CN116042502A

CN116042502A - Genetically engineered bacterium capable of secreting mussel protein extracellularly, construction method and application thereof

Info

Publication number: CN116042502A
Application number: CN202310168298.6A
Authority: CN
Inventors: 李莎; 王鑫沂; 薛瑞; 徐虹; 王瑞; 邱益彬
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2023-02-27
Filing date: 2023-02-27
Publication date: 2023-05-02

Abstract

The invention discloses a genetically engineered bacterium capable of secreting mussel proteins extracellularly, and a construction method and application thereof, and belongs to the technical field of genetic engineering. According to the invention, through signal peptide screening, the signal peptide and the mussel protein gene are connected and transferred into plasmid pET-Duet for fusion expression. Furthermore, the cutinase Tfu-0883 gene is transferred into plasmid pET-Duet, and is co-expressed with signal peptide-mussel protein fusion protein, so that the extracellular secretion of mussel protein is finally realized. In addition, the invention optimizes the fermentation medium by adding the surfactant, thereby improving the extracellular secretion level. The yield of mussel protein in the purified fermentation liquor can reach 20-40mg/L. The invention realizes extracellular secretion of the soluble mussel protein with bioactivity in the escherichia coli, weakens the cytotoxicity caused by product accumulation, promotes the continuous synthesis of the mussel protein, avoids the subsequent cell disruption operation, and optimizes the process.

Description

Genetically engineered bacterium capable of secreting mussel protein extracellularly, construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium capable of secreting mussel proteins extracellularly, and a construction method and application thereof.

Background

Mussels are a common bivalve mollusk. One class of adhesive proteins secreted by mussel foot silk glands is known as mussel foot silk proteins (Mfp). 6 foot proteins (Mfp-1 to Mfp-6) have been identified. Mussel protein has high strength and durable adhesion capability, and can maintain the stability of mussel under repeated flushing of water flow. In addition, the mussel protein has the advantages of good biocompatibility, no sensitization, no toxicity and the like, so that the mussel podophyllotoxin protein has wide application potential in a plurality of industries such as medicine, electronic equipment, automobile and aerospace industries, paint and the like.

The traditional mussel foot protein extraction method is a direct extraction method, and has high production cost and low extraction efficiency. The high price limits the research and utilization of mussel proteins, so people start to obtain Mfps by means of genetic engineering techniques. In recombinant protein expression systems, both prokaryotes and eukaryotes are reported as hosts. Commonly used prokaryotic hosts are mainly bacteria (E.coli), eukaryotic hosts include insects, yeasts (Saccharomyces cerevisiae, pichia pastoris, kluyveromyces lactis) and plants (tobacco, chicory). Expression of Mefp-1, mfp-5, mfp-3A, mcofp-1, -3, etc. in a prokaryotic host; expression of Mefp-3, mefp-1, mcfp-3, mgfp-5, etc., is achieved in eukaryotic hosts. Mussel foot proteins have not been produced on a large scale, mainly due to low heterologous expression yields. In addition, the intracellular reducing environment of the escherichia coli is not beneficial to the formation of disulfide bonds of proteins, so that the soluble proteins with biological activity are expressed less and are mostly expressed in the form of inclusion bodies. The oxidative periplasmic space provides a good environment for proper folding of the peptide chain and fewer proteolytic enzymes make the product less susceptible to degradation. However, the periplasmic space of the cells is limited, and a large volume of fermentation medium may be the best place for recombinant proteins. However, since E.coli has a double membrane and a complex periplasmic space, protein secretion needs to cross both inner and outer membranes, which is more difficult than gram-positive bacteria.

