WO2014092345A1

WO2014092345A1 - Fusaricidin-producing strain, and method for mass producing fusaricidin using same

Info

Publication number: WO2014092345A1
Application number: PCT/KR2013/010231
Authority: WO
Inventors: 박승환; 최수근; 박수영; 김성빈; 김창진; 김하림; 박동진
Original assignee: 한국생명공학연구원
Priority date: 2012-12-11
Filing date: 2013-11-12
Publication date: 2014-06-19
Also published as: KR20140075165A; KR101541446B1

Abstract

The present invention relates to a fusaricidin-producing strain and to a method for mass producing fusaricidin using same. Specifically, fusaricidin is produced at a high yield by deactivating genes of enzymes biosynthesizing polymyxin and tridecaptin in the fusaricidin-producing strain and deactivating a phosphoglucomutase gene affecting the biosynthesis yield of fusaricidin. Thus, the present invention can be effectively used in various fields such as agricultural antibiotics and plant disease resistance inducers.

Description

【Specification】

[Name of invention]

Fuzacidin-producing strains and mass production method of fuzacidin using the same

The present invention relates to the mass production of fuzacidin, specifically, other antibiotics of the polylipinide family (polymyxin and tride) that may act as inhibitors in increasing the production yield in fuzacidine-producing strains. Captin) A method for preparing fuzacidin high productivity mutants through inactivation of biosynthetic enzyme genes and inactivation of phosphoglucomutase genes involved in enhancing the production efficiency of a target substance. The present invention relates to a strain produced by the method and a mass production of fuzacidine using the same.

Background Art

In order to develop sustainable eco-friendly agriculture and produce safe agricultural products, the importance of developing agricultural materials such as environmentally friendly pesticides and fertilizers is increasing greatly. One of them is developing safe and efficient ways to control plant diseases, a major obstacle to growing crops. As a method of replacing or supplementing chemical synthetic pesticides, many methods for controlling plant diseases using functional microorganisms have been developed, and some have entered the commercialization stage, but the application efficiency in the field is lower than that of chemical pesticides, or Many disadvantages such as falling points are raised, and much effort is being made to develop technologies to overcome them. One of them is an attempt to find and use functional materials derived from microorganisms, in addition to using living useful microorganisms.

In an effort to secure useful life resources for agriculture under the background, the present inventors have revealed the genome-wide sequence of genome of F. Bacillus polymyx E681 which has excellent usefulness such as growth promotion and soil disease control on crops and analyzed gene function. Research has led to the production of antibiotics such as polymyxin and fusaricidin, as well as various enzymes such as cellulose, quettintin, xylan, sultin and phosphate degrading enzymes. By revealing a number of genes involved in the production of degrading enzymes and interacting with plants, genetic information was available to support the useful properties of this strain in relation to plants (Choi S.-K. et al. 2008). Biochem. And Biophys.Res. Com / nun. 365: 89-95; Choi S.-K. et al. 2009. J. Bacteriol. 191: 3350-3358; Kim JF et al. 2010. J. Bacteriol. 192 : 6103-6104).

In addition, the inventors of the present invention, the volatile metabolites produced by the E681 bacteria, 3-acetyl-1-propanol, 3-methyl-1-butanol, indole isoamyl acetate and butyl acetate to promote plant growth and resistance to plant diseases The patent was registered for the method of promoting plant growth and plant protection using the induction effect (Reg. No. 10-0946633, registration date March 3, 2010). The present invention relates to fuzacidine among the metabolites produced by the E681 bacterium. First, the conventionally known information on fuzacidine is as follows. Fuzacidin is a lipopeptide antibiotic and has antimicrobial activity against various phytopathogenic fungi and Gram-positive bacteria (Kajimura Y and Kaneda MJ Antibiotics 49: 129-135, 1996). Six amino acid residues form a ring structure and contain guanidino-3-hydroxypentadecanoic acid (15-guanid i ηο-3-hydroxypent adecanoic ac id). So far, fujarycidin LI-F03, LI-F04, LI-F05, LI-F07 and LI-F08 have been identified and reported from Penivacillus polymix (Kurusu K, Ohba, Arai T and Fukushima KJ Antibiotics 40). : 1506-1514, 1987), fuzisidine A, B, C and D have also been reported (Kajimura Y and Kaneda MJ Antibiotics 49: 129-135, 1996; Kajimura Y and Kaneda MJ Antibiotics 50: 220-228, 1997). The amino acid chains of fuzacidin are not encoded by genes and synthesized by ribosomes like conventional polypeptides, but by non-ribosomal peptide synthetase (NRPS) (Marahiel MA, Stachelhaus T and Mootz HD). Chew. Rev. 97: 2651-2673, 1997; Doekel S and Marahiel MA. Metab. Eng. 6: 64-77, 2001). As described above, the present inventors have discovered and discovered the function of fuzacidin biosynthesis gene through the genome detoxification study of the bacterium F. E681 genome. (Coi S.-K. et al. 2007. Biochem. And Biophys.Res. Camun. 365: 89-95). In addition, a patent was registered for the fuzacidine biosynthesis and gene (registration number 10-0762316, registration date September 20, 2007). On the other hand, the present inventors produced six kinds of Fusacidin (LI-F04a, LI-F04b, LI-F05a, LI-F05b, LI-F08a and LI-F08b) by the F. It was found that the combination of the six fuzzy cydins showed excellent disease resistance inducing effects on crops at low concentrations of 10 ng / m £ (0.01 ppm) to 1 β & / ^ 1 (1 ppm). A patent has been filed (Application No. 10-2010-0039500). However, one problem is the fact that the Fusacidin productivity of the F. E681 strain of Penicillus polymix is lower than 0.1 g / L, which needs to be improved in order to secure economic feasibility for industrial use. Accordingly, the present inventors are trying to develop a strain for mass production of fuzacidine and mass production technology using the same, lipopeptide that can act as an inhibitory factor to increase the production yield in the fusicidin-producing strain Fuzacidin is more effective than the parent by inactivating other antibiotics (polymyxin and tridecaptin) biosynthetic enzyme genes in the family and by inactivating the phosphoglucomutase gene, which is involved in improving the production efficiency of the target substance. New strains were produced and selected to exhibit 50 times or more productivity, and the present invention was completed by completing a technique for mass-producing fuzacidin using the strains.

