KR20080111784A - Inhibitor of medium-chain-length-polyhydroxyalkanoic acid synthesis and preparation method of long-chain-length-aromatic polyhydorxyalkanoic acid using the same - Google Patents

Abstract

An inhibitor for the synthesis of medium-chain-length-polyhydroxyalkanoic acid and a preparation method of long-chain-length-aromatic polyhydorxyalkanoic acid using the same are provided to obtain functional aromatic PHA by controlling the PHA side chain length. A preparation method of long-chain-length-aromatic polyhydorxyalkanoic acid comprises (a) a step for cultivating Pseudomonas calcoaceticus in the culture medium containing sugar, a substituted fatty acid and a synthesis inhibitor of a polyhydroxyalkane acid; and (b) a step for collecting the polyhydroxyalkane acid containing the high content chain-lengthened aromatic monomer from the strain cultivated in (a) step.

Description

Inhibitor of medium-chain-length-polyhydroxyalkanoic acid synthesis and preparation method of long-chain-length-aromatic polyhydorxyalkanoic acid using the same}

1 is a graph showing concentration dependent inhibition of incorporation of metabolized aliphatic monomers into PHA from fructose by 11-POU (11-phenoxyundecanoic acid) in Pseudomonas fluorescens BM07.

FIG. 2 shows the molar ratio of total aliphatic and aromatic monomer-units and the growth rate of cells according to the concentration of salicylic acid of PHA synthesized by Pseudomonas fluorescens BM07 cultured in a medium containing 50 mM fructose and 3 mM 11-POU. Graph (A) and 3-hydroxy-5-phenoxyvaleric acid (5-POHV), 3-hydroxy-7-phenoxyheptanoic acid (7POHH) and 3-hydroxy-9 in the aromatic monomer-unit -It is a graph which shows the molar ratio of phenoxy nonanoic acid (9POHN) (B).

Figure 3 shows the effect of salicylic acid or acrylic acid on the monomer composition of PHA synthesized by Pseudomonas fluorescens BM07 cultured in a medium containing fructose and 11-POU.

4 is a graph showing the molar ratio of monomer-units according to the concentration of salicylic acid of PHA synthesized by Pseudomonas fluorescens BM07 cultured in a medium containing 50 mM fructose and 5 mM 11-POU.

FIG. 5 shows a blend between polyester and aliphatic MCL-PHA and aromatic MCL-PHA synthesized by Pseudomonas fluorescens BM07 cultured in medium containing fructose and 11-POU in the absence or presence of 0.7 mM salicylic acid. The organic solvent fractions are compared.

6 shows the glass transition temperature of PHA synthesized by Pseudomonas fluorescens BM07 cultured in medium containing fructose and 11-POU in the absence (A, C) or presence (B, D) of 0.7 mM salicylic acid. The graph shown.

The present invention relates to a synthesis inhibitor of medium-chain-length-polyhydroxyalkanoic acid (hereinafter referred to as MCL-PHA) and a method for producing a long-chain aromatic polyhydroxyalkanoic acid using the same. More specifically, the present invention is an inhibitor composition of the synthesis of MCL-PHA of Pseudomonas spp strain containing salicylic acid or acrylic acid as an active ingredient, the inhibitor composition, a sugar, a medium containing substituted fatty acids A long chain aromatic polyhydroxyalkanoic acid production method characterized by culturing a strain of Pseudomonas sp. And a long chain aromatic monomer of polyhydroxyalkanoic acid, characterized in that the fractional precipitation of the long chain aromatic polyhydroxyalkanoic acid (aromatic and a method for separating monomers).

Polyhydroxyalkanoic acid (hereinafter referred to as PHA) is a polyester that microorganisms accumulate as a carbon source and energy storage material when nutrient deficiencies such as nitrogen, phosphoric acid, sulfur and oxygen are caused in the presence of excess carbon source. polyester) natural polymeric material of structure (Anderson and Dawes, Microbiol. Rev., 54: 450-472 (1990); Lee, Biotechnol. Bioeng., 49: 1-14 (1996)). It is known that ∨ PHA is accumulated in the form of a granular inclusion bodies (granular inclusion body) in a variety of microorganisms (Anderson, AJ et al, Microbiol Rev 1990, 54, 450;.... Madison, LL et al, Microbiol. Mol. Biol. Rev. 1999, 63, 21; Sudeshi, K. et al., Prog.Polym . Sci . 2000, 25, 1503; Lenz, RW et al., Biomacromolecules 2005, 6, 1; P.tter, M. et al., Biomacromolecules 2005, 6, 552). PHAs exist in various forms, chemically having different monomer constituents and exhibiting various useful physicochemical properties. Insertion of genes related to homomorphic or heterophasic PHA synthesis, combinations of different precursor carbon sources, multistage cultures (Choi, MH et al., Biomacromolecules 2003, 4) to improve properties such as melting point, glass transition temperature, crystallinity, degradation rate, mechanical strength, etc. , 38), pathwary routing by inhibitors (Green, PR et al., Biomacromolecules 2002, 3, 208; Lee, H.-J. et al., J. Microbiol. Biotechnol . 2004, 14, 1256 Qi, Q. Steinbuchel, et al., FEMS Microbiol . Lett . Many techniques such as 1998, 167, 89) allow for the design of PHAs.

