CN1733921A - Nikemycin biosynthetic controlling gene and proteins encoded thereof and expression engineering bacteria - Google Patents

Abstract

The invention discloses nikemycin biosynthetic pathway specificity regulatory gene and proteins encoded and application.Gene of the present invention is one of following nucleotide sequences: 1) the SEQ ID № in the sequence table: 1; 2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences; 3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding; 4) under the rigorous degree condition of height can with the SEQ ID № in the sequence table: 1 hybridization sequence.Coded protein is to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 is identical active by SEQ ID №: 2 deutero-protein.The engineering bacteria that structure contains this gene multiple copied can effectively improve the output of bacterial strain nikemycin, and is significant to the production application of nikemycin.

Description

Nikemycin biosynthetic controlling gene and proteins encoded thereof and expression engineering bacteria

Technical field

The present invention relates to microbiotic biosynthetic controlling gene and proteins encoded thereof and application, the engineering bacteria that particularly relates to nikemycin biosynthetic pathway specificity regulatory gene and proteins encoded thereof and this gene of expression, and this gene and the application of engineering bacteria in nikemycin is produced.

Background technology

Nikemycin belongs to the nucleosides peptide antibiotics, is a kind of in acyl group and the glycosyl ucleosides, with the natural substrate UDP-N-acetylglucosamine similar of chitin synthetase, is the strong competitive inhibitor of this enzyme.Because chitin is fungal cell wall and insect and the ectoskeletal important composition composition of other invertebrates, therefore, nikemycin is by competitive inhibition chitinous synthetic effectively suppress fungi, insect and acarid, and nontoxic or toxicity is extremely low and easily degrade at occurring in nature to Mammals, honeybee and plant, be a kind of comparatively ideal agricultural antibiotic.Field test proof nikemycin all has good preventive and therapeutic effect to a lot of Plant diseasess as wheat powdery mildew, cucumber timberrot, leaf muld of tomato, tobacco red spot disease, apple zonate spot etc.Simultaneously, studies show that nikemycin can effectively suppress human body deep infection fungi---Candida albicans (Candida albicans), Nikemycin Z has tangible curative effect as oral pharmaceutical to coccidioidomycosis and blastomycosis.At present, increasing people is faced with the life-threatening fungi infestation that causes owing to autoimmune disease (as AIDS patient, organ transplantation patient and accept the cancer patients of chemotherapy), therefore, as the antifungal drug nikemycin huge application and potentiality to be exploited are arranged.

Studies show that antibiotic biosynthesizing relates to the biochemical reaction of a series of complexity, relevant therewith gene cluster is arranged, and their expression is subjected to multilevel multi-level regulation and control.Usually the biosynthetic regulation and control of microbiotic are mainly carried out three levels: the regulation and control of (1) approach specificity; (2) all relevant with the form differentiation with microbiotic multiple-effect is regulated and control; (3) regulation and control of overall importance.Approach specificity regulatory gene (Antibiotic-Specific Regulatory Genes) is only regulated and control certain antibiotic biosynthesizing specifically, and transcribing of they regulated and control in the general and microbiotic biosynthesis gene cluster existence of these regulatory genes.The most antibiotics biological synthesis gene cluster all comprises approach specificity regulatory gene, has plenty of one, has plenty of two or more.The streptomycete of natural generation nikemycin present known have streptomyces tendae (S.tendae) and streptomyces ansochromogenes (S.ansochromogenes).It once was that research material carries out the nikemycin related work with the streptomyces tendae that beguine university Bormann laboratory is told by Germany; And being material with the streptomyces ansochromogenes, Institute of Microorganism, Academia Sinica doctor Tan Huarong laboratory carried out the biosynthetic molecular biology research of relevant its nikemycin.Though the biosynthesizing to nikemycin has had preliminary research, still belong to blank so far to its Study of Mechanism.

Summary of the invention

The purpose of this invention is to provide a kind of nikemycin biosynthetic pathway specificity regulatory gene and proteins encoded thereof.

Nikemycin biosynthetic controlling gene provided by the present invention derives from streptomyces ansochromogenes (Streptomyces ansochromogenes), and called after sanG is one of following nucleotide sequences:

1) the SEQ ID № in the sequence table: 1;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding;

4) under the rigorous degree condition of height can with the SEQ ID № in the sequence table: 1 hybridization sequence.

The rigorous degree condition of described height is meant at 68 ℃, the strict elution requirement of twice of 15 minutes wash-out among the 0.1XSSC (containing 0.1%SDS).

SEQ ID № in the sequence table: 1 dna sequence dna length is 5073bp, contain a complete open reading frame (ORP), size is 3186-bp, and initiator codon is the ATG of the 1767th beginning of this sequence 5 ' end, and terminator codon is the TGA of the 4952nd end of this sequence 5 ' end.

Modulin SanG by this sanG genes encoding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 is identical active by SEQ ID №: 2 deutero-protein.

SEQ ID № in the sequence table: the 2nd, the protein of forming by 1061 amino-acid residues.

The application of engineering bacteria in nikemycin is produced that another object of the present invention provides the sanG gene and expresses this gene.

Contain expression carrier of the present invention and engineering bacteria and all belong to protection scope of the present invention; utilize existing molecular biological method can obtain containing the expression vector of sanG gene multiple copied; as recombinant plasmid pKC1139 ∷ sanG and utilize expression vector to obtain engineering bacteria by conversion; as streptomyces ansochromogenes (Streptomycesansochromogenes) NK2; this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 07 05th, 2004, preserving number is CGMCC № 1181.

