WO2007073011A1

WO2007073011A1 - Algicidal agent containing prodigiosin and prodigiosin biosynthetic gene cluster

Info

Publication number: WO2007073011A1
Application number: PCT/KR2005/004432
Authority: WO
Inventors: Ji Hyun F. Kim; Tae Kwang Oh; Choong Hwan Lee; Seung-Hwan Park; Hae Young Jeong; Yon Kyoung Park; Hong Kum Lee; Joung Han Yim; Yoo Kyung Lee
Original assignee: Korea Ocean Research And Development Institute; Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2005-12-20
Filing date: 2005-12-21
Publication date: 2007-06-28
Also published as: KR100661175B1

Abstract

The present invention relates to an algicidal agent containing prodigiosin which has algicidal activity against red tide organisms and a gene cluster involved in biosynthesis of prodigiosin, which is a red pigment, produced by Hahella chejuensis, more particularly, to a gene cluster involved in biosynthesis of a red pigment of H. chejuensis, a method for producing prodigiosin using the gene cluster, an algicidal agent containing prodigiosin and a method for removing red tides using prodigiosin. The inventive prodigiosin has an excellent algicidal effect against a red tide dinoflagellate, thus it is useful as an active ingredient of an algicidal agent for removing red tides.

Description

ALGICIDAL AGENT CONTAINING PRODIGIOSIN AND PRODIGIOSIN BIOSYNTHETIC GENE CLUSTER

TECHNICAL FIELD

The present invention relates to an algicidal agent containing prodigiosin which has algicidal activity against a red tide organism and a gene cluster involved in biosynthesis of prodigiosin, which is a red pigment, produced by Hahella chejuensis, and more particularly, to a red pigment biosynthetic gene cluster of H. chejuensis, a method for producing prodigiosin using the gene cluster, an algicidal agent containing prodigiosin and a method for removing red tides using prodigiosin.

BACKGROUND ART

Accounting for more than 98% of the ocean's biomass, marine microbes are the major players of the biogeochemical cycles on earth. Phytoplanktons fix solar energy and provide nutrients to other marine life. On the other hand, unchecked increases in the population of certain dinoflagellates like much-blamed Pfiesteria spp. in the ocean results in blooms that often threaten the marine life. These phenomena called harmful algal blooms or commonly red tides increasingly occur in the coastal waters throughout the world in recent years, affecting not only the health of human and marine organisms but regional economies and marine ecosystem. However, the only practical management strategy being employed in some places is flocculation of microalgae through clay dispersal.

Bacteria known to have algicidal activity include Alteromonas, Cytophaga, Flavobaterium, P seudo alteromonas, Saporospira, Vibrio, and it is reported that such bacteria are directly or indirectly involved in the dissipation of red tides (Lovejoy et al, Appl. Environ. Microbiol., 64:2806-13, 1993). The algicidal bacteria can kill or lyse a variety of algae, and each of such bacteria has species-specific algicidal range. For example, algicidal bacterium, Cytophoga sp. has a various algicidal activities against Bacillariophyceae, Raphydophyceae and Dinophyceae (Imai et al , Mar. Biol. , 116:527-32, 1993).

However, studies on the algicidal mechanism of the algicidal bacteria are yet incomplete, and it is reported that a rapid reduction of red tide organisms is attributed to lysis of organism cells (Imi et al, Mar. Biol., 116:527-32, 1993), death of organisms by their growth inhibition (Imi et al, Fisheries Science, 61 : 628-36, 1995) and death by inhibition of mating process from asexual reproduction to sexual reproduction as in Alexandrium catanella (Sawayama et al., Nippon Suisan Gakkaishi, 59:291-4, 1993).

