CN102533800A - A marine Streptomyces halogenase gene and its product, and a biosynthetic gene cluster of its modified product - Google Patents

Abstract

The present invention relates to the field of genetic engineering. The invention discloses a nucleotide sequence of a marine streptomyces halogenase gene, which is characterized in that nucleic acid containing a nucleotide sequence shown by SEQ ID NO.1 and an amino acid sequence shown by SEQ ID NO. 2; and (b) a nucleic acid having at least 75% sequence identity to the nucleic acid of while retaining the function of a halogenase. The invention adopts a method for establishing a Fosmid genome library, and utilizes a conservative probe of a halogenase gene to carry out PCR screening on the library to obtain a new halogenase gene. Compared with the traditional SuperCos vector, the Fosmid library is constructed without restriction enzyme cutting, so that the preference of enzyme cutting sites is avoided, and the Fosmid library is low in copy number and more stable. And (3) rapidly screening the Fosmid library by utilizing PCR (polymerase chain reaction), and successfully obtaining the full length of the halogenase gene and the gene cluster in which the halogenase gene is located. The gene cluster comprises 11 protein synthesis genes, and the sequences of the protein synthesis genes have higher homology with genes such as non-ribosomal polypeptide synthetase gene, regulatory gene, transfer protein gene and the like.

Description

A marine Streptomyces halogenase gene and its product, and a biosynthetic gene cluster of its modified product

technical field

The invention relates to the field of genetic engineering, in particular to the marine streptomyces halogenase gene and its product, and also to the biosynthetic gene cluster of the halogenase modified product. the

Background technique

Streptomyces xinghaiensis is a new species of Streptomyces xinghaiensis isolated and identified by Dalian University of Technology (a strain of Streptomyces xinghaiensis with broad-spectrum antibacterial activity, Chinese invention patent 200710158478.7), this strain has good resistance to drug-resistant gold Staphylococcus aureus MRSA strain activity, also has good activity to Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Candida albicans etc. prominent among all strains. The 16S rDNA sequence of Streptomyces marine has 98.1% and 97.5% homology with two closely related standard strains , S. flavofuscus NRRL B-8036 ^T and S. albiaxialis DSM 41799 ^T , respectively, but the genomic DNA-DNA hybridization The analysis showed that the correlation between this strain and the two standard strains was only 31.4% and 46.9%. In-depth research on marine Streptomyces found a complete gene cluster of halogenase genes and their modified products.

Halogenase is an important modifying enzyme for modifying many antibiotics, and plays an important role in the activity of many antibiotics. Since FADH _2- dependent halogenases are post-modifying enzymes in many antibiotic biosynthetic gene clusters, the gene sequences encoding such halogenases are coupled to the biosynthetic gene clusters of various antibiotics. In the process of biosynthesis, the halogenation reaction is carried out in the middle of the catalytic synthesis reaction, thereby affecting the biological activity of the final product. Using halogenase as a novel biocatalyst, new drug molecules can be produced for clinical treatment.

Halogenase can modify a variety of antibiotics, including tetracyclines, ANSAs, β-lactams, glycopeptides, etc. Among them, complestatin (chloropeptin II) is a special glycopeptide compound containing a halogenated modified structure. It is different from vancomycin and teicoplanin in that it does not contain a sugar group. It is composed of a linear non-ribosomal polypeptide ( non-ribosomal peptide, NRPS) is further formed by NRPS synthetase for phenol oxidation coupling to form a rigid cross-linked structure. As early as 1980, it was discovered that complestatin has a strong activity of inhibiting the alternative pathway of human complement. As one of the important immune defense systems of the human body, the overactivation of the complement system is related to many diseases such as rheumatoid arthritis, and severe atypical pneumonia (SARS) is also related to the overactivation of the complement system, so the development of high-efficiency and low-toxicity Anticomplement drugs are of great significance for the treatment of related diseases. The researchers also found that complestatin has a strong inhibitory activity of HIV-1 integrase. In addition, complestatin can also promote the autolysis of plasminogen, and also act as a neuroprotective molecule to prevent excitotoxic cell death caused by acute brain injury such as hypoxia-ischemia. Due to the variety of unique biological activities of complestatin, it also attracts people to carry out chemical synthesis research, but its chemical synthesis was not reported until 2010, but the chemical synthesis steps are many and difficult. In addition, the poor solubility of complestatin may also be a factor limiting its drug development, so the development of its analogues has important research significance. the

