JPH07203975A - Gene encoding antimicrobial peptide and production thereof - Google Patents

Abstract

PURPOSE:To provide a novel gene participating the production of antimicrobial peptides such as iturin A or surfactin useful for controlling damping off or wilting. CONSTITUTION:The gone encoding antimicrobial peptide of the formula. The plasmid containing the antimicrobial peptide gene is prepared from the gene library of Bacillus subtilis SD 142 strain and isolated by selecting clones forming harrow around it in the L-medium containing tributyrin.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a fusarium (Fusariu)
m), Rhizoctonia, Xanthomonas and other phytopathogenic fungi have high antibacterial activity against Bacillus subtilis.
A method for producing an antibacterial peptide characterized by culturing a gene encoding an antibacterial peptide produced by SD142 strain (Microtechnology Research Institute No. 13204) and a microorganism transformed with an expression plasmid containing the gene. Regarding

[0002]

2. Description of the Related Art Diseases in which plants wither and die (fall and wilt) are serious diseases in vegetables such as cucumber, spinach and tomato, and grains such as rice, corn and wheat. These diseases are diseases caused by soil-living filamentous fungi such as Fusarium and Rhizoctonia and bacteria such as Xanthomonas. For such diseases, control methods such as soil sterilization and avoidance of continuous cropping of specific crops can be considered, but there are still many technically and economically difficult points for general farmers, and they are effective alternatives. Establishing a control method is desired.

In recent years, microorganisms exhibiting antibacterial activity against plant pathogens have been isolated and expected as environmentally friendly microbial pesticides. Among them, iturin A
Is L-Asn → D-Tyr → D-Asn → L-Gln.
→ L-Pro → D-Asn → L-Ser-consisting of 7 amino acids and having a cyclic structure by β-amino acid bond (Isogai, I., et al., Tetrahedron Lett., 23, 3065 (19
82), Peypoux, F., et al., Biochemistry, 17,3992 (197).
8)) It is an antibacterial peptide, and has better characteristics as a microbial pesticide such as a broad antibacterial spectrum. On the other hand, surfactin is L-Glu → L-
Leu → L-Leu → L-Val → L-Asn → D-L
eu → L-Leu- consists of seven amino acids different from iturin A and has a cyclic structure due to the ester bond of β-hydroxycarboxylic acid (Arima, K., et al., Bioc
hem.Biophys.Res.Commun., 31 , 488, (1968)) peptide and is characterized by having a strong surfactant activity.

Iturin A and surfactin have a biochemical synergistic effect, and it is known that the presence of surfactin further enhances the antibacterial activity of iturin A (Hiraoka, H., et al., J. Gen.Appl.Microbiol.,
38, 635, (1992)). Therefore, microorganisms that produce these substances at the same time are considered to have a high inhibitory effect against plant pathogens, and it is considered that the combination of iturin A and surfactin is an indispensable means for the spread of microbial pesticides.

In 1992, a microorganism (Bacillus subtilis SD142 strain) that simultaneously produces iturin A and surfactin was isolated from nature (Hiraoka, H., e.
T al., J. Gen. Appl. Microbiol., 38 , 635, (1992)). However, the productivity of both substances of Bacillus subtilis SD142 strain is still unsatisfactory for use as a microbial pesticide.

[0006] A 10 Kb fragment (including 4 fragments of HindIII) that transforms a non-surfactin-producing Bacillus subtilis MI113 strain into a surfactin-producing strain was cloned from the chromosome of Bacillus subtilis SD142 (Hiraoka). , H., et al., J.Fer
ment. Bioeng., 74 , 323 (1992)). In addition, the two HindIII two-fragment regions essential for surfactin production were constructed with pC19.
Although the plasmid pC112 subcloned on 4 has been obtained, it is still unsatisfactory and the genes involved in the production of iturin A and surfactin have not been identified.

[0007]

As a means for solving such problems, a gene involved in the production of iturin A and surfactin is isolated from Bacillus subtilis SD142 strain, and the gene is introduced into a microorganism and expressed. As a result, iturin A and surfactin can be produced in large amounts. However, in order to take such genetic engineering means, it is necessary to have knowledge on the genes relating to the antibacterial peptide iturin A and surfactin and the structure of the genes.

