KR101232191B1 - Method for enhancing the production of (R)-3-((R)-3-hydroxyalkanoyloxy)alkanoic acids by using Pseudomonas aeruginosa rhlB mutant strains - Google Patents

Abstract

The present invention is directed to a method of increasing the production of HAAs that can be utilized as biopharmaceuticals as well as pharmaceutical precursors. More specifically, the production of ( R ) -3-(( R ) -3-hydroxyalkanoyloxy) alkanoic acids (HAAs) by culturing medium-chain-length fatty acids in a M1 restriction medium using a rhlB gene mutation strain of Pseudomonas aeruginosa as a carbon source It is about how to increase.

Description

Method of increasing the production of ＡＡｓ [（Ｒ） －３ （（Ｒ） －３－ｈｙｄｒｏｘｙａｌｋａｎｏｙｌｏｘｙ） ａｌｋａｎｏｉｃａｃｉｄｓ] using the Phosphorylated mutant strains (Method--3-) 3-hydroxyalkanoyloxy) alkanoic acids by using Pseudomonas aeruginosa rhlB mutant strains}

The present invention is directed to a method of increasing the production of HAAs that can be utilized as biopharmaceuticals as well as pharmaceutical precursors. More specifically Pseudomonas A method for increasing production of ( R ) -3-(( R ) -3-hydroxyalkanoyloxy) alkanoic acids (HAAs) by culturing medium-chain-length fatty acids in a M1 restriction medium with a carbon source using the rhlB gene variant strain of aeruginosa It is about.

Typically, Pseudomonas Pseudomonas aeruginosa strains have been reported for the first time to isolate bacteria from soil and to study antimicrobial activity.Since 1949, it has been confirmed that FJ Jarvis et al. produce glycolipids, Pseudomonas aeruginosawa Much research has been done on the variants and the types of glycolipid bioemulsifiers (lamnolipids) they produce. Pseudomonas aeruginosa is known as Pseudomonas aeruginosa and is mainly used for wastewater treatment and biological purification of soil oil pollution areas.

The bioemulsifier produced by Pseudomonas aeruginosa is the main component of glycolysis by many researchers, the sugar is rhamnose and the lipid is β-hydroxydecanoic acid. It has been found that the composition, and some variants appear depending on the conditions of the environment, it is reported that the form of the bioemulsifier also changes.

Bio-surfactant (BS) is a compound that is produced on the cell surface or secreted out of cells by living organisms. It is an amphiphilic substance that has both hydrophilic and hydrophobic portions. It has excellent emulsion stability (Gerson and Zajic, 1979).

Bioemulsifiers are categorized based on the microbial species and their biochemical properties they produce.Glycolipids, phospholipids and fatty acids, lipopeptides and lipoproteins, and polymer bioemulsifiers (polymeric surfactants) (Desai and Desai, 1993).

These glycolipid bioemulsifiers, rhamnolipids, are either rhamno lipid-1 (RL-1), rhamno lipid-2 (RL-2), rhamno lipid-3 (RL-3), or rhamno lipid-4 (RL-). Four main types of 4) are known. The most common of the four major forms of rhamnolipid are RL-1 and RL-3, which were found by Hisatsuka et al. (1971) and Itoh et al. (1972), respectively. Syldatk (1985) reported that it is synthesized only in resting cells.

In addition, the morphology of the produced rhamnolipids is reported to be affected by the microbial species and culture conditions, and the constituent sugar of RL is rhamnose without exception. Fatty acids are fatty acid biosynthetic pathway (fatty acid de It is produced as an intermediate of the nove biosynthesis pathway, and it is known that various fatty acids, which form mutant lipids, are derived from this process.

Studies on the mass production of rhamnolipids were carried out using the method of immobilizing Pseudomonas sp. DSM 2874 strain using calcium alginate support followed by continuous culture in a fluidized bed reactor (Siemann and Wagner, 1993) and Bio-emulsifiers are produced by continuous cultivation using a fermenter, followed by recovery and freeze-concentration of bioemulsifiers by ultrafiltration (Mulligan and Gibbs, 1993).

However, there is no known mass production of HAAs [(R) -3-((R) -3-hydroxyalkanoyloxy) alkanoic acids], a precursor of rhamnolipids. HAAs precursor is expected to be very marketable as an alternative material because it can be used not only as a bio surfactant, but also as a pharmaceutical precursor.

