A method for high-yield pyrazole triazine

Abstract

This invention relates to a method for high-yield pyrazole triazine, obtained through the synthesis of a pyrazole triazine synthetic gene cluster, the nucleotide sequence of which is shown in SEQ ID No. 1. The pyrazole triazine synthetic gene cluster consists of psdA, psdB, psdC, psdD, psdE, psdF, and psdG, with nucleotide sequences shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No. 14, respectively. When the strain containing the above genes is fermented in TSB medium at 30°C and 220 rpm for 36 h, the yield of pyrazole triazine can reach 5.14 mg / L. Pyrazole triazine and the 923 strain that produces this antimicrobial substance can be used for the prevention and control of diseases in agriculture and medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for high-yield pyrazole triazine. Background Technology

[0002] Antagonistic substances produced by antagonistic microorganisms serve as important biocontrol factors, enabling the prevention and control of various plant diseases (Li Gong, Liu Na, Zheng Libo. Research progress on plant disease control by Pseudomonas fluorescens [J]. Molecular Plant Breeding, 2018, 16(11): 3693-3697.). Among them, the biosynthesis and regulatory mechanisms of antagonistic substances produced by Pseudomonas have been studied in depth, such as phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (2,4-DAPG). Studies have found that Pseudomonas mainly regulates the expression of antagonistic substance synthesis gene clusters through two pathways: transcriptional and post-transcriptional levels, thereby regulating the production of antagonistic substances (Gross, H., Loper, JEGenomics of secondary metabolite production by Pseudomonas spp [J]. Natural Product Reports. 2009, 26(11): 1408-1446.).

[0003] Pseudomonas mosselii is a species of soil saprophytic bacteria belonging to the group Pseudomonas putida. It is mainly distributed in the rhizosphere soil of plants and is a rare opportunistic pathogen in humans (Dabboussi, F., Hamze, M., Singer, E. et al. Pseudomonas mosselii sp. nov., a novel species isolated from clinical species[J]. International Journal of Systematic and Evolutionary Microbiology. 2002, 52(2):363-376.). Previous studies have shown that Pseudomonas mosselii has the ability to inhibit Agrobacterium tumefaciens and some other pathogens (Li, W., Wang, J., Jiang, X. et al. Identification and Characterization of a Pseudomonas mosselii strain and its Antibacterial Function against Agrobacterium tumefaciens[J].). Recent reports show that P. mosselii can control corn rootworm pests by secreting an antimicrobial protein, PIP-47Aa (Wei, JZ, O'rear, J., Schellenberger, U. et al. A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control[J]. Plant biotechnologyjournal.2018,16(2):649-659.).Studies by Zou Huasong et al. have found that the engineered strain obtained by modifying P. mosselii can express the ripAA gene of Ralstonia solanacearum and has the ability to control tobacco bacterial wilt (Zhuo, T., Chen, S., Fan, X. et al. An improved control efficacy against tobacco bacterial wilt by an engineered Pseudomonas mosselii expressing the ripAA gene from phytopathogenic Ralstonia solanacearum[J].bioRxiv.2019:510628.). Furthermore, *Pseudomonas mosselii* BS011 can also inhibit *Pseudomonas oryzae* by producing a cyclic lipopeptide compound called xantholysins (Wu, L., Xiao, W., Chen, G. et al. Identification of *Pseudomonas mosselii* BS011 gene clusters required for suppression of rice blast fungus *Magnaporthe oryzae* [J]. Journal of biotechnology. 2018, 282: 1-9.). Our study shows that *Pseudomonas mosselii* 923 has significant antagonistic effects on two pathogenic species of *Xanthomonas*: *X. oryzae* epv. oryzicola (Xoc) and *Xanthomonas oryzae* pv. oryzae (Xoo), and also has a certain inhibitory effect on other plant pathogens of the *Xanthomonas* genus. Moreover, it can significantly inhibit the mycelial growth of *Pseudomonas oryzae*, demonstrating broad-spectrum antibacterial ability.

[0004] Chinese patent CN108998389A discloses a Pseudomonas bacterium with antagonistic effects against Xanthomonas oryzae and blast fungus, and its applications. The strain was isolated from the rhizosphere soil of rice paddies in Fengxian District, Shanghai, and its preservation name is *Pseudomonas mosselii* 923, preservation number: CCTCC No: M 2018252. This patent demonstrates that the Pseudomonas bacterium exhibits a significant inhibitory effect on Xanthomonas oryzae, and also has strong inhibitory capabilities against other plant pathogenic bacteria of the *Xanthomonas* genus and blast fungus, thus possessing value for biocontrol applications. However, this patent does not disclose what substance exerts the antagonistic effect against Xanthomonas oryzae and blast fungus.

[0005] Pyrazole triazine is a heterocyclic molecule containing multiple nitrogen atoms. It is purple, polar, and belongs to the pyrazolo[4,3-e][1,2,4]triazine family of natural products.

[0006] To date, research on the biological functions of pyrazole triazine has primarily focused on its derivatives, which exhibit anticancer, antiviral, and antitumor activities. Pyrazole triazine itself also possesses antiviral and antitumor activities and could be used as an anti-sarcoma drug, with predictions of its potential to treat atherosclerosis. However, current chemical synthesis methods for pyrazole triazine are complex and require stringent conditions; its synthetic gene cluster is unknown, and its metabolic regulatory mechanisms are unclear. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for high-yield pyrazole triazine, which avoids the drawbacks of complex chemical synthesis methods and harsh conditions, and improves its bio-fermentation potency and reduces production costs, so as to promote and apply pyrazole triazine in agriculture and medical disease prevention and control in the future.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention provides a method for high-yield pyrazolium triazine, which is synthesized through a pyrazolium triazine synthesis gene cluster, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0010] In one embodiment of the present invention, the synthetic gene cluster of the pyrazolium triazine is composed of psdA, psdB, psdC, psdD, psdE, psdF and psdG, with nucleotide sequences as shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12 and SEQ ID No.14, respectively, and corresponding amino acid sequences as shown in SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 and SEQ ID No.15.

[0011] In one embodiment of the present invention, the synthetic gene cluster of the pyrazotriazine is derived from Pseudomonas mosselii 923, deposited at the China Center for Type Culture Collection (CCTCC) with accession number M2018252, deposited on May 7, 2018, at the Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University). This information is disclosed in patent CN108998389A.

[0012] In one embodiment of the present invention, the rsmY and rsmZ genes contained in the Pseudomonas mosselii 923 can positively regulate the biosynthesis of pyrazolium triazine, and the nucleotide sequences of the rsmY and rsmZ genes are shown in SEQ ID No. 16 and SEQ ID No. 17, respectively.

[0013] In one embodiment of the present invention, Pseudomonas mosselii strain 923 was fermented in TSB medium at 30°C and 220 rpm for 36 h to synthesize pyrazol triazine from the pyrazol triazine synthesis gene cluster.

[0014] The present invention also provides the application of genes psdA, psdB, psdC, psdD, psdE, psdF and psdG, wherein genes psdA, psdB, psdC, psdD, psdE, psdF and psdG coexist in the strain, enabling the strain to produce pyrazolium triazine.

[0015] The present invention also provides the application of the rsmY gene, which can positively regulate the biosynthesis of pyrazolium triazine, and the nucleotide sequence of the rsmY gene is shown in SEQ ID No. 16.

[0016] The present invention also provides the application of the rsmZ gene, which can positively regulate the biosynthesis of pyrazolium triazine, and the nucleotide sequence of the rsmZ gene is shown in SEQ ID No. 17.

[0017] This invention has discovered that Pseudomonas mosselii strain 923 can produce a substance called pydoiodinine after fermentation. Psydoiodinine is a natural product belonging to the pydoiodinine family. Its common biological activities include antiviral and antitumor activities (Dembitsky, VM, Gloriozova, TA, Poroiko, VV Pharmacological and predicted activities of natural azocompounds[J]. Natural products and bioprospecting.2017,7(1):151-169.).

[0018] This application study found that pyrazole triazine is effective against plant pathogens Xanthomonas, especially Xanthomonas oryzae Xoc RS105 and Xoo PX099. A Both exhibited strong antibacterial activity, effectively controlling rice bacterial blight and leaf streak. Furthermore, pyrazole triazine also showed significant antagonistic effects against rice blast fungus, the leading cause of rice disease. This indicates that pyrazole triazine has promising prospects for biocontrol applications.

[0019] This application further investigated and found that the synthetic gene cluster consists of psdA, psdB, psdC, psdD, psdE, psdF, and psdG, and discovered the regulatory mechanism of its metabolic regulation.

[0020] Compared with existing technologies, the method of the present invention synthesizes pyrotriazine by synthesizing gene clusters, resulting in high yield. The pyrotriazine obtained by the method of the present invention provides new ideas and approaches for its promotion and application in agriculture and medical disease prevention and control. Attached Figure Description

[0021] Figure 1 Antimicrobial activity assay and HPLC detection of 7 mutant strains and corresponding complemented strains of pseudoiodinine A against Xoo PXO99 A A: Determination of antibacterial activity; B: HPLC detection of pseudoiodinine produced by 7 mutant strains; C: HPLC detection of pseudoiodinine produced by 7 supplementary strains.

[0022] from Figure 1As can be seen, knockout of the psdA, psdB, psdC, psdD, psdE, psdF, and psdG genes significantly reduced the effect on Xoo PXO99. A It exhibits antibacterial activity and the ability to produce pseudoiodinine.

[0023] Figure 2 Identification of pseudoiodinine biosynthesis gene clusters: A: Intergenetic PCR analysis; B: Drawing a schematic diagram of the gene clusters (psdABCDEFG).

[0024] Figure 3 Determination of antibacterial activity and pseudoiodinine production of different rsmY and rsmZ mutant strains and complemented strains in the genome of *Pseudomonas mosselii* 923. A: Antimicrobial activity and pseudoiodinine production of different strains against Xoo PXO99. A A: Determination of antibacterial activity; B: HPLC peak chromatograms of different strains; C: Determination of pseudoiodinine yield in 7 strains.