The Signal Peptide (SP) is a short peptide chain located at the N-terminus of the target protein and capable of directing the transfer of the protein to the periplasmic space. The signal peptide reaches the lumen of the endoplasmic reticulum via the channel formed by the protein in the membrane, and is then hydrolyzed by the signal peptidase located on the membrane surface. With the aid of signal peptides, nascent polypeptide chains are transferred from the cytoplasm into the periplasmic space of the cell, and folded in the periplasmic space, and finally secreted extracellularly. However, when a protein located in a cell is recombinantly expressed in E.coli, the protein cannot be secreted outside the cell even if a signal peptide is added to the target protein, and thus other means are required to assist in secretion of the target protein. Common means are to increase the export capacity of the target protein (enhancing signal peptide, enhancing transcription regulator, overexpressing transfer RNA, etc.), and to regulate cell membrane permeability (external disruption, key enzyme knockdown, lipid hydrolase, etc.).

Cutinase is a multifunctional enzyme that hydrolyzes various soluble esters and insoluble polymeric cutins, and the like. The cutinase has the hydrolytic activity of phospholipase and can realize the extracellular secretion of target protein through the hydrolytic action of phospholipid component in the cell membrane of colibacillus to raise the permeability of cell membrane. The surfactant can dissolve lipid substances in cell membranes from outside, increase the permeability of the cell membranes, and facilitate the secretion of target proteins to outside, thereby improving the extracellular secretion capacity.

At present, no research on extracellular secretion of mussel protein of a large intestine expression system is reported. In the previous research, the accumulation of the soluble mussel protein can generate toxic effect on thalli, so that the extracellular secretion of the soluble mussel protein is realized, the intracellular accumulation of a product is avoided, the thallus pressure is reduced, and the downstream purification process is simplified, thus having very important significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a genetically engineered bacterium capable of secreting mussel proteins extracellularly, aiming at the defects of the prior art.

The invention also solves the technical problem of providing a construction method of the genetically engineered bacterium for extracellularly secreting mussel proteins.

The invention also solves the technical problem of providing the application of the genetically engineered bacterium for extracellularly secreting mussel protein in the fermentation of mussel protein.

In order to solve the technical problems, the invention adopts the following technical scheme:

a genetically engineered bacterium for extracellularly secreting mussel protein takes E.coli BL21 (DE 3) as an expression host, and sequentially inserts a signal peptide gene, a mussel protein gene and a cutinase gene on an expression vector, wherein the signal peptide gene and the mussel protein gene are subjected to fusion expression and then co-expression with the cutinase gene.

Wherein the expression vector is pET-Duet (purchased in Norflu).

Wherein, the signal peptide is any one of Bla, ompC, mglB, ompA, dsbA and PelB, the nucleic acid sequences of which are shown in SEQ ID NO.3, 5, 7, 9, 11 and 13, and the corresponding amino acid sequences are shown in SEQ ID NO.4, 6, 8, 10, 12 and 14.

The preferred signal peptide is Bla, ompC, mglB, and the most preferred signal peptide is Bla.

Wherein the mussel protein is Mgfp-5, the nucleic acid sequence of the mussel protein is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2.

Wherein, the cutinase is cutinase Tfu-0883, the nucleic acid sequence of which is shown as SEQ ID NO.15, and the corresponding amino acid sequence is shown as SEQ ID NO. 16.

The invention also provides a construction method of the genetically engineered bacterium for extracellularly secreting mussel proteins, which comprises the following steps:

(1) Connecting the signal peptide with the mussel protein gene fragment by PCR, wherein the signal peptide is positioned at the N end of the mussel protein, and introducing the connected fragment into a polyclonal copy site of the plasmid pET-Duet to obtain the plasmid pET-Duet into which the signal peptide and the mussel protein gene are introduced;

(2) Introducing a cutinase gene into the other polyclonal copy site of the plasmid pET-Duet obtained in the step (1) into which the signal peptide and the mussel protein gene are introduced, so as to obtain a recombinant plasmid;

(3) And (3) introducing the recombinant plasmid obtained in the step (2) into E.coli BL21 (DE 3) for expression to obtain recombinant escherichia coli, namely the genetically engineered bacterium capable of secreting mussel proteins extracellularly.

The application of the genetically engineered bacterium for extracellularly secreting mussel protein in preparing the mussel protein by fermentation is also within the scope of the invention.

