WO2023171857A1 - Glycogen operon expression cassette comprising maltose promoter - Google Patents

Abstract

The present invention relates to a glycogen operon expression cassette comprising a maltose promoter. According to a method for producing an expression cassette, a transformed prokaryotic host cell, and glycogen, according to one aspect, highly-soluble glycogen can be mass-produced and thus can be used in various industrial groups.

Description

Glycogen operon expression cassette containing maltose promoter

It relates to a glycogen operon expression cassette containing a maltose promoter.

Glycogen is a homopolysaccharide composed of glucose, like starch, a storage polysaccharide in plants. Glycogen has a polymer structure in which D-glucose is bound by α-1,4 glycosidic bonds, and has a highly entangled network structure with one side chain residue by α-1,6 glycosidic bonds per 8 to 10 glucose residues. has.

Glycogen is known as a storage polysaccharide in animals. In animals, it is known to exist in a granular state (glycogen granules) in most cells, especially in liver and muscle. Muscle glycogen is the energy source for muscle contraction, and liver glycogen is used to maintain blood sugar during fasting. Therefore, these differences in properties lead to differences in the function of glycogen. Muscle glycogen has a molecular weight of 1 to 2 million. Liver glycogen has a molecular weight of about 5 to 6 million, and sometimes has a molecular weight of up to 20 million.

Glycogen is a storage polysaccharide in animals, and is also known to have the effect of improving liver function. There are also reports that glycogen extracted from squid and scallops exhibits strong antitumor activity (Takata, Y. et al., J. Mar, Biotech ., 6:208-213, 1998). This glycogen has utility as a new material for functional foods, and development is underway to apply it. Also as an emollient and hydrating agent as described in JP-A-62-178 505 and JP-A-63-290 809, as an anti-aging agent as described in US 5.093.109 and JP-A-2003-335651, in WO99/47120 It is also known for use in the cosmetic industry as a wetting agent and lubricant in ophthalmic solutions, as described in .

Meanwhile, various systems have been described for heterologous expression of nucleic acids encoding, for example, structural genes in prokaryotic systems. For this purpose, the host is transformed using a vector containing the nucleic acid sequence of the structural gene of interest operably linked to a promoter, which is the nucleic acid sequence that allows the structural gene to be transcribed. By inducing by an appropriate method, the promoter is activated and causes the structural gene to be transcribed.

The present inventors have completed a prokaryotic system capable of mass producing glycogen for use in various industries as described above.

One aspect is to provide an expression cassette comprising a glycogen operon in which the promoter of the glycogen operon is replaced with a maltose promoter or a maltose promoter is inserted.

Another aspect is to provide a prokaryotic host cell transformed with the expression cassette.

Another aspect is to provide a method of producing glycogen by culturing the prokaryotic host cell.

One aspect provides an expression cassette comprising a glycogen operon in which the promoter of the glycogen operon is replaced with a maltose promoter or a maltose promoter is inserted.

As used herein, the term “glycogen operon (glg operon)” refers to a group of genes encoding enzymes involved in synthesizing glycogen, and includes structural genes and promoters.

In one embodiment, the glycogen operon may be derived from Escherichia coli ( E. coli ).

The glycogen operon may be composed of the glgBX and glgCAP operons. Therefore, the promoter of the glycogen operon may be the glgBX or glgCAP promoter.

In one embodiment, the maltose promoter may be the malPQ promoter.

In one embodiment, the maltose promoter may include the base sequence of SEQ ID NO: 1. Specifically, a base having homology or identity of at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 1 It may include a sequence. In addition, if it is a nucleotide sequence that has such homology or identity and exhibits a function corresponding to the maltose promoter, it is obvious that the maltose promoter having a nucleotide sequence in which some sequences are deleted, modified, substituted, or added is also included within the scope of the present application.

As used herein, the term “promoter” refers to a nucleic acid sequence that controls the expression of a transcription unit. 'Promoter region' refers to a regulatory region that can bind RNA polymerase within the cell and initiate transcription of the downstream (3' direction) coding region. Within the promoter region, protein binding regions (consensus sequences) responsible for binding RNA polymerase will be found, such as the putative -35th region and the Pribnow box (-10th region). Additionally, the promoter region may include a transcription initiation site and binding sites for regulatory proteins.