Therefore, an object of the present invention is to prevent the plant diseases such as Fusarium filamentous fungi, Rhizoctonia filamentous fungi, and Xanthomonas spp. And to provide genes involved in surfactin production.
A further object of the present invention is to provide an efficient method for producing iturin A and surfactin encoded by the above genes.

[0009]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to solve these problems. As a result, we succeeded in cloning a gene involved in the production of iturin A and surfactin from the chromosomal DNA of Bacillus subtilis SD142 strain showing a high antibacterial activity against plant pathogens, and contained the structural gene portion of the gene. We determined the open reading frame of the entire 1052 nucleotide sequence and the coding of 224 amino acid residues, and elucidated the entire primary structure of the gene for iturin A and surfactin production. Furthermore, by connecting this gene to an expression vector of Bacillus subtilis and introducing it into Bacillus subtilis lacking the ability to produce iturin A and surfactin, a microorganism producing a large amount of antibacterial peptide is created, and this microorganism is By culturing, a production method for mass-producing iturin A and surfactin derived from Bacillus subtilis SD142 was completed.

That is, the present invention is: 1) a gene encoding the peptide having antibacterial activity shown in SEQ ID NO: 1) 2) a gene containing the base sequence shown in SEQ ID NO: 1 encoding a peptide having antibacterial activity, 3) antibacterial The gene according to 1 or 2 above, wherein the active peptides are iturin A and surfactin, 4) the gene according to 1 above, which is derived from Bacillus subtilis SD142 strain, and 5) any of 1 to 4 above. The present invention provides a method for producing an antibacterial peptide, which comprises culturing a microorganism capable of producing an antibacterial peptide transformed with an expression plasmid containing the gene according to the above item.

Bacillus subtilis SD142 of the present invention
A gene involved in the production of iturin A and surfactin derived from the strain and the amino acid sequence translated by the gene are specified by SEQ ID NO: 1. The nucleotide sequences of the genes involved in the production of iturin A and surfactin of the present invention are:
Orf of Bacillus brevis ATCC9999 which is a gramicidine S-producing bacterium
X region, surfactin producing bacterium Bacillus subtilis A
Sfp gene isolated from TCC21332 and surfactin-producing bacterium B. pumilus
Since the psf-1 gene isolated from the A-1 strain shows homology of about 40 to 70% with each nucleotide sequence, the gene of the present invention is a novel one which is clearly different from the above-mentioned known genes. is there. A plasmid containing the antibacterial peptide gene of the present invention is prepared from a gene library of Bacillus subtilis SD142 strain, and is isolated by selecting a clone formed by a halo around the colony on L medium containing tributyrin. You can

As is well known, there are a plurality of DNA base sequences encoding many amino acids. Therefore, the nucleotide sequence encoding the peptide is not uniquely determined, and there are many possibilities. In the case of the genes involved in the production of iturin A and surfactin of the Bacillus subtilis SD142 strain revealed by the present inventors, the nucleotide sequence of the DNA is not limited to the nucleotide sequence of the natural gene, and many possible sequences are possible. The gene of the present invention is not limited to a natural DNA base sequence, but encodes an amino acid sequence translated from the gene involved in the production of iturin A and surfactin revealed by the present invention. It includes genes of all nucleotide sequences in possible combinations.

In addition, according to the gene recombination technique, a specific portion of the basic DNA is artificially modified without changing the basic characteristics of the one encoded by the DNA or improving the characteristics. Can be mutated. With respect to a gene having a natural base sequence or a gene having a base sequence different from that of the natural gene provided by the present invention, the gene is equivalent to the natural gene by artificially inserting, deleting, or substituting. Improved properties are possible and the invention includes such mutated genes.