[0025] The results showed that the two small RNAs rsmY and rsmZ in 923 can positively regulate the biosynthesis of pseudoiodinine, and that there is functional redundancy between the two.

[0026] Figure 4 The standard curve of pseudoiodinine.

[0027] Figure 5 Effects of different culture media on pseudoiodinine production; A: Growth curves of Pseudomonas mosselii 923 in LB and TSB media; B: Pseudomonas mosselii 923 pseudoiodinine production in LB and TSB media.

[0028] from Figure 5 As can be seen, Pseudomonas mosselii 923 grows relatively quickly in TSB medium, reaching the stable growth phase in 24 hours. When cultured for 36 hours, the yield of pseudoiodinine reaches the highest level of 5.14 mg / L, which is 2.8 times that of LB medium under the same conditions. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0030] The culture medium formulations used in the following examples are as follows:

[0031] (1) LB medium (g / L): 10g tryptone, 10g sodium chloride (NaCl), 5g yeast extract, dissolved in purified water, diluted to 1000mL, pH adjusted to 7.0-7.2, autoclaved at 121℃ for 20min. 15g agar powder needs to be added to LB solid medium.

[0032] (2) TSB medium: used for culturing Pseudomonas (28℃), the formula is as follows: 17g pancreatic digest of casein, 3g soybean digest, 2.5g glucose, 5g sodium chloride, 2.5g dipotassium hydrogen phosphate, dissolved in pure water, diluted to 1000mL, pH adjusted to 7.3±0.2, autoclaved at 121℃ for 15min. For TSB solid medium, 15g agar powder needs to be added.

[0033] (3) Beef extract peptone medium NA (g / L): 3g beef extract, 5g polypeptone, 10g sucrose, 1g yeast extract, 15g agar powder, add water to dissolve, finally bring the volume to 1000mL, adjust the pH to 7.0-7.2, dispense and autoclave (121℃, 20min).

[0034] Example 1: Knockout of the pseudoiodinine biosynthesis gene

[0035] (1) Constructing a knockout vector

[0036] The nucleotide sequences of genes psdA, psdB, psdC, psdD, psdE, psdF, and psdG are shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No. 14, respectively. Primers F1 / R1 and F2 / R2 (sequences shown in Table 1) were designed for the upstream and downstream sequences of genes psdA, psdB, psdC, psdD, psdE, psdF, and psdG, respectively. Using genomic DNA of Pseudomonas molluscum 923 as a template, the following reagents were added sequentially to a 50 μL PCR reaction system to perform PCR reactions and obtain the upstream and downstream fragments, respectively.

[0037]

[0038]

[0039] The procedure for running a PCR reaction is as follows:

[0040]

[0041] By selecting appropriate restriction enzyme sites, the upstream and downstream fragments and the p2P24 vector were double-digested separately, and the three target fragments were purified and recovered. Ligation was performed overnight using T4 DNA ligase. The ligation products were then heat-transformed into DH5α, plated on LB agar plates containing Km antagonist material, and incubated overnight at 37°C. Finally, single clones were selected for colony PCR screening.

[0042] Positive clones obtained from screening were transferred into LB liquid medium containing Km antagonist for amplification culture, and recombinant plasmids were extracted. The recombinant plasmids were verified by double enzyme digestion. Recombinant plasmids with correct enzyme digestion fragments (>100 ng / μL) were selected and sent to Shanghai Platinum Biotech Co., Ltd. for sequencing verification using universal primers M13-47 / RV-M (sequence shown in Table 1).

[0043] (2) First exchange screening of homologous recombination

[0044] The recombinant plasmid obtained above was electroporated into competent cells of *Pseudomonas molluscum 923*, plated on NAN solid plates containing Km antagonist, and incubated at 28°C for 24 h. Positive clones that could grow were then screened. Due to the selection pressure generated by the antagonist Km, the recombinant plasmid carrying the homologous fragment integrated into the chromosome of 923, resulting in the first homologous exchange and producing a single exchanger.

[0045] (3) Second exchange screening for homologous recombination

[0046] Transformants that have undergone the first homologous exchange are selected and inoculated into NBN liquid medium and cultured overnight. The culture medium is then diluted 10 times. 2 -10 4 The samples were then plated on NAS solid plates containing 10% sucrose for screening. Due to the selection pressure generated by the sucrose, the vector containing the sacB gene recombinated from chromosome 923, detached, and died (i.e., committed suicide), resulting in a second homologous exchange and producing double exchangers.

[0047] (4) Screening and validation of mutants

[0048] After culturing at 28℃ for 24 hours, single colonies on NAS plates were individually transfected to NA and NA+Km plates. Single colonies that grew on NA but not on NA+Km were selected for screening. PCR was then used to verify whether the corresponding gene knockout was successful. PCR verification was first performed using F1 / R2 primers, followed by further verification using primers surrounding the gene fragments. Sequencing results showing shorter fragments than the original fragments confirmed successful knockout of genes psdA, psdB, psdC, psdD, psdE, psdF, and psdG.

[0049] Example 2: Complementation of the pseudoiodinine biosynthesis gene mutant

[0050] (1) Cloning of the target gene

[0051] Primers pUFR-ABCDEFG-F / R (sequences shown in Table 1) containing the pyrazolium triazine synthesis gene clusters psdA, psdB, psdC, psdD, psdE, psdF, and psdG, as well as their own promoter sequences, were designed using Primer Premier5. Using genomic DNA as a template, the target fragment was amplified using high-fidelity PCR enzymes. After double digestion with QuickCut restriction enzymes EcoRI and BamHI, the fragment was ligated into the pUFR034 vector. The fragment was then heat-transformed into DH5α for antibiotic resistance selection. Plasmids from positive clones were extracted, verified by enzyme digestion, and sent to Shanghai Platinum Biotech Co., Ltd. for sequencing.

[0052] (2) Transformation of target strain

[0053] The constructed clones were electroporated into mutant strains with deletions of the genes psdA, psdB, psdC, psdD, psdE, psdF, and psdG, respectively. The strains were incubated upside down at 28°C for 20 hours. After single colonies emerged, colony PCR was performed using the universal primers M13-47 / RV-M (sequences shown in Table 1). The PCR products were sent to Shanghai Platinum Biotech Co., Ltd. for sequencing. The correct strains were preserved as glycerol culture at -80°C for subsequent biochemical phenotypic experiments.

[0054] Example 3: Identification of the pseudoiodinine biosynthesis gene cluster

[0055] Total RNA extraction from Pseudomonas

[0056] (1) Transfer the activated single colony to LB liquid medium and incubate overnight at 28°C and 220 rpm.

[0057] (2) Transfer 1 mL of bacterial culture liquid to a 1.5 mL centrifuge tube, centrifuge at 5000 rpm for 5 min, collect the bacterial cells, and discard the supernatant;

[0058] (3) Add 100 μL of lysozyme to resuspend the bacterial cells and let them stand at 37°C for 10 min.

[0059] (4) Add 1 mL of Trizol, vortex to mix, and let stand at room temperature for 5 min;

[0060] (5) Add 200 μL of chloroform to the lysis buffer, vortex to mix, and let stand at room temperature for 5 min.

[0061] (6) Centrifuge at 4℃, 12000rpm for 10min. At this time, the liquid in the centrifuge tube is divided into three layers: the upper colorless supernatant, the middle white protein layer and the lower dark organic phase. Gently aspirate the upper aqueous phase and add an equal volume of isopropanol to a new RNase-free centrifuge tube. After gently mixing, precipitate RNA at -20℃ for several hours.

[0062] (7) Centrifuge at 4℃ and 12000 rpm for 10 min, then discard the supernatant;

[0063] (8) Add 1 mL of 75% ethanol to wash the precipitate;

[0064] (9) Centrifuge at 4℃ and 12000rpm for 2min to remove excess ethanol;

[0065] (10) Add 1 mL of anhydrous ethanol to wash the precipitate;

[0066] (11) Centrifuge at 4℃ and 12000rpm for 2min, and remove excess ethanol.

[0067] (12) Dry the RNA precipitate in a fume hood;

[0068] (13) Add 30-60 μL of DEPC-treated RNase-free water to dissolve the RNA, depending on the amount of white precipitate.

[0069] (14) The concentration of RNA was determined using a Nanodrop spectrophotometer. An A260 / A280 ratio between 1.8 and 2.0 indicates that the RNA quality is qualified and can be used for subsequent experiments. It should be stored at -80℃ for a long time.

[0070] Reverse transcription

[0071] Reverse transcription of RNA was performed according to the instructions of the EasyScript One-step gDNA removal and cDNA Synthesis Supermix kit provided by TransGen Biotech Ltd. After the reaction, the synthesized cDNA was diluted with 80 μL of deionized water for use in RT-PCR. Excess cDNA can be stored at -20℃ for long-term storage.

[0072] RT-PCR

[0073] Using cDNA as a template, the PCR reaction was carried out according to the above-described PCR reaction system and conditions. Primers were designed based on the sequences between genes psdA, psdB, psdC, psdD, psdE, psdF, and psdG (sequences are shown in Table 1) to detect whether the biosynthetic genes of pseudoiodinine are in the same transcription unit. Figure 2 As shown, this indicates that these seven pseudoiodinine synthesis-related genes (psdA-psdG) exist in the same transcriptional unit.

