WO2025079998A1 - Corynebacterium sp. strain with carrageenan degradation and galactose utilization abilities introduced thereinto and method for producing histidine from seaweed using same - Google Patents

Abstract

The present invention relates to a Corynebacterium sp. strain with enhanced histidine production ability and introduced galactose utilization ability, and a method for producing histidine from seaweed using same. The transformed Corynebacterium sp. strain of the present invention exhibits excellent histidine production ability through the enhancement of the galactose utilization and histidine production pathways, thereby producing amino acids on the basis of an eco-friendly process. Additionally, since galactose obtained from the degradation of carrageenan, a major component of seaweed, is utilized, the invention provides the advantage of utilizing seaweed as a biomass resource.

Description

A strain of Corynebacterium spp. with carrageenan decomposition ability and galactose utilization ability and a method for producing histidine from seaweed using the same

The present invention relates to a Corynebacterium strain having improved histidine production ability and a histidine production method using the same.

Currently, research on renewable energy such as bio, hydrogen, and solar energy is being actively conducted worldwide to develop stable and environmentally friendly energy that can replace oil and coal due to the climate change agreement and strengthened environmental regulations. The raw materials for bioenergy production are mainly grains such as sugarcane and corn, and woody resources that are forestry and agricultural byproducts. Research and production on this are being conducted in leading countries in renewable energy production such as the United States, Brazil, and Germany. However, the production of biofuel using grains and woody resources is limited due to food shortage problems, limited cultivation area depending on the type of crop, difficulty in supplying nutrients, and economic feasibility due to additional pretreatment processes for hemicellulose and lignin other than cellulose.

To solve these problems, marine algae, whose cell walls are composed of many fibers and various polysaccharides, have recently attracted attention as a new bioenergy source. The countries that mainly produce seaweed are Asian countries such as Korea, Japan, China, and Indonesia, and the area of the sea that can be cultivated is larger than the land. In addition, seaweed grows faster than wood and plant cellulose, absorbs carbon dioxide in the air through photosynthesis, and thus has the effect of reducing greenhouse gases. In addition, the lignin content in seaweed is low, which simplifies the pretreatment process. In addition, these large seaweeds contain about 60-95% water, and more than 50% of the remaining components are carbohydrates, so they have great potential as a biofuel source. Meanwhile, in Korea and Japan, laver (mainly Porphyra yezoensis ) cultivation among red algae is actively being carried out, and red algae accounts for more than half of the seaweed native to Korea. In addition, since it has a wider habitat than brown or green algae, and grows naturally from shallow waters to deep waters where sunlight reaches, it has a greater number of species than other algae, making it excellent in terms of raw material supply and demand, and it has an advantage in terms of energy conversion as it contains a lot of carbohydrates, that is, monosaccharide conversion substances that microorganisms can convert into ethanol, compared to other seaweeds. Depending on the species, large seaweeds have various types and bonding forms of polysaccharides that they compose. Among them, red algae are composed of cellulose, a structural polysaccharide of the inner layer of the cell wall, and viscous polysaccharides with sulfate groups in the outer layer and between cells, such as agar and carrageenan, as well as xylan and mannan. In particular, agar and carrageenan are representative at 28% and 24%, respectively, and cellulose is not that abundant at 3-16%.

Up to now, agar and carrageenan, which are marine polysaccharides, have been extracted from red algae through processing methods such as alkaline, acid, or enzyme treatment, and have been widely used industrially as food and cosmetic additives and as healthy food resources. However, due to the complex structure of red algae that is difficult to decompose, it is difficult to use it as a substrate for biofuel production, and the disposal of byproducts and wastes other than the currently used materials remains a problem. As a member state of OECD, Korea is obligated to follow the '96 Protocol' adopted by the London Convention, which regulates ocean dumping of wastes, and therefore, it cannot be ruled out that there may be a crisis due to the disposal of discarded seaweed after 2012.

Meanwhile, histidine is one of the twenty standard amino acids present in proteins. Histidine, an essential amino acid for humans, is an essential component of histamine synthesis, which promotes capillary permeability in the circulatory system. As a histamine precursor, histidine enhances sexual function and promotes gastric acid secretion in hypochlorhydria, thereby enhancing digestion, raising blood pressure, and strengthening the muscles of the bronchial tubes in the lungs. It is also essential for the growth and repair of tissues, so it is essential for the growth of infants. Histidine is also an amino acid necessary for patients in the recovery period to enhance immunity, so it is also valuable as an amino acid supplement, like other essential amino acids.

Accordingly, the inventors of the present invention confirmed that a transformed Corynebacterium strain in which genes related to the histidine production pathway, carrageenan decomposition, and galactose utilization pathway are overexpressed or introduced has excellent carrageenan decomposition ability and histidine production ability, and completed the present invention.

The purpose of the present invention is to provide a transformed Corynebacterium strain overexpressing a gene encoding ATP phosphoribosyltransferase, a gene encoding Glucose-6-phosphate 1-dehydrogenase, a gene encoding 6-phosphogluconate dehydrogenase, a gene encoding Ribose-phosphate pyrophosphokinase, a gene encoding Histidinol dehydrogenase, a gene encoding Nucleoside diphosphate kinase, a gene encoding Adenylate kinase, and a gene encoding Bifunctional purine biosynthesis protein PurH.

In addition, another object of the present invention is to provide a composition for producing histidine comprising the transformed Corynebacterium spp. strain.

In addition, another object of the present invention is to provide a method for producing histidine from a Corynebacterium spp. strain, which comprises a step of culturing the transformed Corynebacterium spp. strain.

In addition, another object of the present invention is to provide a composition for producing histidine, comprising seaweed, carrageenan decomposing enzyme and the transformed Corynebacterium spp. strain.

In addition, another object of the present invention is to provide a method for producing histidine using seaweed.

To achieve the above objects, the present invention provides a transformed Corynebacterium strain overexpressing a gene encoding ATP phosphoribosyltransferase, a gene encoding Glucose-6-phosphate 1-dehydrogenase, a gene encoding 6-phosphogluconate dehydrogenase, a gene encoding Ribose-phosphate pyrophosphokinase, a gene encoding Histidinol dehydrogenase, a gene encoding Nucleoside diphosphate kinase, a gene encoding Adenylate kinase, and a gene encoding Bifunctional purine biosynthesis protein PurH.