Bacillus subtilis SD142 of the present invention
Iturin A and surfactin of Bacillus subtilis SD142 strain are produced by Bacillus subtilis by ligating genes involved in the production of iturin A and surfactin of the strain to an appropriate vector, for example, expression vector pC194, pUB110 for Bacillus subtilis. An expression vector can be constructed. It is possible to obtain a microorganism producing a large amount of iturin A and surfactin by introducing the expression vector carrying the gene for iturin A and surfactin production of the present invention into iturin A and surfactin non-producing Bacillus subtilis. .

The transformed microorganism thus produced is treated under appropriate conditions (for example, medium: 30 g of polypeptone, 10 g of glucose, per liter of distilled water,
1 g of KH ₂ PO ₄ , 0.5 g of MgSO ₄ .7H ₂ O
(PH 7.0), culture temperature: 30 ° C, culture time: 8-12
Iturin A and surfactin can be mass-produced by culturing for 0 hours under culture conditions: shaking culture. The isolation of both peptides after culturing can be easily carried out by an operation of concentrating and recovering an aggregate composed of both peptides by, for example, extracting with methanol from the culture supernatant. Further, not only the host-vector system of Bacillus subtilis, but also the host-vector system of Escherichia coli, yeast, Pseudomonas or actinomycetes can be used, and iturin A and surfactin utilizing the characteristics of each host-vector system can be utilized. Mass production is possible. The iturin A and surfactin thus obtained are useful as microbial pesticides.

[0016]

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to the following examples, and can be modified in a usual manner in the technical field of the present invention.

Example 1 Subcloning of pC112 A chromosomal fragment involved in surfactin production of Bacillus subtilis SD142 strain was subcloned on pC194, and the plasmid pC112 was completely digested with the restriction enzyme HpaI to remove the 0.5 Kb HpaI fragment. Plasmid pC113 was prepared. Next, this pC113
Was completely digested with restriction enzymes HpaI and PvuII to remove the 2 Kb HpaI-PvuII fragment from pC113 to prepare a further miniaturized plasmid pC115. The restriction enzyme maps of the plasmids pC112, pC113 and pC115 are shown in FIG. These plasmids (p
C113 and pC115) to Bacillus subtilis MI1
It was introduced into 13 strains, and the surfactin production of these transformants was measured. The results are shown in Table 1. In addition, MI11
Introduction of plasmid DNA into the 3 strains and measurement of surfactin production were carried out as follows.

Traits of Bacillus subtilis MI113 strain
Bacillus subtilis M, a non- converting surfactin producer
Transformation of strain I113 was performed according to the competent cell method of Spizizen, J. et al. That is, 0.5% glucose
Bacillus subtilis MI113 in L plate medium containing
The strain is cultivated at 30 ° C overnight. Scrap young cells, CI
Medium (0.5% glucose, 0.02% casamino acid, 1.4% K
₂ HPO ₄ , 0.6% KH ₂ PO ₄ , 0.2% (NH ₄ ) ₂
SO ₄ , 0.1% sodium citrate, 5 mM MgSO
₄ , 50 μg / ml tryptophan, 50 μg / ml arginine) was inoculated to give A ₆₆₀ = 0.1.
Incubate with shaking at 5 to 36 ° C. until late logarithmic growth. After collecting the bacteria, CII medium (0.5% glucose, 0.01% casamino acid, 1.4% K ₂ HPO ₄ , 0.6% KH ₂ PO ₄ , 0.2%
(NH ₄ ) ₂ SO ₄ , 0.1% sodium citrate, 5 m
M MgSO ₄ , 5 μg / ml tryptophan, 5 μg
/ Ml arginine) and shake culture for about 40 minutes, 1 μg / ml of plasmid DNA is added, and the culture is continued for another 30 to 40 minutes. This culture broth was spread on an L plate containing chloramphenicol (5 μg / ml) and cultured at 37 ° C. overnight to obtain a transformant (MI).
113 / pC194, MI113 / pC112, MI1
13 / pC113 and MI113 / pC115) were obtained. These 4 strains and the parent strain MI113 were treated with No. 3 medium (30 g polypeptone, 10 g glucose, 1 g KH).
₂ PO ₄ , 0.5 g MgSO ₄ .7H ₂ O was suspended in 1 liter of distilled water and adjusted to pH 7.0) and incubated at 30 ° C. for 5 days, and the production amounts of iturin A and surfactin are shown below. It was measured according to the method for detecting iturin A and surfactin.