[0074] Example 4: rsmY and rsmZ positively regulate the biosynthesis of pseudoiodinine

[0075] To verify the effects of small RNAs rsmY and rsmZ on pseudoiodinine production, single mutants ΔrsmY and ΔrsmZ, and a double mutant ΔrsmYZ, were obtained using the homologous recombination double exchange method described above (primer sequences are shown in Table 1). Their corresponding functional complementors C923ΔrsmY, C923ΔrsmZ, and C923ΔrsmYZ were then obtained through in vitro construction and transformation (primer sequences are shown in Table 1). (For Xoo PXO99) A The antibacterial activity assay results showed that single mutations of rsmY and rsmZ did not affect the antibacterial activity. Double mutations of rsmY and rsmZ completely resulted in the loss of antibacterial activity. Figure 3 -A). Further analysis of pseudoiodinine production by HPLC revealed that, compared to the wild-type strain 923, the single mutant rsmY significantly reduced pseudoiodinine production; however, the single mutant rsmZ did not significantly reduce PSD production. In the double mutant strain ΔrsmYZ, pseudoiodinine was completely absent. Figure 3 -BC). Functional complementation results showed that the antibacterial activity of single complementation strains rsmY and rsmZ could partially recover to wild-type levels, while the antibacterial activity of double complementation strains rsmY and rsmZ could completely recover to wild-type levels. However, the production of pseudoiodinine in strain C923ΔrsmY could only recover to a small amount of wild-type levels, while in strain C923ΔrsmZ it could mostly recover to wild-type levels, and in strain C923ΔrsmYZ it could completely recover to wild-type levels. Figure 3 -C). The above results indicate that the two small RNAs rsmY and rsmZ in 923 can positively regulate the biosynthesis of pseudoiodinine, and that there is functional redundancy between the two.

[0076] Table 1 Primer sequences used in the study

[0077]

[0078]

[0079]

[0080] a Restriction enzyme sites are underlined.

[0081] Example 5: Determination of antagonistic activity of different strains

[0082] PXO99, the pathogen causing bacterial blight in rice. A Inoculated into NA liquid medium, cultured at 28°C and 180 rpm for 12 h, then adjusted OD 600 2.0; After thoroughly mixing 200 μL of bacterial suspension with NA solid medium, pour the plate and evenly attach 6 mm diameter filter paper discs to each NA plate. Spot-inoculate each filter paper disc with 5 μL of the tested *Pseudomonas* strains, including different mutant strains, complemented strains, and wild-type strain 923. Each treatment is repeated in triplicate. After drying, incubate upside down in a 28°C biochemical incubator for 24 h and observe for the appearance of inhibition zones. Figure 1 As shown, knockout of the psdA, psdB, psdC, psdD, psdE, psdF, and psdG genes all significantly reduced the inhibition of Xoo PXO99. A Further functional complementation of these seven genes, which enhanced the antibacterial activity and pseudoiodinine production capacity of the wild-type strain 923, showed that the antibacterial phenotype and pseudoiodinine production capacity of the strain were fully restored. This indicates that these seven genes are involved in the biosynthesis of pseudoiodinine.

[0083] Example 6: Effect of different culture media on pseudoiodinine yield

[0084] (1) Plot the standard curve of pseudoiodinine.

[0085] Five concentration gradients of pseudoiodinine were prepared: 0.03 mM, 0.5 mM, 1.5 mM, 2.5 mM, and 4 mM. Peak areas at different concentrations were measured using HPLC (C18, 4.6 x 150 mm, 5 μm) and plotted as the ordinate with concentration on the abscissa to establish a standard curve. Mobile phase: methanol / water. Flow rate: 0.4 mL / min; Detection wavelength: 500 nm; Uniform injection volume: 10 μL. Results are as follows: Figure 4As shown.

[0086] (2) Fermentation by strain

[0087] The culture broth of single colonies of P. mosselii 923 was transferred at a ratio of 1% to Erlenmeyer flasks containing 50 mL of LB and TSB liquid medium, respectively. Fermentation was carried out at 30℃ and 220 rpm. Under the same conditions, the growth curves of P. mosselii 923 and the yield of pseudoiodinine were measured at 12h, 24h, 36h, 48h, and 60h, respectively. Three replicates were set for each medium. Results are as follows: Figure 5 -A is shown.

[0088] (3) Determination of pseudoiodinine yield

[0089] Two mL of bacterial culture at different fermentation time points was extracted three times with an equal volume of ethyl acetate. The ethyl acetate phase was collected, concentrated and dried by centrifugation at 35°C, and then dissolved in 200 μL of methanol. The solution was centrifuged at 12000 rpm for 20 min at 4°C, and 10 μL was injected for HPLC quantitative detection. The measured peak area value was substituted into the standard curve to obtain the corresponding yield of pseudoiodinine. Results are as follows... Figure 5 As shown in Figure B, the highest yield of pseudoiodinine, reaching 5.14 mg / L, was observed in TSB medium after 36 hours of fermentation.

[0090] The sequence involved in this invention is shown below:

[0091] SEQ ID No.1

[0092]

[0093]

[0094]

[0095] SEQ ID No.2

[0096]

[0097]

[0098] SEQ ID No. 3

[0099] MNAQKPVTIYEHADALNSLQGVVDLVKLSDQYRQSAILHSALAQRVFDHTRQPVSAQALSDALGWVPNKGRIFLNALVAMGLLKRTAQGFENLPLSQRFLVSDSAEFIGPIIDHQRLQWQNWPRLDEVLRSRTSLDFQQENRFKHDLAARDAFNDAMVRFSQPMVDVLRDLPLFDKARRVIDLAGGHGTYLAEIASRNPQVKGEVWDLEPTREAAAATFAKYRLDDRLGFHARNLLDLAQYQGERADVVMLNDCLHYFTREQVRTLIAGAAGMVEGDGALLVLSMALEDDEIHPALSADFSLHMMLNTVNGELHPTRYLIDSMASAGLSVEQEPIGRYTLLIGRRAASEQ ID No.4

[0100]

[0101] SEQ ID No.5

[0102] MLRLLALHAAPLGDAAAQALDSLGSAAAAAGFSLQVSQKPAGQGPVDAVCLLLDSATPPVALNTLLNEASGLCRNTALVLVRVQGALAQLPPASGVIAQWQQASQGFLYPYSLDIGAGQAPELAIKDWLAGFAKFAAATKLWRSLDGLGLDEAARAAQRPELNHINILTRDLEASKAFYSDILGANYCYNLGPRKAVMELNGFDFFIEQSESFSYPTGYHIGVRALPGDVRRIAEQVTAAGTIKLVKGNGPAPGYHHGPDNVRSAVYFEDPDGLVIEVYSAEVEMIESNPRLLLDRL

[0103] SEQ ID No.6

[0104]

[0105] SEQ ID No.7

[0106] MNSYKQWHWPRLPSPLPKDVSDRALMGYPEHYQEHPLQAQVATALDDLLQAPLDVLLTLLEQPRQPLARRIAAGEVLALRGDPRIDTFTPQMIDIPAARVHIGLDPRAVEQVMDQFDGLGLDRSWIDKETPRHAVELAAFRIARFPVTHQEYRQFLLDSGHAGIPDGWAFGRYPRHHANHPVYSVSASDADAYAQWLSERTGRRFALPSEAQWEYAAAGPEGRQFPWGDAYQVEHANTAELGYLDSTPVGVFSDAASPFGVLDMAGNVEEYVAEDYRPYPGGPQIEDDLVLDVGTHRVARGGSFTRFRDLARTARRHGRYPRPIYVMGFRLVENHN

[0107] SEQ ID No.8

[0108]

[0109] SEQ ID No.9

[0110] MNKDVSLHTIVPISILGGDVDAHFFGFHGFERDQEHFAVGIGREQCDVPLVRVHSECITGDVFGSQRCDCGPQLQEALRTLKEVGGYLIYLRQEGRGIGLYSKFEAYLLQDKGLDTYEANLRLNHLADSRSFDPAVQILRALGVDQCRLLTNNPEKVAQLRAGGIDVIEQVPTGVFLTNHNRNYLQAKAVKSSHSIKL

[0111] SEQ ID No.10

[0112]

[0113] SEQ ID No.11

[0114] MKHIGPISGIAAHAARFVATAGYDNQVILWNAATGEPIHRVHHDHLANQCAFSSDGKHLVSASSDYTARIWEVPSMRLKAVLQGHNDDVEMAVFSPDSQRVATCSRDHVLRIFDLDGVQLQAFHGHQADVISVVWSPDGQRLISSSDDGTVRQWDTRSGQQCDEVDIGGVETDTIAITREGVVFAGDDEGRISIIAQGQVQTVPAHAAGIKRIVWNDDKRLLVSLSYDRSAILWTFDAARNLVKRRSTALPSIVWPRSCAFVGDEQLVFATFGSRYATWNYEQDQWQVSGIEPAVSINAVVRSEERQYSIGDAGILHRDDQPVTSVGSLCNFLLPFGPLLLTGGQMGQVFDGLSGRMLYQHRSPLNCGATFVRNGEDLALIGTYTGEGLVFGHDGQGGLRLVASIPMHDNAIKGVAADQRHLFSVCASADAALHSIEDFSSVRHIEGAHTRISNGCCPITGGFASIGRDLKLRLWLETGDEVFDSPHQHSIKCIAASADGRVIATAAYNGTVALFDLVSRRWLPMQRPTASGISCLTHDAVSGAFLASSYDGRIYGIDARLAS

[0115] SEQ ID No.12

[0116]

[0117] SEQ ID No.13

[0118] MSKQASDFMAQGDYRKALDTSFVHRYSHGEDEWSWDIGMIQAAQAFLERLDPRADQHVLDIGVGRGRDASTFILAGHRVTGLDIVENSSWPLLRKRWGDRLDLVNKAMQDWQPAPGTVFDAALDNGCFHHQHPDEWGAYLAHVRRLLRPGALVGLNVFGVDAAHPQPGWREMDNQRQGYFFTDDGIRQTLEAHGFTWEGLEVIERQHGEARYLLALVRT

[0119] SEQ ID No.14

[0120]

