KR101250071B1 - Siderophore over-production method from microorganism - Google Patents

Abstract

Present invention ftr1 And it relates to a method for producing a siderofoa from the microorganism comprising the step of culturing in a medium a variant of the cideropoa producing microorganism lacking the ftr2 gene. According to the present invention, the variant can be mass produced 13-23 times as compared to wild wine, and this mass-produced siderofoa can be usefully used to promote the growth of plants and to clean environment-friendly soils contaminated with heavy metals. have.

Description

Siderophore over-production method from microorganism

The present invention relates to a method for mass production of siderophores from microorganisms. More specifically, the present invention relates to a method for producing a large amount of ciderofoa from microorganisms by deleting the ftr1 and ftr2 genes among the genes involved in the iron metabolism process and adjusting the culture conditions.

Iron is an essential nutrient for organisms and is involved in various metabolic processes in vivo. In higher organisms such as humans, iron is mainly absorbed in simple forms and transmitted to various parts of the body. However, microorganisms have a slightly different form of iron absorption mechanism for living in an iron-deficient environment and are known as a unique mechanism of activating iron absorption using ciderropoa.

Siderofoa is a water-soluble, low molecular, ferric iron-chelating compound, generally a yellow-green fluorescent chromophore linked by peptide chains. It is divided into two structural forms according to the composition and size of the peptide chain. The derivatives of catechol (o-dihydroxybenzene) are catechol amides (phenolates, catecholates) and hydroxamic acid. acid, a derivative of RCONHO, is in the form of hydroxamates: catechol forms of cideropoa are enterrobactin, parabactin, acrobactin, anguibactin, and vibriobactin. (vibriobactin), azotobactin, etc., and the form of the hydroxamate form cideropoa includes ferrichrom, ferrioxamine, coprogen, and nocardamine. In recent years, citrate-hydroxamate forms of cideropoa include asthrobactin, aerobactin, schizokinen, and quinoline. (quinoline) form cideroporo is known as pseudo-bactin, inabervertin, and the like.

As described above, sideeropoa is a small molecule material produced by microorganisms, and mainly has a function of binding to iron, and is absorbed by microorganisms in the form of such a complex. Recently, however, as other functions of ciderofoa become known, their uses are diversified. In particular, it has been reported that it helps plant growth by activating the interaction between plants and microorganisms, which is helpful for the growth of microorganisms. In 2004, mung bean experiments showed that root strains increased more than 30% of the microorganisms in the roots that produced a large amount of cederopoa compared to the control group (Plant. Growth.Regulation 2004). It is also reported that the ability of the plant to produce cedrophora to promote plant growth in the soil helps to secure a superior position in competition with phytopathogenic strains of the soil (J. Bac 1988). Pseudomonas is known to determine the activity of microorganisms, whether the production of the sideropoa as a plant growth-promoting bacteria (Nature 1980).

On the other hand, the recent development of a siderofoa technology to control heavy metal pollution in an environmentally friendly manner.

As described above, cideropoa is a substance that helps plant growth and the like, but its practical use has been limited. Chemical synthesis of cedropoa is impossible and is produced from microorganisms because the production method is not established and the amount or production is limited. In addition, in the case of production by microorganisms, the reproducibility is low and it is very difficult to separate each siderofoa.

As a background technology of the present invention, as disclosed in Korean Patent Publication No. 10-0850373 (2008.07.29), there is disclosed a microorganism of the genus Serratia, a method for identifying the same, and a method for promoting plant growth and soil purification using the same. However, there is no mention of mass production of ciderofoa.

In addition, as a background technology of the present invention, Side produced by Fusarium graminearum using Saccharomyces cerevisiae mutant in Park Yong-Sung et al., Current genetics Volume 52, Numbers 3-4 187-190 ISSN 1432-0983 Methods of efficiently separating and identifying ropoa are disclosed, but there is no description of mass production of cideropoa.

Thus, as described above, the present problem remains that the siderofoa is not chemically synthesized and the production by microorganisms is limited. The present inventors have completed the present invention by conducting research aiming at mass production of ciderrofoa.

Republic of Korea Patent Publication No. 10-0850373 (2008.07.29)

PARK et al., New and efficient method using Saccharomyces cerevisiae mutants for identification of siderophores produced by microorganisms, Current genetics Volume 52, Numbers 3-4 187-190 ISSN 1432-0983 PARK et al., Physical and functional interaction of FgFtr1-FgFet1 and FgFtr2-FgFet2 is required for iron uptake in Fusarium graminearum, Biochem. J. (2007) 408, 97-104

The present invention is to solve the problems and demands of the prior art as described above, the object of the present invention is to provide a method for mass-producing a siderofoa from a variant microorganism of a gene involved in iron metabolism.