The present inventors Pseudomonas Using the rhlB variant strain of aeruginosa , we have devised a method for mass production of ( R ) -3-(( R ) -3-hydroxyalkanoyloxy) alkanoic acids (HAAs).

The present invention relates to a method of increasing the production of HAAs. More specifically, using a rhlB gene variant strain of Pseudomonas aeruginosa , by culturing medium-chain-length fatty acid in a M1 restriction medium with a carbon source, a large amount of HAAs (( R ) -3-(( R ) -3-hydroxyalkanoyloxy) It provides a method to increase the production of alkanoic acids).

In order to achieve the above object, the present invention provides a method for increasing the production of HAAs.

More specifically, the present invention relates to

(Iii) preparing a Pseudomonas strain variant by removing the rhamnolipid gene of the Pseudomonas spp. Strain; (Ii) a method of increasing the production of HAAs ((R) -3-((R) -3-hydroxyalkanoyloxy) alkanoic acids) comprising culturing the Pseudomonas genus variant in a medium comprising a heavy chain fatty acid. to provide.

The heavy chain fatty acid is preferably a fatty acid having 6 to 14 carbon atoms.

The content of the heavy chain fatty acid is preferably 20 ~ 100 mM.

The genus Pseudomonas strain may further include Pseudomonas aeruginosa ( Pseudomonas aeruginosa) .

It is preferable that the said Sumodonas aeruginosa company is PA14 or PAO1 of the Sumodonas aeruginosa company.

The variant of the genus Pseudomonas Pseudomonas aeruginosa rhlB It is preferred that it is a variant.

By using rhlB mutant, it is possible to mass-produce highly applicable HAAs precursors, thereby replacing the hydrolytic surfactant. In addition, HAAs can be used as a substitute material as well as bio-surfactant, can be used as a precursor to the drug.

1 shows Pseudomonas grown in 70 mM fructose at 30 ° C. for 120 hours. Schematic of the production of PHA and rhamnolipid in aeruginosa PA14 and PAO1 variants.
2 shows Pseudomonas grown in 30 mM decanoic acid at 30 ° C. for 60 hours. Schematic of the production of PHA and rhamnolipid in aeruginosa PA14 and PAO1 variants.
3 is a schematic of ¹³ C-NMR spectroscopy spectra for chloroform extract of PHA and culture supernatant. The spectra of PHA were determined on CDCl ₃ and the spectra of rhamnolipid on CD ₃ OD. Figure 3a for 30 ℃, 72 sigan 70 mM octanoic- 1 - ¹³ Pseudomonas grown in the acid C ¹³ C-NMR spectra of PHA synthesized from aeruginosa wild type, phaZ mutant, and phaG mutants. Figure 3b for a 30 ℃, 72 sigan 70 mM octanoic- 1 - ¹³ Pseudomonas grown in the acid C ¹³ C-NMR spectra of PHA in supernatant chloroform extracts obtained from aeruginosa wild type, phaZ mutants, and phaG mutants. Figure 3c is extended spectra for the carboxyl carbon and methine carbon region in HAAs and rhamnose ring: a), 70 mM octanoic- 1 for 60 hours - the wild-type PA14 grown at ¹³ C acid; b), 70 mM octanoic- 1 for 84 hours - the wild-type PA14 grown at ¹³ C acid; c), PA14 rhlB mutant grown in 60 mM octanoic acid for 72 hours; d), PA14 wild type grown in 60 mM octanoic acid for 60 hours; e), P. aeruginosa BM114 grown in 70 mM fructose for 120 hours. 3D shows ¹³ C-NMR uptake profiles with incubation time in PA14 rhlB mutants grown in 60 mM octanoic acid: a), 48 h; b), 60 h; c), 72 h. FIG. 3E shows ¹³ C-NMR uptake profiles with incubation time in PA14 biotype grown in 60 mM octanoic acid: a), 48 h; b), 60 h; c), 72 h.
Figure 4 is a schematic of the profile according to incubation time in PA14 wild type cells grown in 60 mM octanoic acid.
5 is a schematic of the pathways for production and HAAs from MCL-fatty acids.

The present invention provides a method of increasing the production of HAAs.