[0121] SEQ ID No. 15

[0122] VSAIDRHYLDMALALASQGLYSTMPNPRVGCVIVNAGQVVGRGWHQRAGQPHAEVHALREAGPAARGATAYVTLEPCGHQGRTPPCADALVAAGVSRVVTASGDVSQSIGADRLREAGIVVEALPCPRARALNRGFFSRIERQRPWVRVLRP AALEIGAIGEGAMLSYCDEQAPLAHWRGRASALLSTCDWVKACDTSLVAQVAGQRAEPVVPLRVLSDQGLDCPASAKMLDGRAPTLVLHGAQARRDGRYARARCQVLEDLGALRILQTLHELDCNEVQVEAEPAWCEALARQGLVDEWLVQV

[0123] SEQ ID No. 16

[0124]

[0125] SEQ ID No. 17

[0126]

[0127] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention. sequence list <110> Shanghai Jiao Tong University <120> A method for high-yield pyrazole triazine <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 6931 <212> DNA <213> Pseudomonas <400> 1 atgaatgccc agaaacccgt gaccatctat gaacacgccg acgcgctgaa cagcctgcaa 60 ggcgtggtcg acctggtgaa gctttccgac cagtaccgcc agtccgcgat cctgcattcg 120 gcgctggccc agcgtgtgtt cgatcacacc cgccagccgg tcagcgccca ggccctgagc 180 gatgcgctgg gttgggtgcc gaacaagggc cgcatcttcc tcaacgcgct ggtggccatg 240 ggcctgttga agcgcaccgc ccaaggtttc gagaacctgc cgctgagcca gcgtttcctg 300 gtcagcgaca gcgccgagtt catcggcccg atcatcgacc accagcgcct gcagtggcaa 360 aactggccgc gcctggacga agtgctgcgc agccgcacct cgctggattt ccagcaggaa 420 aaccgcttca agcacgacct ggcggcccgc gacgccttca acgatgccat ggtccgtttc 480 agccagccca tggttgatgt gctgcgtgac ctgccgctgt tcgacaaggc ccgccgggtg 540 atcgacctgg ccggtggcca cggcacctac ctggccgaga tcgccagccg aaatccgcag 600 gtcaagggcg aggtctggga ccttgagccc acccgcgagg cggccgcggc caccttcgcc 660 aagtaccgtc ttgacgaccg cctgggcttc catgcccgca acctgctgga cctggcgcag 720 taccagggcg agcgggccga cgtggtgatg ctcaacgact gcctgcacta tttcacccgc 780 gagcaggtgc gtaccctgat cgccggcgct gccggcatgg tcgagggcga cggcgcgctg 840 ctggtgctga gcatggccct ggaggatgac gagatccacc cggcgctgtc ggccgacttc 900 tccctgcaca tgatgctcaa cacggtcaac ggcgagctgc atccgacccg ctacctgatc 960 gacagcatgg cgtcggcggg gttgagcgtg gagcaggagc cgatcggccg ctacaccctg 1020 ctgatcggac ggagggctgc ctgatgctgc gcttgttggc attgcatgcc gcgccgcttg 1080 gcgacgcggc ggcccaggcc ctggacagct tgggcagcgc cgcggcggcg gcaggtttca 1140 gcctgcaggt cagccagaag ccggccggcc agggaccggt ggacgcagtc tgcctgctgc 1200 tcgacagcgc cacgccaccg gttgcgttga acacgttgct caatgaggcc agcgggctgt 1260 gtcgcaacac cgcgctggtg ctggtccgcg tgcagggtgc actggcacag ttgccgcccg 1320 ccagcggcgt gatcgcccag tggcagcagg cgagccaggg ctttctctat ccgtactcgc 1380 tggatatcgg cgccgggcag gcgccggagc tggcgatcaa ggactggctg gcgggcttcg 1440 ccaagttcgc ggccgcgacc aagctgtggc gttcgctcga cggcctgggc ctggacgaag 1500 cggcgcgggc cgcgcagcgc cccgagctga accacatcaa tatcctcacc cgcgacctgg 1560 aagcctccaa ggcgttctac agcgacatcc ttggcgccaa ctactgctac aacctggggc 1620 cgcgcaaggc cgtgatggag ctcaacggct tcgacttctt catcgagcag agcgagtcgt 1680 tcagttaccc gaccggctac cacatcggtg tgcgggcact gcccggggat gtgcggcgca 1740 tcgccgagca ggtcaccgct gccggcacca tcaagctggt caagggcaac ggcccggcgc 1800 cgggctacca ccatgggccg gacaacgtgc gcagcgccgt ctacttcgag gacccggatg 1860 gcctggtgat cgaggtctac agcgccgaag tggagatgat cgaaagcaac cctcggttgc 1920 tcctcgaccg cctctgaccc cacgtcgtca caggcctgcc gctcggcggg cctgtgccta 1980 ggcaggtatt cgcatgaaca gctacaaaca gtggcattgg ccacggctgc ccagcccatt 2040 gcccaaggat gtcagcgacc gggcgttgat gggctaccca gagcactacc aggagcatcc 2100 gttgcaggcg caggtcgcca cggcgctcga cgatctgctc caggcgccgc tggacgtctt 2160 gctgacgttg cttgagcagc cacgccaacc cctggcccgg cgcatcgccg ccggcgaggt 2220 gctggcattg cgcggcgacc cgcgcatcga caccttcacc ccgcagatga tcgatatccc 2280 ggcggcgcgg gtgcacatcg gcctcgaccc gcgcgcggtc gagcaggtga tggaccagtt 2340 cgacgggctt ggcctggacc gcagctggat cgacaaggaa acgccgcgcc acgccgtgga 2400 actggcggcg ttccgtatcg cccgcttccc ggtgacgcac caggagtacc gccagttcct 2460 gctcgacagc gggcatgccg gcatccccga cggctgggcg ttcggccggt atccgcggca 2520 ccacgccaac cacccggttt acagcgtctc ggccagtgac gccgatgcct acgcccagtg 2580 gctcagcgag cgcaccggcc gacgctttgc cctgcccagc gaggcgcagt gggagtatgc 2640 cgccgccgga cctgaagggc gtcagttccc ctggggcgac gcctaccagg tcgagcacgc 2700 caacaccgcc gagctgggct atctggacag cacgccggtc ggcgtgttca gcgacgccgc 2760 atcgcccttc ggggtgctgg acatggccgg caacgtcgaa gagtacgtgg ccgaggacta 2820 ccggccatac cccggcggcc cgcagatcga ggacgacctg gtgctcgatg tcggcaccca 2880 ccgcgtggcc cgcggtggca gctttacccg tttccgcgac ctggcgcgca ccgcgcgccg 2940 ccacgggcgt tacccgcgac cgatctacgt catgggtttt cgcctggttg aaaaccacaa 3000 ctgagctgtt ccgtcttccg tgcaagaggt ctgattccat gaacaaggat gtttcgcttc 3060 acaccatcgt gccgatttcc atcctcggcg gcgatgtcga tgcccatttc ttcggcttcc 3120 acggcttcga gcgtgaccag gagcatttcg ccgtcggcat cggccgcgag cagtgcgacg 3180 tgccgttggt gcgggtgcac tccgagtgca tcaccggcga tgtgttcggc tcgcagcgct 3240 gcgattgcgg cccgcaattg caagaggcgt tgcgcaccct caagaggaggtg ggcggctacc 3300 tgatctacct gcgccaggaa gggcgtggca tcggcctgta ctccaagttc gaggcctacc 3360 tgctgcagga caagggcctg gacacctatg aagccaacct gcgcctgaac cacttggccg 3420 actcgcgctc gttcgacccg gccgtgcaga tcctgcgggc cctgggcgtg gaccagtgcc 3480 ggttgctgac caacaacccg gagaaggtcg cccagctgcg cgccggtggc atcgatgtga 3540 tcgaacaggt gcccaccggg gtgttcctga ccaatcacaa ccgcaactac ctgcaggcga 3600 aggccgtcaa gtccagccat tcgatcaagc tctgactcca gcgaaaaagg atctttcgat 3660 gaaacacatt ggccctatca gcggtatcgc cgcccatgcc gcgcgcttcg tcgccaccgc 3720 cggctacgac aaccaggtga tcttgtggaa cgccgccacc ggcgagccga tccaccgtgt 3780 gcaccacgac cacctggcca accagtgcgc cttcagcagt gacggcaagc acctggtcag 3840 cgccagcagc gactacaccg cgcgcatctg ggaagtgccg agcatgcgcc tgaaagccgt 3900 actgcagggg cacaacgacg atgtcgagat ggcggtgttc tcgccagaca gccagcgcgt 3960 ggcgacctgt tcgcgtgacc atgtgctgcg catcttcgac ctcgacggcg tgcagttgca 4020 ggcgttccat ggccaccagg ccgacgttat ctcggtggtc tggtcgccgg acggccagcg 4080 cctgatctcc agcagtgacg acggtaccgt gcgccagtgg gatacccgca gcggtcagca 4140 atgcgacgag gtggacatcg gcggcgtgga gaccgacacc atcgccatca cccgcgaagg 4200 cgtggtgttc gccggggacg acgaggggcg catctcgatc atcgcccagg gccaggtgca 4260 gaccgtgccg gcccacgccg ccgggatcaa gcgcatcgtc tggaacgacg acaagcgcct 4320 gctggtgagc ctgagctatg accgttcggc gatcctctgg accttcgacg ccgcgcgcaa 4380 cctggtcaag cgccgcagta ccgcgctgcc gagcatcgtc tggccgcgca gctgcgcttt 4440 tgtcggcgac gaacaactgg tgttcgccac cttcggctcg cgctacgcca cctggaacta 4500 cgagcaggac cagtggcagg tgtcgggcat cgagccggcg gtaagcatta acgccgtggt 4560 gcgcagcgaa gagcgccagt acagcattgg cgatgctggc attctgcacc gtgacgacca 4620 gccggtgacc agtgtcggca gcctgtgcaa cttcctcctg ccgttcggcc cgctgctgtt 4680 gaccggcggc cagatgggcc aggtgttcga tggcctgtcc gggcgcatgc tgtaccagca 4740 ccgttcgcca ctcaactgcg gcgccacctt cgtcaggaat ggcgaagacc tggcgttgat 4800 cggcacctat accggcgaag gcctggtgtt cggccatgat ggccagggcg ggctgcgcct 4860 ggtggcgtcg atccccatgc atgacaacgc gatcaagggc gtggccgccg accagcgcca 4920 cctgttcagc gtctgcgcct cggccgatgc cgcgctgcac agcatcgagg atttcagcag 4980 cgtgcggcat atcgaggggg cgcatacgcg tatttccaat ggttgctgcc cgatcaccgg 5040 cggtttcgcc agcatcggtc gcgacctcaa gctgcgcctg tggctggaga ccggcgacga 5100 ggtgttcgac tcgccgcacc agcactcgat caagtgcatc gccgcctccg ctgatggccg 5160 ggtgatcgcc accgcggcct acaacggtac cgtggcactg ttcgacctgg tctcgcggcg 5220 ctggctgccc atgcagcggc cgaccgccag cggcatttcc tgcctgaccc acgacgccgt 5280 cagcggcgcc ttcctggcca gttcctatga cgggcgcatc tacggcatcg acgcccgcct 5340 ggcgtcctga ggagcagcac atgagcaagc aagcaagtga cttcatggcc cagggcgact 5400 accgcaaggc cctcgatact tcgttcgtgc accgctacag ccatggcgag gacgagtggt 5460 cgtgggacat cggcatgatc caggcggccc aggctttcct cgagcgtctc gacccccgtg 5520 