Next, the present invention provides a composition for producing histidine comprising the transformed Corynebacterium spp. strain.

Furthermore, the present invention provides a method for producing histidine from a Corynebacterium spp. strain, comprising a step of culturing the transformed Corynebacterium spp. strain.

In addition, a composition for producing histidine is provided, comprising seaweed, carrageenan decomposing enzyme and the transformed Corynebacterium spp. strain.

Finally, the present invention provides a method for producing histidine using seaweed, comprising the steps of: saccharifying seaweed using a carrageenan decomposing enzyme to produce a seaweed hydrolysate; and inoculating and fermenting a transformed Corynebacterium spp. strain into a medium containing the seaweed hydrolysate.

The transformed Corynebacterium spp. strain of the present invention has excellent histidine production ability through strengthening the galactose utilization and histidine production pathways, and thus has the effect of producing amino acids based on an eco-friendly process. In addition, since it utilizes galactose obtained through the decomposition of carrageenan, which is a main component of seaweed, it has the advantage of being able to utilize seaweed as biomass.

FIG. 1 is a diagram simply showing a biosynthetic pathway for histidine production through carrageenan decomposition according to one embodiment of the present invention.

FIG. 2 is a diagram showing the results of confirming the histidine production ability of a transformed Corynebacterium strain with an enhanced histidine production pathway according to one embodiment of the present invention.

FIG. 3 is a diagram showing the results of confirming the carrageenan decomposition ability of Iduronate-2-sulfatase and Kappa-carrageenase in one embodiment of the present invention.

FIG. 4 is a diagram showing the results of confirming the galactose utilization ability and histidine production ability of a transformed Corynebacterium strain into which a galactose utilization pathway has been introduced according to one embodiment of the present invention.

FIG. 5 is a diagram showing the results of confirming the galactose utilization ability of a transformed Corynebacterium strain into which a galactose utilization pathway has been introduced and cultured under optimal culture conditions according to one embodiment of the present invention.

FIG. 6 is a diagram showing the results of confirming histidine production through galactose consumption by a transformed Corynebacterium strain cultured under optimal culture conditions and having a galactose utilization pathway introduced and pentose phosphate, ATP regeneration, and histidine biosynthesis pathways enhanced, in one embodiment of the present invention.

Hereinafter, the present invention will be described in detail with reference to the attached drawings as implementation examples of the present invention. However, the following implementation examples are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description thereof may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

In addition, the terms used in this specification are terms used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definitions of these terms should be made based on the contents throughout this specification. Throughout the specification, when a part is said to “include” a certain component, this does not mean that other components are excluded, but rather that other components can be further included, unless specifically stated otherwise.

Throughout this specification, '%' used to indicate the concentration of a particular substance, unless otherwise stated, is (w/w) % for solid/solid, (w/v) % for solid/liquid, and (v/v) % for liquid/liquid.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention relates to a transformed Corynebacterium strain overexpressing a gene encoding ATP phosphoribosyltransferase, a gene encoding Glucose-6-phosphate 1-dehydrogenase, a gene encoding 6-phosphogluconate dehydrogenase, a gene encoding Ribose-phosphate pyrophosphokinase, a gene encoding Histidinol dehydrogenase, a gene encoding Nucleoside diphosphate kinase, a gene encoding Adenylate kinase, and a gene encoding Bifunctional purine biosynthesis protein PurH.

In one embodiment of the present invention, the gene encoding the ATP phosphoribosyltransferase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 1. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 1, and is capable of expressing a protein exhibiting substantial ATP phosphoribosyltransferase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding Glucose-6-phosphate 1-dehydrogenase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 2. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 2, and is capable of expressing a protein exhibiting substantial Glucose-6-phosphate 1-dehydrogenase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the 6-phosphogluconate dehydrogenase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 3. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 3 and is capable of expressing a protein exhibiting substantial 6-phosphogluconate dehydrogenase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the Ribose-phosphate pyrophosphokinase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 4. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 4 and is capable of expressing a protein exhibiting substantial Ribose-phosphate pyrophosphokinase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the Histidinol dehydrogenase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 5. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 5, and is capable of expressing a protein exhibiting substantial Histidinol dehydrogenase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the nucleoside diphosphate kinase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 6. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 6 and is capable of expressing a protein exhibiting substantial nucleoside diphosphate kinase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the Adenylate kinase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 7. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 7, and is capable of expressing a protein exhibiting substantial Adenylate kinase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the Bifunctional purine biosynthesis protein PurH may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 8. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 8, and is capable of expressing a protein exhibiting substantial Bifunctional purine biosynthesis protein PurH activity, it may be included without limitation.

In one embodiment of the present invention, the transformed Corynebacterium strain may be one in which the tkt promoter of the tkt gene (Genbank ID: BAB98967.1) and the hisD promoter of the hisD gene (Genbank ID: BAB99495.1) are replaced with the H36 promoter, which is a highly expressive synthetic promoter, but is not limited thereto.

In one embodiment of the present invention, the H36 promoter may be represented by the base sequence of SEQ ID NO: 9, but is not limited thereto.

In one embodiment of the present invention, the transformed Corynebacterium strain may have an enhanced pentose phosphate pathway, a histidine synthetic pathway, and an ATP regeneration pathway, thereby increasing histidine production, but is not limited thereto.

In one embodiment of the present invention, the gene encoding the galactose transporter may be derived from Saccharomyces cerevisiae and may consist of a base sequence of SEQ ID NO: 10. In addition, if it is a base sequence that exhibits 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 10 and is capable of expressing a protein that substantially exhibits galactose transporter activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the galactose-1-phophate uridylytransferase may be derived from Escherichia coli and may consist of a base sequence of SEQ ID NO: 11. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 11 and is capable of expressing a protein exhibiting substantial galactose-1-phophate uridylytransferase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding Aldose-1-epimerase may be derived from Escherichia coli and may consist of a base sequence of SEQ ID NO: 12. In addition, if it is a base sequence that exhibits 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 12 and is capable of expressing a protein that substantially exhibits Aldose-1-epimerase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the galactokinase may be derived from Corynebacterium glutamicum and may consist of a base sequence of SEQ ID NO: 13. In addition, if it is a base sequence that exhibits 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 13 and is capable of expressing a protein that substantially exhibits galactokinase activity, it may be included without limitation.

In one embodiment of the present invention, the transformed Corynebacterium strain may have a galactose uptake system introduced, but is not limited thereto.

