KR20230128167A - Novel strain Halomonas shrimpha IBTH01 isolated from salted-fermented shrimp producing polyhydroxyalkanoate - Google Patents

Abstract

The novel strain of the present invention is a strain derived from salted shrimp which can produce polyhydroxyalkanoate (PHA). The strain name is Halomonas shrimpha IBTH01, and it is deposited under the accession number KCTC 14717BP. A method for producing polyhydroxyalkanoate comprising the step of culturing the strain.

Description

Novel strain Halomonas shrimpha IBTH01 isolated from salted-fermented shrimp producing polyhydroxyalkanoate}

The present invention relates to a new strain derived from salted shrimp producing polyhydroxyalkanoate, Halomonas shrimpa IBTH01, and specifically, a novel strain that can effectively produce polyhydroxyalkanoate with excellent growth even at high salt concentration and It's about the technology you use.

Petroleum-based plastics are widely used worldwide for packaging of food, beverages, cosmetics and pharmaceuticals. However, it is slow to decompose, accumulates in the environment, and poses an ecological threat to terrestrial and marine wildlife. For this reason, biodegradable plastics, which provide durability and excellent performance during use and have biodegradable properties at the end of their useful life, are proposed as an alternative.

Biodegradable plastics are plastics that can be degraded by bacteria or living organisms and are expected to potentially contribute to reducing solid waste such as carbon dioxide emissions from microplastics and petroleum-based plastics in the oceans as they decompose without leaving behind contaminants. It is receiving a lot of attention in the field. Bio-based biodegradable plastics are referred to as biodegradable bioplastics, and representative examples include polylactic acid (PLA) and polyhydroxyalkanoate (PHA). Biodegradable bioplastics can be decomposed into water and carbon dioxide in 6 months to 5 years when landfilled. However, physical recycling is still difficult due to physical property limitations. However, it is expected that eco-friendliness can be maximized if biodegradable bioplastics are used for items that are highly likely to leak into the environment, such as food containers that are difficult to recycle, fishing nets, and fishing gear.

Currently, bioplastics are about 2 million tons, or about 1% of the more than 360 million tons of plastics produced annually, of which about 1.2 million tons of biodegradable plastics are produced. However, it is expected to increase to 1.8 million tons in 2025, and the market growth rate of PHA in particular is expected to increase by about 10 times. As demand increases and biopolymers and products with improved performance increase, the bioplastics market is expected to continue to grow and diversify.

PHAs are substances that act as carbon and energy stores and are accumulated by bacteria and archaea. PHA can be stored in high concentrations in cells when an excess carbon source is present and essential nutrients oxygen, phosphorus or nitrogen are limited. PHAs are similar to synthetic petrochemical-based polymers in their mechanical and technical properties. It is also attracting a lot of attention due to its biocompatibility, biodegradability, and ability to be produced from renewable resources. Until now, strains such as E. coli , R. eutropha , P. putida, etc. have been used for PHA production, but recently, research results have been published that produced 69 to 82% by weight of PHA using halophilic strains.

PHAs are named according to the number of carbons in the R group. PHB (polyhydroxybutyrate), one of the most common PHAs, is a homopolymer of 3-hydroxybutyrate and has a simple structure.

To produce PHA from sugar, PhaA (β-ketothiolase), PhaB (acetoacetyl-CoA-reductase), and PhaC (PHA synthase) enzymes are required. The PhaC enzyme, which operates at the very end, is the key enzyme for PHA production, polymerizing the acyl moiety of 3-hydroxybutyryl-CoA into PHA and releasing coenzyme A at the same time.

PHA synthesizing enzymes are classified into four types according to their primary sequence, substrate specificity and subunit composition. Class I and II synthetases are composed of one type of subunit with a molecular weight of 61 to 73 kDa. Class I synthetase consists of only one type of PhaC, and Class II synthetase consists of two types of PhaC1 and PhaC2. Class III and IV synthetases are composed of two different types of subunits. Class III synthetase consists of a 40 kDa PhaC similar to Class I and II and a dissimilar 40 kDa PhaE. Class IV synthase is mainly found in Bacillus , and unlike Class III, it consists of 40 kDa PhaC and 20 kDa PhaR. Class I, III and IV synthetases prefer short-chain-length (scl) monomers with C3 to C5 carbon chains, typically 3HB (3-hydroxybutyrate) with C4 carbon chains. ) is there. Class II synthase prefers medium-chain-length (mcl) monomers with C6 to C14 carbon chains, for example 3HHx with C6 carbon chains.