ctgatcagca tgtactggac atcggcgtgg gccgtggccg tgacgcctcc accttcatcc 5580 tcgccgggca tcgggtcacc ggcctggata tcgtcgagaa ctccagctgg ccgttgctgc 5640 gcaaacgctg gggcgatcgc ctggacctgg tgaacaaggc catgcaggac tggcagccgg 5700 cgccgggcac ggtgttcgat gcggccctgg acaatggctg cttccaccac cagcatccgg 5760 acgagtgggg cgcgtacctg gcccatgtgc gccgtctgct gcgccccggc gcgctggtgg 5820 ggctgaacgt gttcggggtc gacgcggccc atccgcagcc gggctggcgc gagatggaca 5880 accagcgcca aggctacttc ttcaccgacg atggtatccg tcagaccctt gaggcccatg 5940 gcttcacctg ggaggggctg gaggtgatcg agcgccagca cggcgaagcc cgctacctgt 6000 tggcgctggt gcgcacgtga gtgcgatcga ccgccattac ctggacatgg ccctggcgct 6060 cgccagccag ggcctgtaca gcaccatgcc caacccgcgg gtcggttgcg tgatcgtcaa 6120 cgccgggcag gtggtagggc gcggctggca tcagcgcgcc ggccagcccc acgccgaagt 6180 acacgccctg cgcgaggcag gcccggcggc gcgcggggcg accgcctatg tgaccctgga 6240 accttgcggc caccagggtc gcacgccgcc gtgcgccgat gccctggtgg cggcgggggt 6300 aagccgggtg gtcacggcca gcggcgacgt ctcgcagagc atcggcgcgg atcgcctgcg 6360 cgaggcgggc atcgtggtcg aggcattgcc ctgcccacgt gcgcgggcgc tcaatcgcgg 6420 gttcttctcg cgcatcgaac gccagcgccc gtgggtgcgg gtattgcgtc cggcggcgct 6480 ggagatcggc gcgattggcg agggcgcgat gctcagctat tgcgacgagc aggcgcccct 6540 ggcgcattgg cggggcaggg cctcggcgct gctgagcact tgtgactggg tgaaggcctg 6600 cgacacctcg ctggtggcgc aggttgccgg gcaacgcgcc gaaccggtgg tgcccttgcg 6660 ggtattgagc gaccaggggc tggactgccc ggcttcggcc aagatgctcg acgggcgtgc 6720 gccgacgctg gttttgcatg gggcgcaggc gaggcgggac ggtcggtatg cacgggcccg 6780 ttgccaggtg ctggaggatc tgggggcctt gcggatacta cagacgttgc atgagctcga 6840 ctgcaacgaa gtccaggtcg aggccgagcc ggcctggtgc gaggccttgg cgcgccaggg 6900 gttagtggat gagtggttgg tgcaggtttg a 6931 <210> 2 <211> 1044 <212> DNA <213> Pseudomonas <400> 2 atgaatgccc agaaacccgt gaccatctat gaacacgccg acgcgctgaa cagcctgcaa 60 ggcgtggtcg acctggtgaa gctttccgac cagtaccgcc agtccgcgat cctgcattcg 120 gcgctggccc agcgtgtgtt cgatcacacc cgccagccgg tcagcgccca ggccctgagc 180 gatgcgctgg gttgggtgcc gaacaagggc cgcatcttcc tcaacgcgct ggtggccatg 240 ggcctgttga agcgcaccgc ccaaggtttc gagaacctgc cgctgagcca gcgtttcctg 300 gtcagcgaca gcgccgagtt catcggcccg atcatcgacc accagcgcct gcagtggcaa 360 aactggccgc gcctggacga agtgctgcgc agccgcacct cgctggattt ccagcaggaa 420 aaccgcttca agcacgacct ggcggcccgc gacgccttca acgatgccat ggtccgtttc 480 agccagccca tggttgatgt gctgcgtgac ctgccgctgt tcgacaaggc ccgccgggtg 540 atcgacctgg ccggtggcca cggcacctac ctggccgaga tcgccagccg aaatccgcag 600 gtcaagggcg aggtctggga ccttgagccc acccgcgagg cggccgcggc caccttcgcc 660 aagtaccgtc ttgacgaccg cctgggcttc catgcccgca acctgctgga cctggcgcag 720 taccagggcg agcgggccga cgtggtgatg ctcaacgact gcctgcacta tttcacccgc 780 gagcaggtgc gtaccctgat cgccggcgct gccggcatgg tcgagggcga cggcgcgctg 840 ctggtgctga gcatggccct ggaggatgac gagatccacc cggcgctgtc ggccgacttc 900 tccctgcaca tgatgctcaa cacggtcaac ggcgagctgc atccgacccg ctacctgatc 960 gacagcatgg cgtcggcggg gttgagcgtg gagcaggagc cgatcggccg ctacaccctg 1020 ctgatcggac ggagggctgc ctga 1044 <210> 3 <211> 347 <212> PRT <213> Pseudomonas <400> 3 Put Asn Ala Gln Lys Pro Val Thr Ile Tyr Glu His Ala Asp Ala Leu 1 5 10 15 Asn Ser Leu Gln Gly Val Val Asp Leu Val Lys Leu Ser Asp Gln Tyr 20 25 30 Arg Gln Ser Ala Ile Leu His Ser Ala Leu Ala Gln Arg Val Phe Asp 35 40 45 His Thr Arg Gln Pro Val Ser Ala Gln Ala Leu Ser Asp Ala Leu Gly 50 55 60 Trp Val Pro Asn Lys Gly Arg Ile Phe Leu Asn Ala Leu Val Ala Met 65 70 75 80 Gly Leu Leu Lys Arg Thr Ala Gln Gly Phe Glu Asn Leu Pro Leu Ser 85 90 95 Gln Arg Phe Leu Val Ser Asp Ser Ala Glu Phe Ile Gly Pro Ile Ile 100 105 110 Asp His Gln Arg Leu Gln Trp Gln Asn Trp Pro Arg Leu Asp Glu Val 115 120 125 Leu Arg Ser Arg Thr Ser Leu Asp Phe Gln Gln Glu Asn Arg Phe Lys 130 135 140 His Asp Leu Ala Ala Arg Asp Ala Phe Asn Asp Ala Met Val Arg Phe 145 150 155 160 Ser Gln Pro Met Val Asp Val Leu Arg Asp Leu Pro Leu Phe Asp Lys 165 170 175 Ala Arg Arg Val Ile Asp Leu Ala Gly Gly His Gly Thr Tyr Leu Ala 180 185 190 Glu Ile Ala Ser Arg Asn Pro Gln Val Lys Gly Glu Val Trp Asp Leu 195 200 205 Glu Pro Thr Arg Glu Ala Ala Ala Ala Thr Phe Ala Lys Tyr Arg Leu 210 215 220 Asp Asp Arg Leu Gly Phe His Ala Arg Asn Leu Leu Asp Leu Ala Gln 225 230 235 240 Tyr Gln Gly Glu Arg Ala Asp Val Val Met Leu Asn Asp Cys Leu His 245 250 255 Tyr Phe Thr Arg Glu Gln Val Arg Thr Leu Ile Ala Gly Ala Ala Gly 260 265 270 Met Val Glu Gly Asp Gly Ala Leu Leu Val Ser Met Ala Leu Glu 275 280 285 Asp Asp Glu Ile His Pro Ala Leu Ser Ala Asp Phe Ser Leu His Met 290,295,300 Met Leu Asn Thr Val Asn Gly Glu Leu His Pro Thr Arg Tyr Leu Ile 305 310 315 320 Asp Ser Met Ala Ser Ala Gly Leu Ser Val Glu Gln Glu Pro Ile Gly 325 330 335 Arg Tyr Thr Leu Leu And Gly Arg Arg Ala Ala 340,345 <210> 4 <211> 894 <212> DNA <213> Pseudomonas <400> 4 atgctgcgct tgttggcatt gcatgccgcg ccgcttggcg acggcggcggc ccaggccctg 60 zgagcttgg gcagcgccgc ggcggcggca ggtttcagcc tgcaggtcag ccagaagccg 120 gccggccagg gaccggtgga cgcagtctgc ctgctgctcg acagcgccac gccaccggtt 180 gcgttgaaca cgttgctcaa tgaggccagc gggctgtgtc gcacaccgc gctggtgctg 240 gtccgcgtgc agggtgcact ggcacagttg ccgcccgcca gcggcgtgat cgcccagtgg 300 cagcaggcga gccagggctt tcttatccg tactcgctg atacggcgc cgggcaggcg 360 ccggagctgg cgatcaagga ctggctggcg ggcttcgcca agttcgcggc cgcgaccaag 420 ctgtggcgtt cgctcgacgg cctgggcctg gacgaagcgg cgcgggcgc gcagcgccc 480 gagctgaacc acatcaatat cctcacccgc gacctggaag cctccaaggc gttctacagc 540 gacatccttg gcgccaacta ctgctacaac ctggggccgc gcaaggccgt gatggagctc 600 aacggcttcg acttcttcat cgagcagagc gagtcgttca gttacccgac cggctaccac 660 atcggtgtgc gggcactgcc cggggatgtg cggcgcatcg ccgagcaggt caccgctgcc 720 ggcaccatca agctggtcaa gggcaacggc ccggcgccgg gctaccacca tgggccggac 780 aacgtgcgca gcgccgtcta cttcgaggac ccggatggcc tggtgatcga ggtctacagc 840 gccgaagtgg agatgatcga aagcaaccct cggttgctcc tcgaccgcct ctga 894 <210> 5 <211> 297 <212> PRT <213> Pseudomonas <400> 5 Met Leu Arg Leu Leu Ala Leu His Ala Ala Pro Leu Gly Asp Ala Ala 1 5 10 15 Ala Gln Ala Leu Asp Ser Leu Gly Ser Ala Ala Ala Ala Ala Gly Phe 20 25 30 Ser Leu Gln Val Ser Gln Lys Pro Ala Gly Gln Gly Pro Val Asp Ala 35 40 45 Val Cys Leu Leu Leu Asp Ser Ala Thr Pro Pro Val Ala Leu Asn Thr 50 55 60 Leu Leu Asn Glu Ala Ser Gly Leu Cys Arg Asn Thr Ala Leu Val Leu 65 70 75 80 Val Arg Val Gln Gly Ala Leu Ala Gln Leu Pro Pro Ala Ser Gly Val 85 90 95 Ile Ala Gln Trp Gln Gln Ala Ser Gln Gly Phe Leu Tyr Pro Tyr Ser 100 105 110 Leu Asp Ile Gly Ala Gly Gln Ala Pro Glu Leu Ala Ile Lys Asp Trp 115 120 125 Leu Ala Gly Phe Ala Lys Phe Ala Ala Ala Thr Lys Leu Trp Arg Ser 130 135 140 Leu Asp Gly Leu Gly Leu Asp Glu Ala Ala Arg Ala Ala Gln Arg Pro 145 150 155 160 Glu Leu Asn His Ile Asn Ile Leu Thr Arg Asp Leu Glu Ala Ser Lys 165 170 175 Ala Phe Tyr Ser Asp Ile Leu Gly Ala Asn Tyr Cys Tyr Asn Leu Gly 180 185 190 Pro Arg Lys Ala Val Met Glu Leu Asn Gly Phe Asp Phe Phe Ile Glu 195 200 205 Gln Ser Glu Ser Phe Ser Tyr Pro Thr Gly Tyr His Ile Gly Val Arg 210 215 220 Ala Leu Pro Gly Asp Val Arg Arg Ile Ala Glu Gln Val Thr Ala Ala 225 230 235 240 Gly Thr Ile Lys Leu Val Lys Gly Asn Gly Pro Ala Pro Gly Tyr His 245 250 255 His Gly Pro Asp Asn Val Arg Ser Ala Val Tyr Phe Glu Asp Pro Asp 260 265 270 Gly Leu Val Ile Glu Val Tyr Ser Ala Glu Val Glu Met Ile Glu Ser 275 280 285 Asn Pro Arg Leu Leu Leu Asp