The algicidal mechanisms of algicidal bacteria can be divided into the following two categories: (1) a mechanism by contact in which algicidal bacteria adhere to the surface of red tide organisms to lyse the red tide organisms (Imai et al, Mar. Biol, 116:527-32, 1993), and (2) a mechanism in which algicidal bacteria secrete algicidal substances extracellularly to induce the growth inhibition or lysis of red tide organisms. Most of algicidal bacteria are included in the second mechanism, and there are reported algicidal mechanisms caused by antibiotics (Kawano et al. , J. Mar. Biotechnol., 5:225-9, 1997), low molecular weight oligopeptides (Sawayama et al, Nippon Suisan Gakkaishi, 59:291-4, 1993), proteins (Baker and Herson, Appl Environ. Microbiol, 35:791-6, 1978) and heat-unstable extracellular substances (Mitsutani et al, Nippon Suisan Gakkaishi, 58:2159-69, 1992). The reduction of microalgae appears within a period ranging from several hours to several days after inoculation with most of the algicidal bacteria. This difference in the reaction time has a direct connection with the activity of an algicidal substance, and this reaction time varies depending on the difference in the production of algicidal substances and the kind of the inducer. H. chejuensis is a cultivated member of the oceanic j-Protebacteria , which is one of the most prevalent prokayotic groups present in marine environments. None of the members of the Oceanospirillales clade has yet been determined for its genome sequence. Originally isolated from the coastal marine sediment of the southernmost island in Korea, this red-pigmented bacterium is capable of killing Cochlodinium polykrikoides, a major red- tide dinoflagellate problematic in the western coasts of the North Pacific. We disclosed that the algicidal effect of H. chejuensis is caused by red pigment produced by this bacterium (WO 2004/099391).

The present inventors have made extensive efforts to find out the biosynthetic pathway of a red pigment produced by H. chejuensis having algicidal effect, and as a result, determined the complete genome sequence of H. chejuensis KCTC 2396 and identified a gene cluster involved in producing prodigiosin, which is a red pigment, among genome sequence of H. chejuensis, thereby completing the present invention.

SUMMARY OF INVENTION

An object of the present invention is to provide a gene cluster of H. chejuensis, which is responsible for the biosynthesis of prodigiosin.

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

Still another object of the present invention is to provide an algicidal agent containing prodigiosin as an effective ingredient and a method for eliminating a red tide using said prodigiosin.

To achieve the above objects, in one aspect, the present invention provides a gene cluster for prodigiosin biosynthesis, which has base sequence of SEQ ID NO: 1. In another aspect, the present invention provides a recombinant vector containing the gene cluster and a bacterium transformed with the recombinant vector.

In still another aspect, the present invention provides a method for producing prodigiosin, the method comprises: culturing the transformed bacterium; and recovering prodigiosin from the cultured broth of said transformed bacterium.

In still other aspect, the present invention provides an algicidal agent containing prodigiosin as an effective ingredient and a method for removing red tides using prodigiosin.

Other features and embodiments of the present invention will be more fully apparent from the following detailed description and appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows circular representation of the H. chejuensis chromosome. Blue patches on the outermost circle indicate GIs. Circles 2 and 3 show CDSs transcribed clockwise and counter-clockwise, which are color-coded according to the COG functional classes as designated in the inset. Detailed function description for each one-letter classification code is available from ftp://ftp.ncbi.nih.gov/pub/COG/COG/fun.txt.

FIG. 2 shows algicidal activity of the purified red pigment of H. chejuensis against C.polykrikodes strain BWEO 109 at various concentrations.

FIG. 3 shows biosynthesis and structure of the red pigment of H. chejuensis. (A) The genomic region involved in pigment biosynthesis. Genes homologous to those in the S. coelicolor A3 red cluster are indicated by filled arrows. Horizontal lines indicate fosmid clones containing some or all of the pigment-synthetic genes. (B) Structural determination of the red pigment. Upper part, LC-ESI-MS in the positive-ion mode: lower part, MS/MS fragmentation pattern of the base peak(23.73min).

DETAILED DESCRIPTION OF THE INVENTION AND

PPRFERRED EMBODIMENTS

The present inventors have identified a gene cluster involved in the biosynthesis of a red pigment in genome sequence of H. chejuensis showing an algicidal activity against red tide dinoflagellates. Also the present inventors have prepared fosmid clone containing the gene cluster involved in the biosynthesis of a red pigment, and found that the fosmid clone produces the same red pigment as that of H. chejuensis, thereby confirming that the produced red pigment has the same structure as that of prodigiosin by LC-ESI-MS/MS and NMR analysis.