DETAILED DESCRIPTION OF THE INVENTION The present invention aims to inactivate polymyxin and tridegatin biosynthetic enzyme genes, which may be inhibitors in increasing the production yield of a substance, thereby fundamentally blocking their production. ， Inactivating the gene of phosphoglucomutase that acts as an inhibitor in fuzacidin biosynthesis, and using three mutation induction methods (NTG treatment, UV irradiation and gamma irradiation) High to fusaricidin It is to provide a method for producing a strain produced in yield.

Another object of the present invention is to provide a strain produced by this method. Another object of the present invention to provide a method for mass production of fuzacidine using the strain.

Technical Solution In order to achieve the above object, the present invention

Pgm gene as set forth in SEQ ID NO: 1 in a Panibacillus polymyxa 0¾e /? 7½c / 7 / i / s ploymyxa strain; Or high production of fusaricidin which inactivates at least one of the pgm gene of SEQ ID NO: 1, the polymyxin biosynthesis gene of SEQ ID NO: 14, and the tritecaptin biosynthesis gene of SEQ ID NO: 15. Provided is a recombinant Penicillaceae polymyx strain.

In addition, in the present invention, the polymyxin biosynthesis gene of SEQ ID NO: 14 and the tritecaptin biosynthesis gene of SEQ ID NO: 15 are inactivated, and the mutant pgm gene of SEQ ID NO: 3 is introduced into the F. Fuzacidine high-producing recombinant Panibacillus polymyxa strains are provided.

In addition, the present invention

1) the pgm gene as set forth in SEQ ID NO: 1 in a Fanibacilli polymyxa strain; Or inactivating a gene of any one or more of the pgm gene of SEQ ID NO: 1, the polymyxin biosynthesis gene of SEQ ID NO: 14, and the tritegatin biosynthesis gene of SEQ ID NO: 15; And

2) It provides a method for producing a fuzzy cydin high-producing recombinant Fanibacillus polymixa strain comprising the step of selecting the high fuzzy cydin high production strain in the strain of step 1).

In addition, the present invention

1) inactivating the polymyxin biosynthesis gene as set out in SEQ ID NO: 14 and the tritecaptin biosynthesis gene as set out in SEQ ID NO: 15 in a F. 2) inducing a mutation by treating the strain of step 1 with NTG hnethyl- -nitro-nitrosoguanidine); And

3) It provides a method for producing a fuzzy cydin high-producing recombinant Panibacillus polymixa strain comprising the step of selecting the fuzacidine mass-producing strain from the mutated strain of step 2).

In addition, the present invention

1) inducing mutations by treating the selected strains with NTG and UV;

2) It provides a method for producing a fuzzy cydin high-producing recombinant Fanibacillus polymixa strain comprising the step of selecting the fuzacidine mass-producing strain from the mutated strain of step 1).

In addition, the present invention

1) irradiating the selected strains with radiation; And

2) It provides a method for producing a fuzzy cydine high-producing Panicibacillus polymixa strain comprising the step of selecting a fuzzy cydine mass-producing strain from the mutated strain of step 1).

In another aspect, the present invention provides a fusicidin high production Fanibacillus polymixsa strain prepared by the above method. In addition, the present invention

1) culturing the fuzacidin high-producing Pannibacillus polymyxa strain of the present invention; And ^'

2) provides a method for mass production of fuzisidine comprising the step of recovering the fusicidine in the culture medium of step 1).

Advantageous Effects of the Invention The present invention is directed to the production of other antibiotics of the lipopeptide family (purimicin and tridecaptin) that may act as inhibitors in increasing production yield in fuzacidin-producing strains. By inactivating synthetic enzyme genes and inducing high yields of fuzacidin through inactivation of phosphoglucomutase genes, which are involved in improving the production efficiency of target substances, antibiotics for agriculture, inducers of plant disease resistance, etc. It can be usefully used in various fields.

[Brief Description of Drawings]

1 is a view showing a strain development process for the enhancement of the fusicidin productivity of F.

Figure 2 is a diagram showing the selection process of fuzacidin high productivity mutant strains.

Figure 3 is a diagram showing a method for comparing fuzacidin productivity by the antimicrobial activity assay of the culture extract of Panibacillus bacteria.

Figure 4 is a comparison of the antimicrobial activity of strains NU177 and NUR1776 against E681-PT. 5 is a calibration line for fuzacidine quantification

Figure 6 is a comparison of M. luteus bacteria growth inhibitory ring at each concentration of fuzacidin standard sample

Fig. 7 is a diagram showing the reduction of fuzacidin productivity due to the cloning of pgm gene and the introduction into F. bacterium NUR1776.

8 is a diagram confirming the increase in fuzacidin productivity by the introduction of the pgm gene in which the point mutation occurs in the Panibacillus bacteria.

9 is a diagram confirming the increase in productivity of fuzacidin by inactivation of the F. genus pgm gene.

10 is a diagram showing the effect of the mutation of the pgm gene on the viscosity of the culture.

Fig. 11 is a diagram showing the results of a 5-liter scale culture test of F. NUR1776, F.

Fig. 12 is a diagram showing the results of fuzacidin extraction from the F. bacterium culture supernatant cells.

Figure 13 is a diagram showing the fusicidin extraction efficiency using butane and ethane from the fungus Bacillus cells. 14 is a diagram showing the results of a 500-liter scale culture experiment of F. NUR1776 bacteria of the Penivacillus polymix.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in detail. The present invention relates to a pgm gene as set forth in SEQ ID NO: 1 in a strain of Panibacillus polymix (/ ¾e2 / bac /// s ploymyxa); Or high production of fusaricidin which inactivates at least one of the pgm gene of SEQ ID NO: 1, the polymyxin biosynthesis gene of SEQ ID NO: 14, and the tritecaptin biosynthesis gene of SEQ ID NO: 15. Provided is a recombinant Penicillaceae polymyx strain. In addition, the present invention is a polymyxin biosynthesis gene of SEQ ID NO: 14 and the tritecaptin biosynthesis gene of SEQ ID NO: 15 is inactivated in the Pannibacillus polymixa strain, mutant pgm gene described in SEQ ID NO: 3 is introduced Fuzacidine high-producing recombinant Panibacillus polymyxa strains are provided.

