KR100661175B1 - Algicidal agent containing prodigiosin and prodigiosin biosynthetic gene cluster - Google Patents

Abstract

An algicidal agent containing prodigiosin and a prodigiosin biosynthetic gene cluster are provided to kill reddish brown tide-causing bacteria including Cochlodinium polykrikoides, Gyrodinium impudicum and Heterosigma akashiwo. The prodigiosin biosynthetic gene cluster comprises the nucleotide sequence of SEQ ID NO:1. A method for producing the prodigiosin comprises the steps of: producing a vector containing the prodigiosin biosynthetic gene cluster; transforming a cell with the vector; and culturing the transformed cell. The algicidal agent contains prodigiosin.

Description

Algicidal Agent Containing Prodigiosin and Prodigiosin Biosynthetic Gene Cluster

Figure 1 shows the hahel Jejuensis chromosome in a circle. The blue color represents the geenomic islands (GI), and the second and third rings represent CDSs that are transferred clockwise and counterclockwise. Details of the gene function classification system in English are listed at ftp://ftp.ncbi.nih.gov/pub/COG/COG/fun.txt.

FIG. 2 is a graph showing the algae effect of prodgiosin on cocrodinium polycricoides.

Figure 3 shows the structure of the prodigiosin biosynthetic gene cluster and prodigiosin of Hahel Jejuensis. (A) shows a gene region involved in prodigiosin biosynthesis, red indicates genes homologous to S. coelicolor A3 red cluster, and the horizontal line shows a phosphid containing all or part of the pigment biosynthesis gene Represent clones. (B) is the result of determining the structure of prodigiosin, above is the result of LC-ESI-MS, and below is the MS / MS fragment pattern of the base peak (23.73 minutes).

The present invention relates to a gene cluster involved in the biosynthesis of progegiosin, a red pigment produced by Hahella chejuensis and algae preparation containing prodigiosin having a killing effect on red tide. And a red pigment biosynthetic gene cluster of Haella jejuensis, a method for producing prodigiosin using the gene cluster, a algae containing prodigiosin, and a method for removing red tide using prodigiosin.

Marine microbes, which make up more than 98% of marine life, play an important role in ecosystems. Marine plankton anchors solar energy to nourish other marine life, while flagella algae, such as Pfiesteria spp., Also threaten marine life by overproliferating . As such, the seawater turns red due to the rapid proliferation of marine algae. Red tide is a frequent occurrence in recent years near the coast, affecting the local economy and marine ecosystem as well as the health of humans and marine life. However, the actual treatment for this is only to settle the microalgae through the loess spread.

Alteromonas, Cytophaga, Flavobaterium, Pseudoalteromonas, Saporospira, Vibrio, etc. are known to have the ability to kill, and these bacteria have been reported to be directly or indirectly involved in the disappearance of red tide (Lovejoy et al. , Appl. Environ. Microbiol ., 64: 2806-13, 1993). The bactericidal bacteria can kill or melt various species of algae, and each bacterium has a species-specific killing range. For example, the bactericidal bacterium Cytophoga sp. Has various killing abilities against the Bacillariophyceae, Raphydophyceae and Dinophyceae (Imai et al. , Mar. Biol. , 116: 527-). 32, 1993).

Yet, live bacteria of saljo early yet small studies have secluded, an abrupt decrease in the red tide organisms has yongjo of cells (Imi et al, Mar. Biol, 116:.. 527-32, 1993), apoptosis caused by inhibition of growth (Imi et . al, Fisheries Science, 61: . 628-36, 1995) and Al eksan give help category Nella (death by inhibition of the silent mating step color expression sexual reproduction, as in the Alexandrium catanella) (Sawayama et al, Nippon Suisan Gakkaishi , 59: 291-4, 1993).