Meanwhile, prodigiosin, red pigment known for centuries is a cytotoxic compound showing a broad range of activity (Furstner, A, Angew. Chem. Int. Ed. Engl., 42:3582, 2003) and also induces apoptosis in human cancer cells (Perez-Tomas, R. et al, Biochem. Pharmacol., 66: 1447, 2003). However, its activity against dinoflagellates has not been reportd before.

Pigments produced by marine bacteria may function as protective agents against solar radiation or protozoan grazing. Though the biological roles of prodigiosin in the producing organisms have not been defined, in the present invention, we infer that prodigiosin, as well as toxins, TTSS-delivered proteins and other virulence effectors, contribute to the pathogenic lifestyle of H. chejuensis.

Meanwhile, Serratia, Streptomyces are known as bacteria producing prodigiosin, undecylprodigiosin, prodiginin and biosynthetic gene clusters have also been reported (Harris, A.K., et al, Microbiology, 150:3547-60, 2004; Cerdeno, A. M. et al, Chem. Biol. 8:817-29, 2001).

Examples

The present invention will hereinafter be described in further detail by examples. It will however be obvious to a person skilled in the art that these examples are given for illustrative purpose only, and the present invention is not limited to or by the examples.

Examples 1: Genome sequencing and annotation of H. chejuensis

Genome sequence was determined by standard whole-genome shotgun method. The putative origin of replication was determined by GC skew analysis and by identification of genes known to cluster near the prokaryotic oriC site (Grigoriev, A., Nucleic Acids Res., 26:2286, 1998). Putative CDSs of more thanlOO bp were predicted by amalgamating the results from CRITICA (Badger, J.H. et al., MoI. Biol. EvoL, 16:512, 1999) and GLIMMER (Delcher, A. L. et al., Nucleic Acids Res., 27:4636, 1999). Intergenic sequences were reanalyzed for short CDSs by running BLASTX. Metabolic pathways were examined using the KEGG database (Kanehisa, M. et al., Nucleic Acids Res., 32:D277, 2004) and Pathway Tools (Karp, P. D. et al., Bioinformatics, 18:225, 2002).

The genome of H. chejuensis KCTC2396^T contains of one circular chromosome of 7215267 bp (FIG. 1). This makes it the largest among the marine prokaryotic genomes whose genome sequences are available and also among the y-Proteobacterial genomes sequenced. Among the 6783 predicted genes, 76.3% showed significant database matches and 49.6% were assigned a putative function (Table 1). While about a quarter of predicted genes are unique, comparison of broadly conserved H. chejuensis genes with those of other completely sequenced prokaryotes indicated that H. chejuensis is distantly affiliated to Pseudomonas spp. Table 1 : General features of the H. chejuensis genome

Basic metabolic capabilities are well equipped to support a free-living, marine heterotrophic lifestyle. The bacterium has a complete repertoire of enzymes for central carbon metabolism including glycolysis, pentose phosphate pathway and TCA cycle, as well as those required for biosynthesis of nucleotides and 20 amino acids.

The presence of genes for one putative carbon monoxide dehydrogenases and one hydrogenase complex without other genes for autotrophy implies that, when available organic nutrients are scarce, H. chejuensis might rely on the lithoheterophic strategy. Genes for inorganic sulfur oxidation, however, were not identified. We also found genes for the respiratory nitrate reductase complex.

Examples 2: Phylogenetic analysis and adaptation to the marine environment of H. chejuensis

The 16s rDNA sequences or 34 concatenated protein sequences that are conserved as the genetic core of the universal ancestor were retrieved from GenBank and used as common tracers of genome evolution. To identify counterparts of 34 COGs in each species, the retrieved genomes were searched with series of 34 hidden Markov models made up of each COG protein cluster. The 16s rDNA sequences or concatenated universal 34 protein sequences under E- value cutoff (1.0 x 10^"06) were analyzed with neighbor-joining and maximum parsimony methods in CLUSTAL W (Tompson, J. D. et al., Nucleic acids res., 22:4673, 1994) and PHYLIP (Felsenstein, J. PHYLIP 3.6b edn. Dept. of Genetics, University of Washington, Seattle, 2004). To correct multiple substitutions in protein residues, the Kimura 2-parameter model was used, and to evaluate the reliability of the branching patterns, 1000 random bootstrap resamplings were executed.

