KR20190092834A - Expression cassette for producing target protein and strain comprising the same - Google Patents

Abstract

The present invention relates to an expression cassette for producing a target protein and a strain into which the cassette can be introduced to produce a target protein in high yield. By using the expression cassette of the present invention and the strain comprising the same, it is possible to prepare a large amount of high purity target protein at low cost. Therefore, since industrially necessary enzymes and the like can be produced in large quantities, the possibility of industrial utilization thereof is high.

Description

Expression cassette for production of a protein of interest and a strain comprising the same {EXPRESSION CASSETTE FOR PRODUCING TARGET PROTEIN AND STRAIN COMPRISING THE SAME}

The present invention relates to an expression cassette for production of a target protein and a strain into which the cassette can be introduced to produce a desired protein in high yield.

Fungi are very diverse and can secrete a variety of enzymes depending on the metabolism required for survival under a variety of environmental conditions. Some of the enzymes secreted by fungi are of high industrial value, and the more fungi studied, the more new enzymes will be found.

However, even if such enzymes or fungi producing them are found, it is not easy to mass-produce them so that they can be used industrially. Commonly encountered problems include poor production of fungi, or very low production of enzyme proteins even when cultured. For example, shiitake mushrooms can produce a high industrial value of laccase, but these mushrooms are difficult to cultivate and the amount of enzyme produced is very low, making them difficult to produce industrially.

Therefore, various methods for mass production of such enzyme proteins have been attempted, and one of them is a method of manipulating genes of fungi. For example, many genes are manipulated to remove the genes that interfere with the production of enzymes and to enhance the useful genes to produce more enzymes.

In fact, strains that effectively secrete proteins from the genus Aspergillus, Trichoderma, Penicillum, and Rhizopus have been developed and used industrially.

It is an object of the present invention to provide an expression cassette capable of improving the yield of protein to be produced when applied to fungi.

Another object of the present invention is to provide a recombinant microorganism into which the expression cassette is introduced.

Still another object of the present invention is to provide a method for preparing a target protein, which recovers the target protein secreted therefrom by culturing the recombinant microorganism.

One aspect of the present invention provides a expression cassette for a target protein comprising a promoter, a nucleic acid encoding a secretory signal peptide comprising the amino acid sequence of SEQ ID NO: 1, and a gene of the target protein.

Another aspect of the invention includes a promoter, a promoter, a nucleic acid encoding a secretory signal peptide comprising the amino acid sequence of SEQ ID NO: 1, a nucleic acid encoding an effector protein comprising the amino acid sequence of SEQ ID NO: 3 and a gene of the desired protein An expression cassette of the protein of interest is provided.

Another aspect of the present invention provides a recombinant microorganism into which the expression cassette is introduced.

Another aspect of the present invention provides a method of preparing a target protein, comprising culturing the recombinant microorganism and recovering the target protein secreted from the microorganism.

By using the expression cassette of the present invention and the strain comprising the same, it is possible to prepare a large amount of high purity target protein at low cost. Therefore, since industrially necessary enzymes and the like can be produced in large quantities, the possibility of industrial utilization is high.

1 is a schematic of a gene construct comprising a promoter and a pectatease gene.
Figure 2 is a schematic of the gene construct comprising the promoter, secretory signal peptide and pectatease gene.
Figure 3 depicts a gene construct comprising a promoter, secretory signal peptide, effector protein and pectatease gene.
4 is a schematic representation of the expression cassette of the present invention comprising promoters, secretory signal peptides, effector proteins and genes of the desired protein.
Figure 5 shows the primer set used in one embodiment of the present invention.
Figure 6 is a photograph of the results of culturing the strain introduced expression cassette according to an embodiment of the present invention.
7 is a result of SDS-PAGE analysis of a protein expressed from a strain into which an expression cassette is introduced according to an embodiment of the present invention.
8 is a schematic diagram of the total amount of protein expressed from a strain into which the expression cassette according to one embodiment of the present invention is introduced.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

One aspect of the present invention provides an expression cassette of a target protein, including a promoter, a nucleic acid encoding a secretory signal peptide comprising the amino acid sequence of SEQ ID NO: 1, and a gene of the target protein.

As used herein, the term "promoter" refers to a non-translated sequence upstream of the coding region, including a binding site for polymerase and having transcriptional initiation activity of the promoter subgene into mRNA. That is, the DNA region where the polymerase binds to initiate transcription of the gene and is located at the 5 'region of the mRNA transcription initiation site. The promoter may further include an enhancer that may be used for promoting and regulating expression as well as basic factors necessary for transcription.