The killing mechanisms of the killing bacteria are (1) the mechanism by which the killing bacteria adhere to the surface of the red algae and help the red algae (Imai et al. , Mar. Biol ., 116: 527-32, 1993) and (2 ) It can be divided into the mechanism of inhibiting or cultivating the growth of red tide organisms by the algae bacterium secreting algae substances extracellularly. Most algae bacteria are included in the second mechanism, antibiotics (Kawano et al. , J. Mar. Biotechnol ., 5: 225-9, 1997), small molecule oligopeptides (Sawayama et al. , Nippon Suisan Gakkaishi , 59: 291). -4, 1993), proteins (Baker and Herson, Appl. Environ.Microbiol., 35: 791-6, 1978) and heat labile extracellular substances (Mitsutani et al. , Nippon Suisan Gakkaishi , 58: 2159-69, The killing mechanism by 1992) is reported. This decrease in algae occurs within a few hours to a few days after inoculation of most bactericidal bacteria. This difference in reaction time is directly related to the activity of the substance and varies with the substance generation and inducers.

Hahella chejuensis is a type of protozoa, the most protozoan group in the marine environment, and is still a genomic sequence in the Oceanospirillales family. This is a paperless situation. And Greeks Jeju N-Sys is a strain isolated from coastal sediments of the island of Mara of South Korea's southernmost, saljo for the production of a red pigment and the main flagellum algae in Cork Lodi nium poly Cree Koh Death (Cochlodinium polykrikoides) causing red tides on the west coast of the South Pacific Effect. The inventors have found that the killing effect of Hahel Jejuensis is due to the red pigment produced by this strain (WO 2004/099391).

In the present invention, as a result of intensive efforts to determine the biosynthetic pathway of the red pigment produced by Hahela jejuensis having the killing effect, the genome sequence of the hahel jejuensis KCTC 2396 was determined, the red sequence of the genome sequence of haheljuensis Gene clusters involved in the production of the pigment prodigiosin have been identified and the present invention has been completed.

An object of the present invention is to provide a prodigiosin biosynthetic gene cluster of Hahel Jejuensis.

Another object of the present invention to provide a method for producing prodigiosin using the gene cluster.

It is another object of the present invention to provide an algae preparation containing prodigiosin as an active ingredient and a method for removing red tide, characterized in that the use of the prodigiosin.

In order to achieve the above object, the present invention provides a prodigiosin biosynthetic gene cluster containing the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a vector containing the gene cluster, and provides a bacteria transformed with the vector.

The present invention also provides a method for producing prodigiosin, wherein the bacteria are cultured.

The present invention also provides an algae preparation containing prodigiosin as an active ingredient.

The present invention also provides a method for removing red tide, which uses prodigiosin.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In the present invention, a gene cluster involved in the biosynthesis of red pigments in the genome sequence of Hahel Jejuensis, which has a killing effect on the flagella algae causing red tide, was identified. In addition, a phosmid clone comprising a gene cluster involved in the biosynthesis of the red pigment was prepared, and it was confirmed that the phosmid clone produced the same red pigment as that of Hahel Jejuensis, and LC-ESI-MS / MS analysis And MRI confirmed that the red pigment has the same structure as prodigiosin.

On the other hand, prodigiosin is a red pigment known for centuries and is a cytotoxic compound with a wide range of activities (Furstner, A, Angew. Chem. Int. Ed. Engl ., 42: 3582, 2003), and human cancer cells Induction of apoptosis has been reported (Perez-Tomas, R. et al ., Biochem. Pharmacol ., 66: 1447, 2003). However, there have been no reports of the killing effect on flagella algae. Pigments produced from marine bacteria provide a defense against sunlight or protozoa. The biological function of prodigiosin in prodigiosin producing bacteria has not yet been identified, and the present inventors confirmed that prodigiosin is a virulence factor for flagella algae due to the pathogenicity of Hahel jejuensis. On the other hand, Seratia , Streptomyces and the like are known as bacteria that produce prodigiosin, undecylprodigiosin, prodiginin, and biosynthetic genes (Harris, AK, et al. , Microbiology , 150: 3547-60, 2004; Cerdeno, AM et al ., Chem . Biol . 8: 817, 2001).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Determination of the genomic sequence of the hahel jejuensis gene