More specifically, the present invention relates to

(Iii) preparing a Pseudomonas strain variant by removing the rhamnolipid gene of the Pseudomonas spp. Strain; (Ii) a method of increasing the production of HAAs ((R) -3-((R) -3-hydroxyalkanoyloxy) alkanoic acids) comprising culturing the Pseudomonas genus variant in a medium comprising a heavy chain fatty acid. to provide.

The medium-chain-length means 6 to 14 carbon atoms. Fatty acid refers to a saturated or unsaturated monocarboxylic acid having a chain shape as a carboxylic acid chemically or biochemically.

The heavy chain fatty acid may be used as a carbon source for the growth of the genus Pseudomonas.

In one embodiment of the present invention it is preferable that the carbon number 8 octanoic acid (octanoic acid) and 10 decanoic acid (decanoic acid). The content of the octanoic acid and decanoic acid is 60 mM and 30 mM, respectively, is advantageous for the mass production of HAAs.

The medium may be used as long as it is suitable for the growth of the genus Pseudomonas strain, but preferably M1 mineral salt medium (1.0g (NH ₄ ) ₂ SO ₄ , 2.3g KH ₂ PO ₄ , 7.3g Na ₂ HPO ₄ · 12H ₂ O, 0.25g MgSO ₄ · 7H ₂ O, 0.3g NaHCO ₃ , 0.1g CaCl ₂ · 2H ₂ O, 0.03g ferric ammonium citrate and 2 ml microelement solution It is preferable. The trace element solution was 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 in 200 ml of 0.5N hydrochloric acid. 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.

Culture conditions may be cultured at 20 to 37 ℃, preferably 30 ℃ until the strain reaches the maximum growth (maximal growth), the culture time is preferably 48 to 120 hours.

The strain of the genus Pseudomonas Pseudomonas aeruginosa ( Pseudomonas) aeruginosa ). Pseudomonas aeruginosa is a Pseudomonas aeruginosa that is mainly used for wastewater treatment and biological purification of soil oil contaminated areas. Bioemulsifiers produced by Pseudomonas aeruginosa are the main component of glycolysis by many researchers, sugar is rhamnose, lipid is composed of beta-hydroxydecanoic acid (β-hydroxydecanoic acid).

The Pseudomonas aeruginosa may include a Pseudomonas aeruginosa PA14 or PAO1.

The variant of the strain of genus Pseudomonas is Pseudomonas from which the lamnolipid gene of the genus P. aeruginosa Preference is given to rhlB variants.

Hereinafter, the present invention will be described in detail through examples. The following examples are only examples for describing the present invention, and the scope of the present invention is not limited thereto.

< Example  1. Experiment Method>

Bacterial strains and Culture medium

Bacterial strains P. aeruginosa PA14 and PAO1 wild type strains and variants thereof used in the examples of the present invention as shown in Table 1 are shown in PA14 (Liberati et al. 2006) Mutant Library and PAO1 (Jacobs et al. 2003) Mutant Library Purchased from the University of Washington Transposon Mutant Collection. Nutrient-rich (NR) medium containing 1% yeast extract, 1.5% nutrient broth and 0.2% ammonium sulfate was used for seed culture, maintenance and storage of P. aeruginosa cells. .

HAAs synthesis medium includes M1 mineral salt medium (1.0g (NH ₄ ) ₂ SO ₄ , 2.3g KH ₂ PO ₄ , 7.3g Na ₂ HPO ₄ 12H ₂ O, 0.25g MgSO ₄ 7H ₂ O, 0.3g NaHCO ₃ , 0.1 g CaCl ₂ · 2H ₂ O, 0.03 g ferric ammonium citrate and 2 ml microelement solution were used. The trace element solution was 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 in 200 ml of 0.5N hydrochloric acid. 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. Antibiotics (60 μg / ml tetracycline and 30 μg / ml gentamicin) were added to the medium for species culture of the variants.

Cell growth monitoring

Microbial strains were inoculated with 5 ml of NR medium at 30 ° C. at 180 rpm for 12 hours using 70 mM fructose or 30 mM decanoic acid and 1.0 g / liter of ammonium sulfate for the main culture. Transfer to a 2 liter flask containing 500 ml of M1 medium supplemented with. Incubation was until a steady state growth was reached in aerobic. Cell growth was determined by measurement of dry cell weight. Cells were separated by centrifugation (10000 × g, 10 min) of the culture, washed with methanol and dried in vacuo at room temperature for 24 hours. Residual ammonium ions were measured using Nessler reagent. Residual fructose was measured using 3,5-Dinitrosalicylic acid (DNS) method. To determine the concentration of residual decanoic acid, the cell free supernatant was extracted with chloroform and measured by gas chromatography.