Specifically, in the present invention, the promoter may be any one selected from the group consisting of a ToxA promoter, a Trip promoter, or an Ef1a promoter, and preferably, the ToxA promoter or the Trip promoter.

The term "secretory signal peptide" as used herein also refers to up to 30 short peptides present at the N-terminus of the protein so that the newly synthesized protein is secreted to a designated position via the secretory pathway.

Specifically, the secretion signal peptide may be the amino acid sequence of SEQ ID NO: 1, may be a variant that is a modified peptide of the sequence. The modification may be carried out by a method of substituting, modifying, deleting or adding one or more proteins without modifying the secretory activity of the secretory signal peptide. These various peptides comprise the amino acid sequence of SEQ ID NO: 1 and 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or 99% or more identical.

The nucleic acid encoding the secretion signal peptide is not limited as long as it is a base sequence encoding a peptide identical or identical to the secretion signal peptide secretion activity. Preferably, the base sequence of SEQ ID NO: 2 may include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence. It may be the same.

Also, as used herein, the term "target protein" refers to a polypeptide, protein or product that is to be secreted from, or produced in large quantities using, a recombinant microorganism. Specifically, any protein or product that is secreted from or expressed by Alternaria microorganisms may be included without limitation.

Specifically, the target protein in the present invention may be any one selected from the group consisting of pectate lyase, laccase, phytase, and combinations thereof.

As used herein, the term "expression cassette" refers to a unit cassette that contains the gene of the protein of interest and is capable of expressing the protein of interest. According to an embodiment of the present invention, the expression cassette according to the present invention may be introduced into Alternaria brassicicola to produce pectate lyase.

In addition, the expression cassette of the target protein according to the present invention may further include a nucleic acid encoding an effector protein comprising the amino acid sequence of SEQ ID NO: 3. The effector protein is included to increase the production of the target protein, which indicates that the mRNA to which the effector protein is fused is more stable than the mRNA that is not, or the synthesized protein is recognized as a secreted protein, thereby inhibiting degradation.

Specifically, the effector protein may be the amino acid sequence of SEQ ID NO: 3, may be a variant that is a modified peptide of the sequence. The modification may be carried out by a method of substituting, modifying, deleting or adding one or more proteins within the scope of not modifying the activity of the effector protein. These various peptides comprise the amino acid sequence of SEQ ID NO: 1 and 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or 99% or more identical.

The nucleic acid encoding the effector protein is not limited as long as it is a nucleotide sequence encoding a peptide having the same or the same activity as the effector protein. Preferably, the base sequence of SEQ ID NO: 4 may include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence. It may be the same.

In addition, the expression cassette of the present invention may further include a selection marker. Such selection markers allow the selection of transformed or transfected cells and can encode gene products that often provide exogenous specific forms of resistance to untransformed strains. It may generally be resistant to heavy metals, antibiotics or pesticides. Specifically, the selection marker may be a neomycin resistance gene, a hygromycin resistance gene, a geomycin resistance gene, or the like, or a gene associated with a specific amino acid such as a pyrG gene. The pyrG gene is a gene encoding CTP synthetase, and the CTP synthetase is an enzyme involved in pyrimidine biosynthesis, and has an activity of converting UTP to CTP. Cotransformation may be selected if the selection marker is in a separate vector or if the selection marker is in the same nucleic acid fragment as the protein-coding sequence for the heterologous protein.

In addition, another aspect of the present invention may provide a vector comprising the expression cassette. As used herein, the term “vector” refers to an artificial DNA molecule that can be introduced into a suitable host strain and recombined and inserted into a host cell genome, specifically a DNA preparation containing a nucleotide sequence of a gene operably linked to a suitable regulatory sequence. it means. The vector to be used in the present invention is not particularly limited as long as it can replicate in a host cell, and any vector known in the art may be used. Examples of commonly used vectors can be natural or recombinant plasmids, cosmids, viruses and bacteriophages.