The genome sequence of Haella Jeju Nsis was determined by standard whole-genome shotgun method. Chromosome replication origin was determined by GC skew analysis and identifying genes known to be located around the oriC site of protists (Grigoriev, A., Nucleic Acids Res ., 26: 2286, 1998). Anticipated protein coding sequences (CDS) of at least 100 bp are CRITICA (Badger, JH et al ., Mol. Biol. Evol ., 16: 512, 1999) and GLIMMER (Delcher, AL et al ., Nucleic Acids Res ., 27: 4636 , 1999). BLASTX was used for reanalysis of short CDS, and metabolic pathways were determined by KEGG database (Kanehisa, M. et al ., Nucleic Acids Res ., 32: D277, 2004) and Pathway Tools (Karp, PD et al ., Bioinformatics) . , 18: 225, 2002).

Hahel Jejuensis KCTC2396 consists of a 7,215,267 bp monocyclic chromosome, which is the longest sequence of marine protoplasts identified and the longest sequence of the γ-proteobacteria genomes identified. One). The sequence consisted of 6783 predicted genes, of which 76.3% were in good agreement with existing databases and 49.6% were able to predict the function of gene products (Table 1). About a quarter of the predicted genes were unique, and a comparison of the sequence of protozoa with the fully-discovered protozoa with the conserved gene sequence of Haella jejuensis showed a distant relative to Pseudomonas sp.

Characteristics of the Hahel Jeju Nsis Genome Total length (bp) 7,215,267 Number of protein coding sequences (CDS) 6783 Coding percentage 88.3 Average gene length (bp) 940 G + C content (%) 54.8 rRNA operon 5 tRNAs 67 sRNAs 22 CDS Summary    CDS with predicted functionality 3362    CDS unknown function 1132    Conserved Virtual Genes 683    Virtual genes 1616

Haella Jeju Nsis is very well equipped with basic metabolic ability to survive independently in the marine environment. It is not only the pathway required for the biosynthesis of nucleic acids and 20 amino acids, but also the glycolysis, pentose phosphate pathway and Fully equipped with the genes for enzymes required for major carbon metabolism, including the TCA pathway.

It also contains only the genes expected to be carbon monoxide dehydrogenase and one hydrogenase complex without other genes for autotrophs, and relies on lithoheterotrophic methods when there is a shortage of available organic nutrients. I think. A nitrogen reduction complex gene for nitrogen respiration was also identified, and no gene for inorganic sulfur oxidation was shown.

Example 2 Systematic Analysis of Hahel Jeju Nsis and Adaptation to Marine Environment

The 16S rDNA sequence or 34 associated protein sequences, which are genetically well-conserved sequences, were obtained using GenBank and used as universal markers of genomic evolution. To find the corresponding 34 COGs in each species, the genome was searched using 34 hidden Markov models consisting of each COG protein cluster. E-values in CLUSTAL W (Tompson, JD et al ., Nucleic acids res ., 22: 4673, 1994) and PHYLIP (Felsenstein, J. PHYLIP 3.6b edn.Dept. Of Genetics, University of Washington, Seattle, 2004) 16S rDNA sequences or related universal 34 protein sequences below this cutoff (1.0 × 10 ⁻⁰⁶ ) were analyzed using neighbor-joining and maximum parsimony methods. To modify multiple substitutions at the protein residues, a Kimura 2-parameter model was used and 1000 random bootstrap resampling was performed to determine the reliability of the branching pattern.

The number of genes used for the regulation of transcription and environmental sensing in the Hahel Jeju genome genome was about 362, which is less than 5.3% of the total expected genes. This is in line with the trend that the number of regulatory genes increases with increasing genome size. The most common forms of regulators in Hahel Jejuensis included LysR, AraC, TetR and MerR, including four major sigma-70 factors, two extracellular function sigma factors, and one sigma-54 factor. . It also had more than 20 proteins with modules that interact with sigma-54. The expected two-component system (47 sensors, 103 response modulators and 23 sensor-response modulator hybrids) was much more present than the genomes of other bacteria. In addition, 35 genes in the chemical sensing system were very sophisticated enough to encode the sensing transducer protein. However, there was no quorum-sensing system capable of detecting information between living organisms, such as no homology of luxI.