PHA synthesis-related inhibitors can be used to discover metabolic pathways from precursors to PHA synthesis and can be used to deliver intermediate metabolites of specific pathways of PHA synthesis (Green, PR et al., Biomacromolecules 2002, 3 , 208; Lee, H.-J. et al., J. Microbiol.Biotechnol. 2004, 14, 1256; Qi, Q. Steinb Chel, et al., FEMS Microbiol. Lett . 1998, 167, 89). In previous studies, we described the heavy chain length (MCL) -PHA synthesis of Pseudomonas fluorescens BM07 from Pseudomonas fluorescens BM07 from sugars without 2-bromooctanoic acid (2-BrOA) affecting cell growth. Very specific inhibition has been reported (Lee, H.-J. et al., Appl. Environ. Microbiol . 2001, 67, 4963). 2-BrOA is a very useful inhibitor for effective route routing for the production of specifically designed PHAs using cells in an active growth phase (Lee, H.-J. et al. , J. Microbiol. Biotechnol . 2004, 14, 1256; Lee, H.-J. et al., Appl. Environ. Microbiol . 2001, 67, 4963).

On the other hand, a clear competitive relationship has been reported between 2-BrOA and octanoic acid or 11-phenoxyundecanoic acid (hereinafter referred to as 11-POU) (Lee, H.-J. et al., J. Microbiol. Biotechnol . 2004, 14, 1256). 2-BrOA increases the conversion-dependently and most sensitively and effectively the yield of conversion from 11-POU to PHA at a relatively low concentration of 11-POU (similar to 2-BrOA levels). This suggests that 2-BrOA can be used to increase the yield of converting expensive substituted fatty acids to PHA (up to 100% for 11-POU). Wautersia eutropha is known to mix short chain length (SCL) monomers with PHA (Anderson, AJ et al., Microbiol. Rev. 1990, 54, 450; Madison, LL et al., Microbiol.Mol. Biol. Rev. 1999, 63, 21; Sudeshi, K. et al., Prog.Polym . Sci . 2000, 25, 1503; Lenz, RW et al., Biomacromolecules 2005, 6, 1; tter, M. et al., Biomacromolecules 2005, 6, 552). However, acrylic acid, known as a β-oxidation inhibitor of bacteria, induces the synthesis of PHAs containing MCL monomers as copolymer-monomers when culturing wild-type cells using octanoic acid as a carbon source (Green, PR et al., Biomacromolecules 2002). , 3, 208).

The preparation of MCL-PHA from ω-functional substituted MCL fatty acids can be an important tool for obtaining modified 3-hydroxy acids and PHAs by being used as starting intermediates in pharmacology and other related chemistry (Lee, SY et al., Biotechnol.Bioeng. 1999, 65, 363; Song, JJ et al., Appl. Environ.Microbiol. 1996, 62, 536). On the other hand, their physical properties depend on the uniformity and length of the pendant groups. Thus, control of the distribution of side-chain monomer units can be expected to improve physical properties (Nomura, CT et al., Biomacromolecules 2004, 5, 1457; Abe, H. et al., Biomacromolecules 2002, 3, 133; Noda, I. et al., Biomacromolecules 2005, 6, 580). In general, the distribution of monomers in MCL-PHA is specifically dependent on the synthase and intracellular concentration of monomer precursors. On the other hand, due to the high pressure of acetyl-CoA produced through β-oxidative degradation of coenzyme A for growth, the conversion of long-chain monomer-units from short to short in MCL-PHA synthesis is not easy. In some cases, it has been reported that addition of carbon sources that do not induce MCL-monomers can increase the proportion of long chain monomers (Song, JJ et al., Appl. Environ. Microbiol. 1996, 62, 536). For example, Pseudomonas footage is BM01 (Pseudomonas putida BM01) was cultured in a medium containing butyric acid, which only supported cell growth and did not induce the synthesis of PHA monomers, thereby the 3-hydroxy-9-phenoxynonano, the longest monomer-unit in PHA produced from 11-POU. The level of 8 (9POHN) could be increased to 11 mol%. On the other hand, instead of using such a "cosubstrate method " , if one uses an inhibitor that interferes with PHA synthesis by specifically affecting the oxidative degradation pathway of the functional substrate or the enzyme associated with the PHA-synthetic pathway from the substrate, It is expected that it will be possible to convert monomer units to longer monomer-units.