Detection of iturin A and surfactin Detection and confirmation of iturin A and surfactin were carried out by the following three methods. (1) Detection by biosurfactant activity: For detection of iturin A and surfactin, L agar plate containing tributyrin (5 g yeast extract, 5 g sodium chloride, 10 g polypeptone, 20 g agar was dissolved in 1 liter of distilled water, and the pH was adjusted to Adjusted to 7.2). 20 μl
Tributyrin of 1. was plated on L agar plates, and the production of both substances was judged by forming halos around the colonies.

(2) Growth inhibition test of filamentous fungi by bacteria: Fusarium ox which is a plant pathogen
ysporum f.sp.lycopersicirace J1 SUF119) was added to potato dextrose agar medium (potato dextrose agar 39
g, distilled water 1 liter, pH = 5.6), the cells were cultured at 30 ° C. for 5 days, and the cells were suspended in sterile water. L this suspension water
After mixing with an agar medium, the mixture was solidified to obtain a Fusarium plate. Bacteria having the antibacterial activity gene of the present invention were inoculated in the center of this plate, and antibiotics were detected by the presence or absence of formation of an inhibition circle that appears due to the growth of Fusarium.

(3) Analysis of iturin A and surfactin by HPLC: The analysis of iturin A was reversed phase HPL.
This was done by using C. In other words, 30 ℃, 5 days
The culture broth cultivated in No. 3 medium was collected and the supernatant was
The pH was adjusted to 2 with hydrochloric acid. The solution was centrifuged and the resulting precipitate was extracted with 100% methanol for 2 hours. The methanol extract was centrifuged at 10,000 × g for 2 minutes, and the obtained centrifugal supernatant was filtered with a PTFE membrane filter (JP020, manufactured by Advantech) having a pore size of 0.2 μm to remove impurities. 20 μl of this sample is analyzed by HPLC
(Column: ODS-2, φ 4.6 × 250 mm, manufactured by GL Science Co., Ltd.). The developing solution was acetonitrile: 10 mM ammonium acetate = 3: 4 (V /
V) at a flow rate of 1 ml / min. The efflux pattern was monitored at 205 nm. The homologue of iturin A is divided into peaks 1 to 6 depending on the outflow time. Peaks 1 to 6 are, respectively, from the n-C14-β-amino acid,
It corresponds to nteiso-C15-β-amino acids and iso-C15-β-amino acids, n-C16-β-amino acids, iso-C16-β-amino acids and n-C17-β-amino acids. Peak 6
Is usually small and difficult to detect. Further, the amount of iturin A was a value obtained by summing the amounts of the respective components of iturin A.

On the other hand, the analysis of surfactin was carried out by the same HPLC system as iturin A. However, the developing solution was 3.8 mM trifluoroacetic acid: acetonitrile = 1: 4.
(V / V), the flow rate was 1.5 ml / min, and the outflow pattern was detected at 205 nm. A calibration curve was prepared from a standard surfactin sample (manufactured by Sigma), and the amount of surfactin produced was measured.

[0023]

[Table 1]

Example 2: Determination of iturin A and DNA base sequences involved in surfactin production The base sequences of 1.2 kb from HindIII to HpaI of pC115 were determined as follows. From the left to the right of pC113, HindIII-HindIII, PstI-
Three fragments of PstI and HindIII-EcoRI were prepared using various restriction enzymes. Each of these three fragments was subcloned into pUC19 using T4 ligase, and a 373A autosequencer (ABI) was used.
The base sequence of 1.2 Kb was determined. The results are shown in the sequence listing (SEQ ID NO: 1). This region contains an open reading frame (OR that encodes 224 amino acid residues).
F), and this ORF was named lpa-14.