In one embodiment of the present invention, the Corynebacterium strain is Corynebacterium granulosum , Corynebacterium glucuronolyticum, Corynebacterium glutamicum , Coynebacterium glycinophilum, Corynebacterium diphtheriae, Corynebacterium lilium , Corynebacterium renale, Corynebacterium melassecola, Corynebacterium matruchotii , Corynebacterium mcginlay. Corynebacterium macginleyi , Corynebacterium minutissimum , Corynebacterium bovis , Corynebacterium thermoaminogenes , Corynebacterium pseudodiphtheriticum ( Corynebacterium hofmannii ), Corynebacterium pseudotuberculosis , Corynebacterium spec , Corynebacterium striatum , Corynebacterium amycolatum , Corynebacterium acetoacidophilum acetoacidophilum , Corynebacterium acetoglutamicum , Corynebacterium accolens, Corynebacterium aquaticum, Corynebacterium afermentans, Corynebacterium auris , Corynebacterium argentoratense, Corynebacterium alkanolyticum , Corynebacterium ammoniagenes , Corynebacterium efficiens , Corynebacterium ovis ), Corynebacterium urealyticum , Corynebacterium ulcerans, Corynebacterium xerosis , Corynebacterium jeikeium , Corynebacterium callunae , Corynebacterium tenuis, Corynebacterium parvum , Corynebacterium propinquum , Corynebacterium flavescens , Corynebacterium pyogenes , Corynebacterium halopytica The bacterium may be selected from the group consisting of Corynebacterium halofytica , Corynebacterium haemolyticum and Corynebacterium herculis, preferably Corynebacterium glutamicum , but is not limited thereto.

In one embodiment of the present invention, the transformed Corynebacterium strain may be a transformed microorganism in which a recombinant vector including a gene encoding ATP phosphoribosyltransferase, a gene encoding Glucose-6-phosphate 1-dehydrogenase, a gene encoding 6-phosphogluconate dehydrogenase, a gene encoding Ribose-phosphate pyrophosphokinase, a gene encoding Histidinol dehydrogenase, a gene encoding Nucleoside diphosphate kinase, a gene encoding Adenylate kinase, and a gene encoding Bifunctional purine biosynthesis protein PurH is introduced into the Corynebacterium strain; and a recombinant vector including a gene encoding galactose transporter, a gene encoding galactose-1-phophate uridylytransferase, a gene encoding Aldose-1-epimerase, and a gene encoding galactokinase.

The term "recombinant" as used herein refers to a cell that replicates a heterologous nucleic acid, expresses said nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. A recombinant cell can express a gene or gene fragment that is not found in the native form of said cell, either in sense or antisense form. A recombinant cell can also express a gene that is found in the native cell, but which is modified and has been reintroduced into the cell by artificial means.

In addition, the recombinant vector may include, but is not limited to, linear DNA, plasmid DNA, recombinant viral vector, etc., as long as it can be introduced into a host cell to deliver a target gene. The recombinant virus may be any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus, but is not limited thereto.

The term “transformation” used in the present invention means that the genetic properties of a microorganism change when the microorganism accepts DNA provided from outside.

In addition, the transformation may be performed using a suitable standard technique known in the art, such as, but not limited to, electroporation, electroinjection, microinjection, calcium phosphate co-precipitation, calcium chloride/rubidium chloride, retroviral infection, DEAE-dextran, cationic liposome, polyethylene glycol-mediated uptake, gene gun, etc.

In one aspect, the present invention relates to a composition for producing histidine comprising the transformed Corynebacterium spp. strain.

Since the composition for producing histidine of the present invention comprises the above-described transformed Corynebacterium spp. strain, descriptions of overlapping content with the above-described transformed Corynebacterium spp. strain of the present invention are omitted in order to avoid excessive complexity of the present specification due to description of overlapping content.

The term “composition” used in the present invention means a mixture of various substances for producing a target substance, including a microbial culture medium, a carbon source, trace elements, and microbial spawn.

In one aspect, the present invention relates to a method for producing histidine from a Corynebacterium spp. strain, comprising the step of culturing the transformed Corynebacterium spp. strain.

In one embodiment of the present invention, the histidine may be produced through the use of galactose, but is not limited thereto.

In the present invention, the term “cultivation” means growing a target cell or tissue, etc. under artificially controlled environmental conditions. The environmental conditions typically include nutrients, temperature, osmotic pressure, pH, gas composition, light, etc., but what directly affects them is the medium, and the medium can be broadly divided into liquid medium and solid medium. The cultivation of the transformed Corynebacterium strain of the present invention can be performed using a method widely known in the art.

Specifically, the above cultivation is not particularly limited thereto, as long as the transformed Corynebacterium spp. strain can produce histidine, but may be continuously cultivated in a batch process or a fed batch or repeated fed batch process, but is not limited thereto.

The medium used for cultivation must meet the requirements of the specific strain in an appropriate manner. Any medium known as a culture medium for strains of the genus Corynebacterium may be used without limitation, for example, BHI medium, BHIS medium, BHISG medium, CGXII medium, CGAF medium, CN40 medium, etc.

The sources of sugars that can be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture. The sources of nitrogen that can be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The sources of nitrogen can also be used individually or as a mixture. The sources of phosphorus that can be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the culture medium must contain a metal salt such as magnesium sulfate or iron sulfate that is necessary for growth. Finally, in addition to the above materials, essential growth substances such as amino acids and vitamins may be used. In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials may be added to the culture in a batch or continuous manner in an appropriate manner during the culture process.

The pH of the culture can be adjusted by using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid in an appropriate manner. In addition, foaming agents such as fatty acid polyglycol esters can be used to suppress foaming. Oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain an aerobic state. The temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C.

The cultivation is continued until the maximum amount of histidine is produced. Histidine may be released into the culture medium or contained in the cells. The medium may preferably contain glucose, galactose or carrageenan, which may be a substrate for histidine production, and the range of glucose, galactose or carrageenan includes all natural products (particularly, seaweed) containing the substance.

The method for producing histidine of the present invention comprises a step of recovering histidine from cells or a culture medium. Methods for recovering histidine from cells or a culture medium are widely known in the art. The histidine recovery method may use methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., HPLC, ion exchange, affinity, hydrophobicity, and size exclusion).

In one aspect, the present invention relates to a composition for producing histidine comprising seaweed, a carrageenan decomposing enzyme and the transformed Corynebacterium spp. strain.