The number of genes dedicated to transcriptional regulation or environmental sensing amount to 362, which corresponds to -5.3% of total predicted genes. This is in accordance with the tendency that the number of regulatory genes increases as the genome size increases. Most common regulator types in H. chejuensis include LysR, AraC, TetPv and MerR. The bacterium also possesses four major sigma-70 factors, two extracytoplasmic function sigma factors and one sigma-54 factor. There are more than 20 proteins that have the sigma-54 interaction module. The number of putative two-component system (42 sensors, 103 response regulators and 23 sensor- response regulator hybrids) is overrepresented compared with other bacterial genomes. In addition, the bacterium has a complex chemosensory system with 35 genes encoding putative chemotactic sensory transducer proteins. However, the typical quorum sensing system seems absent as no homologs of lux I could be found.

H. chejuensis has a wide range of transporters for sugars, peptides/amino acids, phosphate, manganese, molybdate, nickel and drugs. Sugar transport system, however, appear highly biased to ABC transport systems as there are 11 ABC-type transporters but phosphotransferase system is incomplete. The phenomenon is rather scarce but often observed in some pathogens.

A variety of extracellular hydrolytic enzymes represented by the H. chejuensis genome, such as proteases, lipases, nucleases, chitinases and cellulases could be advantageous once macromolecular nutrients become available. Along with the high portion of regulatory proteins and transporters for a variety of nutrients, these features imply the functional diversity and adaptability of H. chejuensis to changing marine environments.

Like other marine bacteria, H. chejuensis requires 2% NaCl for optimal growth. Na⁺ is essential for marine or halophilic bacteria as transmembrane Na⁺ gradient is utilized for uptake of nutrients and flagellar rotation. In general, Na⁺/H⁺ antiporter generates the sodium motive force for these cellular processes, but Na⁺-translocating respiratory NADH ubiquinone oxidoreducatase is widely distributed among Gram-negative marine bacteria in addition to the primary H⁺ pump and Na⁺/H⁺ antiporter. Genome analysis identified the same type of respiratory complex in H. chejuensis and multiple Na⁺ZH⁺ antiporters including a multi-subunit Na⁺/H⁺ antiporter system.

Example 3: Redundant genes and genomic islands of H. chejuensis

Horizontally transferred genes (HTGs) were inferred from genomic anomalies or phylogenetic context. A gene was considered anomalous if both G+C content and codon usage are aberrant (G+C content more than 1.5σ and Mahalanobis distance as a degree of the codon usage deviation more than 80.23). In addition, genes having orthologs in other prokaryotes but not in y-Proteobacteria were added to the list of HTGs. BLASTP searches were performed to find pairs of reciprocal best hits between in the H. chejuensis CDSs and those in each of the 208 completely sequenced prokaryotes.

An interesting feature of the H. chejuensis genome is the multiplicity of homologous genes encoding functionally equivalent proteins. There are dozens of cases where the same function is redundantly encoded by two to four independent genes. As for gene sets, there are two loci each for F₀Frtype ATP synthesis, flagellar biogenesis and type HI protein secretion. When all-against-all similarity searches were performed to identify recent gene duplication within the genome, overall identities among the homologous genes were far below than those of the closest proteins from other sequenced genomes. While in many cases one member best matches to protein in γ- Proteobactera, the other members are similar to those in various other taxa. These observations support that the origin of multiplicity is likely horizontal gene transfer rather than duplication of genes in the H. chejuensis genome.