Within the cell PHA Quantitative Analysis of

For analysis of PHA in cells, 20 mg of dried cells were collected in 1 ml of chloroform, 0.85 ml of methanol and 0.15 ml of conc. Reaction with a mixture of sulfuric acid. The reactants were reacted in a screw capped tube at 100 ° C. for 3 hours. The organic layer containing the reactants was separated, dried over MgSO ₄ , and analyzed by gas chromatography (GC). Samples were injected in splitless mode. The GC peaks of the samples were normalized by comparing the purified PHAs whose composition was confirmed by quantitative NMR analysis with the GC peaks of 3-hydroxy methyl esters obtained by methanolysis of sulfuric acid. Gas chromatograms were obtained by Hewlett Packard 5890A GC equipped with HP-1 column and Flame Ionization Detector (FID). The column was heated at 8 ° C./min from 80 to 250 ° C. and the injector and detector temperatures were 230 and 280 ° C., respectively (Choi et al., 2009).

Rhamno lipid  analysis

Orcinol assays were used to determine the amount of rhamnose in secreted rhamnoids during incubation in M1 mineral salt medium. 300 μl samples of the culture supernatants were extracted twice with 600 μl diethyl ether. Ether fractions agglomerated and evaporated for drying before addition of 100 μl water. 100 μl of each sample was added to 100 μl of 1.71% orcinol solution and 800 μl of 60% (v / v) sulfuric acid. After heating at 80 ° C. for 30 minutes, the mixture was cooled at room temperature for 15 minutes and the absorbance was measured at 421 nm. The concentration of rhamnose in the rhamno lipid was measured as compared to the standard curve obtained from the orcinol reaction of the rhamnose standard between 0 and 50 mg / ml. Then, by comparing the peaks of the strain and the ¹³ C-NMR signal, the content of rhamno lipid according to the carbon source (g / L) was calculated based on the ratio of mono and diramnolipids. The composition of the acyl group of rhamno lipid was analyzed by GC method. The culture supernatant was acidified to pH 2 and extracted overnight with chloroform. The filtered chloroform extract was concentrated with an evaporator (Eyela N-11 1000, Tokyo Rikakikai Co., LTD, Tokyo, Japan). The concentrated chloroform extract was hydrolyzed with methanol sulfate and analyzed by GC under the same conditions used for GC analysis of intracellular PHA.

PHA  And Rhamno lipid  detach

To investigate the metabolic pathways of the synthesis of rhamnolipids from octanoic acid, the PA14 wild type, PA14 phaZ variant and PA14 phaG variant were incubated with octanoic acid rich in 99% isotope C-13 at the C-1 position. PHA was extracted from the dried cells using hot chloroform for 8 hours in a Pyrex Soxhlet apparatus (Choi et al., 1994). The concentrated solvent extract was precipitated by rapid stirring with cold methanol. The separated PHA was purified by reprecipitation and dried overnight at 50 ° C. vacuum.

Rhamno lipids were isolated from the cell-free culture supernatant by chloroform extraction. The collected culture supernatants were first acidified to pH 2 with 6 N hydrochloric acid and extracted overnight with chloroform at room temperature. Only the chloroform layer was separated and concentrated by evaporation to give a viscous rhamno lipid product. The isolated rhamno lipids were analyzed by NMR spectroscopy.

¹³ C- NMR  analysis

CDCl ₃ in PHA (~ 30mg / ml) at 25 ° C Solution or CD ₃ OD solution of rhamnolipid was measured by 125 MHz ¹³ C-NMR (DRX-500 Bruker, Rheinstetten 4, Germany). Data collection for ¹³ C-NMR spectra was performed using the zgig 30 program, 10 μs pulse width, 30-s pulse repetition, 30000 Hz spectra width, 64K data points and 400 accumulation under gated decoupling mode. It was carried out under the condition of (accumulations). Chemical shifts were designated according to the values reported in the literature for PHA and rhamnolipid.