In one embodiment, a gene selected from the group consisting of glgB, glgX, glgC, glgA, glgP, and combinations thereof may be removed from the glycogen operon.

As used herein, the term "removal" of a gene encompasses mutating, substituting, or deleting some or all of the bases of the gene, or introducing some bases to prevent the gene from being expressed, or preventing it from exhibiting enzymatic activity even if expressed. As a concept, it includes anything that blocks the biosynthetic pathway in which the enzyme of that gene is involved.

The glgB gene encodes glycogen branching enzyme (synonym B3432). The glgB gene (nucleotide positions: 3,571,525 to 3,569,339; GenBank accession number NC_000913.2; gi: 49175990) is located between the glgX and asd genes on the chromosome of E. coli K-12. The base sequence of the glgB gene can be presented as SEQ ID NO: 4.

The glgX gene encodes glycogen phosphorylase-limit dextrin α-1,6-glucohydrolase (synonym B3431, GlyX). The glgX gene (nucleotide positions: 3,569,342 to 3,567,369; GenBank accession number NC_000913.2; gi: 49175990) is located between the glgC and glgB genes on the chromosome of E. coli K-12. The base sequence of the glgX gene can be presented as SEQ ID NO: 5.

The glgC gene encodes glucose-1-phosphate adenylyltransferase (synonym B3430). The glgC gene (gi: 16131304; nucleotides complementary to nucleotides 3566056 to 3567351 under GenBank accession number NC_000913.2) is located between the glgA and glgX genes on the chromosome of E. coli K-12. The base sequence of the glgC gene can be presented as SEQ ID NO: 6.

The glgA gene encodes a subunit of glycogen synthase (synonym B3429). The glgA gene (nucleotide positions: 3,566,056 to 3,564,623; GenBank accession number NC_000913.2; gi: 49175990) is located between the glgP and glgC genes on the chromosome of E. coli K-12. The base sequence of the glgA gene can be presented as SEQ ID NO: 7.

The glgP gene encodes glycogen phosphorylase/glycogen-maltotetraosephosphorylase (synonym B3428, GlgY). The glgP gene (nucleotide positions: 3,564,604 to 3,562,157; GenBank accession number NC_000913.2; gi: 49175990) is located between the yzgL and glgA genes on the chromosome of E. coli K-12. The base sequence of the glgP gene can be presented as SEQ ID NO: 8.

In one embodiment, the glgBX promoter may be replaced with a maltose promoter.

In one embodiment, a maltose promoter may be inserted upstream of the glgCAP operon. Specifically, malPQ , a maltose promoter, may be inserted upstream of the glgCAP operon, or may be inserted between the glgBX operon and the glgCAP operon.

In one embodiment, a gene encoding a glycogen branching enzyme may be further inserted. The gene encoding the glycogen branching enzyme may be inserted downstream of the maltose promoter or upstream of the glgX gene.

In one embodiment, the gene encoding the glycogen branching enzyme may be derived from Vibrio vulnificus .

All of the genes mentioned above, such as genes encoding the maltose promoter, glgB, glgX, glgC, glgA, glgP, and glycogen branching enzymes, may be operably linked. Additionally, the genes may each be heterogeneous.

As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect expression of the encoding nucleic acid sequence. Specifically, such nucleic acids operably linked to each other may be linked directly, i.e., without additional elements or nucleic acid sequences in between, or indirectly with spacer sequences or other sequences therebetween. Operational linkage with a recombinant vector can be made using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be done using enzymes generally known in the art.

As used herein with reference to a gene, nucleotide or amino acid sequence or protein, the term "xenologous" is foreign to a given host cell, i.e., "exogenous," such as not found in nature; or refers to a compound that is naturally found in a given host cell, e.g., “endogenous,” but is “not naturally occurring,” e.g., in the context of a heterologous construct using a heterologous nucleic acid. Heterologous nucleotide sequences, such as those found endogenously, may also be produced non-naturally in cells, e.g., in quantities larger than expected or greater than those found naturally. A heterologous nucleotide sequence, or a nucleic acid comprising a heterologous nucleotide sequence, encodes a protein identical to that found endogenously, although it may possibly differ in sequence from the endogenous nucleotide sequence. Specifically, a heterologous nucleotide sequence is one that is not found in nature in the same relationship to a host cell (i.e., “not naturally associated”). Any recombinant or artificial nucleotide sequence is understood to be heterologous. Examples of heterologous polynucleotides or nucleic acid molecules include nucleotide sequences that are not naturally associated with a promoter, for example, to obtain a hybrid promoter, or are operably linked to a coding sequence as described herein. As a result, hybrid or chimeric polynucleotides can be obtained.