Example 3 Confirmation of Product of lpa-14 Gene Identification of product of lpa-14 gene was carried out as follows. pC115 and pC1 having the lpa-14 gene
P from which the 12 to lpa-14 gene was removed with a PstI fragment
C112Δ (restriction enzyme map is shown in FIG. 1) and vector plasmid pC194 were introduced into iturin A and Bacillus subtilis RΔ1 strain which lost surfactin productivity by high voltage pulse method (electroporation). That is, 150 ml of the culture solution of the RΔ1 strain in the late logarithmic growth phase was collected and well-cooled in 1 mM HEPES buffer (N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid).
The cells were washed 3 times with d, pH 7.0), and the bacterial suspension was concentrated to a final concentration of about 400 μl. This bacterial solution 40μ
5 μl of each plasmid DNA (pC115, pC1
12Δ and pC194) and 40 μl of 50% (w / w)
A sample for pulse application was a mixture of polyethylene glycol 6000. The electroporation device is
Cell PoratorTM with Voltage Booster (BRL Life Technol
ogies, Inc.), and used to connect a mixed solution of 20 μl of the bacterium and each plasmid DNA to a cuvette (distance between electrodes: 0.
15 cm) and 16.0 KV / cm (2.4 KV, 4 KΩ, 2
μF) pulse was applied. Applied sample 0.5 ml
Is added to the L medium and cultured at 37 ° C. for 3 hours. This culture solution was added with L containing chloramphenicol (5 μg / ml).
The transformants (RΔ1 / pC115, RΔ1 / pC115) were plated and cultured overnight at 37 ° C.
112Δ and RΔ1 / pC194) were obtained. After culturing these 3 strains in No. 3 medium at 30 ° C. for 5 days, the production amounts of iturin A and surfactin were measured according to the above-described method for detecting iturin A and surfactin. The results are shown in Table 2. From this result, it was confirmed that the lpa-14 gene is a gene related to the production of iturin A and surfactin.

[0026]

[Table 2]

[0027]

INDUSTRIAL APPLICABILITY The gene of the present invention is a Bacillus subtilis encoding a peptide (Iturin A) having an antibacterial activity against a plant pathogen derived from Bacillus subtilis SD142 strain and a peptide (surfactin) having a strong surfactant activity. It is derived from the SD142 strain. By culturing the microorganism into which the gene of the present invention is introduced, it is possible to efficiently produce iturin A and surfactin, which have a synergistic effect on the antibacterial activity against plant pathogens and are useful as microbial pesticides.

[0028]