The Fanibacilli polymixa strain is preferably a strain deposited with accession number KCTC 8801P, but is not limited thereto.

The gene inactivation is preferably a deletion or mutation of the gene, but is not limited thereto.

The fuzacidin high-producing recombinant Fanibacillus polymyx strains are deposited

Deposited with KCTC 12068BP or KCTC 12323BP is preferred but not limited thereto.

In a specific embodiment of the invention, see. The inventors have found that the parent strain, F. Bacillus polymixes E681, produces five or more antibiotics after analyzing genome information (Kim JF et al. 2010. J. Bacteriol. 192: 6103-6104). In order to block the production of polymyxin and tridecaptin that may act as inhibitors of din production, the PI E gene (GenBank Accession Number: EU371992) and the trdA gene (Patent No. 10—1165247) were introduced by antibiotic resistance genes. After inactivating the strain Penivacillus polymyxa Ε681 (/ υ / ^" 4), the strain was prepared. NTG (A½iethyl-iV'-nitro-i \ ^L ni1: rosoguani (iine) treatment (see FIG. 3), ultraviolet irradiation (see FIG. 4) and gamma ray irradiation (FIG. 5) as parent Mutation) was induced, and as a result of confirming the fusicidin production of the selected strains, it was confirmed that the fusicidin is produced at least 50 times higher than that of the parent strain. It was deposited on November 11, 2011 (Acre) of the Biotechnology Research Institute (ARC) (Accession No. KCTC 12068BP).

In addition, the present inventors have found that the mutation has been revealed through the analysis of genome sequences of the fusicidin high productivity mutant bacterium NUR1776 and comparative analysis with the genome sequence of the bacterium Fanibacillus polymyx E681 (GenBank Accession No. CP000154). A protein consisting of a bp length of phosphoglu ^ mutase gene SEQ ID NO: 1) and a 572 amino acid sequence (SEQ ID NO: 2) encoded by this gene, ie, a phosphoglumutase enzyme, was identified. In addition, a point mutation (SEQ ID NO: 3) occurs at the 509 bp position of the pgn] gene, which causes the 170th amino acid of the phosphoglucomatase enzyme protein to be changed from glutamine to proline (SEQ ID NO: 4) As a result, it was confirmed that fuzacidin productivity was increased (see FIG. 7). In addition, as a result of confirming the productivity of fuzacidin by transferring the pgm gene point mutation on the NUR1776 bacterial chromosome DNA to the F. chromosome as it is or by inactivating the Pgm gene of the F. phylbacillus polymyx bacterium. Fusisidine high-productivity mutant strain Fanibacillus polymixe E681 ^, ^) (hereinafter referred to as PGM1776) and panivacillus polymixe E581 p 'E, trdA, pgm (hereinafter referred to as PGM4441) produced by It was confirmed that significant production of zarycidine was significant (see FIGS. 8 and 9). In addition, the F. bacterium PGM4441 of the F. Panycillus polymix was deposited with the Korea Institute of Bioscience and Biotechnology (KCTC) on November 27, 2012 and received the accession number KCTC 12323BP.

Therefore, inactivation of other biolipase genes (polymyxin and tridegatin) of the lipopeptide family, which may act as inhibitors in increasing the production yield in fuzacidin-producing strains, and the production efficiency of the target substance Of phosphogkicomutase genes involved in enhancement Fuzacidine high production strain prepared by inactivation can be useful in a variety of fields, such as agricultural antibiotics, plant disease resistance inducer by producing fuzacidin in high yield. In addition, the present invention

1) the pgm gene as set forth in SEQ ID NO: 1 in a Fanibacilli polymyxa strain; Or inactivating a gene of any one or more of the pgm gene of SEQ ID NO: 1, the polymyxin biosynthesis gene of SEQ ID NO: 14, and the tritecaptin biosynthesis gene of SEQ ID NO: 15; And

2) provides a method for producing a fuzzy cydin high-producing recombinant Fanibacillus polymixa strain comprising the step of selecting a high fuzisidine-producing strain from the strain of step 1).

In addition, the present invention

1) inactivating the polymyxin biosynthesis gene as set out in SEQ ID NO: 14 and the tritecaptin biosynthesis gene as set out in SEQ ID NO: 15 in a F.

2) inducing a mutation by treating the strain of step 1 with NTG methy 卜 r-nitro- ^ nitrosoguanidine); And

In addition, the present invention

1) inducing mutations by treating the selected strains with NTG and UV; 2) It provides a method for producing a fuzzy cydin high-producing recombinant Fanibacillus polymixa strain comprising the step of selecting the fuzacidine mass-producing strain from the mutated strain of step 1).

In addition, the present invention

1) irradiating the selected strains with radiation; And 2) It provides a method for producing a fujarysidine high production Fanibacillus pulleymic strain comprising the step of screening the fuzacidin mass-producing strain in the mutated strain of step 1).

In addition, the present invention provides a fusicidine high production Fanibacilli polymix "strain prepared by the above method.

The radiation is preferably any one selected from the group consisting of gamma rays, infrared rays, ultraviolet rays, X-rays and protons, and more preferably gamma acid, but is not limited thereto. In addition, the present invention

1) culturing the fuzacidin high-producing Pannibacillus polymyxa strain of the present invention; And

The small-scale (less than 1L) seed culture of the fuzacidin-producing F. genus Bacillus was used to add 30 g / L of glucose to TSB or TSBG TSB. II) using but not limited to medium (TSB, 8 g; glucose,; (NH ₄ ) ₂ S0 ₄ , 2 g; K2HPO4, 0.1 g; MgS0 ₄ , 0.01 g; yeast extract, 1 g / L). The initial ρΗ of the medium is thick (6.5 ~ 7.0), the cultivation degree is 30- 32 ° C, 200 rpm shaking for small scale culture for smooth oxygen supply, 300 rpm stirring for fermenter culture and 1 wm aeration condition. The time is 18 to 72 hours depending on the culture environment such as fermenter scale.