Nikemycin biosynthetic pathway specificity regulatory gene sanG has been cloned in the present invention first from streptomyces ansochromogenes, and it has been carried out sequencing and analysis.Analytical results shows, the product of this coded by said gene and multiple microbiotic biosynthetic controlling albumen (SARPs) have certain homology, but its coded albumen (containing 1061 amino-acid residues) is big more a lot of than general microbiotic biosynthetic controlling albumen (containing 200-400 amino-acid residue), is the new microbiotic modulin of a class.Show that by gene functional research sanG destroys strain and lost the generation ability of nikemycin, and the sanG gene of introducing a copy again can make nikemycin produce again, shows that this gene is a positive regulating gene.The inventor imports the sanG gene of a plurality of copies in the streptomyces ansochromogenes wild strain simultaneously, makes up and has obtained a strain nikemycin high yield engineering bacteria, can make nikemycin output improve 2-3 doubly.The research of gene of the present invention, development and application are extremely important to the production application of nikemycin.

Description of drawings

Fig. 1 is the design of graphics of sanG gene high expression carrier pKC1139 ∷ sanG;

Fig. 2 is that the activity of engineering strain nikemycin detects photo;

Fig. 3 is the production curve of engineering strain nikemycin;

Fig. 4 is the HPLC analysis of spectra of engineering strain nikemycin;

Fig. 5 is the physics spectrogram of plasmid pUC119 ∷ kmr;

Fig. 6 is the physics spectrogram of plasmid pGL001.

Embodiment

The clone of embodiment 1, sanG gene and sequential analysis

1, the preparation of clone's probe

Utilize XhoI-BamHI digested plasmid pZH1300 (Li, W., and Tan, H.Structure and functionof sanV:a gene involved in nikkomycin biosynthesis of Streptomycesansochromogenes.Curr.Microbiol.2003.46 (6): 403-407.), carry out polyacrylamide gel electrophoresis according to a conventional method, reclaim the dna fragmentation (this fragment contains part sanV gene) of 1.7kb then with the DEAE embrane method; The reclaim dna fragmentation that obtains is carried out the on-radiation digoxigenin labeled according to a conventional method, and as the probe of gene clone, the nucleotides sequence of this probe is classified the SEQ ID № in the sequence table as: 3.

2, make up portion gene group library

Inoculation streptomyces ansochromogenes 7100 (Tan Huarong, Wu Wei, Tian Yuqing etc. streptomyces ansochromogenes differentiation and characteristic research thereof. the microorganism journal, 1994,34:398-402) spore suspension is in the YEME liquid nutrient medium, and shaking table was cultivated 48 hours, centrifugal collection thalline, and extract with ordinary method and to obtain the total DNA (Kieser of streptomyces ansochromogenes, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. (2000) PracticalStreptomyces Genetics.Norwich:The John lnnes Foundation.).

The total DNA of digestion with restriction enzyme streptomyces ansochromogenes with different carries out agarose gel electrophoresis, carries out Southern hybridization with above-mentioned clone's probe then, and wherein the XhoI enzyme is cut the signal band that product obtains a 7.5kb.Cut the total DNA of streptomyces ansochromogenes with a large amount of enzymes of XhoI, carry out agarose gel electrophoresis, reclaim the dna fragmentation of size about 7.5kb, get partially recycled dna fragmentation and carry out Southern hybridization once more, the result shows among the DNA that is reclaimed and contains target DNA.

The pBluescriptII carrier (available from Stratagene company, the U.S.) that the DNA and the XhoI enzyme of recovery are cut connects according to a conventional method, adopts electrotransformation Transformed E .coli DH5a then, is built into portion gene group library.

3, the clone of sanV gene downstream 7.5kb dna fragmentation

XhoI-BamHI dna fragmentation with the resulting 1.7kb of step 1 is probe (the SEQ ID № in the sequence table: 3), adopt colony hybridization method that the portion gene library of streptomyces ansochromogenes 7100 is screened, wherein 1 recombinant plasmid obtains positive signal, called after pGL001.This carrier enzyme is cut the result and is shown, wherein contains the XhoI dna fragmentation of the 7.5kb that comprises part sanV gene.

4, sanG Gene Sequence Analysis

Dna sequencing method is routinely carried out sequencing to the 7.5kb dna fragmentation, and it does not comprise that the nucleotides sequence of part sanV gene classifies the SEQ ID № in the sequence table as: 1, and length is 5073bp.With FramePlot software this sequence is analyzed, the result shows that G+C% is about 72.2% in the whole nucleotide sequence, has typical streptomycete codon use characteristic, wherein contain a complete open reading frame (ORP), size is 3186-bp, initiator codon is the ATG of the 1767th beginning of this sequence 5 ' end, and terminator codon is the TGA of the 4952nd end of this sequence 5 ' end, with its called after sanG.

The sanG protein of being made up of 1061 amino-acid residues (SanG) of encoding, its amino acid residue sequence is the SEQ ID № in the sequence table: 2.In the protein sequence database of NCBI, carry out homology relatively, the result shows that the microbiotic biosynthetic controlling albumen (SARPs) of Protein S anG and streptomycete has higher homology, but this albumen (containing 1061 amino-acid residues) is big more a lot of than general microbiotic biosynthetic controlling albumen (containing 200-400 amino-acid residue), also have a typical helix turn helix (helix-turn-helix at its aminoterminal, HTH) DNA is the novel microbiotic modulin of a class in conjunction with territory (from 258 Leu of the 99th Arg to the of N end).