Contents of the invention

The object of the present invention is to provide a kind of marine Streptomyces halogenase gene, also provide its product in addition; The present invention also provides the biosynthetic gene cluster that utilizes halogenase gene modification product, its gene sequence and the biosynthetic gene sequence of complestatin have higher homology, but has its own unique genetic composition. the

The invention discloses a nucleotide sequence of a marine Streptomyces halogenase gene, which is characterized by:

(a), nucleic acid containing the nucleotide sequence shown in SEQ ID NO. 1; and

(b) A nucleic acid having at least 75% sequence identity with the nucleic acid of (a) while retaining the halogenase function.

A marine Streptomyces halogenase protein comprising a coding gene sequence of the present invention:

(a), a protein containing the amino acid sequence shown in SEQ ID NO. 2; and

(b) A protein derived from (a) that undergoes substitution, deletion or addition of one or several amino acids in the amino acid sequence of the protein in (a) and has a halogenase function.

The present invention also discloses the obtained gene cluster sequence of the modified product of marine Streptomyces halogenase, and a method for isolating and obtaining the sequence, including the following steps:

1) Use the Fosmid vector to construct a marine Streptomyces genome library with an insert fragment of about 35-40kb;

2) Transfect bacteria with the obtained library, spread on the plate, and pick the single clone on the plate to culture in the culture medium after the qualified library is identified;

3) Extract the DNA of the cultured single clone, amplify it by PCR, and detect the PCR amplification product to obtain a positive clone containing the halogenase gene; and sequence the positive clone to obtain the full length of the halogenase gene and Modify product gene cluster sequence;

4) Prediction of function of Streptomyces marine complestatin analogue gene cluster, and structure prediction of halogenase modification products: including analysis of non-ribosomal polypeptide synthetase protein functional domains, and analysis of gene cluster sequencing results encoding complestatin analogues.

The method for analyzing the structure prediction of the synthetic product of the modified product gene cluster of Streptomyces marine comprises:

,1) Analysis of the sequencing results of halogenase of Streptomyces marinum;

,2) Sequence analysis of the halogenase-positive plasmid of the marine Streptomyces fosmid library;

,3) Analysis of protein functional domains by non-ribosomal polypeptide synthetase of Streptomyces marine;

4) Analysis of the sequencing results of gene clusters encoding halogenase modified products in Streptomyces marinum.

The invention adopts the method of establishing a Fosmid genome library, uses the conserved probe of the halogenase gene to carry out PCR screening of the library, and obtains a new halogenase gene. Compared with the traditional SuperCos vector, the Fosmid library construction does not use restriction enzymes, which avoids the preference of enzyme cutting sites, and its copy number is lower and more stable. Using PCR to quickly screen the Fosmid library, the full length of the halogenase gene and its gene cluster were successfully obtained. The gene cluster includes a total of 11 biosynthetic enzymes, which are non-ribosomal polypeptide synthetase genes, regulatory genes and transporter genes and other genes with high homology. the

Description of drawings:

Figure 1 is a homologous alignment showing the amino acid sequences of Streptomyces marine and proteins with known crystal structures;

Figure 2 is a phylogenetic tree showing the amino acid sequence of the marine Streptomyces halogenase;

Figure 3 is a comparison of the complestatin-like compound gene cluster in marine Streptomyces and the complestatin gene cluster in S. lavendulae ;

Figure 4 shows the alignment of the structural modules of Streptomyces marine non-ribosomal polypeptide synthase (NRPS) with S. lavendulae ;

Figure 5 shows the prediction of the primary structure of the non-ribosomal polypeptide synthase (NRPS) of Streptomyces marinum.