Haella Jejuensis had a wide range of transfer factors for sugars, peptides / amino acids, phosphoric acid, manganese, molybdate, nickel and drugs. The sugar delivery system has 11 ABC delivery systems, and the phosphotransferase system is incomplete and appears to be heavily biased to the ABC delivery system. This phenomenon is very rare but is often observed in some pathogenic microorganisms.

The variety of extracellular degrading enzymes, such as proteases, lipases, nucleases, chitinases and cellulase, which are found in Hahel Jejuensis, is useful for making nutrients composed of macromolecules available. Due to the high ratio of regulatory proteins and transfer factors for various nutrients, Haella jejuensis strains will be well adapted to changing marine environments.

Like other marine bacteria, Haella jejuensis requires 2% NaCl for optimal growth, Na ⁺ is essential for marine or basophilic bacteria, and nutrient acquisition and flagella are achieved by transmenorrhea Na ⁺ concentration gradients. Used for rotation In general, Na ⁺ / H ⁺ antiporters produce sodium motif forces, and Na ⁺ -potential respiratory NADH: ubiquinone oxidoreductases are widely distributed in gram negative marine bacteria along with Na + / H + antiporters. Through genomic analysis and HeLa same type of respiratory complex and multi-N-Sys in Jeju multi-Na containing subunit Na ⁺ / H ⁺ anti porter system ⁺ / H ⁺ anti porter was confirmed.

Example 3 Gene Excess and Genomic Island of Hahel Jeju Ensis

Horizontally transferred genes (HTGs) were inferred from genetic differences or phylogenetic content.

Genes with abnormal G + C content and codon usage deviations were judged as anomalous (Mahalanobis distance 80.23 or more as a difference between G + C content 1.5δ and codon usage deviations). In addition, genes homologous to protozoa other than γ-proteobacteria were added to the HTG list. BLASTP was performed to compare the CDS of Hahel Jejuensis and the CDS of protists whose 208 sequences were completely determined.

A characteristic of the Hahela jejuensis genome is a variety of homologous genes encoding functionally identical proteins. There have been dozens of cases where the same function is encoded by two to four independent genes. F ₀ F ₁ -type ATP synthase, flagella production, and type III protein secretion systems existed as sets of genes with two loci each. All-against-all similarity tests were performed to determine whether the genes within the genome were amplified. The overall similarity of homologous genes was lower than that of similar proteins in other bacterial genomes. Many of the proteins were similar to those of the γ-proteobacteria, and the rest of the proteins showed similarities to the various proteins. These results suggest that the genetic diversity of Hahel Jejuensis is due to gene duplication within the Hahel Jejuensis genome rather than horizontal gene transfer.

As with other bacteria, horizontal gene transfer has been shown to play an essential role in the shape of the Hahel Jejuensis genome. Based on genetic modifications and phylogenetic backgrounds, Hahel Jejuensis had 69 genome islands (GIs) composed of at least 23.0% of chromosomes (FIG. 1). The gene or gene cluster included in the islands contained genes involved in the biosynthesis, iron utilization, mobility, type III protein secretion or pigmentation of extracellular polysaccharides, toxins, polyketides or nonribosomal peptides. 32 of these had homology with genes associated with mobility elements such as Rhs elements, insertion sequence elements, transposons, bacteriophages and group II introns. The gene encoding the Rhs family protein occupied the largest share in both the quantity and length (more than 1.7% of chromosomes) in the Hahel Jejuensis genome. In most cases, the Rhs protein was very similar to that of Methanosarcina barkeri (an archaeon) or Clostridum thermocellum (firmicute).