Since the extraction of fuzacidin after cultivation exists in the state in which the produced fuzacidin is not secreted into the culture medium but is mostly attached to the cells, the cells are recovered first by centrifugation or the like, or the culture supernatant is removed after standing. An appropriate solvent may be added thereto, and methanol, ethanol, butanol, and the like may be used as the extraction solvent, of which ethanol is most preferred. Collect the recovered cells in an appropriate amount Suspended in water and extracted by adding the same amount (v / v) of ethanol to the supernatant by centrifugation, etc. to obtain fujarycidin in the form of powder through a process such as evaporation under reduced pressure, lyophilization or spray drying. Can be. Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, and the content of the present invention is not limited to the following examples.

<Example 1> Production of high-productivity Fanibacilli strain for mass production of fuzacidine <1-1> Preparation of polymyxin and tridecaptin gene inactivated strain

Fannybacillus polymix yarn E68 used by the inventors ⁵ . polymyxa E681) was used as a plant-use bacterium (Patent No. 0220402, deposited strain No .: KCTC 8801P) provided by Professor Chang-Seok Park. When the strain was deposited, it was named Bacillus polymyx ¾ _{c /} 7 / _{i / s} polymyxa) E681, but was newly introduced by Ash C. et al. (Ash, C. et al., 1993. Antonie van Leeuwenhoek 64: 253-260) was applied to the genus of Panibacillus (/ ¾ //) ac / "s), and it was referred to as the current strain name, 'Panibacillus polymyxa E681'. It was analyzed to produce various kinds of peptide antibiotics such as polymyxin and tridecaptin in addition to fuzacidine, which is a target of the present invention. The strain P. polymyxa E681-PT, which is inactivated polymacxin and tridecaptin biosynthesis gene, was used in the prior art (Korean Patent Registration No. 10-1165247). Briefly, the chloramphenicol-resistant and kanamycin-resistant gene cassette cassettes inside the pmxE gene using fosmid clones with the pmx gene cluster (including / M ?, pmxB, pmxC, pmxD and pmxE) ) Pi was inactivated by inserting the gene, and in the case of the trdA gene, a phosphid clone containing the same was inactivated by inserting a spectinomycin (3 6 101 11) antibiotic resistance gene into the trdA gene. A phosmid clone DNA carrying each inactivated gene was isolated from E. coli transformants, and then introduced into the F. polymyxa E681 strain by electroporation, so that the mutant strains of which each gene was deleted, namely P. polymyxa E681 ( pmxE) and P. polymyxa ^ l (trdA) mutants were prepared. The chromosomal DNA of P. polymyxa E681 (r 4) was isolated and added to the P. polymyxa E681 (pmxE) mutant by electroporation to prepare P. polymyxa E681-PT. _The process of constructing xE mutants is described in detail in a previous study (J. Bacteriol. 2009. 191: 3350-3358). NTG \ ½ethyl- '-ni tro-y ^ ni trosoguanidine) treatment, UV irradiation for the purpose of producing strains with improved productivity of fuzacidin using the parent strain of E.ni.E681-PT produced by the above process Mutation was induced by applying all three methods (Ultraviolet irradiation) and gamma-ray irradiation (Ga 麵 a ray irradiation) (FIG. 1).

<1-2> Development of Fusacidin high-productive Fanibacilli strain by mutation using G (A½ethyl-V'-ni tro-vV-ni trosoguanidine)

Selection of strains with increased mutation induction and productivity by treatment with NTG (^ methyl-^ '-nitro-y ^ nitrosoguanidine) proceeded as follows.

Inoculated with the F. Bacillus polymix E681-PT bacteria prepared in Example <1-1> in TSB 3 mi medium and shake culture (200 rpm) for 15 hours at 30 ^° C, 2% in TSB 3 m £ medium again After inoculation, the OD ₆₀₀ was further cultured to 0.7-1.0, and then 1 was added to add NTG solution, but the final concentration was 400 g / n. In fact, after standing for 10 minutes in a silver centrifuge at 11,000 rpm for 90 seconds using a small centrifuge to remove the supernatant to obtain the cells. 1 m £ of fresh TSB was added to wash the cells and centrifuged to obtain the cells and suspended in 1 TSB. After diluting this suspension sequentially to 10— ^ ICT ⁵ concentration, it was plated in 100 TSA solid medium and incubated for 24 to 48 hours at 30 ^° C. Strains were selected. The strain selection process is shown in Figure 2 and briefly described as follows. Micrococcus / «/ s (ML) bacteria were inoculated in TSB medium and shaken at 30 ^° C for 15 hours, and then 1% in TSA medium (maintained at about 50 ° C after sterilization). The mixture was made to a solid medium (TSA-ML). After NTG treatment, colonies grown in TSA medium were inoculated (toothpicked) in TSA-ML medium (about 2000 colonies), and after 24 hours of cultivation, strains for which growth inhibition rings against ML were larger than the parent were selected first. The same process was repeated again for the first selected strains, and the second selection was performed. The secondary cells were incubated for 24 to 36 hours in 3 ml of TSBG medium (addition of 3% glucose to TSB medium), and 0.5 ml of the culture was extracted with the same amount of butanol and used as a sample. The extraction was performed by mixing butanol thoroughly with the culture solution (about 30 seconds vortexing) and taking the supernatant after centrifugation (11,000 rpm with a small centrifuge for 3 minutes). Butanol extract of each strain was added to 10 ^ by 6 mm paper disk, dried, and then placed on TSA-ML medium and cultured for 24 hours, the excellent activity strain was selected through the comparison of the size of the growth inhibitory ring third. The third selected strains obtained pure culture through a single colony isolation process to extract butane and reconfirmed antimicrobial activity (fourth). The entire process described above was repeated three times, thereby securing the best-acting strain N17. As shown in FIG. 3, the samples were diluted sequentially by 1/2 to compare the activity of the diluents. As a result, N17 bacteria showed more than 10 times the productivity of fuzacidin compared to the parent strain, that is, the F. bacterium E681-PT. It was confirmed (FIG. 3).