Fig. 6 is a functional annotation table of the biosynthetic gene cluster of halogenase modified products of Streptomyces marinum.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. the

In the present invention, the genome of Streptomyces marinum is interrupted by a DNA breaker (Hydro-Shear 0703, GeneMachine, USA) to obtain a fragment with a main band of 35-40kb, and the Klenow fragment is used to fill in the end, and the phenol-chloroform The purified DNA fragments were extracted and precipitated with ethanol, and the purified DNA fragments were confirmed by pulse-field electrophoresis, and then ligated into the Fosmid vector provided by Copycontrol Fosmid Library Production Kit (Epicentre, USA). In vitro packaged with phage packaging protein (Copycontrol Fosmid Library Production Kit, Epicentre), transfected into Escherichia coli EPI300, DNA was extracted by alkaline lysis, identified by Not I enzyme digestion, and the length of the inserted fragment was detected by pulse field electrophoresis. Determine whether the library is qualified, so as to construct and obtain the marine Streptomyces genome library.

The marine Streptomyces genome library was screened with halogenase-specific primers, and 13 positive single clones were finally obtained. The bidirectional end sequencing of these 13 positive single clones was performed, and one of them was selected for full sequencing. After analyzing the sequence results, the streptomyces marinum halogenase gene and its modified Complestatin similar gene cluster were obtained. the

Example 1: Isolation of the halogenase gene of Streptomyces marinum and the acquisition of its modified product gene cluster. the

Extraction of marine Streptomyces genomic DNA:

Use TBS medium (in g/L, tryptone 17, soybean peptone 3, sodium chloride 3, glucose 2.5, dipotassium hydrogen phosphate 2.5, pH 7.5) at 28°C, 150rpm for 48 hours. Collect more than 10ml of actinomycetes cells, resuspend the cells in 5ml SET buffer, add an appropriate amount of 0.1mm glass beads and vortex. Digest with 1ml of 20mg/ml lysozyme at 37°C for more than 2h, then add 500 μl of 15mg/ml proteinase K, and react at 37°C for 30min. Then add 1/10 volume of 20% SDS solution and react at 55°C for 1h. Use 1/3 volume of 5 M NaCl solution, react at room temperature for 30 min, then add saturated phenol/chloroform at a volume ratio of 1:1, mix well, and react at room temperature for 30 min. Centrifuge at 8000 r/min at 4°C for 20 minutes, discard the protein precipitate, and add an equal volume of isopropanol to the supernatant, and place it at room temperature for 30 minutes. Use a pipette tip or capillary to detach the precipitated DNA, and wash the DNA several times with 70% ethanol. Finally, the ethanol was evaporated to dryness, and the obtained DNA was dissolved in ddH2O.

Acquisition of the marine Streptomyces halogenase gene sequence:

Since the sequence of the gene cluster encoding halogenase in the Streptomyces marine genome is unknown, an attempt was made to retrieve the gene with an amalgamative primer, and the amino acid sequence of the published actinomycete halogenase was searched at the NCBI National Center for Biological Information, and the Clustal W software was used to carry out the analysis. homologous comparison. According to the comparison results, the online CODEHOP primer design software http: http://bioinfo.weizmann.ac.il/blocks/codehop.html, Designing degenerate primers.

Table 1 CODEHOP degenerate primers used to amplify Streptomyces marine halogenase gene Primer name Primer sequence Halo-F* 5'-GACGTGGTGGTGGTCggngggnggncc-3' Halo-R* 5'-CATGGCGGAGTAGGTGgcnarrtgnac-3'

*Note: In degenerate primers, y stands for CT; w stands for AT; s stands for CG; r stands for AC; n stands for ACGT.

After synthesizing CODEHOP degenerate primers, Genomic DNA of Streptomyces marine was used as a template to perform Touchdown PCR to amplify the halogenase gene and sequence the PCR product to obtain a sequence of about 960 bp of Streptomyces marine halogenase gene. the

The PCR reaction system is shown in Table 2&3:

Table 2 PCR reaction system

*Note 1: Primer F* stands for Halo-F or sinH-F; Primer R* stands for Halo-R or sinH-R;

*Note 2: The purpose of adding DMSO is to reduce the secondary structure between DNA with high G+C % content and fully denature DNA.