Halophilic bacteria are microorganisms that require NaCl to grow. According to current standards, basophils should grow optimally at NaCl concentrations greater than 5% (w/v) and tolerate salts greater than 10% (w/v). Compared to common bacteria, halophilic bacteria have several advantages as PHA producers. First, because high salt concentrations are used, normal bacteria cannot grow, reducing the risk of contamination. Also, since the risk of contamination is low, a continuous process is possible. Second, since separation is possible using distilled water, the cost of the separation process can be reduced. Finally, it can reduce the cost of carbon substrates by being produced using inexpensive renewable resources such as coffee grounds, sugarcane and corn. In addition, since NaCl is synthesized by HCl and NaOH in the process of pretreatment and hydrolysis to use biomass as a carbon substrate, additional supply of NaCl is not required, which can reduce production costs. Due to these advantages, research on halophilic bacteria and halophilic archaea has received much attention.

Interest in biodegradable plastics and bioplastics is increasing worldwide, and many studies have been conducted on the production of PHA from microorganisms. However, industrialization has not yet been activated due to high production costs. Accordingly, the present inventors attempted to discover new halophilic bacteria for efficient production and cost reduction of PHA. The cost of the carbon substrate accounts for 40 to 50% of the total PHA production cost, and since the production of PHA from halophilic bacteria is possible using an inexpensive carbon source, the cost of the carbon substrate can be reduced. In addition, the cost of the sterilization process is reduced due to the low risk of contamination due to the high salt concentration, and the cost of the separation process can also be reduced due to the relatively easy separation of PHA.

Dan Kucera, Iva Pernicov, Adriana Kovalcik, Martin Koller, Lucie Mullerova, Petr Sedlacek, Filip Mravec, Jana Nebesarova, Michal Kalina, Ivana Marova, Vladislav Krzyzanek and Stanislav Obruca (2018) Characterization of the promising poly(3-hydroxybutyrate) producing halophilic The bacterium Halomonas halophila. Bioresource Technology 256, 552-556.

Therefore, the main object of the present invention is to provide a new halophilic strain capable of producing polyhydroxyalkanoate (PHA).

Another object of the present invention is to provide a method for producing polyhydroxyalkanoate using the strain.

Another object of the present invention is to provide a composition for producing polyhydroxyalkanoate using the strain.

According to one aspect of the present invention, the present invention provides a strain deposited with accession number KCTC 14717BP, Halomonas shrimpha ( Halomonas shrimpha ) IBTH01.

According to another aspect of the present invention, the present invention provides a method for producing polyhydroxyalkanoate comprising culturing the strain of the present invention.

In the polyhydroxyalkanoate production method of the present invention, the step of culturing the strain is preferably a step of culturing the strain at a temperature condition of 30 to 40°C.

In the polyhydroxyalkanoate production method of the present invention, the step of culturing the strain is preferably a step of culturing the strain in an LB medium or a marine medium-based medium.

In the polyhydroxyalkanoate production method of the present invention, the culturing of the strain is preferably a step of culturing the strain in a medium containing 2% (w/v) or more of NaCl.

In the polyhydroxyalkanoate production method of the present invention, culturing the strain is a step of culturing the strain in a medium containing at least one carbon source selected from the group consisting of glucose, glycerol, fructose, and galactose. it is desirable

In the polyhydroxyalkanoate production method of the present invention, the step of culturing the strain is preferably a step of culturing the strain in a medium containing 2% (w/v) or more of the carbon source.

In the polyhydroxyalkanoate production method of the present invention, the polyhydroxyalkanoate is preferably polyhydroxybutyrate.

