Novel strain having excellent polyhydroxyalkanoate-producing ability and use thereof

Abstract

The present invention relates to a novel strain having an excellent polyhydroxyalkanoate-producing ability and a use thereof. A species of Halomonas ixanhensis, which previously has not been reported to have polyhydroxyalkanoate biosynthesis characteristics, was found to produce polyhydroxyalkanoate. A novel HN-1-3-2 strain that produces polyhydroxyalkanoate in a significantly higher content and concentration than existing polyhydroxyalkanoate-producing strains has been selected, and thus can be used for the purpose of producing polyhydroxyalkanoate.

Description

Novel strain with excellent polyhydroxyalkanoate production ability and its uses

[0001] The present invention relates to a novel Halomonas cyanhensis strain having excellent polyhydroxyalkanoate production ability and the use thereof.

[0002] Plastics are polymer materials that have dramatically advanced the convenience of human life. Due to their lightweight nature, moldability, processability, cost-effectiveness, and excellent durability, they are used for a wide range of purposes, from industrial materials to disposable consumables. However, most plastics used universally—such as those for industrial packaging, food packaging, household use, and agriculture / horticulture—exist semi-permanently without decomposing in the natural environment. Consequently, environmental pollution issues arising from the disposal of discarded plastics have persisted. As an alternative to address this, research on biodegradable plastics is actively underway. Biodegradable plastics are plastics that can be broken down by bacteria or living organisms. Because they decompose without leaving behind pollutants, they are receiving significant attention in the field of biotechnology, as they are expected to potentially contribute to reducing solid waste, such as marine microplastics and carbon dioxide emissions from petroleum-based plastics. Bio-based, biodegradable plastics are referred to as biodegradable bioplastics, with representative examples including polylactic acid (PLA) and polyhydroxyalkanoates (PHA). When landfilled, biodegradable bioplastics can decompose into water and carbon dioxide within 6 months to 5 years. Currently, bioplastics account for approximately 1% (2 million tons) of the over 360 million tons of plastic produced annually, with about 1.2 million tons of this being biodegradable plastics. However, this figure is expected to increase to 1.8 million tons by 2025, with the market growth rate for polyhydroxyalkanoates projected to increase tenfold. As demand rises and the availability of high-performance biopolymers and products increases, the bioplastics market is expected to continue growing and diversifying.

[0003] Polyhydroxyalkanoates (PHAs) are intracellular energy storage compounds produced by various microorganisms under limited nutrient conditions and are biodegradable polymers composed of various types of hydroxycarboxylic acids. Polyhydroxyalkanoates have properties similar to existing synthetic polymers derived from petroleum, such as polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), while exhibiting complete biodegradability and excellent biocompatibility. In addition, since mechanical properties and melting points can be controlled by adjusting the type and ratio of polyhydroxyalkanoate monomers, it is being used as an alternative to petroleum plastics in various industrial fields such as medicine, food, and energy.

[0004] [Prior Art Literature]

[0005] [Patent Literature]

[0006] (Patent Document 1) KR 10-2021-0075585 B1

[0007] [Non-patent literature]

[0008] (Non-patent Document 1) Yang, Yung-Hun, et al. “Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha.” Applied microbiology and biotechnology 87.6 (2010): 2037-2045.

[0009] The object of the present invention is to provide a novel halophilic bacterium having the ability to produce polyhydroxyalkanoates.

[0010] In addition, the object of the present invention is to provide a polyhydroxyalkanoate produced by the strain.

[0011] In addition, the object of the present invention is to provide a composition for producing polyhydroxyalkanoates comprising the strain.

[0012] In addition, the objective of the present invention is to provide a method for producing polyhydroxyalkanoates.

[0013] To solve the above problem, the present invention provides the Halomonas xianhensis HN-1-3-2 strain deposited under accession number KCTC19195P.

[0014] In addition, the present invention provides a polyhydroxyalkanoate produced by the strain.

[0015] In addition, the present invention provides a composition for producing polyhydroxyalkanoates comprising the strain.

[0016] In addition, the present invention provides a method for producing polyhydroxyalkanoates.