Example 4 Potential Toxicity-Related Genes of Hahel Jejuensis

Haella Jejuensis produces large amounts of extracellular polysaccharides (EPS). Extracellular polysaccharides are involved in the formation of biofilms and frequently act as toxic factors in pathogens. Hahel Jejuensis identified five gene clusters involved in the synthesis of extracellular polysaccharides, all of which partially or entirely overlapped with genomic islands (GIs). One of these gene clusters is located at the 4.8 Mb region encoding genes of key enzymes such as UDP-glucose dehydrogenase ( ugd ), Wzy type polymerase and Wzx flippase. UGD produces UDP-D-glucuronate, which is used as a basic unit in the production of capsular polysaccharide and colanic acid of E. coli in some pathogenic bacteria.

Hemolysine and RTX toxins that form pores play an important role in many pathogenic Gram-negative bacteria with cytotoxicity. In Hahel Jejuensis, seven RTX toxin homologs and three hemolysine homologues were found and five were included in the genome islands (GIs). One prominent feature found in the Hahel Jejuensis genome is two Type III secretion systems (TTSSs) similar to those found in the genus yersinia, vibrio, Pseudomonas aeruginosa and aeromonas. At 3.34-3.37 Mb and 5.25-5.29 Mb. The two TTSS of Haella Jeju Nsis belonged to the same TTSS subfamily, only one of which was located on the dielectric island (GI). As such, the presence of genes homologous to the virulence factors suggests that Hahel Jejuensis may be pathogenic to marine eukaryotes.

Example 5 Biosynthetic Gene of Algal Pigment Produced by Hahel Jeju Ensis

As a medium for the production of red pigments produced by Haella Jeju Ensis, ZoBell 2216 medium (5% glucose, 0.1% peptone, 0.42 g KH ₂ PO ₄ , 0.34 g K ₂ HPO ₄ , 0.5 g MgSO ₄ , 2.0 g CaCl ₂ , 0.001 g CoCl ₂ .6H ₂ O, 0.001 g MnCl ₃ , 0.001 g ZnSO ₄ and 0.001 g NaMoO ₄ , pH 7.0, finalized to 1 liter using seawater in 250 ml distilled water), incubation 5 liters The fermenter was incubated at 25 ° C. for 72 hours giving 1.5vvm of aeration.

Chloroform was treated in the culture broth to extract crude red pigment (RP10356). The crude extract was purified by silica gel 60 (0.063 mm, Merck, Germany) using chloroform (100, v) and methanol: water with YMC-Pack ODS-A (250 × 10 mm, YMC Co., Japan). Repurification using acetic acid (81: 14: 5, v / v / v).

Microalgae for the determination of the algae effect was incubated in an f / 2 culture medium using an irradiation cycle of 16 hours / 8 hours of light at a light density of 22.5 to 55 μmol / m ² / s or more (Korean Patent Application No. 10-2003- 29526). The purified pigment was dissolved in ethanol, of which 20 μl was added to a tube containing 980 μl of microalgal solution. Surviving cell numbers were stained with 10% Lugol's solution and measured microscopically.

The algae effect of red pigment is represented by the following formula.

                     (Initial cell count-number of surviving cells)

Algae effect (%) = ---------------------------- × 100

                           Initial cell count

As a result of measuring the algae effect on cocrodinium polycricoides using the extract of Haella jejuensis, the rapid cell degradation of cocrodinium polycricoides was observed in both crude extract and red pigment purified extract. (FIG. 2). To find the genes involved in and HeLa biosynthesis of the red dye of the Cheju N-Sys, and HeLa Jeju N-Sys After a genomic search for genes involved in the synthesis of the metabolites in, Streptomyces coelicolor A3 of the red gene (Cerdeno, AM et al., Gene clusters containing genes similar to Chem. Biol ., 8: 817, 2001 were found (SEQ ID NO: 1).