< Example  2. Experiment result>

phaC1 , phaC2 , phaZ , And phaG In variants PHA  And Rhamno lipid  synthesis

Quantitative analysis of the PHA monomer unit via GC analysis showed that the polyester synthesized from fructose was 3-hydroxydecanoate (3HD) as the main component and 3-hydroxyoctanoate (3HO) and 3-hydroxyhexano as the minor component. It was shown that it consists of eight (3HH) (Tables 2 and 3).

On the other hand, PHA accumulated from decanoic acid was the main monomer unit with 3HO having a shorter chain of one ethylene unit than the substrate. (Tables 4 and 5).

The amount of rhamnolipid was calculated using the amount of rhamnose determined by the orcinol method taking into account the molar ratios of mono and di rhamnolipids determined by quantitative ¹³ C-NMR analysis for each carbon source. According to GC analysis, the acyl chain of rhamnolipid contains 3HD (85 mol% or more) as a main component, followed by 3HO as well as a small amount of 3-hydroxydodecenoate (3HDDe) and 3-hydroxydodecanoate (3HDD). ) Units are included (Tables 2 to 5).

The molar ratio of acyl chains in the rhamno lipid synthesized by PA14 and PAO1 was very similar regardless of the type of carbon source. In various P. aeruginosa wild-type strains (Rha-Rha-C ₁₀ -C ₁₀ ) were reported to be the major (more than 70%) rhamnolipids (D18 et al. 1999; Monteiro et al. 2007). As shown in Table 2-5, the 9: 1 average molar ratio of the sum of 3HD and other heavy chain 3-hydroxyalkanoic acids (3HAs) in the HAAs-unit in the rhamno lipid is used to calculate the rhamno lipid content. The total amount of 3HAs measured by GC in the supernatant from fructose medium was found to be rhamnolipid (monoramnolipid: dairamnolipid for PA 14 strains [28: 72]; Nolipide: dairamnolipid] corresponded to the amount of lamnose in [0: -100]) (Tables 2 and 3). However, in the medium chain fatty acid medium, the quantification by GC analysis and orcinol analysis did not coincide with each other, which meant that free 3HAs or free HAAs were present in the supernatant in excess (Tables 4 and 5).

Four pha variant strains that are defective in genes associated with PHA accumulation include PHA synthetic genes ( phaC1 And phaC2 ), PHA depolymerase gene ( phaZ ) and transacylase gene ( phaG ) (Table 1). When cultured in fructose, as shown in Figure 1 P. aeruginosa PA14 and PAO1 phaC1 variants showed slightly reduced PHA accumulation. On the other hand, when cultured in decanoic acid (FIG. 2), PHA accumulation was significantly reduced compared to the wild type. However, phaC2 mutants significantly reduced PHA accumulation in both fructose and decanoate. These results indicate that in fructose, PhaC2 is mainly active in PHA synthesis, while in decanoic acid, both PhaC1 and PhaC2 are active. The difference in PhaC1 and PhaC2 activity depending on the carbon source can be interpreted as the difference of substrate specificity in PHA synthesis.

Table 1 below shows the bacterial strains used in this example. In addition, Table 2 relates to the synthesis of PHA and rhamnolipid in P. aeruginosa PA14 strain cultured in 70 mM fructose under 120 hour one-step culture condition at 30 ° C., Table 3 is a 120 hour one-step culture at 30 ° C. PHA and rhamnolipid synthesis in P. aeruginosa PAO1 strains cultured in 70 mM fructose under conditions.

P. aeruginosa Of PA14 and PAO1 phaC1 And phaC2 For both variants, the production of rhamnolipid was slightly reduced or similar compared to wild type cultured in fructose (FIG. 1) or decanoic acid (FIG. 2). 3-hydroxy fatty acids are used as common precursors for both PHA and rhamnolipid synthesis pathways.

Thus, the fact that PHA synthesis is independent of the synthesis of rhamno lipids means that the production of rhamno lipids can be controlled by the regulation of other cells. P. aeruginosa, as previously reported The production of rhamnolipids in the cell is well regulated by quorum-sensing reactions and environmental conditions in the transcriptional phase (Pearson et al. 1997; Chen et al. 2004; Sober et al. 2005).