[0017] In the present invention, it was confirmed that a species of Halomonas xianhensis, which has not been reported to have polyhydroxyalkanoate biosynthetic properties to date, produces polyhydroxyalkanoate, and a novel Halomonas xianhensis HN-1-3-2 strain was selected that produces polyhydroxyalkanoate with significantly higher content and concentration compared to conventional polyhydroxyalkanoate-producing strains, so it can be utilized for the production of polyhydroxyalkanoate.

[0018] Figure 1 is a figure confirming the intracellular PHA accumulation of Halomonas xianhensisHN-1-3-2, Cobetia marinaHN-1-7-2, Halomonas ventosaeM434, Halomonas litopenaeiYBW-3-4-1 and Halomonas xianhensisYBW-3-4-2 strains by TEM analysis.

[0019] Figure 2 is a figure showing the analysis of the whole genome of Halomonas xianhensisHN-1-3-2.

[0020] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to a person skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description 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 claims set forth below and the equivalent scope interpreted therefrom.

[0021] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0022] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by a person skilled in the art in the relevant field of this invention. Furthermore, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0023]

[0024] In one aspect, the present invention relates to a novel Halomonasxianhensis HN-1-3-2 strain deposited under accession number KCTC19195P.

[0025] In one embodiment, the strain may have the 16s rRNA sequence of SEQ ID NO. 1.

[0026] In one embodiment, the strain may have salt tolerance at a salinity of 1 to 20% (w / v).

[0027] In one embodiment, the strain may have the ability to produce polyhydroxyalkanoates (PHA), and the polyhydroxyalkanoates are poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, It may include P3HU), short-chain or medium-chain length saturated or unsaturated PHA, or any copolymer thereof or any combination thereof.

[0028] In one embodiment, the strain can produce polyhydroxyalkanoate under salinity conditions of 1 to 20% (w / v).

[0029] In one embodiment, the strain may accumulate polyhydroxyalkanoate in the cell at a content of 40 to 90%.

[0030] In one embodiment, the polyhydroxyalkanoate accumulated in the cell may be in the form of micro beads with an average particle size of 10 μm or less, or in the form of micro beads with an average particle size of 400 nm to 5 μm.

[0031] In one embodiment, the strain can produce PHA at a concentration of 0.5 to 10 g / L.

[0032] In one embodiment, the strain may be a formulation selected from the group consisting of a dead cell, a culture medium, a culture medium concentrate, a culture medium dry product, a culture medium extract, a culture supernatant, a culture supernatant concentrate, a culture supernatant dry product, and a culture supernatant extract.

[0033] In one embodiment, the culture medium may be the total culture, culture supernatant, culture supernatant concentrate, pulverized material, filtrate, fractions thereof, freeze-dried material, or supercritical dried material obtained during or after the process of culturing the strain in a medium. In this case, the culture supernatant may be obtained by centrifuging the culture of the strain, the pulverized material may be obtained by physically crushing the strain, and the fractions may be obtained by applying the culture, culture supernatant, pulverized material, etc., to methods such as centrifugation or chromatography.

[0034] In one embodiment, the physical crushing may be achieved by physically crushing with ultrasound or pressure.

[0035] In one embodiment, the culture of the strain may be a fermented product, and the term "fermented product" used in the present invention refers to a substance fermented using the strain of the present invention, and includes not only the fermented product itself, but also all types of substances including a culture medium of the strain in which the strain and the culture coexist, a fermented product obtained by filtering the strain from the culture medium, a fermented product obtained by sterilizing the strain from the culture medium and filtering it, an extract obtained by extracting the fermented product or a culture medium containing it, a diluted solution obtained by diluting the fermented product or the extract thereof, a dried product obtained by drying the fermented product or the extract thereof, a lysate obtained by capturing and crushing the cells of the strain, etc.

[0036] In one embodiment, the extract may be extracted with one or more solvents selected from the group consisting of water, organic solvents, subcritical fluids, and supercritical fluids, and the organic solvent may be any one selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate, methyl acetate, dichloromethane, chloroform, ethyl acetate, acetone, methylene chloride, cyclohexane, petroleum ether, benzene, and mixed solvents thereof.