<1-3> Development of high productivity strains of fuzacidin by repeated UV and NTG treatment (2nd stage development)

Two-stage development was performed to obtain mutant strains that further enhanced fuzacidin productivity for the N17 strain selected in Example <1-2>. This time, a combination of UV and NTG treatment, but the method by NTG treatment is as described in Example <1-2> and mutation selection and productivity improvement strain selection by UV treatment is as follows. After inoculating N17 mutant strain in TSB 3 ml medium for 15 hours shaking culture (200 rpm) at 3C C, inoculated 2¾ in TSB 3 ml medium again to further incubate 0D ₆₀₀ value of 0.7 ~ 1.0, and then took 0.1 ^ 0.1M MgS0 _4, the solution was diluted to ^10-5 - ^10-6 used. After the coating on the TSA medium was irradiated with UV using a UV lamp, but the mortality was about 90%. Antimicrobial activity of 3 colonies (approximately 2000) Strains with increased activity were selected as compared to N17. The excellent active strain selection process was followed the sequence shown in Figure 2 and the details are as described in Example <1_2>. UV and NTG were treated five times and three times by the above method, and as a result, the mutant strain NU177ol having an antimicrobial activity increased about 20 times or more compared to the parent strain ie E681-PT. 4 shows the difference in the antimicrobial activity when 36 hours of direct inoculation of the mother and NU177 bacteria in the TSA-ML solid medium.

<1-4> Development of fuzacidine high productivity strain using gamma ray (γ-ray) (step 3) Fuzacidin productivity is further enhanced for NU177 strain selected in Example <1-3>. Three stages of development were performed to obtain the mutated strains. This time, the gamma irradiation method was used, but it was carried out using the facility of Jeongeup Radiation Science Research Institute of Korea Atomic Energy Research Institute. The NU177 mutants at 30 ^° C was inoculated in TSB 2.5 medium 15 hours with shaking culture (200 rpm) after all of the culture medium TSB 200 mi was inoculated to the medium was 11 hours with shaking culture back to 30 ^° C, 160 rpm condition wherein OD ₆₀₀ The value reached 2.6. The culture solution was centrifuged at 4 ^° C. and 6500 rpm for 15 minutes to recover the cells, washed with the same amount of sterile saline, and the recovered cells were suspended in 20 sterile saline. The NU177 bacterial cell suspension thus obtained was dispensed into 6 plastic tubes (15 ml, Falcon tube) at 3 ra «. The five tubes were irradiated with gamma rays at the actual diameter (about 25 ^° C), but the treatment strengths of the various ribs were varied to 1, 2, 3, 4 and 5 kGy. Antibacterial a cell suspension of each ryubeu targeting 10- ¹ ~ ΚΓ after sequentially with dilution to a concentration of 100 ^ ⁴ by TSA 24-48 hours incubation with grown colony in 30 ^° C and plated on solid medium (about 2000) A strain with increased activity was selected by comparing the activity with NU177. The excellent active strain selection process was followed the sequence shown in Figure 2 and the details are as described in Examples <1-2>. As a result of performing gamma-irradiation once with the selection of excellent active strains from about 4000 strains, NUR1776, a highly productive mutant strain NUR1776, had about 50 times more activity than the parent strain, ie, F. poly68 E681-PT. (FIG. 4). The NUR1776 strain was deposited with the Korea Biotechnology Research Institute (KCTC) on November 11, 2011 and was given accession number KCTC 12068BP. <l-5> Quantitative analysis of fuzacidin

Fuzacidin standard samples were prepared as follows. Penibacilli polymyx E681 was incubated for 3 days at 32C using 8 L of TSBG medium, and the culture medium was gray bear (b rys / sc / nerea) and gram-positive bacteria Micrococcus luteus (/ c /? C Ca / 5 / ews) was confirmed to exhibit excellent antimicrobial activity from this culture medium was purified and purified fuzacidin. First, the culture solution was extracted using butanol (n-BuOH), butanol was removed by evaporation under reduced pressure to obtain a sample, and then silica gel chromatography (chloroform and methanol mixture ratio was changed from 4: 1 to 1: 1). Eluting) and Sepatex column chromatography (Sephadex LH-20 column, eluting with methane). The final step was preparative HPLC (YMC-pack pro C18 250 * 20 mm column, methane 50 ~ 8). Purification by elution using a concentration gradient). Fuzacidine obtained through this purification process was used as a standard sample. Fuzacidine samples were dissolved in methanol at a concentration of 0.1-50 jug / n, and then 5 samples were analyzed by HPLC to obtain the area of the fuzacidin peak. The concentration of fuzacidin sample was determined based on the results. In other words, an unknown sample was dissolved in methanol, diluted appropriately, analyzed by HPLC, and the peak area was determined. Then, the concentration was determined by substituting the ^fuzacidin quantitative assay line. NUR1776 bacteria selected in Example <1>4> were incubated for 24 hours at 32 ° C and 200 rpm shaking conditions using TSBG (II) medium (see Example <4-3>), and then the culture medium was extracted with butanol and completely After evaporation, the solution was dissolved in methanol and quantified by the method described above, and showed a productivity of 6.97 g / L, which was confirmed that the productivity of fuzacidin was improved by 50 times or more compared with the Phenyl Bacillus plymic company E681. In order to know the approximate concentration, as shown in FIG. 6, a comparison method with M. luteus bacterium growth inhibitory ring according to the concentration of fuzacidin (standard sample) was used.

<Example 2> Mutation gene search through analysis and analysis of the genome sequence of NUR1776 bacteria of Panibacillus polymix

Search for the mutant gene of the NUR1776 strain finally selected in <Example 1> and The translation of the entire genome sequence was performed for the purpose of characterization. Genomic sequence translation was performed by whole-genome shotgun method using Illumina HiSeq 2000. The obtained sequencing was submitted to RAST server (http: // r ast.nmpdr.org/) for automatic annotation of the genome sequence, and the nucleotide sequence of the annotated contigs was converted to Fanibacillus polymix E681. The site where the mutation occurred was compared to the standard sequence of bacteria (GenBank accession number CP000154). As a result, it was found that there was a mutation (point mutation) in one nucleotide sequence at the 4,871,643 bp position, and thymine (T) was substituted with guanine (G). As a gene having a base sequence of 1,716 bp (SEQ ID NO: 1) and encoding a protein of 572 amino acids (SEQ ID NO: 2). Searching for protein groups with similarity to these 572 amino acid sequences showed high homology with proteins belonging to the phosphohexomutase super family and the function of phosphoglucomutase. It was expected to have. In addition, the mutation of the 509 bp position of the PPE M441 gene (SEQ ID NO: 3) in the NUR1776 strain revealed that the 170th amino acid of the enzyme protein was changed from glutamine to proline (SEQ ID NO: 4). . Hereinafter, the PPE 34441 gene and the protein produced by the PPE_04441 gene (expected to be phosphoglucomatase) are referred to as PGM.