In one embodiment of the present invention, the seaweed may be at least one selected from the group consisting of red algae, brown algae, and green algae, and may preferably be red algae containing a large amount of carrageenan, but is not limited thereto.

In one embodiment of the present invention, the carrageenan decomposing enzyme may be, but is not limited to, Iduronate-2-sulfatase, Kappa-carrageenase, or a mixture thereof.

In one embodiment of the present invention, the gene encoding Iduronate-2-sulfatase may be derived from Zobellia galactanivorans and may consist of a base sequence of SEQ ID NO: 14. In addition, if it is a base sequence showing 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 14, and is capable of expressing a protein exhibiting substantial Iduronate-2-sulfatase activity, it may be included without limitation.

In one embodiment of the present invention, the gene encoding the Kappa-carrageenase may be derived from Zobellia galactanivorans and may consist of a base sequence of SEQ ID NO: 15. In addition, if the base sequence shows 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically 99% or more homology with the sequence of SEQ ID NO: 15, and is capable of expressing a protein exhibiting substantial Kappa-carrageenase activity, it may be included without limitation.

In one aspect, the present invention relates to a method for producing histidine using seaweed, comprising the steps of: saccharifying seaweed using a carrageenan decomposing enzyme to produce a seaweed hydrolysate; and inoculating and fermenting a transformed Corynebacterium spp. strain into a medium containing the seaweed hydrolysate.

In one embodiment of the present invention, the carrageenan decomposing enzyme may be, but is not limited to, Iduronate-2-sulfatase, Kappa-carrageenase, or a mixture thereof.

In one embodiment of the present invention, the seaweed may be at least one selected from the group consisting of red algae, brown algae, and green algae, and may preferably be red algae containing a large amount of carrageenan, but is not limited thereto.

In one embodiment of the present invention, the red algae may include, but are not limited to, seaweed, laver, cotton, gaedogam, round laver, gaeummu, saebal, champulgasari, gosiraegi, jindubal, champulgae, gasiummu, bidanpul, danbak, dolgasaari, seokmok, jinuari, etc.

In one embodiment of the present invention, brown algae may be used, but are not limited to, seaweed, kelp, sea mustard, sea tangle, sea tangle, sea tangle, sea tangle, sea tangle, gompi, rhubarb, sea tangle history village, mojaban, scurvy mojaban, jichungi, and tangle.

In one embodiment of the present invention, the green algae may include, but are not limited to, blue seaweed, sea cucumber, blue laver, green algae, ginkgo biloba, and yam.

In one embodiment of the present invention, the Corynebacterium spp. strain may be cultured in a BHISG (Brain heart infusion-supplemented with glucose) medium under culture conditions of 25 to 35°C, air 1.5 to 2.5 L/min, and pH 6.0 to 8.0 for 20 to 28 hours, and then cultured in a pre-culture medium under culture conditions of 25 to 35°C, 150 to 250 rpm, air 1.5 to 2.5 L/min, and pH 6.0 to 8.0 for 20 to 28 hours.

In one embodiment of the present invention, the BHISG (Brain heart infusion-supplemented with glucose) medium may contain, but is not limited to, 15 to 25 g/L glucose, 33 to 41 g/L brain heart infusion, 87 to 95 g/LC ₆ H ₁₄ O ₆ , 15 to 25 g/LC ₆ H ₁₂ O _{6 ,} 20 to 30 μg/L kanamycin, and 5 to 15 μg/L chloramphenicol.

In one embodiment of the present invention, the pre-culture medium may contain, but is not limited to, 2 to 4 g/L peptone, 0.9 to 1.5 g/L potassium dihydrogen phosphate, 1 to 3 g/L sodium citrate, 2 to 4 g/L yeast extract, 17 to 23 g/L glucose, 0.5 to 1.5 mg/L biotin, 0.5 to 1.5 mg/L cobalamine, 0.5 to 1.5 mg/L pantothenate, 0.5 to 1.5 mg/L thiamine HCL, and 0.5 to 1.5X trace element.

In one embodiment of the present invention, the medium may be a CN40 medium, a CGXII medium, a CGAF medium, and preferably a CN40 medium, but is not limited thereto.

In one embodiment of the present invention, the CN40 medium may contain, but is not limited to, 4 to 5 g/L peptone, 7 to 9 g/L ammonium sulfate, 1 to 3 g/L potassium dihydrogen phosphate, 17 to 23 g/L corn syrup, 1 to 3 g/L sodium citrate, 7 to 9 g/L yeast extract, 9 to 11 g/L galactose, 1 to 3 g/L MgSO ₄ , 9 to 11 mg/L biotin, 1 to 3 mg/L cobalamine, 1 to 3 mg/L pantothenate, 9 to 11 mg/L thiamine HCL, and 0.5 to 1.5X trace element.

In one embodiment of the present invention, the fermentation may be performed for 40 to 50 hours under culture conditions of 25 to 35°C, 200 to 600 rpm, air 1.5 to 2.5 L/min, and pH 6.0 to 8.0, but is not limited thereto.

In one aspect, the present invention provides a pathway and culture conditions that optimize the utilization of galactose, a main component of seaweed, and the production of histidine.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the attached drawings. However, the following embodiments are only intended to concretize the contents of the present invention, and the present invention is not limited thereby.

<Example 1> Development of Corynebacterium strain with enhanced histidine production pathway

To develop a strain for increased histidine production, a recombinant vector that induces overexpression of genes related to the pentose phosphate pathway, histidine synthetic pathway, and ATP regeneration pathway, which are histidine production pathways, was constructed, and the recombinant vector was transformed into a Corynebacterium strain to produce a transformed Corynebacterium strain with an enhanced histidine production pathway (see Fig. 1).