Arg Leu 290 295 <210> 6 <211> 1011 <212> DNA <213> Pseudomonas <400> 6 atgaacagct acaaacagtg gcattggcca cggctgccca gcccattgcc caaggatgtc 60 agcgaccggg cgttgatggg ctacccagag cactaccagg agcatccgtt gcaggcgcag 120 gtcgccacgg cgctcgacga tctgctccag gcgccgctgg acgtcttgct gacgttgctt 180 gagcagccac gccaacccct ggcccggcgc atcgccgccg gcgaggtgct ggcattgcgc 240 ggcgacccgc gcatcgacac cttcaccccg cagatgatcg atatcccggc ggcgcgggtg 300 cacatcggcc tcgacccgcg cgcggtcgag caggtgatgg accagttcga cgggcttggc 360 ctggaccgca gctggatcga caaggaaacg ccgcgccacg ccgtggaact ggcggcgttc 420 cgtatcgcc gcttccccggt gacgcaccag gagtaccgcc agttcctgct cgacagcggg 480 catgccggca tccccgacgg ctgggcgttc ggccggtatc cgcggcacca cgccaaccac 540 ccggtttaca gcgtctcggc cagtgacgcc gatgcctacg cccagtggct cagcgagcgc 600 accggccgac gctttgccct gcccagcgag gcgcagtggg agtatgccgc cgccggacct 660 gaagggcgtc agttcccctg gggcgacgcc taccaggtcg agcacgccaa caccgccgag 720 ctgggctatc tggacagcac gccggtcggc gtgttcagcg acgccgcatc gcccttcggg 780 gtgctggaca tggccggcaa cgtcgaagag tacgtggccg aggactaccg gccatacccc 840 ggcggcccgc agatcgagga cgacctggtg ctcgatgtcg gcacccaccg cgtggcccgc 900 ggtggcagct ttacccgttt ccgcgacctg gcgcgcaccg cgcgccgcca cgggcgttac 960 ccgcgaccga tctacgtcat gggttttcgc ctggttgaaa accacaactg a 1011 <210> 7 <211> 336 <212> PRT <213> Pseudomonas <400> 7 Met Asn Ser Tyr Lys Gln Trp His Trp Pro Arg Leu Pro Ser Pro Leu 1 5 10 15 Pro Lys Asp Val Ser Asp Arg Ala Leu Met Gly Tyr Pro Glu His Tyr 20 25 30 Gln Glu His Pro Leu Gln Ala Gln Val Ala Thr Ala Leu Asp Asp Leu 35 40 45 Leu Gln Ala Pro Leu Asp Val Leu Leu Thr Leu Leu Glu Gln Pro Arg 50 55 60 Gln Pro Leu Ala Arg Arg Ile Ala Ala Gly Glu Val Leu Ala Leu Arg 65 70 75 80 Gly Asp Pro Arg Ile Asp Thr Phe Thr Pro Gln Met Ile Asp Ile Pro 85 90 95 Ala Ala Arg Val His Ile Gly Leu Asp Pro Arg Ala Val Glu Gln Val 100 105 110 Met Asp Gln Phe Asp Gly Leu Gly Leu Asp Arg Ser Trp Ile Asp Lys 115 120 125 Glu Thr Pro Arg His Ala Val Glu Leu Ala Ala Phe Arg Ile Ala Arg 130 135 140 Phe Pro Val Thr His Gln Glu Tyr Arg Gln Phe Leu Leu Asp Ser Gly 145 150 155 160 His Ala Gly Ile Pro Asp Gly Trp Ala Phe Gly Arg Tyr Pro Arg His 165 170 175 His Ala Asn His Pro Val Tyr Ser Val Ser Ala Ser Asp Ala Asp Ala 180 185 190 Tyr Ala Gln Trp Leu Ser Glu Arg Thr Gly Arg Arg Phe Ala Leu Pro 195 200 205 Ser Glu Ala Gln Trp Glu Tyr Ala Ala Ala Gly Pro Glu Gly Arg Gln 210 215 220 Phe Pro Trp Gly Asp Ala Tyr Gln Val Glu His Ala Asn Thr Ala Glu 225 230 235 240 Leu Gly Tyr Leu Asp Ser Thr Pro Val Gly Val Phe Ser Asp Ala Ala 245 250 255 Ser Pro Phe Gly Val Leu Asp Met Ala Gly Asn Val Glu Glu Tyr Val 260 265 270 Ala Glu Asp Tyr Arg Pro Tyr Pro Gly Gly Pro Gln Ile Glu Asp Asp 275 280 285 Leu Val Leu Asp Val Gly Thr His Arg Val Ala Arg Gly Gly Ser Phe 290 295 300 Thr Arg Phe Arg Asp Leu Ala Arg Thr Ala Arg Arg His Gly Arg Tyr 305 310 315 320 Pro Arg Pro Ile Tyr Val Met Gly Phe Arg Leu Val Glu Asn His Asn 325 330 335 <210> 8 <211> 597 <212> DNA <213> Pseudomonas <400> 8 atgaacaagg atgtttcgct tcacaccatc gtgccgattt ccatcctcgg cggcgatgtc 60 gatgcccatt tcttcggctt ccacggcttc gagcgtgacc aggagcattt cgccgtcggc 120 atcggccgcg agcagtgcga cgtgccgttg gtgcgggtgc actccgagtg catcaccggc 180 gatgtgttcg gctcgcagcg ctgcgattgc ggcccgcaat tgcaagaggc gttgcgcacc 240 ctcaaggagg tgggcggcta cctgatctac ctgcgccagg aagggcgtgg catcggcctg 300 tactccaagt tcgaggccta cctgctgcag gacaagggcc tggacaccta tgaagccaac 360 ctgcgcctga accacttggc cgactcgcgc tcgttcgacc cggccgtgca gatcctgcgg 420 gccctgggcg tggaccagtg ccggttgctg accaacaacc cggagaaggt cgcccagctg 480 cgcgccggtg gcatcgatgt gatcgaacag gtgcccaccg gggtgttcct gaccaatcac 540 aaccgcaact acctgcaggc gaaggccgtc aagtccagcc attcgatcaa gctctga 597 <210> 9 <211> 198 <212> PRT <213> Pseudomonas <400> 9 Met Asn Lys Asp Val Ser Leu His Thr Ile Val Pro Ile Ser Ile Leu 1 5 10 15 Gly Gly Asp Val Asp Ala His Phe Phe Gly Phe His Gly Phe Glu Arg 20 25 30 Asp Gln Glu His Phe Ala Val Gly Ile Gly Arg Glu Gln Cys Asp Val 35 40 45 Pro Leu Val Arg Val His Ser Glu Cys Ile Thr Gly Asp Val Phe Gly 50 55 60 Ser Gln Arg Cys Asp Cys Gly Pro Gln Leu Gln Glu Ala Leu Arg Thr 65 70 75 80 Leu Lys Glu Val Gly Gly Tyr Leu Ile Tyr Leu Arg Gln Glu Gly Arg 85 90 95 Gly Ile Gly Leu Tyr Ser Lys Phe Glu Ala Tyr Leu Leu Gln Asp Lys 100 105 110 Gly Leu Asp Thr Tyr Glu Ala Asn Leu Arg Leu Asn His Leu Ala Asp 115 120 125 Ser Arg Ser Phe Asp Pro Ala Val Gln Ile Leu Arg Ala Leu Gly Val 130 135 140 Asp Gln Cys Arg Leu Leu Thr Asn Asn Pro Glu Lys Val Ala Gln Leu 145 150 155 160 Arg Ala Gly Gly Ile Asp Val Ile Glu Gln Val Pro Thr Gly Val Phe 165 170 175 Leu Thr Asn His Asn Arg Asn Tyr Leu Gln Ala Lys Ala Val Lys Ser 180 185 190 Ser His Ser Ile Lys Leu 195 <210> 10 <211> 1692 <212> DNA <213> Pseudomonas <400> 10 atgaaacaca ttggccctat cagcggtatc gccgcccatg ccgcgcgctt cgtcgccacc 60 gccggctacg acaaccaggt gatcttgtgg aacgccgcca ccggcgagcc gatccaccgt 120 gtgcaccacg accacctggc caaccagtgc gccttcagca gtgacggcaa gcacctggtc 180 agcgccagca gcgactacac cgcgcgcatc tgggaagtgc cgagcatgcg cctgaaagcc 240 gtactgcagg ggcacaacga cgatgtcgag atggcggtgt tctcgccaga cagccagcgc 300 gtggcgacct gttcgcgtga ccatgtgctg cgcatcttcg acctcgacgg cgtgcagttg 360 caggcgttcc atggccacca ggccgacgtt atctcggtgg tctggtcgcc ggacggccag 420 cgcctgatct ccagcagtga cgacggtacc gtgcgccagt gggatacccg cagcggtcag 480 caatgcgacg aggtggacat cggcggcgtg gagaccgaca ccatcgccat cacccgcgaa 540 ggcgtggtgt tcgccgggga cgacgagggg cgcatctcga tcatcgccca gggccaggtg 600 cagaccgtgc cggcccacgc cgccgggatc aagcgcatcg tctggaacga cgacaagcgc 660 ctgctggtga gcctgagcta tgaccgttcg gcgatcctct ggaccttcga cgccgcgcgc 720 aacctggtca agcgccgcag taccgcgctg ccgagcatcg tctggccgcg cagctgcgct 780 tttgtcggcg acgaacaact ggtgttcgcc accttcggct cgcgctacgc cacctggaac 840 tacgagcagg accagtggca ggtgtcgggc atcgagccgg cggtaagcat taacgccgtg 900 gtgcgcagcg aagagcgcca gtacagcatt ggcgatgctg gcattctgca ccgtgacgac 960 cagccggtga ccagtgtcgg cagcctgtgc aacttcctcc tgccgttcgg cccgctgctg 1020 ttgaccggcg gccagatggg ccaggtgttc gatggcctgt ccgggcgcat gctgtaccag 1080 caccgttcgc cactcaactg cggcgccacc ttcgtcagga atggcgaaga cctggcgttg 1140 atcggcacct ataccggcga aggcctggtg ttcggccatg atggccaggg cgggctgcgc 1200 ctggtggcgt cgatccccat gcatgacaac gcgatcaagg gcgtggccgc cgaccagcgc 1260 cacctgttca gcgtctgcgc ctcggccgat gccgcgctgc acagcatcga ggatttcagc 1320 agcgtgcggc atatcgaggg ggcgcatacg cgtatttcca atggttgctg cccgatcacc 1380 ggcggttcg ccagcatcgg tcgcgacctc aagctgcgcc tgtggctgga gaccggcgac 1440 gaggtgtcg actcgccgca ccagcactcg atcaagtgca tcgccgcctc cgctgatggc 1500 cgggtgatcg ccaccgcggc ctacaacggt accgtggcac tgttcgacct ggtctcgcgg 1560 cgctggctgc ccatgcagcg gccgaccgcc agcggcattt cctgcctgac ccacgacgcc 1620 gtcagcggcg ccttcctggc cagttcctat gacgggcgca tctacggcat cgacgcccgc 1680 ctggcgtcct ga 1692 <210> 11 <211> 563 <212> PRT <213> Pseudomonas <400> 11 Met Lys His Ile Gly Pro Ile Ser Gly Ile Ala Ala His Ala Ala Arg 1 5 10 15 Phe Val Ala Thr Ala Gly Tyr Asp Asn Gln Val Ile Leu Trp Asn Ala 20 25 30 Ala Thr Gly Glu Pro Ile His Arg Val His His Asp His Leu Ala Asn 35 40 45 Gln Cys Ala Phe Ser Ser Asp Gly Lys His Leu Val Ser Ala Ser Ser 50 55 60 Asp Tyr Thr Ala Arg Ile Trp Glu Val Pro Ser Met Arg Leu Lys Ala 65 70 75 80 Val Leu Gln Gly His Asn Asp Asp Val Glu Met Ala Val Phe Ser Pro 85 90 95 Asp