The biochemical properties of intracellular PHA degrading enzyme (PhaZ) have been reported to specifically hydrolyze heavy chain-PHA while located on the surface of PHA granules (de Eugenio et al. 2007). Surprisingly, the PA14 phaZ variant accumulated ˜300% more PHA than the PA14 wild type strain when cells were cultured in 30 mM decanoic acid (FIG. 2). Similarly, P. putida KT2442 (Caiet4al.2009) and P. fluorescens BM07 phaZ in (Choi et al. 2010) strain Inactivation of the gene led to increased accumulation of PHA. PAO1 phaZ variants cultured in fructose (FIG. 1) and decanoic acid (FIG. 2) also accumulated much more PHA compared to wild type. However, phaC1 And phaC2 Similar to the variant, rhamnolipid production of two phaZ variants of PA14 and PAO1 is not significantly affected compared to wild-type rhamnolipid production for two carbon sources.

Two phaG variants cultured in fructose (FIG. 1) significantly reduced PHA accumulation and the production of rhamnolipids slightly decreased compared to wild type. On the other hand, almost the same amount of PHA and rhamnolipid were synthesized when incubated in decanoic acid. In a recent study, phaG was reported to compete with RhlA for a common fatty acid precursor for the synthesis of PHA and rhamnolipid. Therefore, the increased production of rhamnolipid in the phaG variant was expected, but the production of rhamnolipid was not increased when incubated in decanoic acid or fructose as a carbon source. This means that the production of rhamnolipid is independent of PHA accumulation and is well regulated by the signaled quorum-sensing response of cells to cells (Pearson et al. 1997; Chen et al. 2004; Soberon-Chaez et al. 2005 ).

rhlA , rhlB , rhlR , And rhlI In variants PHA  And Rhamno lipid  synthesis

rhlA and rhlB genes encode rhlR (electron activator gene) and rhlI (N-butyryl homoserine lactone) that encode proteins involved in the regulation of transcription of rhamnolipid synthesis through a quorum-sensing (QS) reaction (Maier and Sober2000). With autoinducer-synthesizing genes) in operon form. Therefore, P. aeruginosa was used to confirm the correlation between rhamnolipid and PHA synthesis. The production of PHA and rhamnolipid from fructose or decanoic acid in four rhl ( rhlA , rhlB , rhlR , and rhlI ) variant strains of PA14 and PAO1 was investigated.

As expected from the competitive nature of RhlA and PhaG against 3-hydroxyacyl-ACP, PA14 and PAO1 rhl variant strains that do not produce rhamnolipids may be cultured in fructose (FIG. 1) or decanoic acid (FIG. 2). When it showed increased PHA accumulation. When cultured in 70 mM fructose medium, PA14 rhlB and rhlR variants accumulated about 280% more PHA than the PA14 wild type strain. This suggests that fatty acid synthesis is a source for HAA. This is because the (R) -3-hydroxy acid in the HAA of rhamnolipid is consistent with the intermediate in fatty acid biosynthesis. In contrast, the fatty acid β-oxidation (Zhu et al. 12 2008) is inconsistent with the intermediate. Thus, in variant strains in which rhamno lipids are not produced, (R) -3-hydroxy acid precursors produced for HAAs of rhamno lipids can be readily introduced for PHA synthesis via PhaG.

However, the synthesis of PHA and rhamnolipid in PA14 rhlA and rhlI variants did not differ compared to wild type. This may be due to the leaky property of these variant strains.

Pseudomonas incubated in mannitol aeruginosa PG201 produces less than 2% free HAAs within the total amount of lipids (29 mg / L free HAAs) and rhlB variants produce 722 μM (calculated as 256 mg / L) of free HAAs (Deiel et al., 2003). Interestingly, however, when the PA14 and PAO1 strains were incubated in decanoic acid, free 3-hydroxyalkanoic acids (3HAs) were excessively secreted by pha variants with low amounts of wild type and HAAs , whereas free HAAs were 600 in rhlB variants. It was produced up to ˜700 mg / L (Tables 4 and 5). Mass production of free HAAs in rhlB variants cultured in heavy chain fatty acid medium may be due to the high concentration of (R) -3-hydroxy fatty acid precursors during metabolism of heavy chain fatty acids. HAAs are known to play a role in lowering surface tension (Deziel et al. 2003). Thus, high production of free HAAs in variants from heavy chain fatty acids may be important in biotechnology.

Table 4 below relates to PHA and rhamnolipid synthesis in P. aeruginosa PA14 strains cultured at 30 mM decanoic acid under 60 hour one-step culture conditions at 30 ° C., and Table 5 shows 60 hour one-step culture at 30 ° C. PHA and rhamnolipid synthesis in P. aeruginosa PAO1 strains cultured under 30 mM decanoic acid under conditions.