As used herein, the term “expression cassette” may be a gene construct unit containing essential regulatory elements operably linked to the introduced gene so that it is expressed when present in the cells of an individual. The expression cassette may be, for example, in the form of an expression vector, but is not limited thereto, and may include any minimal genetic construct capable of expressing the target gene to be introduced.

The expression cassette can be prepared and purified using standard recombinant DNA techniques. The type of the expression cassette is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells of prokaryotic and eukaryotic cells. The expression cassette may include a promoter, an initiation codon, a gene encoding the target protein, or a termination codon, and in addition, DNA encoding a signal peptide, an enhancer sequence, and untranslated regions on the 5' and 3' sides of the target gene. , selection marker region, or replicable unit, etc. may be appropriately included. In addition, the expression cassette includes a mono-cistronic vector containing a polynucleotide encoding one protein, and a polycistronic vector containing polynucleotides encoding two or more recombinant proteins. It can be done, but is not limited to this.

As used herein, the term “vector” refers to a DNA preparation containing the base sequence of a polynucleotide encoding the target protein operably linked to a suitable control sequence to enable expression of the target protein in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence to regulate such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that regulate the termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.

The vector used in one embodiment is not particularly limited as long as it can be expressed in a host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR, pUC, and pBluescriptII series can be used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used, but are not limited thereto.

The usable vector is not particularly limited, and known expression vectors can be used. Additionally, a gene or polynucleotide encoding a target protein can be inserted into a chromosome using a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome has been inserted. A selection marker is used to select cells transformed with a vector, i.e., to confirm the insertion of the target polynucleotide, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. Markers that give may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so transformed cells can be selected.

Another aspect provides a prokaryotic host cell transformed with the expression cassette.

As used herein, the term “transformation” refers to introducing a vector or expression cassette containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed within the host cell. do. As long as the transformed polynucleotide can be expressed in the host cell, it can include both of these, regardless of whether it is inserted into the chromosome of the host cell or located outside the chromosome. Additionally, the polynucleotide includes DNA and/or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

As used herein, the term “prokaryotic host cell” refers to any bacterial host, and in particular it refers to a bacterial host cell. In principle, there are no restrictions on the choice of bacterial host cell. The bacterial host cell may advantageously be a true bacterium (Gram positive or Gram negative) or a native bacterium as long as it allows genetic manipulation for insertion of the gene of interest for site-specific integration. Bacterial host cells can be cultured on a manufacturing scale, and the host cells have the property of being able to be cultured at high cell densities. Examples of bacterial host cells that have been found to be suitable for recombinant industrial protein production include Escherichia coli , Bacillus subtilis , Pseudomonas fluorescens , as well as variants thereof and Lactococcus lactis . It is a strain. In one embodiment, the prokaryotic host cell may be Escherichia coli.

As used herein, the terms “host,” “host cell,” and “recombinant host cell” are used interchangeably and are used to refer to a prokaryotic cell into which one or more vectors or expression cassettes of one embodiment are introduced. These terms not only refer to specific target cells, but also refer to the progeny or potential descendants of these cells. Such progeny may not actually be identical to the parent cell since certain modifications may occur in the progeny due to mutations or environmental influences, but such cases are also included within the scope of the term used herein.

In one embodiment, the host cell may be capable of mass producing glycogen and protein. The present inventors confirmed that the host cell mass-produces glycogen and protein by replacing the promoter of the glycogen operon with a maltose promoter or inserting a maltose promoter.