The functional study of embodiment 2, sanG gene

1, makes up the recombinant plasmid pGLD020 that is used for gene disruption

With XhoI digested plasmid pGL001, reclaim the dna fragmentation of 7.5-kb size, subclone makes up and obtains recombinant plasmid pGLD021 to the salI site of the pUC19 that has deleted the BamHI site.Cut pGLD021 with the BamHI enzyme, mend flat end with Klenow then, reclaim the fragment of this 6-kb size; Use BamHI and EcoRI double digestion recombinant plasmid pUC119 ∷ kmr (physics spectrogram such as Fig. 5 of this plasmid) simultaneously, mend flat end, reclaim the kalamycin resistance gene fragment of about 1.0-kb through Klenow; Connect above two dna fragmentations, will connect product Transformed E .coli DH5 α, extract plasmid from transformant, enzyme obtains correct recombinant plasmid pGLD022 after cutting checking.Further use HindIII and EcoRI double digestion pGLD022, reclaim the dna fragmentation of 4.4-kb and be cloned into pKC1139 (Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. (2000) PracticalStreptomyces Genetics.Norwich:The John lnnes Foundation.) on the multiple clone site, makes up the recombinant plasmid pGLD020 that obtains being used for gene disruption.Operations such as above enzyme is cut, reclaims, cloned, conversion are all carried out according to the experimental methods of molecular biology of routine.

2, make up the sanG blocked mutant

At first plasmid pGLD020 is transferred among the E.coli ET12567, cultivate, extract the non-methylated DNA of acquisition, transform the protoplastis of streptomyces ansochromogenes 7100 then with this DNA, random choose wherein 6 transformants is forwarded to enrichment thalline among the YEME that contains apramycin (Apramycin), cut checking through plasmid extraction, enzyme, prove that the gained transformant all is correct.One of them transformant is forwarded on the minimum medium flat board that contains apramycin, cultivates after 7 days the preparation spore suspension for 28 ℃.After suitably diluting, with each plate 10 ⁴The concentration of individual spore is coated on the minimum medium flat board that contains kantlex, in 42 ℃ of high-temperature cultivation.PKC1139 has the responsive to temperature type replication orgin, in that be higher than can not normal replication under 34 ℃ of conditions.Therefore, being that the carrier pGLD020 of fundamental construction can not self-replicating in the time of 42 ℃ with pKC1139, having only when homologous recombination has taken place for sanG gene and flanking sequence thereof and streptomyces ansochromogenes 7100 karyomit(e)s---single cross is changed or the bacterial strain of double exchange could be grown containing on the minimum medium of kantlex.If double exchange has taken place, then kmr replaces the sanG gene, and this moment, bacterium colony showed as Km ^rAm ^sIf single cross has taken place changes, then whole pGLD020 is inserted on the karyomit(e), and this moment, bacterium colony showed as Km ^rAm ^rThe bacterium colony that grows xeroxed respectively containing Apramycin and containing on the MM flat board of Kanamycin, those are grown containing on the MM flat board of Kanamycin, and are that double exchange may take place for homologous gene on the karyomit(e) containing the bacterium colony of not growing on the MM flat board of Apramycin.6 plant mutant strains of random screening are seeded on the minimum medium without any selective pressure, cultivate down at 28 ℃, be forwarded to again on the minimum medium that contains kantlex and apramycin respectively after passing for 5 generations, the result still shows as Km ^rAm ^s, illustrate that these sanG deletion mutantion strains that obtain by double exchange are stable in heredity.

3, the Southern blotting of blocked mutant hybridization checking

Stable blocked mutant in the above-mentioned 6 strain heredity is inoculated in the YEME substratum, 28 ℃, the 250rpm shaking culture is extracted total DNA respectively after 30 hours, cut laggard row agarose gel electrophoresis with the XhoI enzyme, reclaim the dna fragmentation of 7.5kb, then dna fragmentation is carried out digoxigenin labeled.

Dna fragmentation with the 7.5kb of digoxigenin labeled is that probe carries out Southern blot hybridization, take place the positive signal band that total DNA presented of the mutant strain of double exchange should little 3.2kb than wild type strain about, theoretically infer that size is respectively 4.3-kb and 7.5-kb.Results of hybridization proves that the hybridization signal band of mutant strain is consistent with the molecular weight size of calculating in theory, illustrates that resulting mutant strain all is correct.

4, nikemycin biological detection and HPLC analyze

Correct blocked mutant and the wild type strain of Southern blotting hybridization checking is seeded in respectively in the YEME substratum, behind 30 ℃ of cultivation 36h, inoculum size with 1% is carried out Secondary Fermentation in the SP substratum, take a sample respectively at different incubation times, detect the rough content of nikemycin in thalline weight in wet base and the supernatant.Behind the filtering with microporous membrane of supernatant liquor through diameter 0.25 μ m, carry out HPLC according to a conventional method and analyze, showing does not have nikemycin in the supernatant, and surveys the thalline weight in wet base and wild type strain does not almost have difference at each time point.The result shows that sanG deletion mutantion strain no longer produces nikemycin, illustrates that the sanG gene is that the nikemycin biosynthesizing is necessary.

5, the complementation analysis of sanG deletion mutantion strain

For the phenotype of getting rid of sanG deletion mutantion strain is because this possibility that other gene point mutation caused on the karyomit(e) has been carried out the genetic complementation experiment of mutant strain.With BamHI digested plasmid pGL001, reclaim the dna fragmentation of 3.3-kb, be connected (Bierman with pSET152, M., Logan, R., O ' Brien, K., Seno, E.T., Rao, R.N., and Schoner, B.E.Plasmid cloning vectors for the conjugal transfer of DNA fromEscherichia coli to Streptomyces spp.Gene.1992.116:43-49), uses BglII and EcoRI double digestion then, reclaim big fragment wherein; With BglII and EcoRI double digestion pGL001, reclaim the dna fragmentation of 4.1kb, two segment DNA fragments are connected, be built into the recombinant plasmid pSET152 ∷ sanG that contains complete sanG gene and upstream promoter thereof.

Changing pSET152 ∷ sanG over to E.coli ET12567/pUZ8002, cultivate, extract the pSET152 ∷ sanG that obtains removing restriction modification, change over to then among the deletion mutantion strain, is selection markers screening transformant with the apramycin.Random screening 6 strain transformants ferment in the SP substratum, carry out the nikemycin biological activity assay then, the result shows that the mutant strain behind the sanG gene complementation has recovered the generation ability of nikemycin, illustrates that from another point of view the sanG gene is that the nikemycin biosynthesizing is necessary.