Like many other bacteria, horizontal gene transfer seems to have had essential roles in shaping the H. chejuensis genome. Based on genomic anomalies and phylogenetic context, the bacterium appears to have at least 69 genomic islands (GIs) constituting -23.0% of the chromosome (FIG. 1). Genes or gene clusters contained in the islands include those involved in biosynthesis of exopolysacchrides, toxins, polyketides or non-ribosomal peptides, iron utilization, motility, type HI protein secretion, or pigmentation. Of them 32 contained homologs of genes associated with mobile elements such as Rhs elements, insertion sequence elements, transposons, bacteriophages and group II introns. Genes encoding Rhs family proteins are the largest group in the H. chejuensis genome in terms of both abundance and length (~1.7% of the chromosome). In most cases, the Rhs proteins are very closely related to those in the archaeon Methanosarcina barkeri or the firmicute Clostridium thermocellum.

Example 4: Potential virulence-associated genes of H. chejuensis

H. chejuensis produces a large amount of extracellular polysaccharides (EPSs). EPSs are responsible for development of biofilms, and often act as a virulence factor in pathogenic bacteria. We found five gene clusters that may be involved in the synthesis of exopolysaccharides, all of which overlap GIs partly or entirely. Among the gene clusters, one is located at the 4.8 Mb region encoding genes for key enzymes such as UDP-glucose dehydrogenase(ug<i), Wzy-type polymerase and Wzx flippase. UGD produces UDP-D-glucuronate, which is known to be a building block for production of capsular polysaccharides in several pathogenic bacteria and colanic acid in E. coli.

Pore-forming hemolysin and RTX toxin play important roles in pathogenic Gram- negative bacteria with their cytotoxic activities. Out of the seven RTX toxin homologs and three hemolysin homologs found in H. chejuensis, five are included in GIs. One of the striking findings from the H. chejuensis genome is the unexpected presence of two type EI secretion systems(TTSSs) located at positions 3.34-3.37 and 5.25-5.29 Mb that are similar to those present in Yersinia spp., Vivrio spp., Psedomonas aeruginosa and Aeromonas spp. While the two TTSSs in H. chejuensis belong to the same subfamily of TTSSs, only one of them is located in a GI. Presence of the homologs of these virulence determinants suggests that H. chejuensis probably is a pathogen of marine eukaryotes.

Example 5; Biosynthesis genome of algicidal pigment produced by H. chejuensis

The medium for production of red pigment was ZoBell2216 medium (5% glucose,

0.1% peptone, 0.42g KH₂PO₄, 0.34g K₂HPO₄, 0.5g MgSO₄, 2.Og CaCl₂, O.OOlg CoCl₂

^• 6H₂O, O.OOlg MnCl₃, O.OOlg ZnSO₄ and O.OOlg NaMoO₄, pH 7.0, where 250ml distilled water and aged seawater were added to the final volume of one liter). Cultivation was carried out in a 5-liter fermentor for 72h at 25 °C with aeration of 1.5vvm after inoculation (2.0%). From the culture broth, crude RP10356 was extracted by chloroform, which was further concentrated. Crude preparation of the pigment was purified with silica gel 60 (0.063mm, Merck, Germany) using chloroform (100, v), and re-purified with YMC-Pack ODS-A (250X10mm, YMC co., Japan) using methanol: water: acetic acid (81 :14.5, v/v/v). All algal strains were maintained in f/2 culture medium at 22.5 ^°C , and under a light intensity of -55 μmol/m²/s using a 16h light/8h dark illumination cycle (KR Publication 10-2004- 95563). Purified pigment was dissolved in ethanol, and aliquots of 20μ£ solution were added to 980μ# of microalgal suspension in test tubes. Cell number was counted with a microscope after 1.0% Lugol's solution staining. Following formula was used to calculate the algicidal effect:

(Number of initial cells - Number of survived)

Algicidal activity(%) = xlOO

Number of initial cells

Experiments with the extract of H. chejuensis indicated that both crude and purified preparations of the red pigment are responsible for the rapid cell lysis of C. polykrikodes (FIG. 2). Therefore, genes possibly involved in synthesis of secondary metabolites were searched for in the genome sequence of H. chejuensis, and a gene cluster harboring genes similar to the red genes of Strptomyces coelicolor A3 (Cerdeno, A.M. et al., Chem. Biol., 8:817, 2001) was suspected to be responsible for the biosynthesis of the red pigment (SEQ ID NO:1).