As an example, the recombinant host with increased glycogen and protein production ability has an increase of about 1% or more, specifically about 1% or more, about 2.5% or more, or about 5% compared to the glycogen and protein production ability of the parent strain or unmodified microorganism before mutation. or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 26% or more, about 27% or more, about 27.5% or more, about 30% or more, about 35% or more, about 40% or more or more, about 45% or more, about 50% or more, about 55% or more, or about 60% or more (the upper limit is not particularly limited and may be, for example, about 200% or less, about 150% or less, or about 100% or less). However, it is not limited thereto as long as it has a positive increase compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain with increased production capacity has a glycogen and protein production capacity of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, or about 1.06 times more than the parent strain or unmodified microorganism before mutation. times or more, approximately 1.07 times or more, approximately 1.08 times or more, approximately 1.09 times or more, approximately 1.10 times or more, approximately 1.11 times or more, approximately 1.12 times or more, approximately 1.13 times or more, approximately 1.14 times or more, approximately 1.15 times or more, approximately 1.16 times or more Two times or more, about 1.17 times more, about 1.18 times more, about 1.19 times more, about 1.20 times more, about 1.5 times more, about 2.0 times more, about 2.5 times more, about 3.0 times more, about 4.0 times more, about 5.0 times more more times, about 10 times more, about 15 times more, about 20 times more, about 25 times more, about 30 times more, about 35 times more, about 40 times more, about 45 times more, about 50 times more, about 55 times more It may be increased by more than two times or more than about 60 times (the upper limit is not particularly limited, for example, it may be about 100 times or less or about 80 times or less), but is not limited thereto. The term "about" is a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range that are equivalent or similar to the value that follows the term "about." Not limited. In addition, even if “about” is not written in front of the number in this specification, it is the same as “about” written in front of the number.

As used herein, the term “non-modified microorganism” does not exclude hosts that contain mutations that may occur naturally in microorganisms, and are either wild-type hosts or natural hosts themselves, or are characterized by genetic mutations caused by natural or artificial factors. It may refer to the host before being changed. The host may be a strain. For example, the unmodified microorganism may refer to a strain in which the genes encoding the maltose promoter and/or glycogen branching enzyme described herein are not introduced or are introduced. The “non-transformed microorganism” refers to “pre-transformed host”, “pre-transformed strain”, “pre-transformed microorganism”, “non-mutated host”, “non-mutated strain”, “non-mutated microorganism”, “non-transformed host”, “ It can be used interchangeably with “unmodified strain” or “reference microorganism.”

In one embodiment, the produced glycogen may have a distribution ratio of side chains having a degree of polymerization (DP) of 5 or less of at least 30% of the total produced glycogen content. Specifically, the distribution ratio of side chains with a degree of polymerization of 5 or less may be 30 to 60%, 30 to 55%, 30 to 50%, 30 to 45%, or 30 to 40% of the total glycogen content produced. As the distribution ratio of side chains with a degree of polymerization of 5 or less increases, water solubility may increase.

Another aspect includes culturing the prokaryotic host cells in a medium; and recovering glycogen from cultured prokaryotic host cells or medium.

As used herein, the term “culture” refers to growing the prokaryotic host cell under appropriately controlled environmental conditions. The culture process can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and/or fed-batch, but is not limited thereto.

As used herein, the term "medium" refers to a material that is mainly mixed with nutrients necessary for cultivating prokaryotic host cells, and supplies nutrients and growth factors, including water, which are essential for survival and development. Specifically, the medium and other culture conditions used for cultivating the prokaryotic host cells may be any medium used for cultivating ordinary microorganisms without particular limitation, but the prokaryotic host cells must be supplied with an appropriate carbon source, nitrogen source, personnel, and inorganic substances. It can be cultured under aerobic conditions in a typical medium containing compounds, amino acids, and/or vitamins, while controlling temperature, pH, etc.

The carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; Sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; Amino acids such as glutamic acid, methionine, lysine, etc. may be included. Additionally, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice bran, cassava, bagasse and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e. converted to reducing sugars). Carbohydrates such as molasses) can be used, and various other carbon sources in an appropriate amount can be used without limitation. These carbon sources may be used alone or in combination of two or more types, but are not limited thereto.

The nitrogen source includes inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used These nitrogen sources may be used individually or in combination of two or more types, but are not limited thereto.

The agent may include monopotassium phosphate, dipotassium phosphate, or a corresponding sodium-containing salt. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and/or appropriate precursors. These components or precursors can be added to the medium batchwise or continuously. However, it is not limited to this.

Additionally, during the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during culturing, foam generation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium, or to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, and is limited thereto. That is not the case.

In the above culture, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

Glycogen and/or protein produced by the culture may be secreted into the medium or remain within the cell.