The structure of embodiment 3, nikemycin high-yielding engineering bacterial strain

1, the structure of the high copy of sanG gene recombinant plasmid pKC1139 ∷ sanG

The process of structure sanG gene high expression carrier pKC1139 ∷ sanG as shown in Figure 1.With XhoI digested plasmid pGL001 (physics spectrogram such as Fig. 6 of this plasmid), reclaim the dna fragmentation of 7.5-kb, this dna fragmentation contains complete sanG gene and promoter sequence thereof, this fragment is inserted the SalI site of vector plasmid pUC19, the plasmid that obtains is carried out double digestion with HindIII and EcoRI, reclaim the HindIII-EcoRI fragment of 7.5-kb; The dna fragmentation of gained is inserted into the multiple clone site of the multiple copied plasmid pKC1139 that can shuttle back and forth in E.coli and streptomycete, makes up and obtain the high copy of sanG gene recombinant plasmid pKC1139 ∷ sanG.

2, transform, screen the nikemycin high-yielding engineering bacterial strain

Change pKC1139 ∷ sanG over to E.coli ET12567/pUZ8002, cultivate, extract the pKC1139 ∷ sanG that obtains removing restriction modification, then pKC1139 ∷ sanG is changed in the wild type strain streptomyces ansochromogenes 7100, with the apramycin is selection markers screening transformant, obtains the nikemycin engineering bacteria.

Therefrom screening 3 strain engineering bacterias ferments in the SP substratum, carry out the nikemycin biological activity assay then, and compare with wild type strain 7100, the result as shown in Figure 2, B, C, D are engineering bacteria among the figure, 7100 is streptomyces ansochromogenes 7100, and 7100/pKC1139 is the control strain that contains empty carrier pKC1139.The result shows that the generation ability of 3 strain engineering bacteria nikemycins all is higher than wild type strain 7100.

Detect the output of engineering bacteria nikemycin with HPLC, and be contrast with streptomyces ansochromogenes 7100, the result as shown in Figure 3, Figure 4, B, C, D are engineering bacteria among Fig. 3,7100 is streptomyces ansochromogenes 7100, and 7100/pKC1139 is the control strain that contains empty carrier pKC1139; Fig. 4 A is engineering bacteria streptomyces ansochromogenes (Streptomycesansochromogenes) NK2 CGMCC № 1181 culture HPLC collection of illustrative plates, B is streptomyces ansochromogenes 7100 culture HPLC collection of illustrative plates (the X peak is a nikemycin X component in the HPLC spectrogram, and the Z peak is a Nikemycin Z component).The result shows that the nikemycin output of engineering bacteria significantly improves, for the 2-3 of wild strain doubly.

Sequence table

<160>3

<210>1

<211>5073

<212>DNA

＜213〉streptomyces ansochromogenes (Streptomyces ansochromogenes)