Therefore, the present inventors studied the method of inhibiting the synthesis of MCL-PHA and converting the monomer unit into a longer chain monomer, and when the salicylic acid was incubated in Pseudomonas fluorescens in a medium containing 11-POU and fructose, The present invention was completed by confirming that the synthesis of MCL-PHA can be suppressed while at the same time converting the aromatic monomer units in the PHA to more long-chain monomer units.

Accordingly, an object of the present invention is to provide a composition for inhibiting the synthesis of heavy chain length polyhydroxyalkanoic acid of Pseudomonas strain comprising salicylic acid or acrylic acid as an active ingredient.

Another object of the present invention is to provide a method for producing long chain aromatic polyhydroxyalkanoic acid, which comprises culturing Pseudomonas strains in a medium containing the inhibitor composition, sugars and substituted fatty acids.

Another object of the present invention is to provide a method for separating long-chain aromatic monomer units of polyhydroxyalkanoic acid, which is characterized by fractional precipitation of the long-chain aromatic polyhydroxyalkanoic acid.

In order to achieve the object of the present invention as described above, the present invention provides a composition for inhibiting the synthesis of heavy chain length polyhydroxyalkanoic acid of Pseudomonas strain comprising salicylic acid or acrylic acid as an active ingredient.

The present invention also provides a method for producing long chain aromatic polyhydroxyalkanoic acid, characterized in that the strain of Pseudomonas genus is cultured in a medium containing the inhibitor composition, sugars and substituted fatty acids.

The present invention also provides a method for separating long-chain aromatic monomer units of polyhydroxyalkanoic acid, characterized in that the long-chain aromatic polyhydroxyalkanoic acid is fractionated.

Hereinafter, the present invention will be described in detail.

The physical properties of MCL-PHA depend on the length of the side-chains of the component copolymer-monomer-units and their distribution. Therefore, control of the monomer-unit ratio is essential for improving their physical properties. On the other hand, the present invention is meaningful to identify a new method for controlling the monomer-unit distribution of MCL-PHA by using a non-metabolic inhibitor.

In one embodiment of the present invention, salicylic acid or acrylic acid inhibits the concentration-dependent synthesis of MCL-PHA from fructose in Pseudomonas strains and changes the distribution of monomer-units derived from MCL fatty acids added together as a carbon source. It was found (see Example 2). Generally 3-hydroxy-5-phenoxyvaleric acid (5-POHV) is the main monomer-unit of aromatic monomers, 3-hydroxy-7-phenoxyheptanoate (7POHH) and 3-hydroxy-9 Longer monomer-units, such as -phenoxynonanonic acid (9POHN), are detected as minor monomers (Lee, H.-J. Rho, JK Noghabi, KA Lee, SE Choi, MH Yoon, SC J. Microbiol.Biotechnol 2004, 14, 1256). On the other hand, attempts have been made to increase the level of monomer units in longer chains by increasing the concentration of 11-POU added (Song, JJ Yoon, SC Appl. Environ. Microbiol . 1996, 62, 536; Song, JJ Choi, MH Yoon, SC Huh, NE J. Microbiol . Biotechnol . 2001, 11, 435). However, in most cases 5POHV was still detected as the main monomer-unit. However, as confirmed in one embodiment of the present invention, the addition of salicylic acid or acrylic acid to cells cultured in medium supplemented with 50 mM fructose and 3 mM 11-POU inhibited aliphatic monomer-units, but aromatic monomer-units. The total content of was increased compared to the control. In particular, it was observed that among the aromatic monomer units, the content of the longer long-chain aromatic monomer-units was significantly increased. More significant conversion was observed in cells cultured at 50 mM fructose and 5 mM 11-POU. Aliphatic PHA synthesis from fructose was extremely inhibited by 11-POU in the absence of salicylic acid (the total amount of aliphatic monomers included was only ˜10 mol%). Therefore, the conversion of salicylic acid or acrylic acid as a result of inhibiting β-oxidase in the accumulation of aromatic PHA in Pseudomonas spp. Transforms the distribution of aromatic monomer-units into longer longer unit and PHA at 11-POU. It was found that the conversion yield to significantly increased.

Therefore, the present invention provides a composition for inhibiting the synthesis of heavy chain length polyhydroxyalkanoic acid of Pseudomonas strain comprising salicylic acid or acrylic acid as an active ingredient. Inhibitor compositions of the heavy chain length polyhydroxyalkanoic acid according to the present invention can be used to discover metabolic pathways from precursors to PHA synthesis or as pathway blockers of intermediate metabolites of specific pathways of PHA synthesis.