Ser Gln Arg Val Ala Thr Cys Ser Arg Asp His Val Leu Arg Ile 100 105 110 Phe Asp Leu Asp Gly Val Gln Leu Gln Ala Phe His Gly His Gln Ala 115 120 125 Asp Val Ile Ser Val Val Trp Ser Pro Asp Gly Gln Arg Leu Ile Ser 130 135 140 Ser Ser Asp Asp Gly Thr Val Arg Gln Trp Asp Thr Arg Ser Gly Gln 145 150 155 160 Gln Cys Asp Glu Val Asp Ile Gly Gly Val Glu Thr Asp Thr Ile Ala 165 170 175 Ile Thr Arg Glu Gly Val Val Phe Ala Gly Asp Asp Glu Gly Arg Ile 180 185 190 Ser Ile Ile Ala Gln Gly Gln Val Gln Thr Val Pro Ala His Ala Ala 195 200 205 Gly Ile Lys Arg Ile Val Trp Asn Asp Asp Lys Arg Leu Leu Val Ser 210 215 220 Leu Ser Tyr Asp Arg Ser Ala Ile Leu Trp Thr Phe Asp Ala Ala Arg 225 230 235 240 Asn Leu Val Lys Arg Arg Ser Thr Ala Leu Pro Ser Ile Val Trp Pro 245 250 255 Arg Ser Cys Ala Phe Val Gly Asp Glu Gln Leu Val Phe Ala Thr Phe 260 265 270 Gly Ser Arg Tyr Ala Thr Trp Asn Tyr Glu Gln Asp Gln Trp Gln Val 275 280 285 Ser Gly Ile Glu Pro Ala Val Ser Ile Asn Ala Val Val Arg Ser Glu 290 295 300 Glu Arg Gln Tyr Ser Ile Gly Asp Ala Gly Ile Leu His Arg Asp Asp 305 310 315 320 Gln Pro Val Thr Ser Val Gly Ser Leu Cys Asn Phe Leu Leu Pro Phe 325 330 335 Gly Pro Leu Leu Leu Thr Gly Gly Gln Met Gly Gln Val Phe Asp Gly 340 345 350 Leu Ser Gly Arg Met Leu Tyr Gln His Arg Ser Pro Leu Asn Cys Gly 355 360 365 Ala Thr Phe Val Arg Asn Gly Glu Asp Leu Ala Leu Ile Gly Thr Tyr 370 375 380 Thr Gly Glu Gly Leu Val Phe Gly His Asp Gly Gln Gly Gly Leu Arg 385 390 395 400 Leu Val Ala Ser Ile Pro Met His Asp Asn Ala Ile Lys Gly Val Ala 405 410 415 Ala Asp Gln Arg His Leu Phe Ser Val Cys Ala Ser Ala Asp Ala Ala 420 425 430 Leu His Ser Ile Glu Asp Phe Ser Ser Val Arg His Ile Glu Gly Ala 435 440 445 His Thr Arg Ile Ser Asn Gly Cys Cys Pro Ile Thr Gly Gly Phe Ala 450 455 460 Ser Ile Gly Arg Asp Leu Lys Leu Arg Leu Trp Leu Glu Thr Gly Asp 465 470 475 480 Glu Val Phe Asp Ser Pro His Gln His Ser Ile Lys Cys Ile Ala Ala 485 490 495 Ser Ala Asp Gly Arg Val Ile Ala Thr Ala Ala Tyr Asn Gly Thr Val 500 505 510 Ala Leu Phe Asp Leu Val Ser Arg Arg Trp Leu Pro Met Gln Arg Pro 515 520 525 Thr Ala Ser Gly Ile Ser Cys Leu Thr His Asp Ala Val Ser Gly Ala 530 535 540 Phe Leu Ala Ser Ser Tyr Asp Gly Arg Ile Tyr Gly Ile Asp Ala Arg 545,550,555,560 Leu Ala Ser <210> 12 <211> 660 <212> DNA <213> Pseudomonas <400> 12 atgagcaagc aagcaagtga cttcatggcc cagggcgact accgcaagc cctcgatact 60 tcgttcgtgc accgctacag ccatggcgag gacgagtggt cgtgggacat cggcatgatc 120 caggcggccc aggctttcct cgagcgtctc gacccccgtg ctgatcagca tgtactggac 180 atcggcgtgg gccgtggccg tgacgcctcc accttcatcc tcgccgggca tcgggtcacc 240 ggcctggata tcgtcgagaa ctccagctgg ccgttgctgc gcaaacgctg gggcgatcgc 300 ctggacctgg tgaacaaggc catgcaggac tggcagccgg cgccgggcac ggtgttcgat 360 gcggccctgg acaatggctg cttccaccac cagcatccgg acgagtgggg cgcgtacctg 420 gcccatgtgc gccgtctgct gcgccccggc gcgctggtgg ggctgaacgt gttcggggtc 480 gacgcggccc atccgcagcc gggctggcgc gagatggaca accagcgcca aggctacttc 540 ttcaccgacg atggtatccg tcagaccctt gaggcccatg gcttcacctg ggaggggctg 600 gaggtgatcg agcgccagca cggcgaagcc cgctacctgt tggcgctggt gcgcacgtga 660 <210> 13 <211> 219 <212> PRT <213> Pseudomonas <400> 13 Met Ser Lys Gln Ala Ser Asp Phe Met Ala Gln Gly Asp Tyr Arg Lys 1 5 10 15 Ala Leu Asp Thr Ser Phe Val His Arg Tyr Ser His Gly Glu Asp Glu 20 25 30 Trp Ser Trp Asp Ile Gly Met Ile Gln Ala Ala Gln Ala Phe Leu Glu 35 40 45 Arg Leu Asp Pro Arg Ala Asp Gln His Val Leu Asp Ile Gly Val Gly 50 55 60 Arg Gly Arg Asp Ala Ser Thr Phe Ile Leu Ala Gly His Arg Val Thr 65 70 75 80 Gly Leu Asp Ile Val Glu Asn Ser Ser Trp Pro Leu Leu Arg Lys Arg 85 90 95 Trp Gly Asp Arg Leu Asp Leu Val Asn Lys Ala Met Gln Asp Trp Gln 100 105 110 Pro Ala Pro Gly Thr Val Phe Asp Ala Ala Leu Asp Asn Gly Cys Phe 115 120 125 His His Gln His Pro Asp Glu Trp Gly Ala Tyr Leu Ala His Val Arg 130 135 140 Arg Leu Leu Arg Pro Gly Ala Leu Val Gly Leu Asn Val Phe Gly Val 145 150 155 160 Asp Ala Ala His Pro Gln Pro Gly Trp Arg Glu Met Asp Asn Gln Arg 165 170 175 Gln Gly Tyr Phe Phe Thr Asp Asp Gly Ile Arg Gln Thr Leu Glu Ala 180 185 190 His Gly Phe Thr Trp Glu Gly Leu Glu Val Ile Glu Arg Gln His Gly 195 200 205 Glu Ala Arg Tyr Leu Leu Ala Leu Val Arg Thr 210 215 <210> 14 <211> 915 <212> DNA <213> Pseudomonas <400> 14 gtgagtgcga tcgaccgcca ttacctggac atggccctgg cgctcgccag ccagggcctg 60 tacagcacca tgcccaaccc gcgggtcggt tgcgtgatcg tcaacgccgg gcaggtggta 120 gggcgcggct ggcatcagcg cgccggccag ccccacgccg aagtacacgc cctgcgcgag 180 gcaggcccgg cggcgcgcgg ggcgaccgcc tatgtgaccc tggaaccttg cggccaccag 240 ggtcgcacgc cgccgtgcgc cgatgccctg gtggcggcgg gggtaagccg ggtggtcacg 300 gccagcggcg acgtctcgca gagcatcggc gcggatcgcc tgcgcgaggc gggcatcgtg 360 gtcgaggcat tgccctgccc acgtgcgcgg gcgctcaatc gcgggttctt ctcgcgcatc 420 gaacgccagc gcccgtgggt gcgggtattg cgtccggcgg cgctggagat cggcgcgatt 480 ggcgagggcg cgatgctcag ctattgcgac gagcaggcgc ccctggcgca ttggcggggc 540 agggcctcgg cgctgctgag cacttgtgac tgggtgaagg cctgcgacac ctcgctggtg 600 gcgcaggttg ccgggcaacg cgccgaaccg gtggtgccct tgcgggtatt gagcgaccag 660 gggctggact gcccggcttc ggccaagatg ctcgacgggc gtgcgccgac gctggttttg 720 catggggcgc aggcgaggcg ggacggtcgg tatgcacggg cccgttgcca ggtgctggag 780 gatctggggg ccttgcggat actacagacg ttgcatgagc tcgactgcaa cgaagtccag 840 gtcgaggccg agccggcctg gtgcgaggcc ttggcgcgcc aggggttagt ggatgagtgg 900 ttggtgcagg tttga 915 <210> 15 <211> 304 <212> PRT <213> Pseudomonas <400> 15 Val Ser Ala Ile Asp Arg His Tyr Leu Asp Met Ala Leu Ala Leu Ala 1 5 10 15 Ser Gln Gly Leu Tyr Ser Thr Met Pro Asn Pro Arg Val Gly Cys Val 20 25 30 Ile Val Asn Ala Gly Gln Val Val Gly Arg Gly Trp His Gln Arg Ala 35 40 45 Gly Gln Pro His Ala Glu Val His Ala Leu Arg Glu Ala Gly Pro Ala 50 55 60 Ala Arg Gly Ala Thr Ala Tyr Val Thr Leu Glu Pro Cys Gly His Gln 65 70 75 80 Gly Arg Thr Pro Pro Cys Ala Asp Ala Leu Val Ala Ala Gly Val Ser 85 90 95 Arg Val Val Thr Ala Ser Gly Asp Val Ser Gln Ser Ile Gly Ala Asp 100 105 110 Arg Leu Arg Glu Ala Gly Ile Val Val Glu Ala Leu Pro Cys Pro Arg 115 120 125 Ala Arg Ala Leu Asn Arg Gly Phe Phe Ser Arg Ile Glu Arg Gln Arg 130 135 140 Pro Trp Val Arg Val Leu Arg Pro Ala Ala Leu Glu Ile Gly Ala Ile 145 150 155 160 Gly Glu Gly Ala Met Leu Ser Tyr Cys Asp Glu Gln Ala Pro Leu Ala 165 170 175 His Trp Arg Gly Arg Ala Ser Ala Leu Leu Ser Thr Cys Asp Trp Val 180 185 190 Lys Ala Cys Asp Thr Ser Leu Val Ala Gln Val Ala Gly Gln Arg Ala 195 200 205 Glu Pro Val Val Pro Leu Arg Val Leu Ser Asp Gln Gly Leu Asp Cys 210 215 220 Pro Ala Ser Ala Lys Met Leu Asp Gly Arg Ala Pro Thr Leu Val Leu 225 230 235 240 His Gly Ala Gln Ala Arg Arg Asp Gly Arg Tyr Ala Arg Ala Arg Cys 245 250 255 Gln Val Leu Glu Asp Leu Gly Ala Leu Arg Ile Leu Gln Thr Leu His 260 265 270 Glu Leu Asp Cys Asn Glu Val Gln Val Glu Ala Glu Pro Ala Trp Cys 275 280 285 Glu Ala Leu Ala Arg Gln Gly Leu Val Asp Glu Trp Leu Val Gln Val 290 295 300 <210> 16 <211> 124 <212> DNA <213> Pseudomonas <400> 16 acggaagtag cgcaggaagc gcgaaaagat agaacaggga cacgaaggat ttcctgccag gacggcgggt tgatagggat gtcgaaggga aacagtctgg aaaaccccgc ttaggcgggg 120 tttt 124 <210> 17 <211> 136 <212> DNA <213> Pseudomonas <400> 17 tgtgtcaagg gacagacaca ggccttcaag gatgacggcc agggacatcg cgggacgcga ttcatcagtg gatgatgatt gggacataca gggactacgg aaaaaaatgt gggcgggtca aaccgcccct tttttt 136