Another object of the present invention is to provide a method for mass-producing ciderropoia using the microorganisms by adjusting the culture conditions.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present invention provides the ftr1 And Provided is a method for producing cideropoa from a microorganism, comprising culturing a variant of a cideropoa producing microorganism lacking the ftr2 gene in a medium.

In the present invention, the microorganism is ftr1 And Microorganisms for the deletion of the ftr2 gene can be used, for example, one selected from filamentous fungi belonging to Fusarium graminearum , for example Aspergillus , Fungus ( Fusarium ) may include fungi, and the like, preferably Fusarium Graminia ( Fusarium graminearum ) is available.

Ftr1 in the present invention And The ftr2 gene encodes iron permease as a gene involved in iron metabolic processes and is known to be located on the chromosome in the manner of Ftr1 / Fet1 and Ftr2 / Fet2 (see FIG. 1, PARK et al., Biochem J. (2007) 408, 97-104).

Ftr1 on the microbial chromosome of the present invention And in a manner that the defect gene is ftr2 known conventional genetic defect in the art, for example, using light, such as chemical or ultraviolet light, or, ftr1 using gene recombination technology by homologous recombination technology And The ftr2 gene region may be mutated to obtain a deleted mutant, and gene pop-out techniques may be used. In a specific embodiment of the present invention, ftr1 And The genetic defect is ftr2 Fujairah Solarium Mini Gras arum (Fusarium graminearum ) variants were prepared (see FIGS. 2A and 2B). Deletion of the ftr1 and ftr2 genes resulted in a sharp increase in the production of sideeropoa compared to the wild type (see FIGS. 4 and 5).

The microbial variant according to the present invention increased significantly more rapidly in the iron-containing medium when cultured in an iron-free medium (see Table 1).

The microbial variant according to the present invention was most increased among filamentous fungi rather than the same strain when cultured in an iron-free medium (see Table 2).

In addition, the variant according to the present invention increased the sideropoa productivity in the medium containing iron (see Fig. 5).

The method for producing ciderrophore from the microorganism according to the present invention may further include the step of separating and purifying the cideropoa produced by the microbial variant using HPLC.

As described above, according to the present invention Δ Fgftr1 , ftr2 By using a variant and adjusting the culture conditions, it is possible to produce a large amount of cideropoa 13-23 times compared to wild strains. This mass-produced siderofoa can be usefully used to promote the growth of plants and to clean the environment contaminated with heavy metals.

1 is a view showing the position of Fgftr1 / 2 on the chromosome of Fusarium graminearum .
Figure 2a and Figure 2b shows a schematic diagram of the production of the Fgftr1, ftr2 Fusarium graminearum variant according to the present invention and the result of the gene deletion confirmed by Southern analysis.
Figure 3 is a schematic diagram showing the HPLC conditions for purification separation of the ciderofoa according to the present invention.
4 is a view showing the results of HPLC using a Hydrospere C18 column according to the present invention.
5 shows Δ Fgftr1 and ftr2 according to the present invention. Figure showing the result of comparing the production of the side of the Fusarium graminearum ( Fusarium graminearum ) with wild wine.
Figure 6 is an illustration showing the kind of siderofoa produced by the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1. Experimental strains and Mutant  making

1) System Strains )

The following 19 strains were used as the tested strains:

Fusarium graminearum ( WT ), Δ Fgsit1 , Δ Fgsit2 , Δ Fgend1 , Δ Fgftr1ftr2 , ΔF gsit1ft r1, Aspergillus niger , Trichoderma longibrachiatum , Trichoderma atroviride, Polyporus bruomalis , Trametes versicolor , Pharero chrysosporium , Irpex lacteus , Phanerochaete sordida Jang Seung , Gloeophyllum trabeum , Phanerochaete sordida (Lily) , Fomitopsis palustris , Rhizopus oryzae , Mucor circinelloides

Among them, two selected strains were found to have sidoforea productivity, and ΔF gftr1ft r2 ( Fusarium graminearum ) and Fomitopsis was palustris .

2) Δ Fgftr1 , ftr2 Fujingum Gramania Room ( Fusarium graminearum ) Mutant  making

By using PCR, 1500bp in front of 5 'gene and 1500bp behind 3' gene of FgFtr gene (Fg05160.1, Fg02143.1) were recombined by double-joint PCR method in front of Hygromycin B gene or Geneticin gene. Gene cassettes were made (FIG. 2A). Each recombinant gene cassette was inserted into the wild strain, and the transformants were selected by Hygromycin B or Geneticin, and the transformants lacking the FgFtrs gene were confirmed through Southern analysis (FIG. 2B) (PARK et al., Functional identification of high). affinity iron permeases from Fusarium graminearum, Fungal Genetics and Biology (2006) 43, 273-282).