The application of the genetically engineered bacterium for extracellularly secreting mussel proteins in preparing the mussel proteins by fermentation comprises the following steps:

(a) Inoculating the genetically engineered bacterium of any one of claims 1-5 into a shake flask for culture after plate activation, and preparing seed liquid;

(b) Inoculating the seed liquid obtained in the step (a) into a liquid culture medium according to an inoculum size of 1-10% v/v for fermentation culture, and collecting fermentation supernatant and thalli.

Wherein in the step (a), the culture conditions are as follows: culturing at 35-40deg.C and 180-220rpm for 8-12 hr.

Preferred culture conditions are: the culture was continued overnight at 37℃with shaking at 200rpm for 10 hours.

In the step (a), the formula of the LB culture medium is as follows: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride and 25-50. Mu.g/mL ampicillin.

Wherein in step (b), the seed solution is 1-10% v/v, preferably 10%.

Wherein in the step (b), the fermentation culture is performed under the following fermentation conditions: culturing at 35-40deg.C and 180-220rpm to OD ₆₀₀ And adding an inducer when the concentration reaches 2-3 and the final concentration of the inducer is 0.1-1mM, and continuously culturing for 6-10h.

Specifically, the inducer is IPTG.

Preferred fermentation conditions are: culturing at 37℃and 200rpm to OD ₆₀₀ IPTG was added to a final concentration of 1mM at 2-3 and the incubation was continued at 37℃and 200rpm for 6h.

Wherein in the step (b), the liquid culture medium is: 10-20g/L peptone, 5-10g/L yeast powder, 10-20g/L sodium chloride, 25-50 mu g/mL ampicillin and 1-5g/L surfactant.

Specifically, the surfactant is any one of Span-80, tween-60, triton-X100, SDS and dimethyl sulfoxide. A preferred surfactant is Triton-X100.

Wherein, in the step (b), the concentration of the extracellular secreted mussel protein can reach 20-40mg/L.

Preferably, the concentration of extracellular secreted mussel protein reaches 35-40mg/L.

The beneficial effects are that:

(1) The research firstly realizes the expression of mussel protein in escherichia coli, and finally realizes the extracellular secretion of the mussel protein through the fusion with signal peptide and the co-expression with cutinase. In addition, the yield of extracellular secretion is improved by optimizing the fermentation medium, which is beneficial to industrial production. The mussel protein prepared by the invention can be widely applied in the fields of medicine, instruments, coatings and the like.

(2) The invention establishes a method for realizing the extracellular secretion of mussel protein, so that the mussel protein can be directly extracted in a fermentation medium, the operation of crushing thalli is avoided, the process is optimized, and the secreted mussel protein is the soluble protein with biological activity.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 is a diagram of an extracellular secretion Mgfp-5 expression vector construction framework;

FIG. 2 shows SDS-PAGE analysis of Mgfp-5, M: protein standard molecular weight, S: intracellular soluble expressed Mgfp-5, IS: mgfp-5, E expressed by intracellular inclusion bodies: extracellular secreted Mgfp-5; (a) example 1: 1% of Triton-X100 was added to the medium, and the strain was a recombinant strain. (b) comparative example 1: 1% Triton-X100 was added to the medium and the strain was Mgfp-5 expressed alone (c) comparative example 2: the culture medium is free of surfactant, and the strain is recombinant strain. (d) comparative example 3: 1% Span-80 was added to the medium and the strain was a recombinant strain. (e) comparative example 4: the culture medium was supplemented with 1% Tween-80 and the strain was recombinant. (f) comparative example 5: the medium was supplemented with 1% SDS and the strain was recombinant.

FIG. 3 shows the growth curve of Mgfp-5 exocrine experiments.

FIG. 4 is a graph showing the yield of Mgfp-5 exocrine experiments.