Claims

1. A method for high-yield pyrazole triazine, characterized in that, It is synthesized through the pyrazolite synthesis gene cluster; The pyrazole triazine synthesis gene cluster is composed of psdA , psdB, psdC, psdD, psdE, psdF and psdG The nucleotide sequences are shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, respectively. The synthetic gene cluster of the pyrazolium triazine originates from *Pseudomonas molluscum*. Pseudomonas mosselii 923, deposited at the China Center for Type Culture Collection, with accession number: CCTCC No: M 2018252; Pseudomonas molluscum Pseudomonas mosselii Strain 923 was fermented in TSB medium at 30°C and 220 rpm for 36 h, and pyrazolium triazine was synthesized by the pyrazolium triazine synthesis gene cluster; the *Pseudomonas moschata* strain was used. Pseudomonas mosselii Strain 923 contains a pyrazolium triazine synthesis gene cluster, the nucleotide sequence of which is shown in SEQ ID No.

1.

2. The method for high-yield pyrazole triazine according to claim 1, characterized in that, The Pseudomonas molluscum Pseudomonas mosselii 923 contains rsmY and rsmZ It can positively regulate the biosynthesis of pyrazolium triazine. rsmY and rsmZ The nucleotide sequences are shown in SEQ ID No. 16 and SEQ ID No. 17, respectively.

3. Genes psdA , psdB, psdC, psdD, psdE, psdF and psdG The application is characterized by, Gene psdA , psdB, psdC, psdD, psdE, psdF and psdG The presence of these genes simultaneously in the strain enables it to produce pyrazolium triazine. psdA , psdB, psdC, psdD, psdE, psdF and psdG The nucleotide sequences are shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14, respectively; Gene psdA , psdB, psdC, psdD, psdE, psdF and psdG It exists in Pseudomonas molluscum Pseudomonas mosselii 923 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number M 2018252.

Images