According to another aspect of the present invention, the present invention provides a composition for producing polyhydroxyalkanoate comprising the strain of the present invention.

In the composition for producing polyhydroxyalkanoate of the present invention, the polyhydroxyalkanoate is preferably polyhydroxybutyrate.

The strain of the present invention shows excellent growth even at high salt concentration and can effectively produce polyhydroxyalkanoate. In addition, the method for producing polyhydroxyalkanoate and the composition for producing polyhydroxyalkanoate of the present invention use the strain of the present invention as described above, and according to this, polyhydroxyalkanoate can be effectively produced even in a high-salinity environment. can produce The use of the new strain of the present invention, the method for producing polyhydroxyalkanoate and/or the composition for producing polyhydroxyalkanoate as described above enables efficient production of polyhydroxyalkanoate and reduction of production cost, thereby enabling bio It is expected to contribute to the development of the plastics field.

Figure 1 shows the microbial accession of Halomonas shrimpha IBTH01, which is the strain of the present invention.
Figure 2 shows the 16S rRNA gene partial sequence of the strain of the present invention.
Figure 3 shows a photograph of culturing salted shrimp samples in the process for screening the strains of the present invention.
4 shows PCR results for confirming whether a candidate strain has a phaC gene in the process of selecting the strain of the present invention. M: molecular weight marker, 1: control strain ( Halomonas halophila ), 2: Halomonas shrimpha ( Halomonas shrimpha ) IBTH01.
Figure 5 shows the results of phylogenetic tree analysis of the strains of the present invention based on the 16s rRNA gene sequence.
6 shows a graph of GC analysis results of PHBV used as a standard material. A: PHBV 20 mg analysis result, B: PHBV 40 mg analysis result, C: PHBV 60 mg analysis result.
7 shows a graph of the GC analysis results of the strains of the present invention.
8 shows NMR peaks of PHBV used as a standard material.
9 shows NMR peaks of PHB used as a standard material.
10 shows NMR peaks of the strain of the present invention.
Figure 11 shows the growth curve according to the culture temperature of the strain of the present invention. Round dot curve: 30°C incubation, triangular dot curve: 37°C incubation.
Figure 12 shows the PHB production according to the incubation temperature of the strain of the present invention.
Figure 13 shows the PHB production according to the culture medium of the strain of the present invention.
14 shows PHB production according to NaCl concentration during cultivation of the strain of the present invention.
15 shows PHB production according to the type of carbon source during cultivation of the strain of the present invention.
16 shows PHB production according to glucose concentration during cultivation of the strain of the present invention.

The novel strain Halomonas shrimpha IBTH01 of the present invention was isolated from salted shrimp and deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (Korean Collection for Type Cultures, KCTC) under accession number KCTC 14717BP (FIG. 1).

The strain of the present invention has a 16S rRNA gene sequence including the nucleotide sequence of SEQ ID NO: 1 (FIG. 2).

The strain of the present invention can be cultured in, for example, marine medium or LB medium. The incubation temperature may be, for example, 30 to 40°C. As the carbon source of the culture medium, for example, at least one selected from the group consisting of glucose, sucrose, glycerol, fructose, galactose and xylose A carbon source may be used.

According to the study results, the strain of the present invention was confirmed as a halophilic strain showing excellent growth ability even at a high salinity of 10% (w / v) NaCl level, and also showed excellent PHA productivity at such a high salinity. Therefore, since culture and PHA production can be performed under high salinity conditions, there is an advantage in that culture and PHA can be produced while reducing contamination of other common bacteria.

According to the study results, the strain of the present invention possesses the phaC gene, which is important for the production of polyhydroxyalkanoate (PHA). Therefore, it is believed that PHA can be produced due to the action of PhaC through the expression of this gene.

The strain of the present invention can effectively produce polyhydroxybutyrate (PHB) among PHA, especially poly(3-hydroxybutyrate) [Poly(3-hydroxybutyrate), P3HB].

The present invention provides a method for effectively producing PHA using the strain of the present invention as described above, comprising culturing the strain of the present invention.