The glycogen production method includes preparing the prokaryotic host cells, preparing a medium for culturing the prokaryotic host cells, or a combination thereof (regardless of the order), for example, before the culturing step. , may be additionally included.

In the step of recovering glycogen and/or protein from the culture medium (medium in which the culture was performed) or the prokaryotic host cells, the recovery may be performed by a method of cultivating the prokaryotic host cells, for example, batch, continuous or fed-batch. Depending on the culture method, etc., the desired glycogen and/or protein may be collected using a suitable method known in the art. For example, centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity. Various chromatographies such as chromatography, HPLC, or a combination of these methods can be used, and the desired glycogen and/or protein can be recovered from the medium or prokaryotic host cells using a suitable method known in the art.

Additionally, the production method may additionally include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the production method includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step. , but is not limited to this.

According to the expression cassette, transformed prokaryotic host cell, and glycogen production method according to one aspect, highly soluble glycogen can be mass-produced and utilized in various industries.

Figure 1 is a schematic diagram of the modified glycogen operon.

Figure 2 is a graph showing the growth rate and glycogen synthesis rate according to culture of the existing parent strain E. coli K-12.

Figure 3 is a graph showing the growth rate according to culture of recombinant strain CMHX1.

Figure 4 is a graph showing the growth rate according to culture of the recombinant strain CMHX2.

Figure 5 is a graph showing the growth rate according to culture of the general strain E. coli MC1061.

Figure 6 is a graph showing the growth rate according to culture of the recombinant strain CMHX2.

Figure 7 is a graph showing the side chain distribution ratio by degree of polymerization of existing polysaccharides Amylopectin (AP) and Cobiosa, polysaccharides produced by the existing parent strain E. coli K-12, and polysaccharides produced by recombinant strains CMHX1 and CMHX2.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of recombinant strain

A recombinant strain with increased expression of glycogen with high water solubility characteristics was produced as follows.

Specifically, as shown in Figure 1, the glycogen promoter and glgB gene of Escherichia coli K-12 strain were removed, and the malPQ promoter from E. coli K-12 and Vibrio vulnificus glycogen branching enzyme were removed. , VvGBE) expression gene was inserted. The linear gene to be inserted is a kanamycin cassette containing the malPQ promoter, VvGBE, FRT flanking region (FLP recognition target), and homology, amplified by polymerase chain reaction (PCR) from pKD 13 plasmid, and cloned into the p6xHis_119 vector using an infusion cloning kit. It was recombinant and amplified using PCR. Removal and insertion of genes were carried out according to the one step knock-out principle. To insert the target gene, an electroporation machine was used by transforming the PKD 46 plasmid into the strain to induce expression of the λ-red-recombinase enzyme. Constructed mutants were selected by plates containing antibiotics. The FLP region was removed by pCP20 plasmid. The target site where the gene is inserted is the glg operon ( glgBXCAP ), which is directly involved in glycogen formation. This operon consists of two operons, glgBX and glgCAP . The CMHX1 strain is a mutant strain in which the promoters of the glgB and glgBX operons in the glgBXCAP operon, as shown in Figure 1, are replaced with the malPQ promoter, and in the case of CMHX2, the promoter of the glgCAP operon is additionally replaced with the malPQ promoter, as shown in Figure 1. am. The nucleotide sequence of the malPQ promoter is shown in SEQ ID NO: 1, the nucleotide sequence of VvGBE is shown in SEQ ID NO: 2, the nucleotide sequence of the kanamycin resistance gene is shown in SEQ ID NO: 3, and the nucleotide sequence of glgB is shown in SEQ ID NO: 4. , the base sequence of glg

Experimental Example 1. Glycogen production analysis

The glycogen production of the recombinant strain prepared in Example 1 was confirmed.

Specifically, high-concentration cells were cultured using maltodextrin carbon source suitable for promoter activity replaced with R2-restricted medium and supplying carbon source by exponential feeding method at optimal specific growth rate. During the culture process, 50ml of culture medium was harvested and mixed with 50mM sodium acetate buffer solution, pH 4.0. Afterwards, DNA and proteins were inactivated through ultra sonicator and heat treatment steps, and glycogen was precipitated using an acetate-ethanol precipitation method using over 95% ethanol and completely dried. Afterwards, glycogen was completely dissolved in distilled water at a concentration of 10 mg/ml. After reaction using 5% phenol solution and 95% sulfuric acid, the total sugar was measured using the phenol sulfuric method by measuring the absorbance at 470 nm with a microplate reader device. Then, the same amount of culture medium was harvested, the bacteria were separated, washed with NaCl, and dry cell weight (DCW) was measured. Glycogen production was measured using DCW compared to glycogen quantitative analysis.