The salicylic acid is preferably contained in a concentration of 0.4 ~ 1.5 mM and acrylic acid is contained in 0.5 ~ 2.0 mM. When salicylic acid and acrylic acid are contained less than 0.4 mM and 0.5 mM, respectively, there is a disadvantage in that the inhibition of aliphatic monomer-unit and the increase of aromatic monomer-unit are not effective in the synthesis of polyhydroxyalkanoic acid, respectively, 1.5 mM At a concentration exceeding and 2.0 Mm, the inhibitor-induced effect on the synthesis of polyhydroxyalkanoic acid does not increase anymore, but rather inhibits the growth of Pseudomonas spp.

Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas fluorescens BM07 ( Pseudomonas fluorescens BM07, Accession No .: KCTC 10005BP) may be preferably used.

The Pseudomonas fluorescens BM07 strain has been deposited with KCTC 10005BP in the Gene Bank (Korean Collection for Type Cultures) as a strain that the inventors have previously isolated and identified from activated sludge in a wastewater treatment plant (Lee, H. -J. et al., Appl . Environ . Microbiol . 2001, 67, 4963).

On the other hand, the present invention provides a method for producing a long-chain aromatic polyhydroxyalkanoic acid, characterized in that culturing Pseudomonas strains in a medium containing the medium-chain polyhydroxyalkanoic acid synthesis inhibitor, sugar, and substituted fatty acid to provide.

Preferably, the method comprises the steps of: (a) culturing a strain of Pseudomonas in a medium containing a sugar, a substituted fatty acid, and an inhibitor of synthesis of the polyhydroxyalkanoic acid;

(b) recovering the polyhydroxyalkanoic acid containing a high content of the long chain aromatic monomer from the strain cultured in step (a).

Pseudomonas fluorescens ( Pseudomonas fluorescens ), more preferably Pseudomonas fluorescens BM07 ( Pseudomonas fluorescens BM07, Accession No .: KCTC 10005BP).

The sugar of step (a) can be used as long as it can be used as a carbon source for the growth of Pseudomonas strains. For example, fructose, glucose, galactose, mannose, and the like. Preferably fructose can be used. The sugar content in the medium is preferably 50 to 70 mM.

The substituted fatty acid of step (a) is not particularly limited, but it is preferable to use an aromatic substituted carboxylic acid, more preferably carboxylic acid substituted with a phenyl group, a substituted phenyl group, a phenoxy group, or the like. Can be used. In one embodiment of the present invention 11-phenoxyundecanoic acid (11-phenoxyundecanoic acid, hereinafter referred to as 11-POU) was used. The content of the substituted fatty acid in the medium is not particularly limited, but is preferably included in the content of 3 ~ 5 mM.

The synthesis inhibitor of the polyhydroxyalkanoic acid of step (a) is a composition containing salicylic acid or acrylic acid as an active ingredient as described above, the content of the medium is contained in a concentration of salicylic acid 0.4 ~ 1.5 mM and acrylic acid 0.5 It is preferably included at ˜2.0 mM.

The medium of step (a) can be used as long as it is suitable for the growth of Pseudomonas strains, in one embodiment of the present invention M1 mineral salt medium (1.06g (NH ₄ ) ₂ SO ₄ , 2.3g KH ₂ PO a _{4, 7.3g Na 2 HPO 4 ·} 12H 2 0, 0.25g MgSO 4 · 7H 2 0, 0.3g NaHCO 3, 0.1g CaCl 2 · 2H 2 0, 0.03g ferric ammonium citrate and 2 ml microelement solution) was used . The microelement solution contained 0.556 g FeSO ₄ .7H ₂ O, 0.396 g MnCl ₂ · 4H ₂ O, 0.034 g CuCl ₂ · 2H ₂ O, 0.06 g H ₃ BO ₃ , 0.006 g NaMoO _4. 2H ₂ O, 0.562g CoSO ₄ .7H ₂ O, 0.058g ZnSO ₄ .7H ₂ O, and 0.004g NiCl ₂ .6H ₂ O were added.

Culture conditions may be cultured at 10 ~ 30 ℃, preferably 30 ℃ until the strain reaches the maximum growth (maximal growth).

Step (b) is a step of recovering the long chain aromatic polyhydroxyalkanoic acid from the strain cultured in step (a). Recovering polyhydroxyalkanoic acid containing a high content of long-chain aromatic monomer from the strain cultured in step (a). That is, the strain culture is recovered and the cells are obtained by centrifugation and washing and then dried. The dried cells are transferred to an extraction cylinder (28 × 100 mm, advantec), and the polyhydroxyalkanoic acid in the cells is extracted with a chloroform solution using a soxhlet extraction device. Concentrate the solvent extract extracted for 8 ~ 10 hours using a rotary vacuum concentrator to about 1/5 and precipitate the polymer by dropping the concentrate dropwise to the stirred cold methanol solution. In this case, the ratio of chloroform and methanol is preferably about 1:10. Precipitated polyhydroxyalkanoic acid is separated by centrifugation at 10,000 ° C. at 4 ° C. and vacuum dried at room temperature for 24 hours.