[0037] The sequences used in the present invention are interpreted to include sequences that exhibit substantial identity with the sequences listed in the sequence list, provided that variations having biologically equivalent activity are taken into account.

[0038] As used in the present invention, the term "culture medium" refers to a product obtained by culturing the strain in a culture medium. For example, the culture medium of the present invention may include components remaining in the medium after obtaining the strain from the culture medium of Halomonas xianhensis HN-1-3-2 (accession number KCTC19195P).

[0039] As used in the present invention, the term "substantial identity" means a sequence in which, when the sequence of the present invention is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art, at least 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

[0040] In one aspect, the present invention relates to a polyhydroxyalkanoate produced by the strain of the present invention.

[0041] In one aspect, the present invention relates to a composition for producing polyhydroxyalkanoates comprising the strain of the present invention.

[0042] In one aspect, the present invention relates to a method for producing polyhydroxyalkanoate comprising the step of culturing a strain of the present invention.

[0043] In one embodiment, the strain can be cultured in a medium containing 1 to 15% (w / v) of NaCl, and it is more preferable for PHA production to culture it in a medium containing 2 to 10% (w / v) of NaCl.

[0044] In one embodiment, the method may include the step of culturing the strain in a medium that is free of a carbon source and contains 1 to 15% (w / v) of NaCl; and the step of culturing in a medium that contains a carbon source and contains 1 to 15% (w / v) of NaCl.

[0045] In one embodiment, the carbon source may be included in an amount of 1 to 5% (w / v), and the carbon source may be one or more selected from the group consisting of glucose, sucrose, glycerol, fructose, galactose, and xylose.

[0046] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0047]

[0048] Example 1. Primary screening of halophilic strains capable of PHA production

[0049] Among the eight newly identified halophilic strains (Table 1), five strains capable of producing PHA were selected using dry cell weight (g / L), PHA content (%), and PHA concentration (g / L) as indicators. Specifically, each strain was cultured at 37°C and 180 rpm in a basic medium of Marine Broth (salinity 3% (w / v)) supplemented with 30 g / L of glucose. To calculate direct biomass before and after lysis, cells were uniformly suspended in the culture medium using a vortex mixer; 1 mL of the sample was placed into a 1.5 mL microtube, and the cells were precipitated using a small centrifuge at 5000 × g for 20 minutes at 4°C. The supernatant was removed as much as possible, and the cells were dried at 65°C for 24 hours. The dry cell weight was determined by subtracting the weight of the empty tube from the total weight. In addition, for the analysis of intracellular PHA content (%), 20 mL of culture medium was centrifuged at 3000 xg for 20 minutes at 4°C to obtain cells, 5 mL of sodium hypochlorite was added and reacted at 37°C and 180 rpm for 1 hour, and 5 mL of chloroform was added to the pellet containing PHA obtained by centrifuging at 3000 xg for 20 minutes at 4°C and reacted at 37°C and 180 rpm for 1 hour. 5 mL of iced-cold 70% methanol was added and vortexed for 2 minutes, and the mixture was centrifuged at 3000 xg for 20 minutes at 4°C to completely separate only the chloroform containing PHA from the bottom, and dried at 40°C for 48 hours to obtain a PHA film. The mass of the dried PHA film was converted into a percentage relative to DCW to calculate the PHA content (%) accumulated within the cell.In addition, for the analysis of PHA concentration (g / L), a standard solution was prepared by adding 10 mL of sulfuric acid (Deajung, Sulfuric acid 98%, GR) to 20 g / L of Crotonic acid (Sigma-Aldrich, 98%, 107-93) and reacting at 100 °C for 10 minutes. This solution was diluted with triple-purified water to 2.5, 5, 7.5, 10, 15, 20, 25, 30, 40, 50, 60, and 80 μg / L, and the absorbance was measured at 230 nm using a spectrophotometer (TECAN). Subsequently, the coefficient of determination R was calculated using samples of concentrations where the absorbance value fell within the range of 0.1000–0.9999. 2 A standard curve with a value of 0.99 or higher was derived. Then, 10 mL of sulfuric acid was added to the PHA dried after extraction and reacted at 100°C for 10 minutes. The sample was then diluted with tertiary purified water to within the range of the standard curve, the absorbance was measured at the same wavelength, and the results were substituted into the standard curve to calculate the Crotonic acid content in the sample.