The culture temperature conditions of the culturing step may be, for example, 28 to 40 ° C, but are preferably 30 to 40 ° C, more preferably 35 to 39 ° C, and more preferably 36 to 38 ° C. According to this, PHA can be produced more efficiently, for example, in a larger amount within the same amount of time.

The culture medium in the culturing step may be, for example, a medium based on LB medium or marine medium, but is preferably a medium based on LB medium. According to this, PHA can be produced more efficiently, for example, in a larger amount within the same amount of time. In this case, the base medium may be a medium having the same composition as the base medium or a medium in which the type and/or content of salt and/or carbon source is changed.

Culture salinity in the culturing step may be, for example, NaCl is 1% (w / v) or more. Preferably, the level of NaCl is 2% (w / v) or more, more specifically, the level of 2 to 10% (w / v), more preferably the level of NaCl is 4 to 10% (w / v), more preferably Preferably, the level of NaCl is 6 to 10% (w / v), more preferably the level of NaCl is 9 to 10% (w / v). According to this, PHA can be produced more efficiently, for example, in a larger amount within the same amount of time. The above level of salinity can be achieved by adding a corresponding level of salt, for example, NaCl, to the medium.

The culture medium in the culturing step may be a medium containing at least one selected from the group consisting of, for example, glucose, glycerol, fructose, and galactose as a carbon source. Preferably, it is a medium containing at least one selected from the group consisting of glucose, glycerol and galactose as a carbon source, more preferably a medium containing glucose as a carbon source. According to this, PHA can be produced more efficiently, for example, in a larger amount within the same amount of time.

In addition, the culture medium in the culturing step may be a medium containing 0.5% (w / v) or more of the carbon source as described above, more specifically, a medium containing 0.5 to 4% (w / v) of the carbon source. . Preferably, a medium containing 1% (w / v) or more of the carbon source, more specifically, a medium containing 1 to 4% (w / v) of the carbon source, more preferably 2% (w / v) of the carbon source /v) or more, more specifically, a medium containing 2 to 4% (w/v) of the carbon source. According to this, PHA can be produced more efficiently, for example, in a larger amount within the same amount of time.

The PHA production method of the present invention as described above is particularly suitable for the production of PHB (polyhydroxybutyrate), particularly P3HB [poly(3-hydroxybutyrate), Poly(3-hydroxybutyrate)].

The present invention also provides a composition for producing PHA comprising the strain of the present invention as a composition for use in effectively producing PHA using the strain of the present invention as described above.

The composition for producing PHA of the present invention may be, for example, a microbial preparation used to produce PHA through microbial culture.

The composition for producing PHA of the present invention can be used, for example, by adding the composition of the present invention to a medium for producing PHA. The composition for producing PHA of the present invention in an appropriate medium described above in connection with the PHA production method, for example, an LB-based medium containing about 6% (w/v) NaCl and about 2% (w/v) glucose PHA may be produced in cells by adding and then culturing at an appropriate temperature, for example, about 37° C. for an appropriate period of time, for example, about 72 hours. PHA can be purified by applying a conventional PHA purification method conventionally used from the PHA-produced cells.

The composition for producing PHA of the present invention needs to maintain the strain of the present invention in the composition until use. Therefore, additives necessary for maintaining the viability of the strain may be further included. The additives include additives used in common probiotics, such as lyophilization protection agents.

The composition for producing PHA of the present invention may include the strain of the present invention in a lyophilized form, for example. In addition, the composition for producing PHA of the present invention may include, for example, 0.01 to 100% by weight of the strain of the present invention, but is not limited thereto.

The composition for producing PHA of the present invention as described above is preferably a composition for producing PHB (polyhydroxybutyrate), particularly P3HB [poly(3-hydroxybutyrate), Poly(3-hydroxybutyrate)].

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

Example 1. Strain selection

1-1. method

1-1-1. Sample preparation and candidate strain acquisition

I bought salted shrimp at the traditional market in Jochiwon, Sejong City. The obtained samples were stored at 4°C. After sufficiently grinding the salted shrimp sample in a blender, only the liquid was used for plate culture. Samples obtained by diluting the sample stock solution with distilled water (10 ^-1 , 10 ^-2 ) were incubated on a marine 8% agar plate (37°C, 24 hours), and the morphology and color of colonies were compared to candidate strains. has been secured.