On the other hand, prior to the step (a) may further comprise the step of seed culture (seed culture) strain Pseudomonas.

Preferably, the species culture of the Pseudomonas genus strain is carried out by incubating for 12 hours at 30 ° C., 180 rpm in NR medium (containing Nutrient rich medium) (containing 1% yeast extract, 1.5% nutrient broth and 0.2% ammonium sulfate). Can be.

The polyhydroxyalkanoic acid prepared according to the process of the invention is characterized by containing a high content of long chain aromatic monomers. That is, the process of the present invention enables the production of PHAs with a high content of aromatic monomers and a minimum content of aliphatic monomers. Preferably, the polyhydroxyalkanoic acid according to the invention contains from 40 to 70 mol% of long chain aromatic monomer units. In the present invention, the long chain aromatic monomer unit may be 3-hydroxy-7-phenoxyheptanoic acid (7POHH) or 3-hydroxy-9-phenoxynonanonoic acid (9POHN).

Furthermore, the present invention provides a method for separating long-chain aromatic monomers of PHA, characterized by fractional precipitation of long-chain aromatic polyhydroxyalkanoic acid prepared by the above method.

Preferably the process comprises the steps of: (a) mixing the long chain aromatic polyhydroxyalkanoic acid prepared by the process of the present invention with PHA-aliphatic and PHA-aromatic polymers and dissolving in chloroform;

(b) precipitating the polymer solution of step (a) in a mixed solvent of methanol and chloroform and fractionating the solution into a solution and a precipitate; And

(c) separating the aromatic monomer unit from the precipitate of step (b).

In the step (b), the mixing ratio of methanol and chloroform is preferably 1: 1.5.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example 1>

Inhibition of Incorporation of Aliphatic Monomers into PHA with Different Concentrations of 11-Phenoxyundecanoic Acid

Incorporation of Pseudomonas fluorescens BM07 (KCTC 10005BP) into the PHA of the metabolized aliphatic monomer from Pructose according to the concentration of 11-POU while culturing Pseudomonas fluorescens BM07, KCTC 10005BP The degree was analyzed.

Nutrient rich medium (containing 1% yeast extract, 1.5% nutrient broth and 0.2% ammonium sulfate) was used for spawn culture, maintenance and preservation of the strain, and the modified M1 mineral was used as the PHA synthesis medium. Salt medium (1.06 g (NH ₄ ) ₂ SO ₄ , 2.3 g KH ₂ PO ₄ , 7.3 g Na ₂ HPO ₄ 12H ₂ 0, 0.25 g MgSO ₄ 7H ₂ 0, 0.3 g NaHCO ₃ , 0.1 g CaCl ₂ 2H ₂ 0, 0.03 g ferric ammonium citrate and 2 ml microelement solution) were used (Choi, MH Yoon, SC Appl. Environ. Microbiol. 1994, 60, 3245). 50 mM fructose was fed together (Co-fed) as a carbon source, incubated at 30 ° C. for 72 hours, and then the monomer composition of intracellular PHA was determined by methyl ester analysis. That is, cells were recovered from sulfuric acid / methanol treatment and analyzed using Hewlett-Packard HP5890 Series II gas chromatography equipped with an HP-5 capillary column and flame ionization detector (Choi, MH Yoon, SC Appl. Environ.Microbiol. 1994, 60, 3245). GC conditions are as follows: initial temperature 80 ° C., 2 min, heating rate 8 ° C./min, final temperature 250 ° C., 1.75 min, carrier gas (He) flow rate 3 ml / min, injector temperature 250 ° C., detector temperature 300 ° C. Standardize each GC peak against known structures of PHA (Lee, H.-J. Choi, MH Kim, T.-U. Yoon, SC Appl. Environ. Microbiol . 2001, 67, 4963) by quantitative NMR analysis It was.

As a result of the experiment, when the BM07 strain was cultured in fructose-containing M1 medium to which 11-POU was added, incorporation of PhaG-mediated coenzyme A monomers derived from fructose into PHA was suppressed with increasing concentration of 11-POU. In particular, 11-POU at concentrations of 5 mM or more inhibited the incorporation of aliphatic monomers into PHA at 20 mol% or less. The concentration dependent S-type graph of the incorporation of the monomer unit from 11-POU into PHA indicates that the PhaG enzyme is specifically inhibited by the 3-hydroxy monomer precursor (FIG. 1).

<Example 2>

Effect of Salicylic Acid or Acrylic Acid on the Distribution of PHA Monomers

<2-1> Effect of Salicylic Acid Addition

Effect of Salicylic Acid Addition of Salicylic Acid on the PHA Monomer Distribution in Pseudomonas fluorescens BM07, KCTC 10005BP in 11-POU (3 mM or 5 mM) and 50 mM Fructose-Containing M1 Medium Was investigated.