Example  2. Cultivation of Experimental Strains

The first strains were fungi grown at room temperature (25 ° C) for 5 days on PDA (Potato dextrose agar; Bifco PD broth 24 g, Agar 20 g / liter) plates (90x15mm petri Dish).

Next, put a hyphae of about 4 cm _{2 into} CMC (Carboxymethyl cellulose 1.5 g, Yeast extract 1 g, Magnesium sulfate 7H ₂ O 0.5 g, Ammonium nitrate 1 g, Photassium phosphate 1 g / liter) broth 40ml (25 ° C.) for 3 days.

Hyphae grown on the CMC broth medium were filtered with filter paper (125 mm), and the iron-free medium (Low iron broth; 6 g Sodium nitrate, 0.52 g Potassium chloride, 0.52 g Magnesium sulfate 7H ₂ O, Potassium dihydrogen phosphate) 1.52 g, Glucose 10 g, 100 mM BPS 1 ml / liter, pH 6.5) or medium with iron (Iron broth; Sodium nitrate 6 g, Potassium chloride 0.52 g, Magnesium sulfate 7H ₂ O 0.52 g, Potassium dihydrogen phosphate 1.52 g, Glucose 10 g, 1.5 M Ferric chloride 1 ml / liter, pH 6.5) 2 liters were grown in a plastic flask at room temperature (25 ℃) for one week.

Example  3. Siderofoa  Complex Siderophore mixture Purification and separation

One) Column  through Siderofoa  Purification of the Complex

After filtering the sample prepared in Example 2 once with gauze, the medium from which the mold was removed was removed to a small spore of the mold by using a pump on a 0.45 μm filter paper.

Next, 20 g of Amberlite XAD16 resin (Sigma) was charged to the column, washed with 200 ml of 50 mM Potassium phosphate buffer (pH 7.5) to allow sufficient equilibration, and the above sample from which the mold was removed was passed through a resin. Spilled.

The resin turned red was washed with 50 ml of 50 mM Potassium phosphate buffer (pH 7.5) and 20 ml of Methanol (HPLC grade) was eluted from the column.

At this stage, the siderophore mixture was eluted with 1/1000 H ₂ O of the culture volume and stored at 4 ° C.

2) HPLC Through Siderofoa  Separation tablets

The elution solution was concentrated by blowing methanol (methanol) at 100 ℃ / hg, 20 ℃ in a vacuum concentrator (Vacuum concentrator). The concentrated siderophore complex was dissolved in 500 μl of methanol (HPLC grade), followed by centrifugation (12000 rpm, 4 ° C., 1 min) to remove undissolved residues, and then the supernatant was transferred. The transferred supernatant was filtered with a 0.22 μm Nylon membrane syringe filter.

100 μl of the prepared Siderophore complex (Siderophore mixture, stored at 4 ° C.) was purified by separation by HPLC (High Performance Liquid Chromatography) (Agilent Eclipse XDB-C18 column).

The first fraction was carried out under the following conditions.

Solvent Condition: Water 90% (w / 0.1% TFA): Acetonitrile 10% (w / 0.1% TFA) (TFA: Triflouroacetic acid)

Detection & gradient conditions: Absorbance at 435 nm (Sample volume: 100 μl) (see Fig. 3)

0 min-10%,

15 min-20%,

30 min-50%,

40 min-60%,

45 min-10%,

65 min-10%

Fractionated ciderrophore was blown into a vacuum concentrator at 100 mm / hg and 20 ° C, and then dissolved in 50 μl H ₂ O (HPLC grade) to spin down, followed by fractionation of the supernatant. Purification by fractionation was repeated one more time under the same conditions to refine the peak.

The second and third fractions were then subjected to the following conditions.

Solvent Condition: Water 90% (w / 0.1% TFA): Acetonitrile 10% (w / 0.1% TFA)

Detection & gradient condition: Absorbance at 435nm (Sample volume: 50 μl)

20 min-10%

60 min-20%

The results are shown in FIGS. 4 to 6 and Tables 1 and 2.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

Claims (4)

A method of producing cideropoa from a microorganism, comprising culturing a variant of a cideropoa producing microorganism lacking the ftr1 and ftr2 genes in a medium. The method of claim 1, wherein the microorganism is one selected from the group of filamentous fungi belonging to Fusarium graminearum . The method of claim 1, wherein the culturing the variant of the microorganism is culturing in an iron-deficient medium. 4. The method of claim 3, wherein the medium is an iron-free medium.

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