FIG. 5 is an SDS-PAGE electrophoretic analysis of Mgfp-5 under different signal peptide conditions, M: protein standard molecular weight, S: intracellular soluble expressed Mgfp-5, IS: mgfp-5, E expressed by intracellular inclusion bodies: extracellular secreted Mgfp-5; example 1: the signal peptide is Bla, 1% of Trition-X100 is added to the culture medium, and the strain is recombinant strain. Example 2: the signal peptide was OmpC, 1% of Trition-X100 was added to the medium, and the strain was recombinant. Example 3: the signal peptide is MglB, 1% of Trition-X100 is added into the culture medium, and the strain is recombinant strain.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

In the following examples, the extracellular secreted mussel protein Mgfp-5 concentration was measured using the Bradford protein concentration assay kit.

Example 1: construction of genetically engineered bacteria for producing extracellular mussel protein Mgfp-5 and shake flask fermentation to prepare extracellular mussel protein Mgfp-5.

1. Obtaining Bla-Mgfp-5 Gene fragment

(1) Designing an upstream primer and a downstream primer according to the sequence of the coding region of Mgfp-5, wherein the designed primers are as follows:

the upstream primer 5-Bla-F:

5’-GGTGTTCGCCAAGCTTGAATTCAGCAGCGAAGAATA-3’

downstream primer 5-R:

5’-CGCAAGCTTGTGGTGATGATGGTGATGGGACGAACCG-3’

the PCR reaction system was 25. Mu.L: mgfp-5 genome template DNA 2. Mu.L, upstream and downstream primers 2. Mu.L, 2X Phanta Max Master Mix 12.5.5. Mu.L, ddH, respectively ₂ O6.5. Mu.L. 2X Phanta Max Master Mix was purchased from Northenzan (Nanjing, china). The PCR reaction conditions were 94℃for 5min, followed by 94℃for 30s,60℃for 30s, and 72℃for 9s for 30 cycles, and finally 72℃for 10min. The PCR product was subjected to 1% agarose gel, and the result showed that there was a specific band below 250bp, and Mgfp-5 gene fragment was recovered by cutting gel.

Wherein, the nucleic acid sequence of the mussel protein Mgfp-5 is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2.

(2) Designing an upstream primer and a downstream primer according to the coding region sequence of the signal peptide Bla, wherein the designed primers are as follows:

the upstream primer Bla-F:

5’-AGATATACCATGGgcATGGGCATGAGCATTCAGCA-3’

downstream primer Bla-R:

5’-TATTCTTCGCTGCTGAATTCAAGCTTGGCGAACACC-3’

the PCR reaction system was 25. Mu.L: bla genome template DNA 2. Mu.L, upstream and downstream primers 2. Mu.L, 2X Phanta Max Master Mix 12.5.12.5. Mu.L, ddH, respectively ₂ O6.5. Mu.L. 2X Phanta Max Master Mix was purchased from Northenzan (Nanjing, china). The PCR reaction conditions were 94℃for 5min, followed by 94℃for 30s,60℃for 30s, and 72℃for 3s for 30 cycles, and finally 72℃for 10min. The PCR product is subjected to 1% agarose gel, and the result shows that about 80bp has a specific band, and the Bla gene fragment is recovered by cutting gel.

Wherein, the nucleic acid sequence of the signal peptide Bla is shown as SEQ ID NO.3, and the corresponding amino acid sequence is shown as SEQ ID NO. 4.

(3) The gene fragments of Bla and Mgfp-5 were ligated by PCR, the upstream primer Bla-F and the downstream primer 5-R. The PCR reaction system is the same as in step 1 (1). The PCR reaction conditions were 94℃for 5min, followed by 94℃for 30s,60℃for 30s, and 72℃for 10s for 30 cycles, and finally 72℃for 10min. The PCR product is subjected to 1% agarose gel, and the result shows that a specific band exists at 300bp, and the Bla-Mgfp-5 gene fragment is obtained by cutting gel and recycling.