Pseudomonas fluorescens BM07, incubated for 12 hours at 30 ° C., 180 rpm in NR medium (5 ml) containing 11-POU (3 mM or 5 mM), 50 mM fructose, various concentrations of salicylic acid and 1.06 g / L ammonium sulfate Transfer to 500 ml of M1 mineral salt medium and incubate at 30 ° C. until the accumulation of polyhydroxyalkanoic acid is maximized. Sodium hydroxide was added to dissolve salicylic acid and the pH of the solution was adjusted to 7.2 before addition to the medium.

Cell growth was observed by measuring optical density at 660 nm. The remaining amount of fructose was measured by DNS method, and the remaining amount of 11-POU or other carboxylic acid was measured by gas chromatography (Choi, MH Yoon, SC Appl. Environ. Microbiol . 1994, 60, 3245). . Cells were separated by centrifugation (7000 rpm, 10 minutes), washed with methanol and dried under vacuum at room temperature for 2 days. Inhibition rate and cell growth rate of PHA synthesis in the presence of salicylic acid were determined according to previously reported procedures (Lee, H.-J. Choi, MH Kim, T.-U. Yoon, SC Appl. Environ. Microbiol . 2001 , 67, 4963). Salicylic acid levels were observed by measuring the UV absorption of the culture supernatant at 298 nm using a culture medium without the addition of salicylic acid as a blank. The residual amount of NH ₄ ⁺ was measured using Nessler's reagent (Choi, MH Yoon, SC Appl. Environ. Microbiol . 1994, 60, 3245). The monomer distribution of PHA was measured in the same manner as in Example 1. The content of PHA was calculated by dividing the PHA weight obtained by gas chromatography analysis by the dry cell weight used for gas chromatography analysis. Also, The total conversion yield of 11-POU to aromatic monomer was calculated according to the following formula.

[Sum of moles of each monomer unit in recovered PHA] / [Moles of 11-POU initially supplied].

Experimental results showed that the stepwise addition of salicylic acid to the 3 mM 11-POU / 50 mM fructose system reduced the total amount of aliphatic monomer-units (from 67 mol% to 33.4 mol%) and increased the total amount of aromatic monomer-units (33 mol). % To 66.6 mol%) (A and FIG. 3 of FIG. 2). Some promotion of cell growth was found at low concentrations of salicylic acid (0.1-0.3 mM). However, increased salicylic acid inhibited cell growth. In addition, the addition of salicylic acid significantly changed the composition of the aromatic monomer-units derived from 11-POU (FIG. 2B). That is, 5POHV and 7POHV showed the same occupancy when 1.0 mM salicylic acid was added (˜40 mol%, restandardized in terms of aromatic monomer units).

A total of ˜10 mol% aliphatic monomer-units were detected in the 5 mM 11-POU / 50 mM fructose system regardless of the level of salicylic acid. Fructose in the feed was mostly used for cell growth and was consumed within 24 hours and 11-POU was consumed within 40 hours. On the other hand, the addition of 0.5 mM salicylic acid to 5 mM 11-POU / 50 mM fructose medium, compared to the salicylic acid-free 11-POU / fructose system, increased the specific consumption ratio of fructose from 1.67 to 1.25 mM / L · h. It did not affect the consumption ratio of 11-POUs (results not shown). Despite the slightly delayed consumption of fructose, the two carbon sources were consumed within 48 hours.

The monomer ratio of aromatic units derived from 11-POU was shown to be strongly dependent on the level of salicylic acid (FIGS. 3 and 4). That is, increased levels of salicylic acid significantly changed the proportion of aromatic monomers: a decrease in 5POHV levels in PHA and an increase in 7POHH and 9POHN (FIG. 3). In particular, the addition of 1.5 mM salicylic acid significantly converted the monomer-unit composition to longer chain monomer units: at a concentration of 1.5 mM salicylic acid, 5POHV was 66 to 31 mol%, 7POHH was 30 to 48 mol%, 9POHN was 4 Conversion to 21 mol% at. The longer chain monomer-units 7POHH and 9POHN constituted ˜70 mol% of the aromatic monomer-units. Thus 7POHH was found to be included as the major monomer-unit in the 5 mM 11-POU / 50 mM fructose system in the presence of 1.0 mM or higher salicylic acid. In addition, salicylic acid was found to increase the conversion yield of PHA significantly in the 11-POU up to 60-80%.

In conclusion, salicylic acid was found to effectively control the distribution of aromatic monomer-units.

<2-2> Effect of Addition of Acrylic Acid

In addition, Pseudomonas fluorescens BM07 was incubated under the same conditions as in Example <2-1>, but 11-POU was added at a concentration of 3 mM, and incubated by adding 2 mM of acrylic acid in place of salicylic acid, followed by synthesis. The compositional changes of the monomers in the PHA were investigated.