PHA  And Rhamno lipid  In synthesis rpoS  The role of genes

In E. coli , sigma factor S ( rpoS ) is directly or indirectly involved in rhlAB expression during the stationary phase of growth. When rpoS variant strains of PA14 and PAO1 were cultured in 70 mM fructose, the production of PHA and rhamnolipids was reduced by 10% and 40% less than in wild type, respectively (FIG. 1). These results indicate that reduced PHA production in rpoS variants may be affected by reduced expression of phaC2 .

Similarly, reduced rhamnolipids cultured in fructose appear to be due to low expression of RhlAB. PA14 rpoS variant did not accumulate PHA from 30 mM decanoic acid (FIG. 2). In contrast, PAO1 rpoS variants accumulated a similar amount of wild-type PHA (FIG. 2). This difference may be due to differences in transcriptional dependence of the two strains on the RpoS sigma factor. However, the two variant strains did not show a clear difference in the production of rhamnolipid from decanoic acid (FIG. 2).

Heavy chain  From fatty acids PHA  And Rhamno lipid  Metabolic Pathways for Synthesis

Metabolic pathways for PHA and rhamnolipid synthesis from heavy chain fatty acids were investigated by using 99% isotope C-13 rich octanoic acid (70 mM) at the C-1 position. NMR spectra of PHA were found to be rich in carbonyl carbon (˜170 ppm region) in monomer-unit 3HO for all three strains tested (PA14 wild type, PA14 phaZ, and phaG variants) (FIG. 3A). It has thus been shown that monomer-unit 3HO is derived directly from the octanoic acid substrate molecule and synthesized with PHA without any rearrangement of the carbon backbone.

However, two strong C-13 NMR migration regions of ˜175 and ˜75 ppm were observed from chloroform extraction of the culture supernatant containing rhamnolipid as main constituent (FIG. 3B). Similarity between the spectra of the PA14 wild type and PA14 phaG is suggested as the independence of the phaG gene in the synthesis of rhamnolipids from heavy chain fatty acids. Because it gives the most useful information, the uptake for carbonyl carbon and O-bound methine carbon has been analyzed in detail. The expanded carbonyl region showed uptake, 176.9, 175.0, 173.4, 172.1, 171.54 and 171.84 ppm (FIG. 3C). The peaks were designated as follows according to the chemical shift reported in the literature and the chemical shift obtained from standard substances (3HO acid, 3HD acid and diramnolipid synthesized in BM114 strain cultured in fructose): Octanoic acid ( OA) carbonyl carbon (C-1), 176.9 ppm; C-1, 175 ppm of 3HO acid and 3HD acid; A1, A'1 and A''1, 173.4 ppm; B''1, 172.1 ppm; B'1, 171.54 ppm; B1, 171.48 ppm. The designated non-primed, primed and double primed match the dairamnolipid, monoramnolipid and liberated HAAs, respectively, and the dairamnolipid Rha-Rha-C10-C10 The numbering system of the carbon skeletons (A, B, C and D) is shown in FIG. 3b. One oxygen atom comprises -CH (OH)-in the Rhamnose ring and -CH (R) -O- or 3-hydroxyalkanoic acid (3HA) (FIG. 3C) in HAAs It is associated with methine carbon bound to. The peaks at 74.5 and 74.3 ppm are B'3 and B3 β in 3HD-units that are continuous from the oxide-linkage of the rhamnose ring to the anomeric carbon in the dioramnolipid and monoramnofeed, respectively. It is associated with carbon. Strong absorption at 71.4 ppm is associated with β-carbons in 3HD-units with free carboxyl groups (A3 and A'3) in diramnolipid and monoramnofeed. Further strong absorption is also found at 68.4 ppm, which has not yet been reported in the literature. Absorption at 68.4 ppm was found to be consistent with the uptake of β-carbon in 3HO and 3HD acids. For the PA14 rhlB variant, only two major signals of 71.3 and 68.4 ppm were found in the O-bound methine carbon region. The variant produced only free HAA. Thus, two peaks of 71.3 and 68.4 ppm can be designated esters linked to β-carbons (A ″ 3) and hydroxyl groups attached to methine carbon (B ″ 3) of HAAs. The 71.3 ppm peak associated with HAAs slightly overlapped with A3 and A'3 in rhamno lipid (FIG. 3C).