1-1-2. Selection of phaC gene-bearing strains

Strains having the phaC gene were selected using polymerase chain reaction (PCR).

Genomic DNA of the obtained candidate strain was purified using the Solg ^TM Genomic DNA Prep Kit (Solgent, Korea). The primers in Table 1 were used, and the PCR conditions were [95°C 5 minutes] × 1, [95°C 1 minute, 60°C 90 seconds, 72°C 2 minutes] × 30, [70°C 5 minutes] × 1. . Halomonas halophila was used as a control strain. After completion of the PCR reaction, amplification was confirmed by electrophoresis using a 1% agarose gel.

primer order forward 5'-CCN CCN TGG ATC AAY AAG TAY TAC-3' reverse 5'-CCA RTG NGG CCA CCA GGA RCC YTC-3'

1-1-3. Confirmation of PHA production

Candidate strains were cultured using LB, Marine and Mineral media as a culture medium. The composition of each medium is listed in Table 2. Cultivation proceeded in two stages. In order to increase the number of cells, pre-culture was first performed. The strain was cultured in 50 ml of medium at 37°C and 200 rpm for 20 hours. The culture was inoculated at 3% by volume to the main culture and cultured in 100 ml of medium for 72 hours at 37 ° C. and 200 rpm. In this culture, NaCl (2-6%) and glucose (2%) were additionally added.

badge composition LB badge 5g Yeast extract, 10g Tryptone, 10g NaCl in 1L distilled water marine badge 19.45 g NaCl, 5.9 g MgCl ₂ , 5 g Peptone, 3.24 g MgSO ₄ , 1 g Yeast extract, 1.8 g CaCl ₂ , 0.55 g KCl, 0.16 g NaHCO ₃ , 0.1 g Ferric citrate, 0.08 g KBr, 34 mg SrCl in 1 liter of distilled water ₂ , 22mg H ₃ BO ₃ , 4mg Na-silicate, 2.4mg NaF, 1.6mg NH ₄ NO ₃ , 8mg Na ₂ HPO ₄ mineral badge 3g (NH ₄ ) ₂ SO ₄ , 1g KH ₂ PO ₄ , 11.1g Na ₂ HPO ₄ 12H ₂ O, 0.2g MgSO ₄ 7H ₂ O in 1 liter of distilled water, 1㎖ micronutrient solution (micronutrient solution: 0.1M 9.7 g FeCl ₃ , 7.8 g CaCl ₂ , 0.156 g CuSO ₄ 5H ₂ O, 0.119 g CoCl ₂ , 0.118 g NiCl ₂ , 0.062 g CrCl ₂ in HCl)

After culturing for 72 hours, cells were harvested by centrifugation at 4000 rpm for 30 minutes. Thereafter, the cells were washed with distilled water and centrifuged again to obtain only clean cells. After drying the cells at high temperature for 1 to 2 days, 2 ml of chloroform and 1 ml of PHA solution (Methanol 970 ml, H ₂ SO ₄ 30 ml, benzoic acid 8 g) were added to about 40 mg of the dried sample and incubated overnight at high temperature. did Then, after slowly cooling at room temperature, 1 ml of distilled water was added, and the mixture was allowed to stand until the layers were separated. When the layer separation was clear, only 1 ml of the bottom layer was aliquoted and used for GC analysis.

PHA quantitative analysis was performed using GC (shimadzu GC-2014, Japan). DB-WAX (length 30m, diameter 0.25mm, film 0.25μl, J&W scientific, USA) was used as the column. The initial temperature of the analysis was set to 100 °C in the oven, 250 °C in the injector, and 250 °C in the detector. Helium (He) was used as a carrier gas, and hydrogen (H ₂ ) was used as an auxiliary gas. PHBV [Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)] (Sigma Aldrich) was used as a standard material.