2. Acquisition of the cutinase Tfu-0883 Gene fragment

Designing an upstream primer and a downstream primer according to the coding region sequence of Tfu-0883, wherein the designed primers are as follows:

the upstream primer Tfu-F:

5’-TAAGAAGGAGATATACATATGGCAGTTATGACCCCGCGCC-3’

downstream primer Tfu-R:

5’-GTTTCTTTACCAGACTCGAGTTAAAACGGGCAGGTACTGC-3’

the PCR reaction system was 25. Mu.L: tfu-0883 genomic template DNA 2. Mu.L, upstream and downstream primers 2. Mu.L each, 2 XPhantaMaxMasterMix 12.5. Mu.L, ddH ₂ O6.5. Mu.L. 2 x PhantaMaxMasterMix was purchased from Northenzan (Nanjing, china). The PCR reaction conditions were 94℃for 5min, followed by 94℃for 30s,60℃for 30s,72℃for 30s, and finally 72℃for 10min. The PCR product was subjected to 1% agarose gel, and the result showed that there was a specific band below 1000bp, and the Tfu-0883 gene fragment was recovered by cutting.

Wherein, the nucleic acid sequence of the cutinase Tfu-0883 is shown as SEQ ID NO.15, and the corresponding amino acid sequence is shown as SEQ ID NO. 16.

3. Construction of expression vector for extracellular secretion of mussel protein Mgfp-5

(1) The pET-Duet empty plasmid was extracted in small amounts. The PCR recovered product (Tfu-0883) was digested with restriction enzymes NdeI and XhoI together with plasmid pET-Duet (purchased from Yu Nuowei ZA), and the digested product was purified by 1% agarose gel and recovered for use.

(2) The cleavage product Tfu-0883 was ligated with the linearized pET-Duet plasmid. The reaction system is as follows: 5. Mu.L of linearization vector, 3. Mu.L of insert, 2. Mu.L of ExnaseII, 4. Mu.L of 5 XCEII Buffer, 6. Mu.L of ddH ₂ O. ExnaseII and 5 XCEII Buffer were purchased from Novozan (Nanjing, china). After ice-bath reaction of 20. Mu.L of the reaction system for 30 minutes, 100. Mu.L of the reaction system was addedThe ice bath was continued for 30 minutes in the competent (Yu Nuowei praise) bacteria solution of the color DH 5. Alpha. After heat shock at 42℃for 60 to 90 seconds and recovery culture with 5-fold volume of LB liquid medium (no resistance) for 1 hour, a proper volume of plating plate (with 50. Mu.g/mL of ampicillin resistance) was used and cultured at 37℃for 12 hours. Screening monoclonal, culturing and extracting plasmid for sequencing to finally obtain the corresponding expression vector pET-Duet-Tfuu-0883. The recombinant plasmid is transferred into E.coli BL21 (DE 3) for expression.

(3) The pET-Duet-Tfu-0883 plasmid was extracted in small amounts. The PCR recovery product (Bla-Mgfp-5) and plasmid pET-Duet-Tfu-0883 are subjected to double digestion by restriction enzymes NcoI and HindIII, and the digested product is recovered for later use after purification by 1% agarose gel.

(4) The cleavage product Bla-Mgfp-5 was ligated with the linearized pET-Duet-Tfu-0883 plasmid. The reaction system is as follows: 5. Mu.L of linearization vector, 3. Mu.L of insert, 2. Mu.L of ExnaseII, 4. Mu.L of 5 XCEII Buffer, 6. Mu.L of ddH ₂ O. ExnaseII and 5 XCEII Buffer were purchased from Novozan (Nanjing, china). After the reaction system of 20. Mu.L was ice-bathed for 30 minutes, 100. Mu.L of E.coli DH 5. Alpha. Competent bacteria solution was added thereto, and the ice-bath was continued for 30 minutes. After heat shock at 42℃for 60 to 90 seconds and recovery culture with 5 volumes of LB liquid medium (no resistance) for 1 hour, a proper volume of plating plate (with ampicillin resistance) was used and cultured at 37℃for 12 hours. Screening monoclonal, culturing, extracting plasmid and sequencing to obtain the expression vector pET-Duet-Bla-Mgfp-5-Tfu-0883 capable of realizing extracellular secretion of mussel protein Mgfp-5. The recombinant plasmid is transferred into E.coli BL21 (DE 3) for expression to obtain recombinant escherichia coli.