[0050] No.Isolate numberEzBioCloud Closest matchDry Cell Weight (g / L)PHA content (%)PHA concentration (g / L)1YBW-3-4-1Halomonas litopenaei1.07353.33 ±1.070.57 ±± 0.012M434Halomonas ventosae1.8972 ± 2.311.36 ± 0.043HSS-4Halomonas hydrothermalis1.05228.53 ± 1.460.30 ± 0.014HN-1-3-2Halomonas xianhensis2.64471.5 ± 2.151.76 ± 0.165YBW-3-4-2Halomonas xianhensis1.41953.6 ± 3.880.76 ± 0.056HSS-13Cobetia amphilecti0.281320.097HSS-16Cobetia marina0.31528.40.098HN-1-7-2Cobetia marina0.363420.15

[0051]

[0052] Through this, strains HN-1-3-2, HN-1-7-2, M434, YBW-3-4-1, and YBW-3-4-2, which have excellent PHA production ability under a salinity of 3% (w / v), were selected from the eight newly identified natural PHA-producing strains.

[0053]

[0054] Example 2. Final selection of strains with excellent PHA production capacity

[0055] In order to quantitatively confirm the PHA production ability of the five strains selected in Example 1 above and to select strains with excellent PHA production ability, culture for strain growth was carried out in ASS medium (Soy peptone 5 g / L, Yeast extract 1 g / L, Artificial sea salt 30 g / L) (3% (w / v) salt concentration). Then, for the purpose of PHA production, a carbon source of Glucose 30 g / L was added to the medium and the strains were cultured. Subsequently, PHA accumulation was analyzed by TEM, and PHA was extracted to analyze the PHA production of each strain. Afterward, strains were selected based on cell density (OD 600 nm), dry cell weight (g / L), PHA content (%), and PHA concentration (g / L). Specifically, the five strains mentioned above were each inoculated into ASS medium (Soy peptone 5 g / L, Yeast extract 1 g / L, Artificial sea salt 30 g / L) at a concentration of 2% (v / v) with an optical density (600 nm) of 1.5-1.8 and cultured overnight at 37°C and 180 rpm (seed- or pre-culture), and then cultured into a glucose-supplied medium (Soy peptone 5 g / L, Yeast extract 1 g / L, Artificial sea salt 30 g / L, and Glucose 30 g / L) at a concentration of 2% (v / v) with an optical density (600 nm) of 4.5-5.0 and cultured at 40°C and 180 rpm (production-culture). In this case, when a flask was used, the culture was performed at 1 / 5 of the flask's volume. Cell density of the culture medium diluted with a 0.85% NaCl aqueous solution was measured at a wavelength of 600 nm using a spectrophotometer (TECAN), and dry cell weight, PHA content, and concentration were analyzed as in Example 1 above. In addition, to confirm the accumulation of PHA particles within the cells, 1 mL of the culture medium was 1.Cells were placed in a 5 mL microtube and centrifuged at 5000 xg for 3 minutes to precipitate them. The cells were recovered and washed three times with PBS, then reacted with 2% Glutaraldehyde (pH 7.2) at room temperature for 2 hours. After washing three times, secondary fixation and primary staining were performed using Osmium Tetroxide, followed by TEM analysis.

[0056] Isolate numberCell density(OD 600nm)Dry cell weight(g / L)PHA Content(%)PHA Concentration(g / L)HN-1-3-216.74 ± 0.268.70 ± 0.3554.84 ± 3.658.70 ± 0.35HN-1-7-24.54 ± 0.742.98 ± 0.5840.95 ± 8.152.98 ± 0.58M43414.02 ± 1.206.56 ± 0.7849.60 ± 5.406.56 ± 0.78YBW-3-4-115.72 ± 2.497.43 ± 1.9652.22 ± 2.597.43 ± 1.96YBW-3-4-215.54 ± 2.178.46 ± 1.0151.47 ± 1.568.46 ± 1.01