Table 3 Touchdown PCR reaction program temperature time Cycles 94°C 3min 1 94°C 30sec 64-56°C* 40sec 30 72°C 1min 72°C 10min 1

*Note: The Touchdown annealing temperature starts at 64°C and decreases by 2°C every 6 cycles until it reaches 56°C.

Prepare Genomic Library Inserts:

Take an appropriate amount of genomic DNA of Streptomyces marinum, and use a DNA fragmentation instrument (Hydro-Shear 0703, GeneMachine, USA) to break up the genomic DNA, then separate the fragmented genomic DNA by pulse-field electrophoresis, and recover by cutting the gel under the condition of avoiding ultraviolet radiation 35 ~ 40kb fragments.

The ends of the DNA fragments are blunted and ligated with the Fosmid vector:

The recovered DNA fragments were blunted with Klenow fragments, and the DNA fragments were purified by phenol-chloroform extraction and ethanol precipitation. The purified DNA fragments were confirmed by pulse-field electrophoresis and connected to Copycontrol Fosmid Library Production Kit (Epicentre, USA). Fosmid carrier.

Library packaging, transfection, plate coating, identification:

In vitro packaged with phage packaging protein (Copycontrol Fosmid Library Production Kit, Epicentre), transfected into Escherichia coli EPI300, and spread on LB plates containing chloramphenicol. Randomly pick 24 single clones to inoculate, cultivate overnight, extract DNA by alkaline lysis, then use NotI for enzyme digestion identification, and pulse field electrophoresis to detect the length of the inserted fragment. Judge whether the library is qualified or not according to the results of plate plating and the length of the inserted fragment.

Screening of Genomic Libraries:

Since the degenerate primers have a certain chance of non-specific binding to the template DNA and producing a dimerization structure, a pair of specific primers for genomic library screening was redesigned. The size of the PCR product is about 580bp, which can reduce the time spent in PCR amplification while improving the sensitivity and accuracy of screening.

Table 4 Specific primers used for screening of Streptomyces marine genome library Primer name Primer sequence sinH-F 5'-TGACGGCCATGCAGGGACA-3' sinH-R 5'-TCGAACGCCACCGACAGGA-3'

Pick a single clone with a sterile toothpick, add 1.5 mL of LB medium containing chloramphenicol 12.5 μg/mL and shake overnight at 37 °C. Take an appropriate amount of each sample, add glycerol with a final concentration of 20%, and store at -70°C. At the same time, take another 25 μL of each sample, mix every 24 single clones into a group, and then mix every 8 groups into a large group, boil the mixed sample at 99°C for 10 minutes to destroy the bacteria, take an appropriate amount as a template, and follow the table The PCR reaction systems in Table 2 and Table 3 were subjected to Touchdown PCR, and the PCR products were detected by gel electrophoresis.

Among the 15 large groups of samples, 10 sample groups capable of amplifying PCR products were detected, and the 8 groups under the large group of positive samples were further tested by PCR, and the 24 groups of the positive samples were tested separately. The clones were tested by PCR, and finally 13 positive single clones were obtained, and the two-way terminal sequencing of these 13 positive single clones was performed, and one of them was selected for full sequencing. the

Example 2: Analysis of sequencing results of gene clusters encoding Complestatin-like Compound in marine Streptomyces