First, to strengthen the pentose phosphate pathway and histidine synthetic pathway, the tkt promoter of the tkt gene (Genbank ID: BAB98967.1) and the hisD promoter of the hisD gene (Genbank ID: BAB99495.1) were replaced with the H36 promoter (SEQ ID NO: 9), a highly expressive synthetic promoter, and the start codon of the pgi gene (Genbank ID: 69621401) was substituted from ATG to GTG. CRISPR Vector was constructed to replace the promoter of each gene and change the base sequence. For construction of the above vector, the RA, LA sequences of the tkt and hisD genes were amplified by PCR using primers tkt LA F, tkt LA R, tkt RA F, tkt RA R, hisD LA F, hisD LA R, hisD RA F, hisD RA R, respectively, and the genes of tkt RA: 600 bp, tkt LA: 587 bp, hisD RA: 600 bp, hisD LA: 600 bp were amplified. Afterwards, for introduction of the H36 promoter, the related promoter was amplified by PCR using primers H36 F and R, and a 74 bp gene sequence was secured. Afterwards, each gene was inserted into the pJYS2 vector to construct a recombinant vector. To change the start codon of the pgi gene, the relevant gene was amplified by PCR using crRNA pgi F, R primers, and a 75 bp base sequence was obtained. The relevant base sequence was then cloned into pJYS2 to construct a recombinant vector. Afterwards, overexpression vectors containing the genes of hisG (ATP phosphoribosyltransferase, SEQ ID NO: 1), zwf (Glucose-6-phosphate 1-dehydrogenase, SEQ ID NO: 2), gnd (6-phosphogluconate dehydrogenase, SEQ ID NO: 3), prs (Ribose-phosphate pyrophosphokinase, SEQ ID NO: 4), hisD (Histidinol dehydrogenase, SEQ ID NO: 5), ndk (Nucleoside diphosphate kinase, SEQ ID NO: 6), adk (Adenylate kinase, SEQ ID NO: 7), and purH (Bifunctional purine biosynthesis protein PurH, SEQ ID NO: 8) were constructed to enhance the pentose phosphate pathway, histidine synthetic pathway , and ATP regeneration pathway. For the construction of the overexpression vector, the hisG gene was amplified by PCR using forward and reverse primers (HisG F, HisG R) containing restriction enzyme sequences from the genomic DNA of Corynebacterium glutamicum ( C. glutmiaum), and a sequence of 605 bp was obtained. The Zwf gene was amplified by PCR using forward and reverse primers (Zwf F, Zwf R) containing restriction enzyme sequences from the genomic DNA of Corynebacterium glutamicum ( C. glutamiaum), and a sequence of 1455 bp was obtained. The Gnd gene was amplified by PCR using forward and reverse primers (Gnd F, Gnd R) containing restriction enzyme sequences from the genomic DNA of Corynebacterium glutamicum (C. glutamiaum) , and a sequence of 1479 bp was obtained. The Prs gene was amplified by PCR using forward and reverse primers (Prs F, Prs R) containing restriction enzyme sequences from the genomic DNA of Corynebacterium glutamicum (C. glutmiaum ), resulting in a sequence of 978 bp. The HisD gene was amplified by PCR using forward and reverse primers (HisD F, HisD R) containing restriction enzyme sequences from the genomic DNA of Corynebacterium glutamicum (C. glutamiaum) , resulting in a sequence of 1329 bp. The amplified genes and the pEKEx2 vector were treated with BamH1, EcoR1, and Kpn1 restriction enzymes, and then ligated with T4 ligase to construct a recombinant vector. Thereafter, the purH, adk, and ndk genes were amplified by PCR using forward and reverse primers (purH F, purH R, adk F, adk R, ndk F, ndk R) containing restriction enzyme sequences, respectively, to obtain sequences of 411, 546, and 1563 bp, respectively. Afterwards, the amplified genes and the pMT-tac vector were treated with BamH1 restriction enzyme and ligated with T4 ligase to construct a recombinant vector. Afterwards, the constructed vectors were transformed into Corynebacterium glutamicum by electroporation (2.5 kV), and the histidine production ability of the transformant was confirmed. Specifically, the histidine production ability was measured through o-phthaladehyde (OPA) derivatization based on the Agilent Zorbax Eclipse AAA column using high-performance liquid chromatography (HPLC).

The recombinant vectors and strains used in the present invention are shown in Tables 1 and 2, and the primer information used in this experiment is shown in Table 3. All transformed strains were cultured in a 250 ml shaking Erlenmeyer flask containing 50 ml of CN40 medium (containing 20 g/L glucose) at 30°C and 200 rpm for 72 hours.

As a result, as shown in Fig. 2, it was confirmed that the histidine production of the TDPH4 strain was improved 1.44-fold and that of the TDPH6 strain was improved 2.15-fold compared to the TDP strain (Fig. 2a, b). In addition, in the case of the TDP strain (5.25 ng/gdcw) with an enhanced pentose phosphate and histidine pathway, the NADPH amount was confirmed to increase 4.14-fold compared to the wild type strain (1.27 ng/gdcw). Additionally, in the case of the TDPH4 strain, a strain with an enhanced pentose-phosphate pathway in which hisG ^* , zwf , gnd , and prs were overexpressed, 7.7 ng/gdcw of NADPH was produced, confirming that the NADPH production was increased 1.48-fold compared to the TDP strain (Fig. 2c). In addition, we confirmed that the mRNA expression levels of related genes were increased in the TDPH6 strain in which hisD , ndk , adk , and purH were additionally expressed (Fig. 2d, e).

vector characteristic pEKEx2 C. glutamicum - E. coli shuttle vector, P _tac , lacI, Kan ^R , pBL1ori pEKEx2:: hisG*::zwf::gnd pEKEx2, carrying hisG* (C-terminal area of hisG was deleted), zwf , and gnd genes originating from C. glutamicum originating from C. glutamicum pEKEx2:: hisG*::zwf::gnd::prs pEKEx2:: hisG*::zwf::gnd , carrying prs gene originating from C. glutamicum pMT- tac C. glutamicum - E. coli shuttle vector, P _tac ,Amp ^R ,Cm ^R ,pCG1ori pMT- tac::purH::adk::
ndk pMT-tac, carrying purH, adk, ndk genes originating from C. glutamicum

Strain characteristic C. glutamicum ATCC13032 wild type TDP C. glutamicum ATCC13032 with replacement of the native promoter of tkt gene and hisD gene by H36 synthetic promoter and the start codon of pgi gene was modified from atg to gtg, harboring pEKEx2 TDPH3 TDP harboring pEKEx2 ::hisG*::zwf::gnd TDPH4 TDP harboring pEKEx2 ::hisG*::zwf::gnd::prs TDPH6 TDP harboring pEKEx2 ::hisG*::zwf::gnd::prs::hisD, pMT- tac::purH::adk::ndk