[0057] TEM analysis results showed that among the five strains, HN-1-7-2 exhibited the lowest PHA accumulation, followed by M434 with the next lowest accumulation rate, and HN-1-3-2 with the highest PHA accumulation, while YBW-3-4-1 and YBW-3-4-2 showed similar levels of PHA accumulation (Fig. 1). In addition, analysis of cell density (OD 600 nm), dry cell weight (g / L), PHA content (%), and PHA concentration (g / L) revealed that HN-1-3-2 demonstrated the most superior effect (Table 2). Notably, it exhibited a PHA accumulation rate superior to that of YBW-3-4-2, which belongs to the same Halomonas xianhensis genus. Accordingly, the Halomonas xianhensis HN-1-3-2 strain was selected.

[0058] Example 3. Genomic analysis of the Halomonas xianhensis HN-1-3-2 strain

[0059] Because the industrial production potential of short-chain PHA and 4HB monomer compositions is high, the whole genome information of the Halomonas xianhensisHN-1-3-2 strain, which was finally selected in Example 2 as an efficient producer of PHA, was analyzed using the Illumina platform, a PacBio Sequal II system. It was analyzed that its genome consists of a 4.4 Mb circular chromosome and contains a total of 4,182 predicted coding genes (Table 3 and Figure 2). In addition, its 16S rRNA sequence (Sequence No. 1) was analyzed, and it was confirmed to be a novel strain and deposited at the National Institute of Biotechnology and Bioengineering (NIBB) under accession number KCTC19195P.

[0060] Genome size4,406,532 bpContig 14,042,016 bpContig 2342,090 bpContig 322,426 bpGenes (total)4182Number of CDSs (total)4090Number of CDSs (matched protein)3826Genes (RNA)91Number of tRNA76Number of rRNA15G+C61.3%

[0061]

[0062] The Halomonas xianhensis HN-1-3-2 strain according to the present invention and the polyhydroxyalkanoate (PHA) production technology using the same can be applied to the manufacturing process of biodegradable bioplastics. The strain of the present invention can produce a high content of PHA and can be reproducibly applied under various culture conditions, allowing for expansion into industrial-scale fermentation processes. Therefore, the present invention has clear industrial applicability as it can be commercially utilized in PHA-based material industries, such as eco-friendly packaging materials, agricultural films, and medical polymer materials.

[0063]

[0064] [Consignment Number]

[0065] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0066] Trustee Number: KCTC19195P

[0067] Date of Trust: 20240524

[0068]

[0069] [Correction pursuant to Rule 91 25.11.2025]

Claims

1. Halomonas xianhensis HN-1-3-2 strain deposited under accession number KCTC19195P.

2. The strain having the 16S rRNA of SEQ ID NO. 1 in claim 1.

3. A strain according to claim 1, having salt tolerance at a salinity of 1 to 20% (w / v).

4. A strain having the ability to produce polyhydroxyalkanoates (PHA) according to claim 1.

5. In paragraph 4, the polyhydroxyalkanoate is poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), short-chain or medium-chain length, saturated or A strain comprising unsaturated PHA, or any copolymer thereof, or any combination thereof.

6. A strain according to claim 1, wherein polyhydroxyalkanoate accumulates in the cell at a content of 40 to 90%.

7. A strain according to claim 1 that produces polyhydroxyalkanoate at a concentration of 0.5 to 10 g / L.

8. Polyhydroxyalkanoate produced by the strain of claim 1.

9. A composition for producing polyhydroxyalkanoates comprising the strain of claim 1.

10. A method for producing polyhydroxyalkanoate comprising the step of culturing the strain of claim 1.

11. A method for producing polyhydroxyalkanoate according to claim 10, wherein the method involves culturing in a medium containing 1 to 15% (w / v) of NaCl.

12. A method for producing polyhydroxyalkanoate according to claim 10, wherein the method involves culturing in a medium containing 1 to 5% (w / v) of a carbon source.

13. A method for producing a polyhydroxyalkanoate according to claim 12, wherein the polyhydroxyalkanoate is cultured in a medium comprising a carbon source comprising one or more selected from the group consisting of glucose, sucrose, glycerol, fructose, galactose, and xylose.

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