Sequencing Results and Analysis of Halogenase from Streptomyces marinum

Halogenase gene sequence

> sinH SEQ ID NO. 1

ATGACCCGCCGGGTGACAAGGGGAGGAGGGATGGCCTTGCCGGATTCCGAGGAATTCGATGTGGTGGTCGTCGGTGGAGGGCCCGCCGGATCGACGCTGGCCGCGTTGACGGCCATGCAGGGACACCGGGTGCTGGTCCTGGAGAAGGAGTTCTTCCCCCGTCACCAGATCGGGGAGTCGCTCCTGCCGGCCACCGTGCACGGCGTGTGCCGGCTGACCGGCGTGGCGGACGAGCTCGCCGCCGCGGGCTTCCCGCGCAAGCGCGGCGGCACGTTCAAGTGGGGCGCCAACCCCGAGCCGTGGACCTTCTCCTTCTCCGTCTCCCCGCGCATGACCGGGCCGACGTCCTACGCCTACCAGGTCGAGCGGGCCAAGTTCGACGAGATCCTGCTCAACAACGCCCGCCGGGTGGGCGCCGAGGTGCGCGAGGGCTGTGCCGCCGTCGACGTCGTCGAGGACGGGGAGCGGGTCCGGGGCGTCCGGTACACCGACGCCGACGGCCGCGAGCACCGGGCGTCGGCCACGTTCGTCGTGGACGCCTCCGGCAACGGAAGCCGGCTGTACCGGCGGGTGGGCGGAACCCGGGAGTACTCGGAGTTCTTCCGCAGCCTGGCCCTGTACGGCTACTTCGAGGGCGGCAAGCGGCTGCCGGAACCGAACTCGGGCAACATCCTGTCGGTGGCGTTCGAGAGCGGCTGGTTCTGGTACATCCCGCTGAGTCCGGACCTCACCAGCGTCGGTGCCGTGGTCCGCCGGGAGATGGCCGGCAAGATCCGGGGCGACTCCGGCAAGGCGCTGGCGGCGCTCATCGCCGAGTGCCCCCTGATCTCCGAGTACCTGGCGGACGCGCGGCGGGTCACCGAGGGCCCGTACGGGAAGCTCCGGGTCCGCAAGGACTACTCGTACCACCACACGACCTTCTCGCGGCCCGGCATGATCCTGGTCGGCGACGCTGCCTGCTTCGTGGACCCGGTGTTCTCCTCCGGCGTCCACCTGGCCA CCTACAGCGCCCTGCTGGCGGCCCGCTCCATCAACAGCGTGCTCGCCGGGCTGGTCGGCGAGGACCGGGCCCTGCGGGAGTTCGAGTCCCGTTACCGCCGCGAGTACGGCGTCTTCTACGAGTTCCTGCTCTCCTTCTACGAGATGCACCAGGACGAGAACTCCTACTTCTGGCAGGCCAAGAAGGTCACCCGGGCCAACCGCCCGGAGCTGGAGTCGTTCGTCGAGCTCATCGGCGGGGTCTCCTCCGGCGAGCGGGTCCTGACGGACGCCGAGGTGCTGGCGAAGCGCTTCAGCTCGGGCTCCGCGGAGTTCGCCGCGGCCGTCGACGAACTCGCGGGCAGCGAGGACGGCAGCATGGTGCCGCTGTTCAAGTCCTCGGTGGTGCGCGAGGTCATGCAGGAGGGCGGCCAGGTCCAGATGCGCGCCCTGCTCGGCGAGGACGCCGAACCCGAGGCCCCCCTGTCCGCGGACGGCCTGGTGCCGTCCCCCGACGGCATGTTCTGGCTGCCCGCCCAGGGCACCGGCGAGTAG

Amino acid sequence encoded by halogenase

>SinH SEQ ID NO. 2

MTRRVTRGGGMALPDSEEFDVVVVGGGPAGSTLAALTAMQGHRVLVLEKEFFPRHQIGESLLPATVHGVCRLTGVADELAAAGFPRKRGGTFKWGANPEPWTFSFSVSPRMTGPTYAYQVERAKFDEILLNNARRVGAEVREGCAAVDVVEDGERVRGVRYTDADGREHRASATFVVDASGNGSRLYRRVGGTREYSEFFRSLALYGYFEGGKRLPEPNSGNILSVAFESGWFWYIPLSPDLTSVGAVVRREMAGKIRGDSGKALAALIAECPLISEYLADARRVTEGPYGKLRVRKDYSYHHTTFSRPGMILVGDAACFVDPVFSSGVHLATYSALLAARSINSVLGLVGEDRALREFESRYRREYGVFYEFLLSFYEMHQDENSYFWQAKKVTRANRPELESFVELIGGVSSGERVLTDAEVLAKRFSSGSAEFAAAVDELAGSEDGSMVPLFKSSVVREVMQEGGQVQMRALLGEDAEPEAPLSADGLVPSPDGMFWLPAQGTGE

