at S.b ΔCUP1 and S.b ΔCUP1ΔURA3

Picking S.b ΔCUP1/pCUP Hyg-eGFP, S.b ΔCUP1ΔURA3 +.

PCUP1Ura3-eGFP single colonies were inoculated into 10mL of liquid medium, cultured overnight at 37℃and then inoculated into a new 100mL of liquid medium at 1% of the inoculum size the next day, and then added with Cu ²⁺ as an inducer at a final concentration of 0.1mM, and cultured at 180rpm at 37℃for 3 days.

The bacterial pellet was collected by centrifugation at 10000rpm for 10min, resuspended in 0.01M PBS, and analyzed by flow cytometry for the fluorescence intensity (MFI) of eGFP, thereby semi-quantifying the protein expression levels of S.b. DELTA.CUP1 and S.b. DELTA.CUP1. DELTA.URA3. The flow cytometry analysis results showed (see fig. 20) that the expression level of eGFP was significantly higher at S.b Δcup1Δura3 than at S.b Δcup1 (P < 0.0001). The result shows that S.b delta CUP1 delta URA3 has higher capability of expressing exogenous protein than S.b delta CUP1 under experimental conditions, and the constructed Cu ²⁺ -induced uracil auxotroph Saccharomyces boulardii expression system has ideal exogenous protein expression level.

EXAMPLE 5 construction of bDeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ Strain and in vitro induced expression

(1) Construction of pCUP Ura3-cf-hlyZ plasmid

The pGSI-cf-hlYZ plasmid stored in a laboratory is used as a template to amplify the cf-hlYZ sequence. The reaction system and conditions were as follows:

hLYZ-F(5’-3’):

GCAATATCATATAGAAGTCATCGAAATAGACGGATTCTAGAACTAGTATGAGGT

hLYZ-R(5’-3’):

ATCTTAGCTAGCCGCGGTACCCGTGACATAACTAATTACATGACTC

The PCR amplification system (50. Mu.L) was: 2X PhantaMax MasterMix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, extension at 68℃for 30s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.0% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

The vector backbone portions other than αf-eGFP were amplified using the pCUP Ura3- αf-eGFP plasmid stored in the laboratory as a template (see FIG. 21). The reaction system and conditions were as follows:

Pcup1-F(5’-3’)：GGTACCGCGGCTAGCTAAGAT

Pcup1-R(5’-3’):TCTATTTCGATGACTTCTATATGATATTGC

The PCR amplification system (50. Mu.L) was: 2X PhantaMax MasterMix (Dye Plus) 25. Mu.L, 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 2. Mu.L of the template, and 21. Mu.L of double purified water. The reaction conditions for PCR amplification were: pre-denaturation at 95℃for 1min; denaturation at 95℃for 10s, annealing at 55℃for 15s, elongation at 68℃for 1min 30s for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product was separated by 1.0% agarose gel electrophoresis, and then the target fragment was recovered using a DNA purification recovery kit.

According to the Invirginiane homologous recombination enzyme Clon Express II instruction, the vector and fragment usage amounts are calculated, cf-hLYZ recovery fragments, vector frame recovery fragments, 4. Mu.L of 5 XCE II Buffer and 2. Mu. LExnase II are respectively added on ice, double distilled water is added to the total volume of 20. Mu.L, the reaction is carried out at 37 ℃ for 37min, and the homologous recombination products are immediately placed on ice for cooling.

h-F(5’-3’):TAGAAGCAAAAAGAGCGATGCGT

h-R(5’-3’)：TGGGTCTCTAACGACTCTCTTAGC

The PCR amplification system (50. Mu.L) was: green Taq Mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, template 1. Mu.L, double pure water 21. Mu.L. The reaction conditions for PCR amplification were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 68℃for 1min for 35 cycles; final extension at 68℃for 5min; preserving at 4 ℃. The PCR product is identified by 1% agarose gel electrophoresis, a single colony which is successfully identified is amplified and cultured at 37 ℃ and 180rpm, plasmid extraction is carried out by referring to an OMEGA plasmid extraction kit instruction book, the extracted plasmid is sent to the Shanghai biological engineering Limited company for sequencing and identification, and the sequencing result shows that the constructed pCUP Ura3-cf-hlyZ plasmid and no-load mutation sites thereof exist.

(2) PCUP1 transformation and expression of Ura3-cf-hLYZ plasmid

The S.b delta CUP1 delta URA3 strain constructed before the YPD resuscitation laboratory is prepared into competence by referring to Coolabe Super yeast competence preparation and transformation kit Plus instruction, the constructed pCUP Ura3-cf-hlyZ plasmid and empty vector thereof are transferred into the prepared S.b delta CUP1 delta URA3 competence, and transformation solution is coated on SD/-Ura culture medium for constant temperature culture for 4d at 37 ℃ to obtain S.b delta CUP1 delta URA3/pCUP1Ura3-cf-hlyZ and empty vector thereof.