Primer (restriction enzyme) Base sequence (5'-3') tkt LA F(EcoR1) ATATGAATTCTTAAGTTGTGAGTCCTTAT tkt LA R(Xma1) ATACCCGGGCAATCTTAAGTCTGGGA tkt RA F(Xba1) ATATCTAGATTGACCACCTTGACGCTGTCACCTG tkt RA R(Sal1) ATAGTCGACCAGCTGCTGGGTGCCAGCGA H36 F (BamH1) ATAGGATCCTCTATCTGGTGCCCTAAACG H36 R (Xba1) ATATCTAGACATGCTACTCCTACCAACCA crRNA pgi F CTAGGTATAATGGATCGAATTTCTACTGTTGTAGATATGGCGGACATT crRNA pgi R TTTATTTAAATGGATCTGGGTGTGTCGAAATGTCCGCCATATCTACAAC hisD LA F (Sal1) ATAGTCGACTCATCGGCATTGTTCGTGTCG hisD LA R (BamH1) GGGGGGGGATCCAGAGTTCATTTGAATTAGACTTAAAACTTAAAATGACC hisD RA F (BamH1) ATAGGATCCTGCGGGCTTAAGAACTTGT hisD RA R (EcoR1) ATAGAATTCGCCAGCCACCGTGCTCAG crRNA hisD F AAGCTTGCATGGCGGCCGGAATTTCTACTGTTGTAGATCCTATTGTATTCC crRNA hisD R TAAATGGATCTGGCGGCCGTGTTACGTGGGGAATACAATAGGATCTACAACAGTAG hisG F (BamH1) ATAGGATCCATGTTGAAAATCGCTGTCCC hisG R (Kpn1) ATAGGTACCCTAGATGCGGGCGATGC zwf F GCATCGCCCGCATCTAGGGTACAAGGAGATATAGATGGTGATCTTCGGTGTC zwf R (Kpn1) CCAGTGAATTCGAGCTCGGTACCTTATGGCCTGCGCCAGG gnd F CCTGGCGCAGGCCATAAGGTACAAGGAGATATAGATGCCGTCAAGTACGATCAA gnd R (Kpn1) CCAGTGAATTCGAGCTCGGTACCTTAAGCTTCAACCTCGGAGCG prs F GCTCCGAGGTTGAAGCTTAAGGTACAAGGAGATATAGATGACTGCTCACTGGAAACA prs R (Kpn1) GGCCAGTGAATTCGAGCTCGGGTACCTTAGGCCTCGCCCTCG hisD F TCGAGGGCGAGGCCTAAGGTACAAGGAGATATAGATGTTGAATGTCACTGACCTG hisD R (Kpn1) CAGTGAATTCGAGCTCGGGTACCTTAGGCCTCGTCGGTGG ndk F GGAGATATCATATCGATGGATCCATGACTGAACGTACTCTCATC ndk R (BamH1) GTACGAGTCGCATCTATATCTCCTTTTACAGGTTAGGGAACCAGAT adk F CTCCATCTGGTTCCCTAACCTTGTAAAAGGAGATATAGATGCGACTCGTACTCCT adk R CGCGGTACCCGGGGATCTTATTTGCCCAGTGCCTTG purH F GGCAAATAAGATCCCCGGAAGGAGATATAGATGAGCGATGATCGTAAGG purH R AACAGGCGGCCGCGGTACCCGGGTTAGTGAGCGAAGTGTCGCG

<Example 2> Confirmation of carrageenan decomposition ability of Iduronate-2-sulfatase and Kappa-carrageenase

2-1. idsA3 Genes and cgkA Production of transformants expressing genes

To decompose carrageenan, a main component of red algae, the idsA3 gene (Iduronate-2-sulfatase) and cgkA gene (Kappa-carrageenase), which are related to the carrageenan hydrolysis pathway, were expressed through the pColdII vector. To construct this overexpression vector, the idsA3 gene was amplified by PCR using forward and reverse primers ( IdsA F, IdsA R) containing restriction enzyme sequences from the genomic DNA of Zobellia galactanivorans , and a sequence of 1531 bp was obtained (SEQ ID NO: 14). The cgkA gene was amplified by PCR using forward and reverse primers (CgkA F, CgkA R) containing restriction enzyme sequences from the genomic DNA of Zobellia galactanivorans , and a sequence of 1641 bp was obtained (SEQ ID NO: 15). To perform in vitro experiments by expressing kappa-carrageenase and iduronate-2-sulfatase for carrageenan degradation, the constructed recombinant vector was transformed into E. coli BL21 by the heat shock method.

vector characteristic pColdII E. coli expression vector, P _CSPA ,TEE,lacI,Amp ^R ,Pbr322ori pColdII:: cgkA PColdII, carrying cgkA gene originating from Zobellia galactanivorans pColdII:: idsA3 PColdII, carrying idsA3 gene originating from Z. galactanivorans

Strain characteristic E. coli BL21 pColdII:: cgkA E. coli BL21 harboring pColdII:: cgkA vector E. coli BL21 pColdII:: idsA3 E. coli BL21 harboring pColdII:: idsA3 vector

Primer (restriction enzyme) Base sequence (5'-3') idsA3_F ATATGAGCTCTGCAGCTCGGCACA idsA3_R GCGCAAGCTTTTAGTTTAGGGAATGTTGCTCTTT CgkA_F ATATGAGCTCTATGAAAAAACCAAATTTTTATGGC CgkA_R GCGCAAGCTTTTACTCCACGAGTATCTTTTTT

2-2. idsA3 Genes and cgkA Confirming gene expression

To confirm the protein expression of the transformant obtained in Example 2-1, purification using His-Tag and SDS-PAGE were performed. The E. coli transformant was shaken and cultured at 16°C for 12 hours with a concentration of 1 mM IPTG, and then centrifuged to obtain cells. The cells were disrupted using ultrasonication and centrifuged to prepare the supernatant, which was concentrated to obtain the Iduronate-2-sulfatase protein for the idsA3 gene and the Kappa-carrageenase protein for the cgkA gene, and then loaded onto SDS-PAGE. As a result, it was confirmed that bands appeared at the same positions as the sizes of Iduronate-2-sulfatase and Kappa-carrageenase (Fig. 3a).