As a result, as shown in Figures 2 to 4 and Table 1, the CMHX1 mutant showed cell lysis due to acetate overflow due to cell growth and glycogen accumulation. On the other hand, the final CMHX2 mutant had an improved acetate excess phenomenon and grew stably up to OD 211, which was higher than the OD 600nm value of 171 for the CMHX1 mutant. In addition, it grew to an intracellular glycogen accumulation rate of more than 50% and a cell mass (dry cell weight: DCW) of more than 55 g/L. This indicates a glycogen accumulation rate that is approximately 68% higher than that of the CMHX1 mutant, and an accumulation rate that is more than 40 times that of the parent strain, E.coli K-12.

host	Sample	Glycogen (g)/DCW (g)*100 (%)	Glycogen (g/L)	DCW(g/L)
E.coli K-12	Before N-limit	1.12 ± 0.48	0.48 ± 0.18	43.21 ± 0.61
	AfterN-limit	1.4 ± 0.61	0.56 ± 0.68	39.75 ± 0.73
CMHX1	Before N-limit	35±0.51	17.07 ± 0.13	48.5 ± 1.12
	AfterN-limit	33 ± 0.24	15.02 ± 0.05	45.94 ± 0.38
CMHX2	Before N-limit	51 ± 0.13	27.89 ± 0.73	56.29 ± 0.54
	AfterN-limit	45±0.11	26.28 ± 0.24	58.33 ± 0.38

Experimental Example 2. Protein production analysis

To confirm whether the recombinant strain prepared in Example 1 can be used as a protein expression strain, an experiment was performed as follows.

Specifically, first, the CMHX2 strain, which showed excellent results in terms of growth and glycogen accumulation, was transformed using a p6xHis 119 expression vector containing a gene expressing a specific protein using heat treatment. Colonies were selected using solid plate medium containing antibiotics. Afterwards, the culture was cultured to an O.D. of 600 nm of 56 under the established culture conditions, and then the culture medium and bacteria were separated using centrifugation. After dissolving the isolated bacteria in a buffer solution, the cells were disrupted using an ultra sonicator to extract the target protein. And, taking advantage of the heat-resistant nature of the target protein, the enzyme was purified through step-by-step heat treatment. After purification, the protein was quantitatively analyzed using the bradford assay, and the protein activity was calculated according to the absorbance after reacting with amylopectin as a standard using lugol solution.

As a result, as shown in Figures 5, 6, and Table 2, it was confirmed that the specific activity was approximately twice as different compared to the existing protein expression strain, and the yield was also confirmed to be increased. These results indicate that even as a protein expression strain, it is superior to existing strains.

host	purification steps	Total protein (mg)	Total activity (units)	Specific activity (units/mg)	transference number (%)
MC1061	cell extract	1253.3	1416.12	1.13	100
	heat treatment	112.12	431.35	3.85	30.46
CMHX2	cell extract	1782.46	2491.36	1.40	100
	heat treatment	200.38	1449.97	7.24	58.2

Experimental Example 3. Analysis of side chain distribution of produced glycogen

The side chain distribution of glycogen produced by the recombinant strain prepared in Example 1 was analyzed as follows.

Specifically, the reaction was performed at 40°C for 72 hours in 50mM sodium acetate buffer at pH 4.0 to a final concentration of 2mg/ml using isoamylase enzyme (1 unit/ml), which cuts the side chain of glycogen. Cobiosa, glycogen derived from mussel, was also treated in the same way, and amylopectin was dissolved and reacted in DMSO solution. Afterwards, the supernatant was separated by centrifugation through a heat treatment process to inactivate the enzyme, filtered through a 0.2μm membrane, and analyzed using a high performance anion-exchange chromatography (HPAEC) device. Carbopac PA-1 (250x4 mm, Dionex, Sunnyvale, CA, USA) was used as the stationary phase column, and the column was equilibrated with 150mM NaOH buffer solution, and a buffer solution with a concentration of 150mM NaOH and 600mM sodium acetate was added at 1 ml/min. The sample was analyzed while flowing.