<400>1

ggatcctttg?cctccccgac?ggcacctgat?cgagcgggtg?ccgtgcgtgc?ggctgcgggt 60

cacgcgtccg?gttcggctcc?ggtcatcagg?gcgagcaggg?gtgtaagggg?cgtggtcgtc 120

gatcatggtg?gtgtagaagc?cgtcgtcgtc?cgcttccacc?tcggcggcgc?gggagaacgt 180

cacctgttcg?tgcgaggcga?gcgcggcttc?ggtgtcgccg?gggtgcgtgg?cgagggcgcg 240

gccgagtccg?gcgccgtccg?gcatggccgg?gttggcgccc?ttgccgttcg?gtgcccgcag 300

gcgggcggcg?tccccgagca?gcgtcacccc?ggccacgcga?cgcctcaccg?cctaccgtgc 360

acgaactcgc?cagggccggc?cgggccggct?tttacgcctg?caacgctccc?gaagggcagt 420

cggacctccg?aaaccgacaa?tatccacatt?tgttctgttt?tgttcttcct?ggtgcgtcaa 480

cctcatcccg?aatggccagg?tcgcggatac?atggagccac?tttaagtcac?ctggctcatt 540

cgcgttcgcc?cagctcagga?gaatgctcga?tacactgatt?ccggcgaggc?ataccagcct 600

cagaaaagca?ggggcatcgc?gaacaccatg?aacgcaaaca?tgtccgagcg?gaatggaaac 660

atcagccgac?tcaccacagc?cgacccgacc?gtccgtgccg?cagaccgcac?ccatttcccc 720

cctcccaccc?ccgaggggaa?gaggacacgc?aaggcaacca?cgacacaaca?ggaacccagg 780

ccgtccgggc?cctccgtacc?cgcccagaac?caggccgccg?caccggacgc?caccttcacg 840

accgcgcccg?tccccctctc?acgcggcaca?gcagtccgga?tacgacctcg?cggggcccgg 900

caagcccggc?cggccgggac?gcgctccgag?accgtgacga?aaggcgaccc?ctggggaggg 960

tgggctgtgc?gccagcggca?cagggctgag?gcgaccgctc?tcaggcaggc?agcgaggtca 1020

ctggctgaat?accaggcgct?ggcaagaaga?ggcactcgac?tatcaggcga?gacggcgtaa 1080

tggccggcag?ccgcaggggc?cgctggggcc?tccttgtcgc?gatgcggttc?ggcgtcggca 1140

atggctggta?ttggctgtgc?tcgggtgcgg?acgttgacac?gttaatcgtt?tccggcaatt 1200

ccgatttggc?caatcggcgt?ttgcgagccg?ggttgccgcc?gctccaggcg?gcagtagacg 1260

gcctttcggc?catttcagct?catgagctga?tcgagcggct?cgtcgcccat?ggccgcgttc 1320

ggatcaccca?cgctgatgat?gacgaggtcg?cggagtcccg?gcgcgtcgtc?gaccacgcga 1380

aacggcacgg?cctgagacca?gcgggcaagc?gcattgagaa?agttccgtac?ggcgggtcgg 1440

ggctggagct?cgtcataggg?ggttgttccg?gtcgccgcgg?gccggccgga?agcagaaccg 1500

ggcggcccgg?gtgttccgcg?agcccgggct?ccgcgcgcat?caggccgttc?ctgccctccg 1560

cgcccgccgg?tccctccggc?cggcacagcc?gcgcccctcc?tgaacgcggg?accggacgag 1620

cgggcacggc?cagctcactg?gccggtatcg?aaatcgccgt?gtcatggcgt?gttcagtgcg 1680

tcacttgatc?ggcaccgttt?cagcacctgc?cccgccctac?gaccgaccgc?ccgtcaagtg 1740

accgtcgaaa?ggcaagaacc?cccggcatgg?cgtacacagc?tcaagagcaa?caaaccgcgt 1800

ccctgacaga?tctgtcgttc?gaggctttcg?gtgtgatgac?ggctcggcgc?ggggccgagc 1860

aggtgcctct?cggtccccca?cggcaccggg?ccgtgctggg?actgctgctc?atccggctgg 1920

gccaagtggt?gcccgccgat?cagctcatcg?acgaattatg?gggcgacagc?ctccccagac 1980

gcccgcagcc?accctgcaga?acgtacctgt?cccacctgcg?gcgggtgctg?acggggcggc 2040

acgggccgat?cgccccgctg?cgctaccagg?cccccgggta?cgtcctgacg?gtggagccgg 2100

gcacgttcga?cctgtcccgg?ttcgagaccc?tcgtctccca?ggggcagcgc?catgtgtccg 2160

acggccggct?ccaggacgcc?cgcgacgcgt?tcgacaacgc?gctgcggtta?tggcgggcgg 2220

accccttcct?ggacctcgcg?gcctacaccc?cgctcgcgga?ggaggccgcc?cgcctgagcc 2280

tcctgcggac?caccgccgtc?gcggcccaag?ccgacacgct?cctcacgctc?ggcgaggcct 2340

gcgacgccgt?cgcgctgctg?cgccgcgagg?tgtccctcca?gcccatcgac?gaacgcctgg 2400

tcggcagcct?catgacgggc?ctgtaccgcc?tgggccggca?ggccgaggcg?ctccagctgt 2460

acgaccggac?gcgcgcctac?ctctccgagg?agctcggggt?cgccccggcc?cgcgagctcc 2520

agcgcgtcca?cctcgccctg?ctgcgccacg?aactcgacga?ccagacgtcc?gccccggccg 2580