As a result, even when acrylic acid was added instead of salicylic acid, it was confirmed that the total amount of the aliphatic monomer-unit was decreased and the total amount of the aromatic monomer-unit was increased (FIG. 3).

<Example 3>

Fractional precipitation of separated PHA

Two polyesters accumulated in Pseudomonas fluorescens BM07 strain cultured in the same manner as in Example <2-1> in a medium containing 50 mM fructose and 3 mM 11-POU in the presence or absence of 0.7 mM salicylic acid ( In the absence of salicylic acid: polyester A, in the presence of salicylic acid: polyester B) was fractionated. In addition, Pseudomonas puti in medium containing 20mM 11-POU and polyester (PHAs) containing only aliphatic monomer units without containing aromatic monomer units obtained by culturing Pseudomonas fluorescens BM07 in a medium containing 70 mM fructose In addition, blend polymers containing polyester (POPHAs) containing only an aromatic monomer unit without the aliphatic monomer unit obtained by culturing BM01 were fractionated.

Pseudomonas fluorescens BM07 incubated for 12 hours at 30 ° C. and 180 rpm in NR medium (5 ml) was transferred to 500 ml of M1 mineral salt medium containing 70 mM fructose and 1.06 g / L ammonium sulfate and incubated at 30 ° C. for 48 hours. After that, only the cells were collected and dried to separate aliphatic copolyesters containing no aromatic monomer units by chloroform. In addition, Pseudomonas putida BM01, which was also incubated for 12 hours at 30 ° C. and 180 rpm in NR medium (5 ml), was transferred to 500 ml of M1 mineral salt medium containing 20 mM 11-POU and 1.06 g / L ammonium sulfate for 30 hours for 72 hours. After incubation at 占 폚, an aromatic copolyester containing no aliphatic monomer unit was isolated.

First, to determine whether each polyester is a homogeneous random copolymer-polymer or a heterogeneous blend of polymers (ie, two or more separate polymer mixtures: for example aromatic and aliphatic copolyesters), Fractional precipitation of each polyester sample in methanol and chloroform) was performed (Song, JJ Yoon, SC Appl. Environ. Microbiol. 1996, 62, 536). Two polymer poly (5.5 mol% 3-hydroxyoctanoate-co-38 mol% 3-hydroxydecanoate-co-18 mol% 3-hydroxy in an appropriate proportion to approximate the total monomer composition of the test polymer. Roxydodecanoate-co-31 mol% 3-hydroxy-cis-5-dodecanoate) [PHA-aliphatic] and poly (72 mol% 3-hydroxy-5-phenoxyvaleric acid-co-28 mol% 3- Hydroxy-7-phenoxyheptanoate) [PHA-aromatic] was mixed and dissolved in chloroform. The mixture dissolved in chloroform was reprecipitated in a mixed solvent (final volume ratio: 1.5 volumes methanol; 1 volume chloroform) and the polymer composition (precipitate and solution) in the two phases was analyzed using gas chromatography.

As a result of fractional precipitation, the mixed two polyesters were almost completely separated into their constituent polymers. PHA-aromatics precipitated out of solution with 37% PHA-aliphatic and most of the PHA-aliphatic remained in solution (Figure 5). PHA synthesized by Pseudomonas fluorescens BM07 from 3 mM 11-POU and 50 mM fructose without salicylic acid was fractionated into each phase. The soluble fraction consisted mainly of PHA-aliphatic, but the main component of the precipitation fraction was PHA-aromatic. Therefore, it was found that PHA synthesized in the absence of salicylic acid was not a homogeneous random copolymer.

On the other hand, slightly different fractional patterns were observed for PHA prepared in the presence of salicylic acid. The addition of salicylic acid significantly increased the aromatic monomer-units in the precipitated fraction.

<Example 4>

Glass transition temperature of separated PHA

After culturing Pseudomonas fluorescens BM07 in the same manner as in Example <2-1> by adding or without adding 1 mM salicylic acid to a medium containing 11-POU (3 mM or 5 mM) and fructose (50 mM), The glass transition temperature of PHA synthesized by was measured by the following method.

Thermal transfer of each PHA was measured using DSC Q10 V6.21 (TA Instruments) equipped with a data station system. For DSC characterization, 10 mg of dried PHA samples were prepared by placing them in a DSC sample pan for at least one month. PHA samples annealed at room temperature were heated from −100 to 100 ° C. at a rate of 10 ° C./min under a dry nitrogen purge (50 ml / min).