[Sequence listing] SEQ ID NO: 1 Sequence length: 1052 Sequence type: Nucleic acid and amino acid Number of chains: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bacillus subtilis
SD 142 Sequence HpaI ATACGCGATCTCCGGGCGGGCAACGTCCGTTCATTAAAAACAAAGCCGAAACGGTTTTG 65 TTTTTAATGA ACGGACAGCT TTCGTCTGAT ATGATAGGAT GGTTTTGACA ATATTTTCAG 125 rg Arg Crb GRA T CG CG Tactiles CG AC GTG ATC CG Tactiles CGAC GCT GAG TACT ATG ATG GCG GCC GTG TCC GCC GAA AAG 223 Ser Ala Gly Glu Glu Asp Arg Met Met Ala Ala Val Ser Ala Glu Lys 15 20 25 CGG GAA AAA TGC CGG CGC TTT TAC CAT AAG GAG GAT GCT CAC CGC ACC 271 Arg Glu Lys Cys Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr 30 35 40 TTG ATC GGC GAC ATG CTG ATC CGC ACC GCT GCG GCG AAG GCT TAT GGA 319 Leu Ile Gly Asp Met Leu Ile Arg Thr Ala Ala Ala Lys Ala Tyr Gly 45 50 55 60 CTT GAT CCG GCC GGG ATT TCA TTC GGC GTC CAG GAA TAC GGA AAG CCG 367 Leu Asp Pro Ala Gly Ile Ser Phe Gly Val Gln Glu Tyr Gly Lys Pro 65 70 75 TAC ATC CCC GCG CTT CCG GAC ATG CAC TTT AAC ATT TCC CAC TCC GGG 415 Tyr Ile Pro Ala Leu Pro Asp Met His Phe Asn Ile Ser His Ser Gly 80 85 90 CGC TGG ATC GTG TGC GCC GTT GAT TCA AAA CCG ATC GGC ATT GAT ATT 463 Arg Trp Ile Val Cys Ala Val Asp Ser Lys Pro Ile Gly Ile Asp Ile 95 100 105 GAA AAA ATG AAG CCC GGC ACG ATT GAT ATC GCC AAA CGG TTT TTT TCG 511 Glu Lys Met Lys Pro Gly Thr Ile Asp Ile Ala Lys Arg Phe Phe Ser 110 115 120 CCG ACG GAA TAC AGT GAT CTG CAA GCG AAA CAC CCC GAT CAG CAG ACC 559 Pro Thr Glu Tyr Ser Asp Leu Gln Ala Lys His Pro Asp Gln Gln Thr 125 130 135 140 GAT TAT TTT TAC CAC CTG TGG TCG ATG AAA GAA AGC TTT ATC AAA CAG 607 Asp Tyr Phe Tyr His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln 145 150 155 GCC GGA AAA GGG CTT TCC CTG CCG CTT GAT TCA TTC AGC GTC CGC CTC 655 Ala Gly Lys Gly Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu 160 165 170 AAA GAC GAC GGC CAT GTG TCC ATT GAG CTT CCG GAC GGG CAT GAA CCT 703 Lys Asp Asp Gly His Val Ser Ile Glu Leu Pro Asp Gly His Glu Pro 175 180 185 TGT TTC ATC CGC ACA TAT GAT GCG GAC GAG GAG TAT AAG CTG GCC GTT 751 Cys Phe Ile Arg Thr Tyr Asp Ala Asp Glu Glu Tyr Lys Leu Ala Val 190 195 200 TGT GCG GCG CAT CCC GAT TTT TGT GAC GGG ATT GAG ATG AAA ACG TAT 795 Cys Ala Ala His Pro Asp Phe Cys Asp Gly Ile Glu Met Lys Thr Tyr 205 210 215 220 GAA GAG CTG TTA TAAGCAATCC GTCAGCATTT TGATGCCTCG 851 Leu Leu * 224 CCTGTCTTGA CGTTGGACAC GTTGAGTTTT ATGATATTTT CTTTCGGAAA ATCCGATAGG 911 TAATGGCTGT CAATCGGCTC CACCAGCACG CCTTGCGCTT TCAGACGCTG CACAACTCTT 971 GAAGCGGAAA ACGGCTTTCTTCCAGCACGAGA TGGG2TGTCATT

[Brief description of drawings]

FIG. 1 Plasmids pC112, pC112Δ, pC
113 is a restriction enzyme map of 113 and pC115.

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[Procedure amendment]

[Submission date] March 11, 1994

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

[0027]

Gene of the present invention exhibits, those derived from Bacillus subtilis SD142 strain that encodes a peptide (surfactin) with peptide (iturin A) and strong surface activity with antibacterial activity against plant pathogens is there. By culturing the microorganism into which the gene of the present invention is introduced, it is possible to efficiently produce iturin A and surfactin, which have a synergistic effect on the antibacterial activity against plant pathogens and are useful as microbial pesticides.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

[0028]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // (C12N 15/09 ZNA C12R 1: 125) (C12P 21/02 C12R 1: 125) C12R 1 : 125)

Claims (5)

[Claims] 1. A gene encoding a peptide having antibacterial activity shown in SEQ ID NO: 1. 2. A gene comprising the nucleotide sequence shown in SEQ ID NO: 1 encoding a peptide having antibacterial activity. 3. The gene according to claim 1, wherein the peptides having antibacterial activity are iturin A and surfactin. 4. The gene according to claim 1, which is derived from Bacillus subtilis SD142 strain. 5. A method for producing an antibacterial peptide, which comprises culturing a microorganism capable of producing an antibacterial peptide transformed with an expression plasmid containing the gene according to any one of claims 1 to 4. .