Western Blot shows (see FIG. 22) that S.b ΔCUP1ΔURA3/pCUP1Ura3-cf-hlYZ broth has a specific band at about 15.5kDa, consistent with the expected protein size, and no empty load appears without obvious band, indicating that S.b ΔCUP1ΔURA3/pCUP1Ura3-cf-hlYZ successfully secretes and expresses hlYZ.

EXAMPLE 6 verification of the expression Effect of the bDeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ Strain

And (3) performing in-vitro activity verification on the expressed hlyZ through a double-layer agar method bacteriostasis experiment.

S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlYZ and no-load bacteria were induced and cultured for 3 days respectively according to the method in example 1, the fermentation supernatant was collected by centrifugation at 10000rpm for 10min at high speed, filtered with a 0.22 μm filter membrane, 10mL of the filtered supernatant was dried with a freeze-dryer, and resuspended in 500. Mu.L of 0.1M sulfate buffer (pH 6.8) to give a protein sample;

Staphylococcus aureus ATCC 29213 and escherichia coli ATCC 25922 grown on LB plates are respectively picked up, inoculated in 10mL of LB liquid medium and cultured for 6 hours at 37 ℃ and 180 rpm;

Respectively sucking 10 mu L of staphylococcus aureus ATCC 29213 and escherichia coli ATCC 25922 bacterial liquid, placing in 10mL of 0.5% LB agar cooled to 50-55 ℃ and uniformly mixing; sucking 2-3 mL of agar after uniformly mixing, spreading on a 1.5% agar plate, and cooling and solidifying;

mu.L of the prepared protein samples were pipetted onto a double-layered agar plate containing Staphylococcus aureus ATCC 29213 (gram positive bacteria) and Escherichia coli ATCC 25922 (gram negative bacteria), each sample was preset with 3 replicates, and incubated at 37℃for 6 hours, and the zone of inhibition was measured.

As human lysozyme can hydrolyze bacterial cell walls, thereby producing antibacterial effect, a protein sample prepared from S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlYZ expression supernatant can respectively produce 12.35mm (see figure 23) and 8.45 mm-sized antibacterial rings (see figure 24) on staphylococcus aureus ATCC 29213 and escherichia coli ATCC 25922, and no obvious antibacterial ring appears on empty expression supernatant, the results show that the S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlYZ engineering yeast constructed by the invention meets experimental expectations, and the protein expressed by the exogenous genes can produce obvious antibacterial effect on staphylococcus aureus ATCC 29213 and escherichia coli ATCC 25922.

EXAMPLE 7S.bDeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ Strain oral delivery Effect validation

16 SPF-class BALB/C mice were randomly divided into 4 groups and 5 days after being housed for gastric lavage. Group 1: gastric lavage PBS, group 2: gastric lavage 6.4mg/kg Cu ²⁺, group 3: gastric lavage S.b ΔCUP1ΔURA3/pCUP1Ura3-cf-hLYZ strain, group 4: gastric lavage S.b. DELTA.CUP1DELTA.URA 3/pCUP1Ura3-cf-hlyZ strain +6.4mg/kg Cu ²⁺ (experimental group). The total volume of the lavage was 200 uL/day, with group 3 and group 4 lavage yeasts 2X 10 ⁹ CFU/day, followed by 7d of continuous lavage. Both group 2 and group 4 had 6.4mg/kg Cu ²⁺ (calculated as element) added to the drinking water for free uptake of Cu ²⁺ by the mice (see FIG. 25 for experimental flow).

During the experiment, the weight of the mice is measured at fixed time every day, and the daily gain is calculated

The analysis result of the weight gain rate of mice after stomach filling shows that the weight gain of the group 1 (Cu group) is the most obvious, but has no significant difference compared with the other groups, the weight of the group 3 (hLYZ group) and the weight of the group 4 (CuhLYZ group) are both in an increasing trend, and have no significant difference compared with the PBS group, which indicates that the S.b delta CUP1 delta URA3/pCUP1Ura3-cf-hLYZ engineering bacteria constructed by the invention has no obvious influence on the weight gain of the mice and can not influence the normal vital activities of the mice (see figure 26).

Mice continuously perfused for 7 days were sacrificed two hours after the last lavage, cecal and colonic contents were collected under sterile conditions and weighed, and the content of hLYZ in group 3 and group 4 cecal and colonic contents was detected using the southern genipin biological limited human lysozyme (hLYZ) ELISA kit.