ccgtcgaggt?acgcgtccgg?cccggcaccg?tggccgagcg?ggaggcggag?gggcccgcgc 2640

cggcagacgg?tccggaggcc?gtccccgagg?ccggacagcg?gggtacggca?ccggcggcct 2700

ggttcccctg?ccgcggcgac?gcccttgacg?ctctgcggtc?gtccatcacc?ggcgccctgc 2760

gcggcagcgg?gcacaccacc?gccgtcgtcg?gcgaggcggg?cgtcggcaag?accgaactgg 2820

tggcccgtgc?cacggatcag?acgcagccgc?cgcaaccgca?ggcggaggtc?atccgtgtca 2880

actgccgcag?cgacgagggg?attcccgtcg?gctgggtgtg?gcagcaggtg?ctgcgccggc 2940

tcgacatcct?gttcggggag?tccttcgacg?acctgcgcaa?gcagcgcgcc?gaagggcatc 3000

cggtgacaac?gctgtcggaa?cccccctcgg?accagatgga?cttcctcgcc?caggacgccc 3060

tgtgcgaagt?gatcctccag?tacgccgacg?gcaggcccct?cctgctcgtc?ctcgaagaca 3120

gtgcaccggc?ggaccgcctc?acgctcgacg?tgctggggct?gttctgcagc?cgcacgcagg 3180

ggcggcccgt?cagcgtggtg?atcacggtcc?gcgaacccgg?tctcggggcc?gggcccgaga 3240

ccgacggccc?gctcgtcgac?ctcctggccg?actcccgcac?caaggtcgtg?cacctggaca 3300

acctcaccga?ggactacgtc?cgggccgcgg?tcacggccca?ggcggggccc?ggcgtcgaag 3360

cgtccgtcgt?acgggcgctg?tacgagcgca?gcgccggcaa?cccctatctc?ctcgaccagt 3420

tgatcgccga?cgccggcggg?gcgcggcgcc?tgcacgatcc?ccgtgcggcc?gatgtcgtcc 3480

gcgccgggat?cccgacgggc?gtccgcagca?tgctgcgccg?gcagttcgcc?gggctgcccc 3540

cgcgcgtcct?gcacctcctc?cagtgctgcg?ccgtgctcgg?cggcgaggcg?acgctggcct 3600

cgctgaccgc?catgctggac?gacgaaggcg?ccggccacga?cgtcctggaa?gaggtcctgg 3660

cgaccgggct?catgcacagc?gaccgcgacg?acccgcaccg?gctcgtcttc?cgctacggcc 3720

tcatccgcga?cgtcctcctg?gcggagctgc?gccgccagga?gcgggcggcc?ctgcacgcgc 3780

gcgccgtcgg?cgtgctcggt?gcccgccacg?gcgactcccc?cgaggcgagc?gagcagatcg 3840

ccgagcacgc?ctggcaggcc?gtgctggccc?tgccggccga?ggacgtgctg?ccgcacctca 3900

gacgcgccgg?cgaacaggcc?gtcgtcgagg?gcgactaccg?gcgcgcgcag?acctggttcg 3960

agcacgcgca?caccttgctc?tgcgccctgc?cccgggacgg?tggcgcgcca?tcgaagcaga 4020

cgctggacct?gcgcagcaag?ctcctctaca?cggcgagcat?cacccgcggc?tacaccaacc 4080

gggaaggtga?aggccgaggc?cgggcgcatc?ccggaagctg?tacgccgcca?cgggcgggcc 4140

ggaccatcga?gcagggtgcc?ttgctgctgt?ggcagttcgt?ggccgaactg?gtcaccgccc 4200

ggcacaccga?gtgcgcccgg?cacgcggagc?agctgcgcgc?tctcaccgag?cggtccgacg 4260

ccccggaggt?ccggttctac?gagcggatcg?cccgcggtct?gctccagttg?cccagcgatg 4320

ccgccgacgc?gctcaccgcc?ctgaccgagg?cggtgcggat?cgccgagaag?ctgacccccg 4380

cacagcggga?gcgcatgacg?cgccgcgccc?agcaggatcc?gcggttcatg?gccgcgaacc 4440

atcgcgtcct?caccctgagg?ctgctcggcg?ccaccgacga?ggcgcgggcg?ctgtgcgagg 4500

agctgctggt?agccaccggc?ctggacggca?cgccggtcga?ccaggcgagc?gcgtactact 4560

tccactccct?gacagcggcc?ctgggcgagg?accccgacgg?ggcggcggcc?tccagcggcc 4620

ggggcctgga?catcgcccgc?gcccacgggc?tgagccactg?gaccgccatg?ctccaggtgt 4680

gccggagctg?ggcactccac?cagagcggcg?aggccgccgc?actcgacagc?ctggaatcgg 4740

ccgtcgccga?gctgcgcgag?cggcggctgc?tcatccggct?gccgctccac?ctgggcctgc 4800

tggcccacgc?ccagcacagc?agcggggcgg?ccgaagacgc?cagacggacg?ctgcggtcgt 4860

cgaccgcgga?ggccagatcc?cgcggggagt?tggcctatgt?gagccgcact?ctgccgttca 4920

cccggctgtg?tcccctgacg?tccgcttcgt?agcggccgga?tcgtcggttc?cttcgcctct 4980

cagccgggcg?cgggcctggc?cggcctcggg?gtggccgagt?ccgtcgaggg?tccgcgccgc 5040

ctcccgccac?agggcagggc?ggcggggcgg?tct 5073

<210>2

<211>1061

<212>PRT

＜213〉streptomyces ansochromogenes (Streptomyces ansochromogenes)