4. Preparation of extracellular mussel protein Mgfp-5 by shake flask fermentation

The recombinant E.coli strain obtained in step 3 was streaked on a plate. Single colonies grown on the plates were picked, inoculated in shake flasks with 5mL LB medium and incubated at 37℃overnight with shaking at 200rpm for 10h. Transfer to 1L baffle shake flask containing 200mL of liquid medium at 10% v/v inoculum size, and culture at 37℃at 200rpm to OD ₆₀₀ IPTG was added to a final concentration of 1mM at 2-3 and the incubation was continued at 37℃and 200rpm for 6h. And after the culture is finished, respectively collecting fermentation supernatant and thalli. Purifying the fermentation supernatant by nickel column affinity chromatography to collect extracellular secretionMgfp-5 of (F). The thalli are subjected to ultrasonic crushing, and are divided into soluble Mgfp-5 and inclusion bodies after crushing. The expression of the recombinant protein was then determined by SDS-PAGE electrophoresis.

Wherein, the formula of the LB culture medium is as follows: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride and 25-50. Mu.g/mL ampicillin; the formula of the liquid culture medium is as follows: 10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride, 25-50. Mu.g/mL ampicillin and 1g/L Triton-X100.

Comparative example 1: other embodiments are the same as example 1 without introducing signal peptide and cutinase.

Comparative example 2: other embodiments are the same as example 1 without adding a surfactant to the liquid medium.

Comparative example 3: 1g/L Span-80 was added to the liquid medium, and the other embodiments were the same as in example 1.

Comparative example 4: tween-80 was added at 1g/L to the liquid medium, and the other embodiments were the same as in example 1.

Comparative example 5: 1g/L SDS was added to the liquid medium, and the other embodiments were the same as in example 1.

The results are shown in fig. 2, 3 and 4. In the embodiment 1 of the invention, the yield of extracellular secreted soluble Mgfp-5 reaches 35mg/L due to the adoption of the genetically engineered bacterium and the culture medium added with 1g/L of Trition-X100. The extracellular secretion expression level of comparative examples 2 to 4 was significantly lower than that of example 1, whereas comparative examples 1 and 5 were not secreted at all. The addition of surfactants affects cell growth, resulting in reduced biomass. Comparative example 2 was best grown since no surfactant was added, but the biomass of experimental example 1 was less decreased compared to comparative example 2 and grown better compared to other surfactants. In conclusion, the optimal extracellular secretion of Mgfp-5 is obtained by applying the recombinant genetically engineered bacteria and the optimized culture medium of the research.

Example 2: the signal peptide of example 1 was replaced with OmpC, and the other conditions were the same as those of example 1.

Wherein, the upstream primer 5-OmpC-F corresponding to Mgfp-5 is:

5’-GCAAATGCAAAGCTTGAATTCAGCAGCGAAGAATA-3’

the downstream primer 5-R corresponding to Mgfp-5 is unchanged.

Wherein, the upstream primer OmpC-F corresponding to the signal peptide OmpC is:

5’-GAGATATACCATGGgcATGGGCATGAAGGTTAAGG-3’

the downstream primer OmpC-R corresponding to OmpC is:

5’-TATTCTTCGCTGCTGAATTCAAGCTTTGCATTTGC-3’。

wherein the nucleic acid sequence of the signal peptide OmpC is shown as SEQ ID NO.5, and the corresponding amino acid sequence is shown as SEQ ID NO. 6.

Example 3: the signal peptide of example 1 was changed to MglB, and the other conditions were the same as in example 1.

Wherein, the upstream primer 5-MglB-F corresponding to Mgfp-5 is:

5’-CATGCCAAGCTTGAATTCAGCAGCGAAGAATACAA-3’

the downstream primer 5-R corresponding to Mgfp-5 is unchanged.