<Example 3> Development of fuzacidin high production strain by pgm gene inactivation <3-1>; Analysis of the Effect of Gene Overexpression on Fusacisidine Productivity

Complementation tests were performed to determine whether the increase in fuzacidin productivity of the NUR1776 strain is related to the point mutation of the pgm gene identified in <Example 2>. As a result of the point mutation, one amino acid was changed from glutamine to plinine. If this is the factor that increases the productivity of fuzacidin, the introduction of normal pgm gene into NUR1776 will reduce the productivity of fuzacidin. Specifically, pgm was not generated from the parent strain at the BamHI and Smal restriction enzyme positions of the pHPspac plasmid vector expressed by the promoter Pspac. The gene was cloned by PCR using forward primer 4441F (5-CGGGATCCGCTCTGC CGACATAAGC-3: SEQ ID NO: 5) containing BamHI and reverse primer 4441R (5-TCCCCGGGCGTTGCC GGAATAAGCC-3: SEQ ID NO: 6) containing Stnal. PCR was followed by a 3 min predenaturation at 95 ^° C followed by 30 seconds at 95 ^° C for denaturation, 30 seconds at 50 ^° C for annealing step and 72 ^° C at extension step 72 ^° C. The reaction was performed 30 times for 2 minutes and finally carried out under the condition of 5 minutes of termination reaction at 72 ^° C. Both ends were cut with the same restriction enzyme and recombinant with the vector to prepare a PHR4441 plasmid, which was then introduced into NUR1776, and transformants were selected by erythromycin resistance (FIG. 7). Transformants (polymyxa NUR1776 (pHR4441)) colonies were shaken for 48 hours in TSBG medium. The same amount of butanol was added to the culture solution to obtain a fusarisidine extract sample, and 10 ^ extracts were placed on a paper disk to compare the inhibition of micrococcus luteus ^ growth.

As a result, as shown in Figure 7, NUR1776 (pHR4441) was confirmed that the antimicrobial activity is significantly reduced compared to the parental NUR1776 (pHPspac) having only a vector. Therefore, it was confirmed that the point mutation of the pgm gene is one of the factors that increase the productivity of fusaricidin.

<3-2> Analysis of Effect of Mutation of pgm Gene on Productivity of Fuzarizidine

In order to confirm that the point mutation of the pgm gene of NUR1776 is a factor that increases the productivity of fusaricidin, the analysis of the productivity of the fusarisid was induced by inducing the mutation of the pgm gene on the chromosome of the parent strain, ie, E681-PT. The high productivity of fuzacidin in NUR1776 may be the result of a combination of several genetic variations, so it is intended to see the effects of mutations in the pgm gene alone. To this end, two types of mutations were introduced into the parent strain, first of which introduced a point mutation identical to that of the NUR1776 strain (FIG. 8) and second of completely inactivating by removing a part of the pgm gene (FIG. 9). )to be. First, the point mutation introduction process is as follows. To obtain the point mutation of NUR1776, the mutant DNA of NUR1776 was used as a template, and forward primer 4441F (SEQ ID NO: 5) and reverse primer 4441R-em (5-CACACTCTTAAGmGCTTCctUgccgtgagaggtcacc-3: SEQ ID NO: 7) were obtained. PCR was performed. The erythromycin (em) resistance gene was then plasmid pDG1664 [Bacillus Genetic Stock Center (BGSCID: ECE117)] with a template of forward primer emF (5-GMGC ACTTMGAGTGTG-3: SEQ ID NO: 8) and reverse primer emR (5). -TCCTTGGMGCTGTCAGTAG-3: Amplified by PCR using SEQ ID NO: 9) and then mixed with the pgm e PCR product, followed by fusion PCR method (Horton RM et al., Gene 77: 61-68 1989) to _P gm _{177 (r} em cassette). Fusion PCR was followed by a 3 minute predenaturation step at 94 ^° C, followed by 20 seconds at 94 ^° C denaturation ion and 20 seconds at 45 ^° C annealing step (0.3 per time). Upwards), extension step 15 times at 68 ^° C for 4 minutes, 20 seconds at 94 ^° C, 20 seconds at 501 ：, 20 times for 4 minutes at 68 ° C, and last Was run under conditions of 5 minutes termination reaction at 68 ^° C. Pgm subpart 2 kbOW ^ i?) By forward primer 4440F-era (5-

CTACTGACAGCTTCCAAGGAggcagt acgccgt ggaggt c-3: SEQ ID NO: 10 and amplified by PCR using reverse primer 4440R (5-GATAATAGCCGTGCGATAA-3: SEQ ID NO: 11), and then linked to pgm e-emcassette by fusion PCR again to 4.85kb A pgmn em-N4440 fragment of size was obtained. This 4.85kb fragment was cloned into pGEM T-easy vector (Promega) to make pHR4441 _P plasmid, which was introduced into F. E681-PT strain of F. polymycosa, and point mutations in the gene through recombination on chromosomes. PGM1776 bacteria were obtained from Fanibacilli polymix. The inoculated strains were inoculated in TSBG medium and incubated for 48 hours. Butane in the culture was compared with the parent strain for M. // i «/ s growth inhibition. It was confirmed (FIG. 8).