3C shows a chloroform extract containing substrate OA, intermediates (3HO acids, 3HD acids, and free HAAs) and rhamnolipids. Two pairs of peaks (B1 and B'1) and (B3 and B'3) in approximately equal proportions indicate that there is much more production of monoramnolipids in the heavy chain fatty acid medium than in the irrelevant carbon source medium (Figure 3C). As shown in FIG. 3C, the abundant carboxyl carbon peaks in the rhamno lipid (A1 and B1 series) and the β-carbon peaks (A3 and B3 series) of the HAAs-unit are CHD 3HD units in the acyl chain of the rhamno lipid. It means that the conversion is directly from the octanoic acid-1- ¹³ C acid substrate after chain elongation with one ethylene unit at the −1 position. Of the six carbons of the Rhamnose ring, only carbons 3 and 4 show increased absorption as shown in FIG. 3C. Other Rhamnose carbon 1 (˜100 ppm), 2, 5 and 6 show weak and almost insignificant signals (FIGS. 3B and 3C). This means that the carbon 3 and 4 of the lamnose portions of mono and diramnolipids are almost derived from carbon 1 in the octanoic acid. Thus, the glucose used in the dTDP-L-rhamnose biosynthetic pathway (Sober et al. 2005) may have been derived through the rearrangement of octanoic acid-1- ¹³ C acid through the glucose synthesis pathway.

To understand the migration correlation of metabolism between OA, intermediates (free acids: 3HO, 3HD and HAAs), and rhamnolipids, profiles of the time-dependent profiles of PA14 wild type in 60 mM octanoic acid were investigated. (FIG. 4). In addition, the culture supernatants of PA14 rhlB variants and PA14 wild type cultured in 60 mM octanoic acid were analyzed at three intervals of 48, 60 and 72 hours in ¹³ C-NMR spectra (FIG. 3D, e). However, the maximum production of rhamnolipids occurred at 70 hours. A 1: 1 mixture of mono and diramnolipids was used for the calculation of the amount of rhamnolipids in the supernatant (FIG. 3E). Among the GC-measured 3HAs such as 3HO, 3HD, 3HDDe and 3HDD, the major constituents 3HO and 3HD acids are shown in FIG. 4. The liberated acid 3HO was separated in large quantities by 55 hours and then reselected for integration into rhamnoid after conversion to 3HD. However, the 3HAs measured in the supernatant were significantly attributed to the free (R) -3HAs (associated with the 175.0 ppm signal) as in FIG. 3E. Gradual increase in fractions of HAAs compound (signal associated with 172.1 ppm, B ”1) was found in the supernatant: 13, 27, and 42 mol% (peak region moles in FIG. 3E) in total 3HAs amounts at 48, 60 and 72 hours. Recalculated in terms of ratio). In addition, the optical rotation of the three chloroform extracts for the PA14 culture supernatant (prepared under optimal conditions without pigment) was determined to be -8.5, -25.1 and -13.8 ^o at 55, 65 and 75 hours. Wherein each supernatant is 0.15 3HO and 0.01 3HD (g / L); 0.43 and 0.05 (g / L); 0.35 and 0.15 (g / L). Negative values of light rotation indicated that the free acid 3HAs were (R) -form. This is because (R) -3-hydroxybutyl acid has [α] D = -17.5o (0.31 g / L in chloroform). However, in PA14 rhlB variants, progressively modified OA → 3HAs → HAAs can be seen more clearly with incubation time. The molar ratio of 3HAs to HAA (calculated from the carboxyl carbon peaks 175.0 and the area ratio of 173.4 or 172.1 ppm) was 15:85 at 72 hours. Thus, the PA14 rhlB variant produced a large amount of HAAs more effectively than wild-type cells cultured in fructose.

Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (6)

Rugi Pseudomonas Oh removing the gene RhlB labor (pseudomonas aeruginosa), glass HAAs ((R) to the variant, wherein the incubation in a medium containing a fatty acid having 8 to 10 20 to 100mM of 3 - (( R) -3-hydroxyalkanoyloxy) alkanoic acids). delete delete delete The method of claim 1,
The Pseudomonas aeruginosa is Pseudomonas aeruginosa PA14 or PAO1 to increase the production of HAAs characterized in that.


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