The sequencing results were analyzed, and the DNA sequence analysis open reading frame (ORF) analysis was performed using the ORF Finder function provided by the National Center for Biological Information (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) . The DNA homology analysis was compared with various DNA databases using the worldwide Blast engine (http://www.ncbi.nlm.nih.gov/BLAST/) provided by the National Center for Bioinformatics of the United States.

For amino acid sequence analysis, the amino acid sequence homology analysis of the target protein is carried out using the worldwide Blast engine (http://www.ncbi.nlm.nih.gov/BLAST/) provided by the National Center for Biological Information of the United States and each amino acid The amino acid sequences in the sequence database were compared. The analysis of the conserved domain of the amino acid sequence of the target protein was carried out through the NCBI Conserved Domain Database search of the National Center for Bioinformatics (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The amino acid sequence alignment analysis among different proteins uses the software BioEdit. the

The comparison between the streptomyces marinum halogenase SinH and the protein with known crystal structure (Fig. 1), which is very similar to the amino acid of FADH ₂ -dependent halogenase, and its 30th-35th amino acid is the FAD binding site ( GGGXXG), the 243-249th amino acid is the tryptophan residue binding site (GWTWXIP). Thus, it can be confirmed that it is a fragment of the FADH _2- dependent halogenase gene. According to the homology comparison with the amino acid sequence of halogenase with known crystal structure, the halogenase of Streptomyces marinum has the highest homology with the halogenase CndH in the chondrochloren biosynthesis gene cluster, with 75% homology. The homology between Streptomyces marinum halogenase and the halogenase gene CmlS in the chloramphenicol biosynthesis gene cluster is not high, only 55%, and the homology with the more studied PrnA subclass halogenase is even lower, only 37.6% %. Therefore, the halogenase sequence of Streptomyces marinum S187 is generally different from the known crystal structure halogenase sequence, and it is presumed to be a new halogenase gene.

The phylogenetic tree of the amino acid sequence of Streptomyces marinum halogenase is shown in Figure 2. The selected halogenase is the expression product of the halogenase gene in the recently reported antibiotic biosynthesis gene cluster, and the black color represents the protein with crystal structure. It can be seen that SinH is most similar to the protein ComH in the complestatin gene cluster. the

Example 3

The gene and product thereof of embodiment 2 are detected and analyzed as follows:

(1) Sequence analysis of the halogenase-positive plasmid of the marine Streptomyces fosmid library:

Combining the halogenase-positive Fosmid plasmid sequencing results with the 454 and Solexa high-throughput sequencing of the aforementioned marine Streptomyces, the sequence was analyzed, and the DNA sequence analysis open reading frame (ORF) analysis was provided by the National Center for Bioinformatics of the United States. ORF Finder function (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The DNA homology analysis was compared with various DNA databases using the worldwide Blast engine (http://www.ncbi.nlm.nih.gov/BLAST/) provided by the National Center for Bioinformatics of the United States.

After annotating the 42.81kB sequencing information (Table 2) and comparing it with the gene cluster of complestatin in S. lavendulae (Figure 3), it can be predicted that there is a gene cluster homologous to the compound complestatin that synthesizes and has anti-complement activity in Streptomyces marine Highly sexed gene clusters.

Gene sequences of compounds modified by halogenase from Streptomyces marinum

>Complestatin-like gene cluster from S. xinghaiensis SEQ ID NO. 3

TAG

(2) Analysis of protein functional domains by Streptomyces marine non-ribosomal polypeptide synthetase:

Submit the sequences encoding non-ribosomal polypeptide synthetases in the Complestatin-like Compound gene cluster of Streptomyces marine to the online analysis system of the Pfam protein family database of Sanger College, UK (http://pfam.sanger.ac.uk/search), Protein domain analysis of a non-ribosomal polypeptide synthase from Streptomyces maritimus. By comparing with the non-ribosomal polypeptide synthetase in the Complestatin gene cluster in Streptomyces lavender S. lavendulae (Figure 4), it was found that the SinC gene in the non-ribosomal polypeptide complex in Streptomyces marine The ComC gene lacks a methylation functional domain, and it is preliminarily predicted that the non-ribosomal polypeptide synthase in Streptomyces marine is different from the non-ribosomal polypeptide synthase in the Complestatin gene cluster in S. lavendulae , and the primary structure may be different from that of Complestatin-like Compound of Complestatin.