2-3. Confirmation of carrageenan decomposition ability of Iduronate-2-sulfatase and Kappa-carrageenase

In order to specifically confirm the activity of the above Iduronate-2-sulfatase and Kappa-carrageenase, hydrolysis activity analysis using carrageenan as a substrate was performed using the purified Iduronate-2-sulfatase and Kappa-carrageenase. Specifically, for the measurement of hydrolysis activity, 10 mg/ml of each enzyme was treated and 1% (w/v) of kappa-carrageenan was used. Each enzyme and substrate was reacted at 30 degrees in 1.0 mM MgCl ₂ , 1.0 mM NaCl ₂ , 100 mM sodium acetate buffer (pH 5.0). After that, the reaction was performed at 60 degrees for 5 minutes, and the reaction was stopped through heat treatment at 99 degrees. Then, the hydrolysis activity was measured by measuring the amounts of galactose and sulfate, respectively. In Figure 3, Control represents 50 mM citric acid buffer, IdsA3 represents single treatment with Iduronate-2-sulfatase, CgkA represents single treatment with Kappa-carrageenase, and CgkA and IdsA3 represent combined treatment with Iduronate-2-sulfatase and Kappa-carrageenase.

As a result, as shown in Fig. 3, when Iduronate-2-sulfatase and Kappa-carrageenase were treated simultaneously, it was confirmed that the hydrolysis activity increased by about 2 times compared to when each enzyme was treated alone when carrageenan was used as a substrate (Fig. 3b). In addition, it was confirmed that about 2 mM sulfate was produced when Iduronate-2-sulfatase was treated alone on carrageenan (Fig. 3c). Through the above results, it was confirmed that the treatment with Iduronate-2-sulfatase successfully removed sulfate, known as a residue of carrageenan, and the hydrolysis of Kappa-carrageenase was significantly increased through the removal of sulfate.

These results imply that Iduronate-2-sulfatase and Kappa-carrageenase have carrageenan-decomposing activity and can decompose carrageenan, a main component of seaweed, into galactose, and confirm the production of d-galactose through the decomposition of carrageenan, confirming the usability of marine biomass.

<Example 3> Development of a transformed Corynebacterium strain with a galactose utilization pathway introduced

According to the results of Examples 1 and 2 above, the inventors of the present invention created a recombinant strain capable of galactose metabolism, added Iduronate-2-sulfatase and Kappa-carrageenase to the medium, and then cultured the recombinant strain using carrageenan as a single carbon source, and then confirmed the ability to utilize galactose and produce histidine.

Specifically, to introduce the galactose uptake system into a transformed Corynebacterium strain (TDPH6) with an enhanced histidine production pathway, a galactose transporter gene from Saccharomyces cerevisiae and the Leloir pathway in E. coli K-12 strain for galactose metabolism were introduced (see Fig. 1). Each gene was introduced into the pEKEx2 vector, an overexpression vector. The gal2 gene (SEQ ID NO: 10), known as a galactose transporter, was amplified by PCR using forward and reverse primers (Gal2_F, Gal2_R) containing restriction enzyme sequences from the genomic DNA of S. cerevisiae , and a sequence of 1725 bp was obtained. In addition, to introduce and strengthen the galactose utilization pathway, the galT (SEQ ID NO: 11, galactose-1-phophate uridylytransferase) and galM (SEQ ID NO: 12, aldose-1-epimerase) genes were amplified by PCR using forward and reverse primers (GalT_F, GalT_R; GalM_F, GalM_R) containing restriction enzyme sequences from the genomic DNA of E. coli , and the galK (SEQ ID NO: 13, galactokinase) gene was amplified by PCR using forward and reverse primers (GalK_F, GalK_R) containing restriction enzyme sequences from the genomic DNA of Corynebacterium glutamicum , obtaining sequences of 1296 bp, 1041 bp, and 1047 bp, respectively. A transformant Corynebacterium strain with an enhanced histidine production pathway was constructed by introducing an overexpression vector containing gal2, galT, galM, and galK into the strain, and its galactose utilization and histidine production abilities were confirmed. All transformants were cultured in a 250 ml shaking Erlenmeyer flask containing 50 ml of CN40 medium (containing 10 g/L galactose) at 30°C and 200 rpm for 36 h. The recombinant vectors and strains used in this experiment are shown in Tables 7 and 8, and the primers are shown in Table 9.

As a result, as shown in Fig. 4, it was confirmed that the strain into which the galactose utilization pathway was introduced consumed about 23% of galactose and produced about 150 mg of histidine in 36 hours (Fig. 4a). In addition, it was confirmed that the histidine yield in the 6-hour period was the highest at about 3 mg/g/h (Fig. 4b). Through these results, it was confirmed that the transformed Corynebacterium strain into which the galactose utilization pathway was introduced can produce histidine by consuming galactose.

vector characteristic pEKEx2 C. glutamicum - E. coli shuttle vector, P _tac , lacI, Kan ^R , pBL1ori pEKEx2:: hisG*::zwf::gnd pEKEx2, carrying hisG* (C-terminal area of hisG was deleted), zwf and gnd genes originating from C. glutamicum pEKEx2:: hisG*::zwf::gnd::prs pEKEx2 ::hisG*::zwf::gnd , carrying prs gene originating from C. glutamicum pEKEx2::h isG*::zwf::gnd::prs::hisD pEKEx2 ::hisG*::zwf::gnd::prs , carrying hisD gene originating from C. glutamicum pMT- tac C. glutamicum - E. coli shuttle vector, P _tac ,Amp ^R ,Cm ^R ,pCG1ori pMT- tac::purH::adk::ndk pMT-tac, carrying purH, adk, ndk genes originating from C. glutamicum pMT- tac::purH::adk::ndk::gal2KTM pMT- tac::purH::adk::ndk , carrying gal2, galK, galT and galM ( gal2 is originating from Saccharomyces cerevisiae , galT and galM are originating from E. coli K-12, and galK is originating from C. glutamicum )

Strain characteristic TDPH6G2 TDP harboring pEKEx2 ::hisG*::zwf::gnd::prs::hisD, pMT- tac::purH::adk::ndk::gal2KTM

Primer Base sequence (5'-3') Gal2_F CCAAGCTTGCATGCCTGCAATGGCAGTTGAGGAGAACAA Gal2_R TGGCCATGTATATCTCCTTGTCGACTTATTCTAGCATGGCCTTGTACC GalK_F ATAGTCGACAAGGAGATATACATGGCCATTTGGCAGCCAA GalK_R ATAGGATCCCTAGCCCTGTTCCGCGAC GalT_F CCCGGGTACCGAGCTCGAATTAAGGAGATATACATGACGCAATTTAATCCCGT GalT_R GTAAAACGACGGCCAGTGAATTCTTACACTCCGGATTCGCGA GalM_F ACGTCGCGGAACAGGGCTAGAAGGAGATATACATGCTGAACGAAAACTCCCGC GalM_R TCTCATCCGCCAAAACAGGCGGCCGCTTACTCAGCAATAAACTGATATTCCGT