As a result, the blend-like properties of the two PHAs were confirmed in their thermal transfer analysis (FIG. 6). The two PHAs exhibited at least two glass transitions associated with each component polymer (aliphatic rich chain and aromatic rich chain). PHA-aliphatic and PHA-aromatic polymers show glass transitions at -52 and 14 ° C (Lee, H.-J. Choi, MH Kim, T.-U. Yoon, SC Appl. Environ. Microbiol . 2001, 67, 4963; Song, JJ Yoon, SC Appl. Environ.Microbiol . 1996, 62, 536). Thus, lower glass transitions are believed to be due to domains composed of aliphatic monomer-rich chains and higher transitions are due to aromatic monomer-rich chains. The Tg of PHA produced in the presence of salicylic acid was significantly lower than that of PHA-aromatic polymer at -5.4 ° C. Thus, PHA synthesized by salicylate treated cells is more Copolymerization It appears to have a polymer-like nature.

MCL-PHA synthesis inhibitors according to the invention can be used to discover metabolic pathways from precursors to PHA synthesis or as pathway blockers of intermediate metabolites of specific pathways of PHA synthesis. In addition, it is possible to produce functional aromatic PHA by adjusting the PHA side chain length using the method of the present invention, which can be utilized especially in the pharmaceutical industry.

Claims (20)

Inhibitor composition of the medium chain length polyhydroxyalkanoic acid of Pseudomonas strain comprising salicylic acid or acrylic acid as an active ingredient. The composition of claim 1, wherein the salicylic acid is included at a concentration of 0.4 to 1.5 mM and acrylic acid is contained at 0.5 to 2.0 mM. The composition of claim 1, wherein the Pseudomonas genus strain is Pseudomonas fluorescens . The composition of claim 1, wherein the Pseudomonas genus strain is Pseudomonas fluorescens BM07 (Accession No .: KCTC 10005BP). (a) culturing the Pseudomonas spp. strain in a medium containing a sugar, a substituted fatty acid, and the inhibitor of synthesis of the polyhydroxyalkanoic acid of claim 1; (b) a method for producing a long chain aromatic polyhydroxyalkanoic acid comprising recovering a polyhydroxyalkanoic acid containing a high content of the long chain aromatic monomer from the strain cultured in step (a). The method of claim 5, wherein in step (a), the Pseudomonas strain is Pseudomonas fluorescens . 7. The method of claim 6, wherein the Pseudomonas fluorescens is Pseudomonas fluorescens BM07 (Accession Number: KCTC 10005BP). The method according to claim 5, wherein the sugar contained in the medium in step (a) contains 50 to 70 mM. 6. The method of claim 5, wherein the sugar of step (a) is fructose. 6. The method of claim 5, wherein the substituted fatty acid contained in the medium in step (a) is 3 to 5 mM. The method of claim 5, wherein the fatty acid substituted in step (a) is an aromatic substituted carboxylic acid. The method of claim 10, wherein the aromatic substituted carboxylic acid is a carboxylic acid substituted with a phenyl group, a substituted phenyl group, or a phenoxy group. 13. The method of claim 12, wherein it is 11-POU (11-phenoxyundecanoic acid). The method of claim 5, wherein the inhibitor contained in the medium in step (a) comprises salicylic acid at a concentration of 0.4 to 1.5 mM and acrylic acid at 0.5 to 2.0 mM. The method of claim 5, wherein the medium of step (a) is M1 mineral salt medium (0.66g (NH ₄ ) ₂ SO ₄ , 2.3g KH ₂ PO ₄ , 7.3g Na ₂ HPO ₄ · 12H ₂ 0, 0.25g MgSO ₄ · 7H ₂ 0, 0.3g NaHCO ₃ , 0.1g CaCl ₂ · 2H ₂ 0 and 1 ml microelement. The method of claim 5, wherein the long-chain aromatic monomer of step (b) is 3-hydroxy-7-phenoxyheptanoic acid (7POHH) or 3-hydroxy-9-phenoxynonanonoic acid (9POHN). . 6. The method according to claim 5, wherein the polyhydroxyalkanoic acid of step (b) contains 40 to 70 mol% of long chain aromatic monomer units. A polyhydroxyalkanoic acid prepared by the method of claim 5 and containing a long chain aromatic monomer unit in an amount of 40 to 70 mol%. (a) mixing the polyhydroxyalkanoic acid of claim 18 with a PHA-aliphatic and PHA-aromatic polymer and dissolving in chloroform; (b) precipitating the polymer solution of step (a) in a mixed solvent of methanol and chloroform and fractionating the solution into a solution and a precipitate; And (c) separating the long chain aromatic monomer of polyhydroxyalkanoic acid comprising the step of separating the aromatic monomer unit from the precipitate of step (b). 20. The method of claim 19, wherein the long chain aromatic monomer is 3-hydroxy-7-phenoxyheptanoic acid (7POHH) or 3-hydroxy-9-phenoxynonanonoic acid (9POHN).

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