To examine that this genome cluster effects pigment biosynthesis, fosmid library of H. chejuensis is made by library building system (EPICENTRE, USA). Five fosmid clones of Escherichia coli EPI300, ΗC81010E03, HC81008E02, HC81002H12, HC81006F09 and HC81004F05 containing either part or the whole of the pigment gene cluster were plated and incubated overnight at 37^°C on Luria-Bertani agar containing 20μg/m£ chloramphenicol, CopyControl Induction Solution.

When fosmid clones carrying the complete gene cluster were grown on agar plates, colonies located close to those of H. chejuensis or grown in a medium containing the H. chejuensis extract turned red, indicating that this genomic region has the gene set required for pigment biosynthesis (FIG. 3A). HC81006F09-R clone always produce red pigment and other fosmid clone also produces red pigment under suitable gene expression factor. Transposon mutagenesis on a variant of HC81006F09 that constitutively expresses the red pigment was carried out using EZ::TN <KAN- 2>Insertion Kit (Epicentre). Further supporting the observation, transposon insertions in the red homologs resulted in loss of colony color when tested with the variant clone that constitutively produces the red pigment, thereby confirming that the gene cluster is a gene cluster biosynthesizing a red pigment. A maximum absorbance of the purified red pigment at 535 and 470 nm in acidic and basic conditions, respectively, suggested that the pigment is a prodigiosin-like compound.

Meanwhile, a maximum absorbance of the red pigment purified from culture broth of HC81006F09-R clone was 535nm and 470nm in acidic and basic conditions, suggesting that the pigment is the same pigment as the red pigment purified from culture broth of H. chejuensis. From this result, it was confirmed that the gene cluster of SEQ ID NO: 1 was involved in biosynthesis of red pigment.

For LC-ESI-MS/MS analysis, red pigment was extracted with a mixture of methanol/ IN HC1(24: 1) from the supernatant of H. chejuensis culture which was grown on Marine Broth (Difco) for 24-48h at 30 °C with vigorous shaking. Red- colored fraction was purified through HPLC. Following LC using acetonitrile and water (with 0.1% formic acid) as the mobile phase at a flow rate of 0.2 mβ/min, ESI- MS was carried out with a Finnigan LCQ Advantage MAX ion trap mass spectrometer equipped with a Finnigan electrospray source. Through LC-ESI-MS/MS analysis, the fragmentation pattern of a base peak [23.73 min; m/z 324.2, (M+H)⁺] from the red pigment was shown to be identical to that of the antibiotic prodigiosin.

To determine the molecular structure, ¹H NMR (CD₃OD, 300 MHz) and ¹³C NMR (CD₃OD,75 MHz) analyses were performed, resulting in the raw data of 6.94 (m, IH), 6.71 (m, IH), 6.66 (s, IH), 6.39 (s, IH), 6.21 (m, IH), 6.01 (s, IH), 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 (¹³C NMR). From the above results, it was further confirmed that the red pigment was prodigiosin (FIG. 3B).

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is solely for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

As described above, the present invention provides a gene cluster involved in biosynthesis of a red pigment of H. chejuensis, a method for producing prodigiosin using the gene cluster, an algicidal agent containing prodigiosin and a the method for removing red tides using prodigiosin. The inventive prodigiosin has an excellent algicidal effect against a red tide dinoflagellate, such as Cochlodinium polykrikoides, Gyrodinium impudicum and Heterosigma akashiwo, thus it is useful as an active ingredient of an algicidal agent for removing red tides.

Claims

THE CLAIMSWhat is Claimed is:

1. A gene cluster for prodigiosin biosynthesis, which has base sequence of SEQ ID NO:1.

2. A recombinant vector containing the gene cluster of claim 1.

3. A bacterium transformed with the recombinant vector of claim 2.

4. A method for producing prodigiosin, the method comprises: culturing the transformed bacterium of claim 3; and recovering prodigiosin from the cultured broth of said transformed bacterium.

5. An algicidal agent containing prodigiosin as an effective ingredient.

6. A method for removing red tides, the method comprises: treating said red tides with prodigiosin.