1-1-4. Strain identification and phylogenetic tree analysis

16s rRNA analysis was performed to identify the selected strain. Analysis was commissioned by Cosmogenetech (http://www.cosmogenetech.com/main.jsp). Universal primers, forward primer 27F (5'-AGA GTT TGA TCM TGG CTC AG-3') and reverse primer 1492R (5'-GGT TAC CTT GTT ACG ACT TC-3') were used for 16s rRNA gene amplification.

NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for sequence analysis. For sequence analysis, blastn was used, and the search range was designated as 16s ribosomal RNA sequence (Bacteria and Archaea) in the rRNA/ITS database. The 16s rRNA analysis results were entered into NCBI BLAST and similarity comparisons were made. For the phylogenetic tree analysis, Fast Minimum Evolution was used, and the max sequence difference was set to 0.05.

1-2. result

1-2-1. Isolation of strains from samples

As a result of culturing the sample on a marine 8% agar plate for 24 hours, colonies as shown in FIG. 3 appeared. When comparing the appearance of these colonies, there are various colors such as light yellow, dark yellow, and slightly transparent white, and the difference in size can also be confirmed. Candidate strains were obtained by comparing the shape and color of colonies on each plate.

1-2-2. Selection of phaC gene-bearing strains

From the candidate strains, a strain having the phaC gene, that is, a strain showing a phaC amplification band was obtained through PCR (FIG. 4).

1-2-3. Confirmation of PHA production

Culture was performed to confirm whether the strain obtained in Example 1-2-2 could produce PHA. Two mediums, marine and mineral, were used, and NaCl (2-6%) and glucose (2%) were additionally added. Incubation was carried out for 72 hours at 200 rpm at 30 °C. Halomoans halophila was used as a control strain to compare yields.

As a result of GC analysis, the obtained strain was confirmed to produce PHB (polyhydroxybutyrate) among PHA.

1-2-4. Strain identification and phylogenetic tree analysis

Microbial identification and phylogenetic tree analysis were performed on the strains having the phaC gene and producing PHA in Examples 1-2-2 and 1-2-3.

Microbial identification was confirmed by 16s rRNA analysis, and taxonomic identification and phylogenetic tree analysis were performed using NCBI BLAST based on the 16s rRNA results. The partial sequence of 16s rRNA is shown in SEQ ID NO: 1, and the phylogenetic tree analysis results are shown in FIG. 5 . In addition, the 16s rRNA sequence of the obtained strain was registered in the NCBI 16s rRNA database (NCBI registration number: MW479458).

The strain IBTH01 isolated from salted shrimp was identified as the genus Halomonas of the family Halomonadaceae. 98% concordance with Halomonas alimentaria , 98% concordance with Halomonas halodenitrificans , and 98% concordance with Halomonas nitroreducens . It was registered as a new species, and the name was named Halomonas shrimpha IBTH01.

Example 2. Characterization of PHA production of strains

2-1. Classification of types of PHAs produced

GC analysis was performed to confirm PHA production of IBTH01. PHBV [poly(3-hydroxybutyrate-co-3-hydroxyvalerate)] and PHB [poly(3-hydroxybutyrate)] were used as standard materials. The assay was performed using about 40 mg of dry IBTH01 cell sample. When the graph was drawn with the resultant value of the standard material, it was confirmed that the R ² value was 0.9992, which was a reliable result.

As a result of standard material analysis, 3HB was detected at 5.8 components and 3HV was detected at 6.7 components (FIG. 6). Chloroform and benzoic acid were detected at 2.2 and 7.7 minutes, respectively.

As a result of the analysis of IBTH01, a peak was detected in the 3HB time zone, 5.8 minutes, but nothing was detected in the 3HV time zone, 6.7 minutes (FIG. 7). Thus, IBTH01 was confirmed to produce PHB.

NMR (Jeol, Japan) analysis was further conducted to reconfirm PHB production. Chloroform-d (CDCl ₃ ) (Sigma Aldrich) was used as the analysis solvent, and PHBV (Sigma Aldrich) and PHB (Sigma Aldrich) were used as standard materials. After mixing 10 mg/ml of the dry sample with the analysis solvent, only the liquid was used for NMR analysis.