<Example 6> Optimal culture conditions for galactose utilization of strains with galactose utilization ability introduced

To increase galactose utilization, optimal culture conditions for strains with galactose utilization introduced through fermentation were established. The strain was cultured on BHISG (20 g/L glucose, 37 g/L brain heart infusion, 91 g/LC _{6H 14} O _6, 20 g/LC _{6H 12} O _6, 25 μg/L kanamycin, and 10 μg/L chloramphenicol) solid medium at 30°C for 24 h, and then cultured in pre-culture medium (3 g/L peptone, 1.2 g/L potassium dihydrogen phosphate, 2 g/L sodium citrate, 3 g/L yeast extract, 20 g/L glucose, 1 mg/L biotin, 1 mg/L cobalamine, 1 mg/L pantothenate, 1 mg/L thiamine HCL, 1x trace element, pH 7) at 30°C and 200 rpm for 24 h. Afterwards, it was cultured in 2 L of CN40 medium (4.54 g/L peptone, 8.0 g/L ammonium sulfate, 2.0 g/L potassium dihydrogen phosphate, 20 g/L corn syrup, 2 g/L sodium citrate, 8 g/L yeast extract, 40 g/L galactose, 2 g/L MgSO ₄ , 10 mg/L biotin, 2 mg/L cobalamine, 2 mg/L pantothenate, 10 mg/L thiamine HCL, 1x trace element) at 30 ℃, 200-600 rpm for 45 h. During the culture, air was adjusted to 2 L/min, pH was 7.0, and when DO dropped below 20, rpm was increased by 20 and cultured at 200 to 600 rpm for the maximum.

As a result, as shown in Fig. 5, it was confirmed that the galactose utilization ability increased to 100% 24 hours after the strain cultured in BHISG medium and pre-culture medium was inoculated into CN40 medium, thereby establishing the optimal galactose utilization culture conditions.

In addition, as shown in Fig. 6, it was confirmed that 100% of galactose was consumed and approximately 0.4 g/L of histidine was produced, thereby establishing optimal galactose utilization culture conditions.

As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other backward inventions or other embodiments included within the scope of the spirit of the present invention by adding, changing, deleting, etc. other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are exemplary and not restrictive in all respects. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modifications derived from the meaning and scope of the claims and the equivalent concept should be interpreted as being included in the scope of the present invention.

Claims (20)

A transformed Corynebacterium strain, wherein a gene encoding ATP phosphoribosyltransferase, a gene encoding Glucose-6-phosphate 1-dehydrogenase, a gene encoding 6-phosphogluconate dehydrogenase, a gene encoding Ribose-phosphate pyrophosphokinase, a gene encoding Histidinol dehydrogenase, a gene encoding Nucleoside diphosphate kinase, a gene encoding Adenylate kinase, and a gene encoding Bifunctional purine biosynthesis protein PurH are overexpressed. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the above ATP phosphoribosyltransferase is represented by the base sequence of sequence number 1. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the Glucose-6-phosphate 1-dehydrogenase is represented by the base sequence of sequence number 2. A transformed Corynebacterium strain, characterized in that the gene encoding the above 6-phosphogluconate dehydrogenase is represented by the base sequence of SEQ ID NO: 3. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the ribose-phosphate pyrophosphokinase is represented by the base sequence of sequence number 4. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the histidinol dehydrogenase is represented by the base sequence of SEQ ID NO: 5. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the nucleoside diphosphate kinase is represented by the base sequence of SEQ ID NO: 6. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the above adenylate kinase is represented by the base sequence of SEQ ID NO: 7. In the first paragraph, A transformed Corynebacterium strain, characterized in that the gene encoding the bifunctional purine biosynthesis protein PurH is represented by the base sequence of SEQ ID NO: 8. In the first paragraph, The above-mentioned transformed Corynebacterium strain is a transformed Corynebacterium strain characterized in that the tkt promoter of the tkt gene (Genbank ID: BAB98967.1) and the hisD promoter of the hisD gene (Genbank ID: BAB99495.1) are replaced with the H36 promoter, which is a highly expressive synthetic promoter, and the start codon of the pgi gene (Genbank ID: 69621401) is substituted from ATG to GTG. In Article 10, A transformed Corynebacterium strain, characterized in that the above H36 promoter is represented by the base sequence of sequence number 9. In the first paragraph, The above-mentioned transformed Corynebacterium strain is a transformed Corynebacterium strain in which the pentose phosphate pathway, the histidine synthetic pathway, and the ATP regeneration pathway are enhanced. In the first paragraph, The above-mentioned transformed Corynebacterium strain is a transformed Corynebacterium strain into which a gene encoding a galactose transporter, a gene encoding galactose-1-phophate uridylytransferase, and a gene encoding aldose-1-epimerase have been introduced, and a gene encoding galactokinase has been overexpressed. In Article 13, A transformed Corynebacterium strain, characterized in that the gene encoding the galactose transporter is derived from Saccharomyces cerevisiae . In Article 13, A transformed Corynebacterium strain, characterized in that the gene encoding the galactose-1-phophate uridylytransferase and the gene encoding aldose-1-epimerase are derived from Escherichia coli . In Article 13, The above-mentioned transformed Corynebacterium strain is a transformed Corynebacterium strain into which a galactose uptake system has been introduced. A composition for producing histidine, comprising a transformed Corynebacterium strain according to any one of claims 1 to 16. A method for producing histidine from a Corynebacterium spp. strain, comprising the step of culturing a transformed Corynebacterium spp. strain according to any one of claims 1 to 16. A composition for producing histidine, comprising seaweed, a carrageenan decomposing enzyme and a transformed Corynebacterium strain of any one of claims 13 to 16. A step of producing a seaweed hydrolysate by saccharifying seaweed using a carrageenan decomposition enzyme; and A method for producing histidine using seaweed, comprising the step of inoculating and fermenting the transformed Corynebacterium spp. strain into a medium containing the seaweed hydrolysate.

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