In order to confirm whether the gene cluster is a gene cluster involved in pigment biosynthesis, first, a phosphide library of Hahel Jejuensis was prepared using a library construction system (EPICENTRE, USA). Fosmid clones of Escherichia coli EPI300, HC81010E03, HC81008E02, HC81002H12, HC81006F09 and HC81004F05Z5 (FIG. 3A) containing all or part of the gene cluster sequence included 20 μg / ml of chloramphenicol and CopyControl Induction Solution Was inoculated in LB agar medium and incubated at 37 ° C.

When the fosmid clone comprising the gene cluster is incubated in a solid medium, the colonies are located in close proximity to the colonies of Hahel Jejuensis or a medium containing Hahel Jejuensis extract (20% crude extract). Produced a red pigment. It was found that the genetic site is a set of genes required for pigment biosynthesis (FIG. 3A). In addition, HC81006F09-R clones always produce red pigment, and the remaining phosmid clones also produce red pigment in the presence of suitable gene expression regulators. Meanwhile, when the transposon was inserted into the pigment biosynthesis gene site using the HC81006F09 clone which always produces the red pigment, the colony disappeared. We proved that this gene group is a gene group that synthesizes red pigment.

The maximum absorbance of the red pigment purified from the Hahel jejuensis culture was 535 nm under acidic conditions and 470 nm under basic conditions, indicating that the red pigment is a prodigiosin-like compound.

On the other hand, as a result of measuring the maximum absorbance of the purified red pigment from the culture medium of the HC81006F09-R clone in the same manner as described above, it was measured at 535nm in acidic conditions and 470nm in basic conditions, purified red pigment in Hahel Jejuensis culture medium It was confirmed that the same pigment as. From this result, it was confirmed that the gene clotor of SEQ ID NO: 1 according to the present invention is involved in the synthesis of red pigment.

For LC-ESI-MS / MS analysis of the red pigment, the Haella jejuhensis culture supernatant shaken with Marine Broth (Difco) for 24 to 48 hours at 30 ℃ was extracted using methanol / 1N HCl mixture. The red fraction was purified using HPLC, and LC was performed at a rate of 0.2 ml / min using acetonitrile and water (0.1% formic acid) as the mobile phase. ESI-MS performed a Finigan LCQ Advantage MAX ion trap mass spectrometer using a Finnigan dlectrospray source.

Base peak from red pigment via LC-ESI-MS / MS analysis [23.73 min; m / z 324.2, (M + H) ⁺ ] We confirmed that fragmentation pattern is consistent with antibiotic prodigiosin, and ¹ H NMR (CD ₃ OD, 300 Hz) and ¹³ C NMR (CD ₃ OD, 75 MHz) analysis were also performed. As a result, 6.94 (m, 1H), 6.71 (m, 1H), 6.66 (s, 1H), 6.39 (s, 1H), 6.21 (m, 1H), 6.01 (s, 1H), 3.89 (s, 3H), 2.37 (t, 2H), 2.27 (s, 3H), 1.53 (m, 2H), 1.33 (m, 4H), 0.90 (t, 3H) (1H NMR) and 169.6,160.2, 141.0, 135.8, 129.9, 129.2, 125.1, 123.0, 120.6, 115.9, 113.1, 110.9, 95.9, 58.9, 32.7, 31.8, 26.6, 23.6, 14.4, 11.5 (80 13C NMR) to reaffirm prodigiosin (FIG. 3B).

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

The present invention has the effect of providing a red pigment biosynthetic gene cluster of Haella jejuensis, a method for producing prodigiosin using the gene cluster, an algae containing prodigiosin, and a method for removing red tide using prodigiosin.

Prodigiosin according to the present invention is useful for removing red tide because it has a high algal effect on cochrodinium polycricoides, gyrodinium impeducum, heterosigma acacio, and the like, which is a flagella algae causing red tide.

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Claims (6)

Prodigiosin biosynthetic gene cluster containing the nucleotide sequence of SEQ ID NO: 1. A vector containing the gene cluster of claim 1. A bacterium transformed with the vector of claim 2. A method for producing prodigiosin, comprising culturing the bacterium of claim 3. Algae containing prodigiosin as an active ingredient. A method of removing red tide, characterized by using prodigiosin.

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