<400>2

Met?Ala?Tyr?Thr?Ala?Gln?Glu?Gln?Gln?Thr?Ala?Ser?Leu?Thr?Asp?Leu

1 5 10 15

Ser?Phe?Glu?Ala?Phe?Gly?Val?Met?Thr?Ala?Arg?Arg?Gly?Ala?Glu?Gln

20 25 30

Val?Pro?Leu?Gly?Pro?Pro?Arg?His?Arg?Ala?Val?Leu?Gly?Leu?Leu?Leu

35 40 45

Ile?Arg?Leu?Gly?Gln?Val?Val?Pro?Ala?Asp?Gln?Leu?Ile?Asp?Glu?Leu

50 55 60

Trp?Gly?Asp?Ser?Leu?Pro?Arg?Arg?Pro?Gln?Pro?Pro?Cys?Arg?Thr?Tyr

65 70 75 80

Leu?Ser?His?Leu?Arg?Arg?Val?Leu?Thr?Gly?Arg?His?Gly?Pro?Ile?Ala

85 90 95

Pro?Leu?Arg?Tyr?Gln?Ala?Pro?Gly?Tyr?Val?Leu?Thr?Val?Glu?Pro?Gly

100 105 110

Thr?Phe?Asp?Leu?Ser?Arg?Phe?Glu?Thr?Leu?Val?Ser?Gln?Gly?Gln?Arg

115 120 125

His?Val?Ser?Asp?Gly?Arg?Leu?Gln?Asp?Ala?Arg?Asp?Ala?Phe?Asp?Asn

130 135 140

Ala?Leu?Arg?Leu?Trp?Arg?Ala?Asp?Pro?Phe?Leu?Asp?Leu?Ala?Ala?Tyr

145 150 155 160

Thr?Pro?Leu?Ala?Glu?Glu?Ala?Ala?Arg?Leu?Ser?Leu?Leu?Arg?Thr?Thr

165 170 175

Ala?Val?Ala?Ala?Gln?Ala?Asp?Thr?Leu?Leu?Thr?Leu?Gly?Glu?Ala?Cys

180 185 190

Asp?Ala?Val?Ala?Leu?Leu?Arg?Arg?Glu?Val?Ser?Leu?Gln?Pro?Ile?Asp

195 200 205

Glu?Arg?Leu?Val?Gly?Ser?Leu?Met?Thr?Gly?Leu?Tyr?Arg?Leu?Gly?Arg

210 215 220

Gln?Ala?Glu?Ala?Leu?Gln?Leu?Tyr?Asp?Arg?Thr?Arg?Ala?Tyr?Leu?Ser

225 230 235 240

Glu?Glu?Leu?Gly?Val?Ala?Pro?Ala?Arg?Glu?Leu?Gln?Arg?Val?His?Leu

245 250 255

Ala?Leu?Leu?Arg?His?Glu?Leu?Asp?Asp?Gln?Thr?Ser?Ala?Pro?Ala?Ala

260 265 270

Val?Glu?Val?Arg?Val?Arg?Pro?Gly?Thr?Val?Ala?Glu?Arg?Glu?Ala?Glu

275 280 285

Gly?Pro?Ala?Pro?Ala?Asp?Gly?Pro?Glu?Ala?Val?Pro?Glu?Ala?Gly?Gln

290 295 300

Arg?Gly?Thr?Ala?Pro?Ala?Ala?Trp?Phe?Pro?Cys?Arg?Gly?Asp?Ala?Leu

305 310 315 320

Asp?Ala?Leu?Arg?Ser?Ser?Ile?Thr?Gly?Ala?Leu?Arg?Gly?Ser?Gly?His

325 330 335

Thr?Thr?Ala?Val?Val?Gly?Glu?Ala?Gly?Val?Gly?Lys?Thr?Glu?Leu?Val

340 345 350

Ala?Arg?Ala?Thr?Asp?Gln?Thr?Gln?Pro?Pro?Gln?Pro?Gln?Ala?Glu?Val

355 360 365

Ile?Arg?Val?Asn?Cys?Arg?Ser?Asp?Glu?Gly?Ile?Pro?Val?Gly?Trp?Val

370 375 380

Trp?Gln?Gln?Val?Leu?Arg?Arg?Leu?Asp?Ile?Leu?Phe?Gly?Glu?Ser?Phe

385 390 395 400

Asp?Asp?Leu?Arg?Lys?Gln?Arg?Ala?Glu?Gly?His?Pro?Val?Thr?Thr?Leu

405 410 415

Ser?Glu?Pro?Pro?Ser?Asp?Gln?Met?Asp?Phe?Leu?Ala?Gln?Asp?Ala?Leu

420 425 430

Cys?Glu?Val?Ile?Leu?Gln?Tyr?Ala?Asp?Gly?Arg?Pro?Leu?Leu?Leu?Val

435 440 445

Leu?Glu?Asp?Ser?Ala?Pro?Ala?Asp?Arg?Leu?Thr?Leu?Asp?Val?Leu?Gly

450 455 460

Leu?Phe?Cys?Ser?Arg?Thr?Gln?Gly?Arg?Pro?Val?Ser?Val?Val?Ile?Thr

465 470 475 480

Val?Arg?Glu?Pro?Gly?Leu?Gly?Ala?Gly?Pro?Glu?Thr?Asp?Gly?Pro?Leu

485 490 495

Val?Asp?Leu?Leu?Ala?Asp?Ser?Arg?Thr?Lys?Val?Val?His?Leu?Asp?Asn

500 505 510

Leu?Thr?Glu?Asp?Tyr?Val?Arg?Ala?Ala?Val?Thr?Ala?Gln?Ala?Gly?Pro

515 520 525

Gly?Val?Glu?Ala?Ser?Val?Val?Arg?Ala?Leu?Tyr?Glu?Arg?Ser?Ala?Gly

530 535 540

Asn?Pro?Tyr?Leu?Leu?Asp?Gln?Leu?Ile?Ala?Asp?Ala?Gly?Gly?Ala?Arg

545 550 555 560

Arg?Leu?His?Asp?Pro?Arg?Ala?Ala?Asp?Val?Val?Arg?Ala?Gly?Ile?Pro

565 570 575

Thr?Gly?Val?Arg?Ser?Met?Leu?Arg?Arg?Gln?Phe?Ala?Gly?Leu?Pro?Pro

580 585 590

Arg?Val?Leu?His?Leu?Leu?Gln?Cys?Cys?Ala?Val?Leu?Gly?Gly?Glu?Ala

595 600 605

Thr?Leu?Ala?Ser?Leu?Thr?Ala?Met?Leu?Asp?Asp?Glu?Gly?Ala?Gly?His

610 615 620

Asp?Val?Leu?Glu?Glu?Val?Leu?Ala?Thr?Gly?Leu?Met?His?Ser?Asp?Arg

625 630 635 640

Asp?Asp?Pro?His?Arg?Leu?Val?Phe?Arg?Tyr?Gly?Leu?Ile?Arg?Asp?Val

645 650 655

Leu?Leu?Ala?Glu?Leu?Arg?Arg?Gln?Glu?Arg?Ala?Ala?Leu?His?Ala?Arg

660 665 670

Ala?Val?Gly?Val?Leu?Gly?Ala?Arg?His?Gly?Asp?Ser?Pro?Glu?Ala?Ser

675 680 685

Glu?Gln?Ile?Ala?Glu?His?Ala?Trp?Gln?Ala?Val?Leu?Ala?Leu?Pro?Ala

690 695 700

Glu?Asp?Val?Leu?Pro?His?Leu?Arg?Arg?Ala?Gly?Glu?Gln?Ala?Val?Val

705 710 715 720

Glu?Gly?Asp?Tyr?Arg?Arg?Ala?Gln?Thr?Trp?Phe?Glu?His?Ala?His?Thr

725 730 735