The next step was to prepare a 1.4 kb PCR fragment (fc) containing the N-terminal part of the pgm gene by forward fusion PCR method as described above in order to construct a mutant lacking the pgm gene. Forward primer 4441FF (5-GMMGAGCAGTAGGCATTA- 3: SEQ ID NO: 12) and reverse primer 4441FR-em (5-

PCR amplification was performed using CACACTOTAAGTTTGOTCcggt cagcat cagggt ctg-3 SEQ ID NO: 13). This Npgm PCR fragment was linked with the em-N4440 PCR fragment to obtain 4.2 kb of Npgm ^to em-N4440 fragments, which were TA cloned into a pGEM T-easy vector (Pr omega) to pHR4441 _D. Plasmids were prepared. This Fannie Bacillus poly miksa introduced in the E681-P bacteria was produced _P gm gene is completely inactivated the mutant Fannie Bacillus poly miksa PGM4441 bacteria through recombination on the chromosomes, PGM4441 strains Life Resource Center, Korea Research Institute of Bioscience and Biotechnology (KCTC) Was deposited on November 27, 2012 and was granted the accession number KCTC 12323BP. In addition, the PGM4441 strain was inoculated in TSBG medium and cultured for 48 hours, and the growth inhibition activity of M. luteu growth of butanol extract of the culture medium was compared with the parent strain, and the growth inhibition activity was confirmed 10 times higher than that of the parent strain (FIG. 9). <3-3> Analysis of the Effect of Gene Mutations on the Viscosity of Culture Media

The strains of _pgm gene were examined after incubating the parent strain E681-PT and the fusicidin high-producing strains, ie, PUR1776, PGM1776 and PGM4441, in TSBG medium for 24 hours. Viscosity was significantly reduced for the strains that occurred (FIG. 10). This may be due to the decrease in the production of viscous substances such as extracellular polysaccharides due to the mutation of the pgm gene, thereby facilitating cell recovery by centrifugation.

<3-4> Analysis of the function of the genome P68 (PPE-04441) gene of E.ni. p68 (PPE-04441) of Pannibacilli polymyxa It was predicted that the function of the protein encoded by the PPE 04441 gene is phosphoglucomutase (PGM) In order to confirm that the predicted function is correct, the incubated fungi, ie, F. bacterium E681-PT and point mutation PGM1776, were incubated in TSB 3ml at 30 ° C for 15 hours, and the cells were recovered by centrifugation. After suspension in phosphate buffer (pH 7.0), ultrasonic disruption (Branson 450 Sonifier, output 3.0, duty cycle 30¾) was used as an enzyme sample. The phosphoglucomatase titer was measured by Rick et al. (J. Bacteriol. 176: 4851-4857, 1994), and the enzyme was converted to glucose-1-phosphate substrate by glucose and then converted into glucose-6 phosphate. glucose-6 一: NADPH produced during oxidation by phosphate dehydrogenase is measured as absorbance at 340 nm. Absorbance measurements at 340 nm The result obtained by dividing by 0¾ ₀₀ value of the strain culture medium and multiplying by 1000 is shown in Table 1. The E681-PT sample shows a high AU value of 303.7 (Arbitrary Unit), whereas the point mutation has an AU value of 7.7 in the culture of PGM1776 bacteria. In the case of PGM4441 bacteria in which the pgm gene was completely inactivated, a value of 2.0 AU was shown. As a result, it was confirmed that the ^ (PPE_04441) gene encodes phosphoglucomutase as predicted.

Table 1

Analysis of Phosphoglucomatase Activity by PGM1776 and PGM4441

<Example 4> mass culture and production of fuzacidin of the fungus Bacillus polymix NUR1776

<4-1> 5L culture experiment

A culture test using a 5 L fermenter was performed to produce fuzacidine using the fuzacidin high-producing strain Fanibacillus polymyx NUR1776. Colonies obtained by culturing 1-2 days in TSA solid medium were inoculated in 10 TSB medium and shaken at 32 ^° C for 15 hours at 200 rpm, and then the culture medium was added to 350 mi TBSG medium (added 3% glucose to TSB medium). Was inoculated to 1%. It was incubated for 48 hours at 32 ^° C, 300rpm stirring, 1 vvm aeration conditions. 0.5 hours of culture was sampled and extracted with the same amount of butanol, and 10 ^ extracts were placed on paper discs to track M. lutein growth inhibition. As a result, as shown in FIG. 11, the production was greatly increased after 9 hours of culture, reaching a maximum at 27 hours. <-2> Extract with fuzacidin from the culture medium of Panibacillus After culturing NUR1776 bacteria in the same manner as in Example <4-1>, the culture medium 0.5 was sampled at 2 hours or at appropriate time intervals to obtain the cells and the culture supernatant by centrifugation (11,000 rpm, 1 minute). As a result of preparing the sample by extracting butane by the method described in Example <1-2> and examining the antibacterial activity, as shown in FIG. On the other hand, in extracting fuzacidine, in order to select an appropriate solvent, the cells were extracted with two solvents, ie, butane and ethanol, and as a result, the extraction efficiency by ethane was excellent as shown in FIG. It was. Therefore, it was confirmed that it is preferable to use fuethanidine for extracting fuzacidin from the fungus Bacillus cells.

<4-3> Fuzacidin Production by 500 L Scale Culture

For mass culture of NUR1776 strain, TSBG (II) medium (TSB, 8g; glucose, 3g; (NH ₄ ) ₂ S04, 2g; K ₂ HP0 ₄ , 0.1 g; MgS0 ₄ , 0.01 g; yeast extract, 1 gl L ) Was used.

Colonies of NUR1776 were inoculated in 10 ml of TSBG medium and shaken at 32 rpm for 15 hours at 200 rpm, and then inoculated in 500 ml of TSBG medium and incubated for 10 hours under the same conditions. The culture was inoculated 1¾ in 35 L TBSG (II) medium and incubated for 6 hours at 32 ^° C, 300 rpm stirring, 1 vvm aeration conditions. It was confirmed that the well grown and inoculated 10% in 350 L TBSG (II) medium and incubated for 20 hours at 32 ^° C, 300rpm stirring, 1 wm aeration conditions. After 12 hours of cultivation, the cell growth reached a maximum, and fuzacidin production was actively carried out from 9 hours after the start of cultivation (Fig. 14). It was. An equal amount of ethanol was added to the cell suspension, and the mixture was left to stand overnight, followed by extraction. The supernatant was collected by centrifugation, concentrated by evaporation under reduced pressure, and finally, fuzacidine was obtained by lyophilization. The analysis of fuzacidine in the culture by the method described in Example <1-5> resulted in a level of 7 g / L.