The NMR analysis results of PHBV and PHB are shown in FIGS. 8 and 9 . As a result of the analysis, it was confirmed that the peak pattern of IBTH01 coincided with that of PHB (FIG. 10).

2-2. Determining the effect of temperature on PHB production

To set the growth temperature of IBTH01, two temperature conditions of 30 ° C and 37 ° C were tested. IBTH01 showed about 3 times higher growth at 37°C than at 30°C (FIG. 11). It was confirmed that PHB production increased by about 5 times (FIG. 12).

2-3. Determining the effect of media on PHB production

The effect of the medium on the production of PHB by IBTH01 was confirmed. A total of three LB, marine and mineral media were tested, and 6% NaCl and 2% glucose were equally introduced. Incubation was carried out for 72 hours at 200 rpm at 37 °C.

IBTH01 showed about 57% yield with 4.12±0.10g/ℓ PHB out of 7.13±0.09g/ℓ DCW (dry cell weight) in LB medium, and 1.07±0.06g out of 2.32±0.10g/ℓ DCW in marine medium. /L PHB showed about 45% yield (FIG. 13). Mineral media were found to be unsuitable for PHB production. As a result of the experiment in the three media, it was confirmed that both strain growth and PHB production were highest in the LB medium, and then the experiment was conducted in the LB medium.

2-4. Comparison of PHB production at various NaCl concentrations

After confirming the effect of the medium, the effect of the concentration of NaCl on PHB production was confirmed. LB was used as the medium, and 2 to 10% of NaCl was additionally added. 2% glucose was used as a carbon source. Incubation was carried out for 72 hours at 200 rpm at 37 °C.

It was confirmed that the concentration of NaCl in IBTH01 did not significantly affect the growth of the strain. However, PHB production showed little difference, among which 7.66 ± 0.10 g / ℓ PHB in 10% NaCl and 4.47 ± 0.01 g / ℓ PHB in DCW showed the highest accumulation with about 58% production (FIG. 14).

2-5. Comparison of PHB production from different carbon substrates

Experiments were conducted using various carbon substrates to confirm the effect of carbon substrates on PHB production. Glucose, sucrose, glycerol, fructose, galactose and xylose were used. Equal amounts of 6% NaCl and 2% carbon substrate were added to LB medium. Incubation was carried out for 72 hours at 200 rpm at 37 °C.

IBTH01 could confirm PHB accumulation in glucose, glycerol, fructose, and galactose, but it was difficult to confirm accumulation in sucrose and xylose (FIG. 15). Glucose showed the highest PHB production at 57%, and strain growth also showed the highest growth in glucose with glucose >> glycerol > fructose > galactose (FIG. 15).

Growth of the strain in glycerol gave quite interesting results. 5.46 ± 0.64 g / ℓ of biomass was produced, which showed a fairly valuable result with about 76% of biomass production compared to glucose (FIG. 15). However, PHB production showed a big difference, only about 21% of total DCW was produced, and it was very reduced to about 28% of glucose (FIG. 15).

In conclusion, it was confirmed that glucose is the most suitable for the growth of IBTH01 and PHB production, and glycerol is also quite useful when only strain growth is required without PHB production.

2-6. Comparison of PHB production at various glucose concentrations

After confirming that glucose is most suitable for PHB production, strain growth and PHB production were compared using various concentrations. 0 to 4% glucose was added to LB medium supplemented with 6% NaCl. Incubation was carried out for 72 hours at 200 rpm at 37 °C.

IBTH01 did not produce PHB in a glucose-free medium, and 0.5% and 1% were also insufficient to say that it was produced (FIG. 16). PHB production could be confirmed from the time 2% glucose was added, and strain growth and PHB production increased up to 3%, but a tendency to decrease from 4% was confirmed (FIG. 16). An excess of carbon substrate is thought to hinder strain growth and inhibit accumulation as well. The highest production was shown when 3% glucose was added, and 4.54 ± 0.3 g / ℓ PHB was produced in 8.63 ± 0.05 g / ℓ DCW, showing about 52% production (FIG. 16).