As a result, as shown in Figure 7 and Table 3, it was confirmed that it had a short branched structure with a degree of polymerization of 5 or less compared to other polysaccharides. These results indicate that the recombinant strain can produce structurally changed polysaccharides.

Sample	DPn *	Side chain distribution (%)
		degree of polymerization
		DP ≤ 5	5 < DP ≤ 12	13 ≤ DP
Amylopectin (AP)	14.90	0.26	25.51	72.19
Cobiosa	8.26	18.51	39.9	41.59
E.coli K-12	9.08	9.7	54.66	41.59
CMH1	7.31	19.21	68.9	11.86
CMHX1	6.37	29.78	62.85	7.37
CMHX2	6.21	31.44	63.56	5

* Number-average degree of polymerization: DPn = ∑ (peak area of each chain)/∑ (peak area of each chain/chain length of that chain)

Experimental Example 4. Analysis of water solubility of produced glycogen

In order to confirm the change in water solubility of the glycogen produced by the recombinant strain prepared in Example 1 due to the structural form as shown in Experimental Example 3, the water solubility of the produced glycogen was analyzed as follows.

Specifically, the excess glycogen produced was dissolved in distilled water at a concentration of 10 mg/ml for 3 hours at room temperature. After reaction using 5% phenol solution and 95% sulfuric acid, the total sugar was measured using the phenol sulfuric method by measuring the absorbance at 470 nm with a microplate reader device.

As a result, as shown in Table 4, the glycogen of the parent strain, E. coli K-12, was more than 200 times more water-soluble than amylopectin (AP) present in starch. Glycogen produced from the final mutant, CMHX2, showed a water solubility that was 68% higher than that of the parent strain, and a value that was more than 40,000 times higher than that of AP. These results indicate that the glycogen produced by the recombinant strain has high water solubility and can be utilized in various industries based on these characteristics.

Sample	Water solubility (mg/ml)
Amylopectin (AP)	0.81 ± 0.12
E.coli K-12	220 ± 0.49
CMHX2	343.85 ± 0.87

Claims (17)

1. An expression cassette containing the glycogen operon in which the promoter of the glycogen operon is replaced with a maltose promoter or a maltose promoter is inserted.
2. The expression cassette according to claim 1, wherein the glycogen operon is derived from Escherichia coli ( E. coli ).
3. The expression cassette according to claim 1, wherein the maltose promoter is derived from Escherichia coli ( E. coli ).
4. The expression cassette according to claim 1, wherein the glycogen operon is composed of the glgBX and glgCAP operons.
5. The expression cassette according to claim 1, wherein the promoter of the glycogen operon is a glgB , glgBX or glgCAP promoter.
6. The expression cassette of claim 1, wherein the maltose promoter is a malPQ promoter.
7. The expression cassette according to claim 1, wherein the maltose promoter consists of the base sequence of SEQ ID NO: 1.
8. The expression cassette according to claim 1, wherein a gene selected from the group consisting of glgB, glgX, glgC, glgA, glgP, and combinations thereof has been removed.
9. The expression cassette of claim 5, wherein the glgB , glgBX or glgCAP promoter is replaced with the malPQ promoter.
10. The expression cassette according to claim 4, wherein a maltose promoter is inserted upstream of the glgCAP operon.
11. The expression cassette according to claim 1, wherein a gene encoding a glycogen branching enzyme is further inserted.
12. The expression cassette of claim 11, wherein the gene encoding the glycogen branching enzyme is derived from Vibrio vulnificus .
13. A prokaryotic host cell transformed with the expression cassette of claim 1.
14. The prokaryotic host cell according to claim 13, wherein the prokaryotic host cell is Escherichia coli.
15. The prokaryotic host cell according to claim 13, wherein the host cell is capable of mass producing glycogen and protein.
16. The prokaryotic host cell of claim 15, wherein the produced glycogen has a distribution ratio of side chains having a degree of polymerization (DP) of 5 or less of at least 30% of the total produced glycogen content.
17. Culturing the prokaryotic host cell of claim 13 in a medium; and recovering glycogen from cultured prokaryotic host cells or medium.

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