Wherein, the upstream primer MglB-F corresponding to MglB is:

5’-AGGAGATATACCATGGgcATGGGCATGAACAAGAA-3’

the downstream primer MglB-R corresponding to MglB is:

5’-TTGTATTCTTCGCTGCTGAATTCAAGCTTGGCATG-3’

wherein, the nucleic acid sequence of the signal peptide MglB is shown as SEQ ID NO.7, and the corresponding amino acid sequence is shown as SEQ ID NO. 8.

As a result, as shown in FIG. 5, in example 1, 1 g/LTtion-X100 surfactant was added instead of the signal peptide as compared with examples 2 and 3. As is clear from the figure, the three signal peptides Bla, ompC and MglB all can realize extracellular secretion of Mgfp-5, but Bla has the best secretion efficiency, the band is the most clear, and the secretion promoting effect of the signal peptides OmpC (20.5 mg/L) and MglB (24.5 mg/L) is superior.

The invention provides a genetically engineered bacterium for extracellular secretion of mussel proteins, a construction method and an application idea thereof, and a method and a way for realizing the technical scheme are more specific, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by a person skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A genetically engineered bacterium for extracellularly secreting mussel protein is characterized in that E.coli BL21 (DE 3) is taken as an expression host, and a signal peptide gene, a mussel protein gene and a cutinase gene are sequentially inserted into an expression vector, wherein the signal peptide gene and the mussel protein gene are subjected to fusion expression and then co-expression with the cutinase gene.

2. The genetically engineered bacterium of claim 1, wherein the expression vector is pET-durt.

3. The genetically engineered bacterium of claim 1, wherein the signal peptide is any one of Bla, ompC, mglB, ompA, dsbA and PelB, the nucleic acid sequence of which is shown in SEQ ID nos. 3, 5, 7, 9, 11, 13, and the corresponding amino acid sequences of which are shown in SEQ ID nos. 4, 6, 8, 10, 12, 14.

4. The genetically engineered bacterium of claim 1, wherein the mussel protein is Mgfp-5, the nucleic acid sequence of which is shown in SEQ ID No.1, and the corresponding amino acid sequence of which is shown in SEQ ID No. 2.

5. The genetically engineered bacterium of claim 1, wherein the cutinase is cutinase Tfu-0883, the nucleic acid sequence of which is shown in SEQ ID No.15, and the corresponding amino acid sequence of which is shown in SEQ ID No. 16.

6. The construction method of the genetically engineered bacterium for extracellularly secreting mussel proteins is characterized by comprising the following steps:

7. Use of the genetically engineered bacterium that exogenously secretes mussel proteins according to any one of claims 1-5 in the fermentative preparation of mussel proteins.

8. The use according to claim 7, characterized by the steps of:

(a) Inoculating the genetically engineered bacterium of any one of claims 1-5 into a shake flask containing an LB culture medium for culture after plate activation, and preparing seed liquid;

(b) Inoculating the seed solution obtained in the step (a) into a liquid culture medium according to an inoculum size of 1-10% v/v for fermentation culture to obtain extracellular secreted mussel protein.

9. The use according to claim 8, wherein in step (a), the culturing is performed under the following conditions: culturing at 35-40deg.C and 180-220rpm for 8-12 hr; in the step (b), the fermentation culture is carried out under the following culture conditions: culturing at 35-40deg.C and 180-220rpm to OD ₆₀₀ And adding an inducer when the concentration reaches 2-3 and the final concentration of the inducer is 0.1-1mM, and continuously culturing for 6-10h.

10. The use according to claim 8, wherein in step (b), the liquid medium is: 10-20g/L peptone, 5-10g/L yeast powder, 10-20g/L sodium chloride, 25-50 mu g/mL ampicillin and 1-5g/L surfactant; wherein the surfactant is any one of Span-80, tween-60, triton-X100, SDS and dimethyl sulfoxide.