[Accession number]

Depositary Name ： Korea Research Institute of Bioscience and Biotechnology Accession number ： KCTC12068BP Accession date ： 20111111 Depositary name ： Korea Research Institute of Bioscience and Biotechnology Accession number ： KCTC12323BP Accession date ： 20121127

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Preface

？. Number of recurrences

TO: Park Seung

Guk Gong Fu ¾¾¾ S48-1B

31 -92E

BUOAMiST TREATY ON THE I TERNATiONAL MSXXJNITK OF THE DEPOSIT

OF MICROORGANISMS OIi THE PURPOSE OF PATENT PHOCGPUliE

IMTERNATIO AL FOiiM

RECEIPT I THE CASE OF AN ORIGINAL DEPOSIT

issued pursuant to Rule 7.1 O ： PARK Setmg * H 5

Korea Research Institute of Bioscience and Biotechnology

548-16 Wonbong— ri, Geumnam-m eon, Sejong-si 339-837

epub! ic of Korea

Π, SCIENTIFIC DESCRIPTION AKD / OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identifi ^! under！ above was accompanied by:

ί x] a scientific description

[] A proposed ta 難麵 ic designation

(Mark with cross where applicable)

EL RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I above, which was received by it on November 27, 2012 _*

{V, RECEIPT OF REQUEST FOR CONVERSIO

The microorganism identified under I above was received by this International Depositary Authority on md a request to convert the originai deposit Co a deposit under the Budapest Treaty was received by it on

V. INTERNATIONAL DEPOSITARY AUTHORITY

Signatured s) of person (s) having the power

Name: Korean Collection lor Type Cultures to represent the Internationa! Depositary

Address- Korea Research Institute of

Bioscience and Biotechnology (R1BB)

125 Gwahak-ro, Yuseong-gu,

Daejeon 3 (»ᅳ 806 BAE, Kyung Sook, Director Republic of Korea Date: December 05 2012

Fom BP / 4 <CC Foiw Ϊ.7) sole page

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Claims

[Range of request]

[Claim 1]

A pgm gene as set forth in SEQ ID NO: 1 in a F. paniscillus polymyx (/ ¾e / 2 / 0ac /// i / s ploymyxa) strain; Or a fuzisidine inactivating at least one of the pgm gene of SEQ ID NO: 1, the polymyxin biosynthesis gene of SEQ ID NO: 14, and the tritecaptin biosynthesis gene of SEQ ID NO: 15; Producing Recombinant Penicillaceae Polymyx Strains.

[Claim 2]

According to claim 1, wherein the Fanibacillus polymixa strain is fuzisidine high production recombinant Favaibacillus polymixa strain, characterized in that the strain deposited with the accession number KCTC 8801P.

[Claim 3]

According to claim 1, wherein the gene inactivation is fusicidin high production recombinant Favacillus polymyxa strain, characterized in that for deletion or mutation.

[Claim 4]

According to claim 1, wherein the fuzacidin high production recombinant panic Bacillus polymixa strain is characterized in that deposited with the deposit No. KCTC 12068BP or KCTC 12323BP fuzacidin high production recombinant Panic Bacillus polymixa strain.

[Claim 5]

Fuzacidin, in which the polymyxin biosynthesis gene as shown in SEQ ID NO: 14 and the tritetaptin biosynthesis gene as shown in SEQ ID NO: 15 are inactivated and the mutant pgm gene as shown in SEQ ID NO: 3 is introduced in the F. High Production Recombinant Penivacillus Polymyx Strains.

[Claim 6] According to claim 5, wherein the Fanibacillus polymixa strain is a fuzzy cydin high production recombinant Favabacillus polymixa strain, characterized in that the strain deposited with the accession number KCTC 8801P.

[Claim 7]

2) A method for producing a fuzzy cydin high-producing recombinant F. ni. Polymyxa strain, comprising the step of selecting the high fuzisidine-producing strain from the strain of step 1).

[Claim 8]

The method of claim 7, wherein the strain of the Panibacillus polymix "of step 1) is a strain deposited with accession number KCTC 8801P.

[Claim 9]

2) inducing mutations by treating NTGO methyl— —nitro— ^ nitrosoguanidine) to the strain of step 1; And

3) a method for producing a fuzzy cydin high-producing recombinant panibacillus polymixsa strain comprising the step of selecting the fuzacidine mass-producing strain from the mutated strain of step 2)

[Claim 101]

10. The method according to claim 9, wherein the strain of Panicibacillus polymixsa of step 1) is deposited No. Method for producing a fujarysidine high production recombinant paniva silus polymyxa strain, characterized in that the strain deposited with KCTC 8801P.

[Claim 11]

1) treating the strain of claim 9 with NTG and UV to induce mutations; And

2) a method for producing a fuzzy cydin high-producing recombinant panibacillus polymixsa strain comprising the step of selecting the fuzacidine mass-producing strain from the mutated strain of step 1)

[Claim 12]

1) irradiating the strain of claim 11; And

2) a method for producing a fuzzy cydin high-producing recombinant F. ni. Polymyxa strain comprising the step 1) and the step of selecting a fuzacidine mass-producing strain from the mutated strain.

[Claim 13]

13. The method according to claim 12, wherein the radiation of step 1) is any one selected from the group consisting of gamma rays, infrared rays, ultraviolet rays, X-rays, and protons. Manufacturing method.

[Claim 14]

A fusicidine high-producing recombinant Fanibacillus polymyxa strain prepared by the method of any one of claims 7 to 13.

[Claim 15]

1) culturing the strain of claims 1 to 6; And

2) a method for mass production of fuzacidine comprising recovering fuzacidine from the culture medium of step 1).

[Claim 16]

1) culturing the strain of claim 14; And

2) a method for mass production of fuzacidine, comprising recovering fuzacidine from the culture solution of step 1).

[Claim 17]

The recombinant Fanny Bacillus polymyxa strain of claims 1 to 6 used for mass production of fuzacidine.

[Claim 18]

Recombinant Fenbacillus polymyxa strain prepared by the method of any one of claims 7 to 13 used for mass production of fuzacidine.