PHB production according to the concentration of glucose did not show a clear difference from 2% or more, and 3% showed slightly higher accumulation (FIG. 16). However, since the cost of the carbon substrate is the biggest problem for PHB production, if there is not a big difference, using 2% glucose will be more economically effective.

Korea Research Institute of Bioscience and Biotechnology KCTC14717BP 20210929

<110> Sejong Industry-Academia Cooperation Foundation Hongik University <120> Novel strain Halomonas shrimpha IBTH01 isolated from salted-fermented shrimp producing polyhydroxyalkanoate <130> ALP22001 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1193 <212> DNA <213> unknown <220> <223> Halomonas shrimpha IBTH01 <400> 1 ccctgggcta ccagcctaca catgcagtcg agcggaacga tggaagcttg cttccaggcg 60 tcgagcggcg gacgggtgag taatgcatag gaatctgccc gatagtgggg gataacctgg 120 ggaaactcag gctaataccg catacgtcct acgggagaaa gcaggggatc ttcggacctt 180 gcgctatcgg atgagcctat gtcggattag ctagttggtg aggtaacggc tcaccaaggc 240 cgcgatccgt agctggtctg agaggatgat cagccacatc gggactgaga cacggcccga 300 actcctacgg gaggcagcag tggggaatat tggacaatgg gcgaaagcct gatccagcca 360 tgccgcgtgt gtgaagaagg ccctcgggtt gtaaagcact ttcagtgggg aagaaatcct 420 cagggccaat acccctgagg gaggacatca cccacagaag aagcaccggc taactccgtg 480 ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg 540 cgcgtaggcg gcgtgatcag ccggttgtga aagccccggg ctcaacctgg gaacggcatc 600 cggaactgtc acgctagagt gcaggagagg aaggtagaat tcccggtgta gcggtgaaat 660 gcgtagagat cgggaggaat accagtggcg aaggcggcct tctggactga cactgacgct 720 caggtgcgaa agcatgggta gcaaacagga ttagataccc tggtagtcca tgccgtaaac 780 gatgtcgact agccgttggg gtccttgtga cctttgtggc gcagttaacg cgataagtcg 840 accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgacggg ggcccgcaca 900 agcggtggag catgtggttt aattcgatgc aacgcgaaga accttaccta ctcttgacat 960 cgtgcgaact ttctagagat ggattggtgc cttcgggaac gcacagacag gtgctgcatg 1020 gccgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc cgtaacgagc gcacccctat 1080 ccctatttgc cagcgattcg gtcgggaact ctagggagac tgccggtgac aaccggagga 1140 aggggggatg acgtcaggtc atcatggccc ttacgagtag ggctaccacg tgc 1193

Claims (10)

Halomonas shrimpha, a strain deposited with accession number KCTC 14717BP, IBTH01. A method for producing polyhydroxyalkanoate comprising culturing the strain of claim 1. According to claim 2,
The step of culturing the strain is a step of culturing the strain at a temperature condition of 30 to 40 ° C., polyhydroxyalkanoate production method. According to claim 2,
The step of culturing the strain is a step of culturing the strain in a medium based on LB medium or marine medium, polyhydroxyalkanoate production method. According to claim 2,
Culturing the strain is a step of culturing the strain in a medium containing 2% (w / v) or more of NaCl, polyhydroxyalkanoate production method. According to claim 2,
The step of culturing the strain is a step of culturing the strain in a medium containing at least one carbon source selected from the group consisting of glucose, glycerol, fructose and galactose, polyhydroxyalkanoate production method. According to claim 6,
The step of culturing the strain is a step of culturing the strain in a medium containing 2% (w / v) or more of the carbon source, polyhydroxyalkanoate production method. According to claim 2,
The method of producing polyhydroxyalkanoate, wherein the polyhydroxyalkanoate is polyhydroxybutyrate. A composition for producing polyhydroxyalkanoate comprising the strain of claim 1. According to claim 9,
The polyhydroxyalkanoate is polyhydroxybutyrate, a composition for producing polyhydroxyalkanoate.