Further, another aspect of the present invention provides a recombinant microorganism into which the expression cassette of the target protein or the vector is introduced. The microorganism is preferably of the genus Alternaria, specifically, may be alternaria japonica (Alternaria brassicicola). The expression cassette or the vector of the protein of interest may be introduced into the strains of the genus Alternaria through transformation. Specifically, when the cultivation of an Alternaria Brassiciola strain introduced with an expression cassette comprising the above-described promoter, the secretion signal peptide nucleotide sequence of SEQ ID NO: 2, the effector protein nucleotide sequence of SEQ ID NO: 4, and the target protein The yield of the protein of interest is the best.

In addition, another aspect of the present invention may provide a method for producing a target protein comprising culturing the above-mentioned recombinant microorganism and recovering the target protein secreted from the microorganism. An example of the specific production method will be described by way of examples.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Transformed Strains Using Construct

Each construct was prepared by PCR using an expression cassette as a template. The primers used were p1, p4, p5, and p6 primers in common, and P2 and p3 primers were prepared according to each construct. A first construct (FIG. 1) comprising a promoter and a pectetiria gene thus prepared, a second construct (FIG. 2) and a promoter comprising a promoter, a secretory signal peptide and a pectatitriase gene, Three kinds of recombinant strains were prepared by transforming the Alternaria ja Brassica strain into a third construct including a secretion signal peptide, an effector protein, and a pectatease gene.

Experimental Example  1. Recombinant strains Colony  Growth comparison

Colonies were observed after incubating three kinds of recombinant strains prepared in Example 1 in a PDA medium and 0.2 x PDA medium containing an additional carbon source and pectin (FIG. 6).

When cultured in the optimum medium of PDA medium, all three different strains were confirmed to exhibit similar colony size. Looking at the middle and lower panels, which contain low nutrients such as major carbon sources or pectins, it can be seen that the colonies are of different sizes. At this time, it can be seen that the strain produced by the third construct is most actively grown.

Experimental Example  2. from recombinant strains Obtained  Protein SDS -PAGE analysis

SDS-PAGE analysis of the protein expressed from the three strains prepared in Example 1. (Fig. 7). Lane 1 relates to the strain prepared with the first construct, which expressed the pectateriase protein without secretion signal peptide but secreted less than 1% of the total amount of secreted protein. Lane 2 relates to a strain prepared from the second construct, which expressed a pectate triase protein having a secretory signal peptide at the N-terminus. Most of the purified protein corresponds to the target protein, but most of it was degraded.

Lane 3 expressed the pectateriase protein with secretory signal peptide and effector protein at the N-terminus. The size of the protein was larger than the protein without the effector domain, and the protein bands were separated, indicating that the protein was stably expressed in the culture medium.

Experimental Example 3 Analysis of Total Amount of Protein Obtained from Recombinant Strain

Three strains prepared in Example 1 were grown for 3 days with replacement of fresh medium every 24 hours. After 72 hours of incubation, 1x GYEB medium was replaced with concentrated culture medium and incubated for 72 hours. As shown in FIG. 8, it can be seen that the protein production of the strain into which the third construct is introduced is significantly higher.

As mentioned above, although an embodiment of the present invention has been described, those of ordinary skill in the art may add, change, delete or add components within the scope not departing from the spirit of the present invention described in the claims. The present invention may be modified and changed in various ways, etc., which will also be included within the scope of the present invention.

Claims (9)

Promoter; Nucleic acids encoding secretory signal peptides comprising the amino acid sequence of SEQ ID NO: 1; And an expression cassette of the protein of interest, comprising a gene of the protein of interest.
The method of claim 1,
The nucleic acid encoding the secretion signal peptide comprises the nucleotide sequence of SEQ ID NO: 2, the expression cassette of the protein of interest.
The method of claim 1,
An expression cassette of a protein of interest, further comprising a nucleic acid encoding an effector protein comprising the amino acid sequence of SEQ ID NO: 3.
The method of claim 3,
The nucleic acid encoding the effector protein comprises a nucleotide sequence of SEQ ID NO: 4, the expression cassette of the protein of interest.
The method of claim 1,
The target protein is any one selected from the group consisting of pectate lyase, laccase, phytase, and combinations thereof, expression cassette of the target protein.
The method of claim 1,
An expression cassette of the target protein, further comprising the gene of the selection marker.
A recombinant microorganism into which the expression cassette according to any one of claims 1 to 6 is introduced.
The method of claim 7, wherein
The microorganism is Alternaria brassicicola (Alternaria brassicicola), recombinant microorganism.
Culturing the recombinant microorganism according to claim 8; And
Recovering the target protein secreted from the microorganism, a method for producing a target protein.

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