(3) Structure prediction of the synthetic product of the Complestatin-like Compound gene cluster of Streptomyces marinum

In order to further determine the structure of the Complestatin-like Compound produced by the gene cluster in Streptomyces marine, we submitted the sequence encoding the non-ribosomal polypeptide synthase in the Complestatin-like Compound gene cluster of Streptomyces marine to the online NRPS&PKS product structure prediction website http ://dna.sherman.lsi.umich.edu/, the primary structure prediction of the possible synthetic product structure of the marine Streptomyces Complestatin-like Compound gene cluster. And open and view the predicted results through ChemDraw software (Figure 5).

The prediction results show that the amino acid sequence of the polypeptide synthesized by the Complestatin-like Compound gene cluster of Streptomyces marine is Hpg→Trp→Ala→Hpg→Leu→Tyr→Hpg, which is different from the Hpg→Trp of the Complestatin primary polypeptide structure (Figure 6). →Hpg→Hpg→Hpg→Tyr→Hpg. This may indicate that the gene cluster in marine Streptomyces may encode a primary structure different from Complestatin. the

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Claims (5)

1. marine streptomyces chlB4 gene nucleotide series is characterized in that:

(a), the nucleic acid that contains nucleotide sequence shown in the SEQ ID NO. 1; With

(b), have at least 75% sequence homogeny, kept the nucleic acid of chlB4 function simultaneously with (a) said nucleic acid.

2. marine streptomyces chlB4 protein that comprises coding gene sequence is characterized in that:

(a), the protein that contains the aminoacid sequence shown in the SEQ ID NO. 2; With

(b), in the proteinic aminoacid sequence of (a) through replacement, lack or add one or several amino acid and have the chlB4 function by (a) deutero-protein.

3. obtain marine streptomyces halo enzyme full length gene with and the method for modified outcome gene cluster sequence, it is characterized in that: may further comprise the steps:

Insert the marine streptomyces genomic library that fragment is about 35-40kb with the Fosmid vector construction;

With the library transfection bacterium that is obtained, dull and stereotyped coating, the mono-clonal after the evaluation library is qualified on the picking flat board is cultivated in substratum;

Extract the monoclonal DNA that cultivates, PCR increases, and the PCR amplified production is detected, and obtains to contain the positive colony of halo enzyme gene; With this positive colony is checked order, obtain this halo enzyme full length gene and modified outcome gene cluster sequence;

Marine streptomyces complestatin analogue gene bunch function prediction, and chlB4 modified outcome structure prediction: comprise the analysis of non-ribosomal polypeptide synthetase albumen domain, and the gene cluster sequencing result of coding complestatin analogue is analyzed.

4. acquisition marine streptomyces halo enzyme full length gene according to claim 3 with and modified outcome gene cluster sequence method, it is characterized in that, comprising:

Marine streptomyces chlB4 sequencing result is analyzed;

The sequence results analysis of marine streptomyces Fosmid library halo enzyme positive plasmid;

Marine streptomyces non-ribosomal polypeptide synthetic enzyme carries out the analysis in protein function territory;

The gene cluster sequencing result of coding chlB4 modified outcome is analyzed in the marine streptomyces.

5. the gene cluster of coding chlB4 modified outcome in the marine streptomyces is characterized by:

(a), the nucleic acid that contains nucleotide sequence shown in the SEQ ID NO. 2; With

(b), have at least 75% sequence homogeny, kept the nucleic acid of synthetic similar structures compound simultaneously with (a) said nucleic acid.

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