The content of hLYZ in the cecum content of group 4 was measured to be 2.2ng/g, the content of hLYZ in the colon content was measured to be 0.6ng/g, and the detected hLYZ in group 3 was consistent with the expected experimental result (see fig. 27), which shows that S.b Δcup1Δura3/pCUP1URA3-cf-hLYZ engineering bacteria constructed in the present invention can successfully induce expression of hLYZ in the small intestine and the intestinal tract through Cu ²⁺.

The cecal contents of the 4 groups were simultaneously subjected to 16S sequencing analysis. Intestinal microbial sequencing analysis showed that group 2 (Cu group) had significantly increased α diversity compared to group 1 (PBS group), group 3 (hLYZ group) had little change over group 1 as a whole, and group 4 (CuhLYZ group) had decreased α diversity compared to group 1, indicating that S.b Δcup1 Δura3/pCUP1URA3-cf-hLYZ produced hLYZ under Cu ²⁺ induction and that hLYZ had an effect on mice intestinal microorganisms, thereby eliminating the increase in mice intestinal α diversity due to gastric lavage Cu ²⁺ (see fig. 28).

Analysis of the mice intestinal microbiome at the gate level revealed that the microbiome at the gate level consisted mainly of firmicutes (Fimicutes) and bacteroides (Bacteroidota) (see fig. 29), and the ratio of firmicutes (F/B) to bacteroides (see fig. 30) was visually analyzed, wherein no significant difference in F/B values between group 2 (Cu) and group 1 (PBS) indicated that no significant increase in F/B values was effected after Cu ²⁺ feeding, group 3 (hLYZ) compared to group 1 (P < 0.05), indicated that no significant difference in F/B values was effected after feeding engineering bacteria, but group 4 (CuhLYZ) compared to group 1, indicated that no significant increase in F/B values due to feeding engineering bacteria occurred after simultaneous feeding of Cu ²⁺ and engineering bacteria, indicating that S.b Δcup1Δura3/pCUP1URA3-cf-hLYZ engineering bacteria produced no significant increase in F/hLYZ under Cu ²⁺ induction, and that no significant increase in F/hlyzobacteria after feeding engineering bacteria, thereby eliminating the strain F/h of bacteroides.

All groups of mice were analyzed for species abundance at the microbial genus level and visualized as a heat map, and for convenience of the results display, it was seen from fig. 31 that group 1 (PBS group) had higher species abundance, group 1 decreased after oral administration of Cu ²⁺ as compared to group 1 (hLYZ group) with no significant effect on each cluster species, while oral administration of Cu ²⁺ and engineered yeast as compared to group 4 (CuhLYZ group) had significantly decreased species abundance of group 2, increased species abundance of group 1, indicating that Cu ²⁺ induced engineered yeast to produce hLYZ and Cu ²⁺ induced species changes. This result is consistent with LEfSe abundance difference analysis (see figure 32). Group 2 (Cu group) with significantly increased microbial species changes compared to the remaining 3 groups focused mainly on Adlercreutzia, low-halophilic bacteria (Dehalobacterium) and Bacteroides (Bacteroides), suggesting that feeding Cu ²⁺ alone would cause an increase in the species abundance of these microbial species; while group 4 (CuhLYZ) showed significantly altered microbial species distributed in clusters 1-3, but no significant increase in microbial species, indicating that after simultaneous feeding of Cu ²⁺ and engineered bacteria, the increase in microbial abundance of Adlercreutzia, low halophilic bacteria and Bacteroides (Bacteroides) due to feeding of Cu ²⁺ was restored, indicating that S.b DeltaCUP1DeltaURA 3/pCUP1Ura3-cf-hlyZ engineered bacteria produced hlyZ under Cu ²⁺ induction and had an effect on Adlercreutzia, low halophilic bacteria and Bacteroides (Bacteroides) and other microorganisms.

The results prove that the S.b delta CUP1 delta URA3/pCUP1Ura3-cf-hlyZ engineering bacteria constructed by the invention can successfully induce and express hlyZ in intestinal tracts of small intestines through Cu ²⁺, and the expressed hlyZ has certain functional activity to cause the change of intestinal microbiome.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims

1. A bradyyeast system for expressing human lysozyme, which is characterized in that the preparation method of the expression system comprises the following steps:

s3, constructing pCUP Ura3cf-hlyZ plasmid: amplifying cf-hLYZ sequence, and amplifying a carrier skeleton part except for alpha f-eGFP by using pCUP Ura 3-alpha f-eGFP plasmid as a template; homologous recombination is adopted to the cf-hlyZ sequence and the vector skeleton fragment to obtain pCUP Ura3-cf-hlyZ plasmid;

2. Use of the buddyyeast system of claim 1 for the preparation of lysozyme.

3. Use of the buddyyeast system of claim 1 for the preparation of a feed additive.

4. A feed additive comprising the buddyyeast system of claim 1.

5. Use of the buddyyeast system of claim 1 for improving the intestinal microbial abundance in an animal.