Leu?Leu?Cys?Ala?Leu?Pro?Arg?Asp?Gly?Gly?Ala?Pro?Ser?Lys?Gln?Thr

740 745 750

Leu?Asp?Leu?Arg?Ser?Lys?Leu?Leu?Tyr?Thr?Ala?Ser?Ile?Thr?Arg?Gly

755 760 765

Tyr?Thr?Asn?Arg?Glu?Gly?Glu?Gly?Arg?Gly?Arg?Ala?His?Pro?Gly?Ser

770 775 780

Cys?Thr?Pro?Pro?Arg?Ala?Gly?Arg?Thr?Ile?Glu?Gln?Gly?Ala?Leu?Leu

785 790 795 800

Leu?Trp?Gln?Phe?Val?Ala?Glu?Leu?Val?Thr?Ala?Arg?His?Thr?Glu?Cys

805 810 815

Ala?Arg?His?Ala?Glu?Gln?Leu?Arg?Ala?Leu?Thr?Glu?Arg?Ser?Asp?Ala

820 825 830

Pro?Glu?Val?Arg?Phe?Tyr?Glu?Arg?Ile?Ala?Arg?Gly?Leu?Leu?Gln?Leu

835 840 845

Pro?Ser?Asp?Ala?Ala?Asp?Ala?Leu?Thr?Ala?Leu?Thr?Glu?Ala?Val?Arg

850 855 860

Ile?Ala?Glu?Lys?Leu?Thr?Pro?Ala?Gln?Arg?Glu?Arg?Met?Thr?Arg?Arg

865 870 875 880

Ala?Gln?Gln?Asp?Pro?Arg?Phe?Met?Ala?Ala?Asn?His?Arg?Val?Leu?Thr

885 890 895

Leu?Arg?Leu?Leu?Gly?Ala?Thr?Asp?Glu?Ala?Arg?Ala?Leu?Cys?Glu?Glu

900 905 9l0

Leu?Leu?Val?Ala?Thr?Gly?Leu?Asp?Gly?Thr?Pro?Val?Asp?Gln?Ala?Ser

915 920 925

Ala?Tyr?Tyr?Phe?His?Ser?Leu?Thr?Ala?Ala?Leu?Gly?Glu?Asp?Pro?Asp

930 935 940

Gly?Ala?Ala?Ala?Ser?Ser?Gly?Arg?Gly?Leu?Asp?Ile?Ala?Arg?Ala?His

945 950 955 960

Gly?Leu?Ser?His?Trp?Thr?Ala?Met?Leu?Gln?Val?Cys?Arg?Ser?Trp?Ala

965 970 975

Leu?His?Gln?Ser?Gly?Glu?Ala?Ala?Ala?Leu?Asp?Ser?Leu?Glu?Ser?Ala

980 985 990

Val?Ala?Glu?Leu?Arg?Glu?Arg?Arg?Leu?Leu?Ile?Arg?Leu?Pro?Leu?His

995 1000 1005

Leu?Gly?Leu?Leu?Ala?His?Ala?Gln?His?Ser?Ser?Gly?Ala?Ala?Glu

1010 1015 1020

Asp?Ala?Arg?Arg?Thr?Leu?Arg?Ser?Ser?Thr?Ala?Glu?Ala?Arg?Ser

1025 1030 1035

Arg?Gly?Glu?Leu?Ala?Tyr?Val?Ser?Arg?Thr?Leu?Pro?Phe?Thr?Arg

1040 1045 1050

Leu?Cys?Pro?Leu?Thr?Ser?Ala?Ser

1055 1060

<210>3

<211>1691

<212>DNA

＜213〉streptomyces ansochromogenes (Streptomyces ansochromogenes)

<400>3

ggatccgcgg?ttcatggccg?cgaaccatcg?cgtcctcacc?ctgaggctgc?tcggcgccac 60

cgacgaggcg?cgggcgctgt?gcgaggagct?gctggtagcc?accggcctgg?acggcacgcc 120

ggtcgaccag?gcgagcgcgt?actacttcca?ctccctgaca?gcggccctgg?gcgaggaccc 180

cgacggggcg?gcggcctcca?gcggccgggg?cctggacatc?gcccgcgccc?acgggctgag 240

ccactggacc?gccatgctcc?aggtgtgccg?gagctgggca?ctccaccaga?gcggcgaggc 300

cgccgcactc?gacagcctgg?aatcggccgt?cgccgagctg?cgcgagcggc?ggctgctcat 360

ccggctgccg?ctccacctgg?gcctgctggc?ccacgcccag?cacagcagcg?gggcggccga 420

agacgccaga?cggacgctgc?ggtcgtcgac?cgcggaggcc?agatcccgcg?gggagttggc 480

ctatgtgagc?cgcactctgc?cgttcacccg?gctgtgtccc?ctgacgtccg?cttcgtagcg 540

gccggatcgt?cggttccttc?gcctctcagc?cgggcgcggg?cctggccggc?ctcggggtgg 600

ccgagtccgt?cgagggtccg?cgccgcctcc?cgccacaggg?cagggcggcg?gggcggtctc 660

cccgggactc?gtgggtgtct?cccatgtgat?ggacccagcg?ggtactgcgt?gctcctccgg 720

ggcagcagcg?gcaggaccgg?gaagggtgtg?ccgggcgccg?gcatcaggtc?ggcgccctca 780

cgcagggcgg?ccaggtgttc?gctgtcgacg?gcggaaaaca?gggggtgcac?ccggattacc 840

gggggactcc?ccgcgctgct?tctccgcgcc?ggagggagag?ggcctgggcg?tagagctcgg 900

cggcggcgca?ccggcagggc?ccccgggggc?ggagcgggga?gtcgggcttc?agcggtttcc 960

gcaaggagcc?gatgtggcgg?tggacgacgt?ccacggtgct?gttcggcggg?tcgccgtgcc 1020

acaggacgtc?gacgatctgg?ctcaggccga?cggtctgtcc?ggcgtgcaac?ggcaacgccg 1080

gcaaggcgcg?ctgcttcggt?ggtccgaggg?cgactcggcg?cctgaccgcc?acactgcgac 1140

cggccccagg?accatggtgg?tcctggtgtt?cgtgatcttc?ctcgccgtgc?ggcccgtcaa 1200

cgagggtggc?tggtccggga?cgggtggtcg?tagcgccgtt?cgttgtaact?gagcatgtcg 1260

atgaggcccc?gggcggtgag?ccgctgccgg?ttccgggcca?gatcgcgcgc?ccggggaatc 1320

ggcacccctg?cggggtcggc?ccaggtgagc?cggccacggt?cgtcgatgcg?ggcccggcag 1380

cggttcgcgt?tgtcctggtg?caggcagaac?gggatgtcga?ggtgtccgag?ggcgaacgcg 1440

cgaacgagcg?cctttccgac?atcgctgccg?agggtcagcg?tgtgcgtgat?gatcgcctgg 1500

gcctcctcgt?agatcccgga?gtccggaatt?ccggccggtt?cggccaccgc?ctcccgctcg 1560

gccgcccacg?cggcgcgttc?gagggcgtcg?acgttctccg?tgatcgacgg?gattctgctg 1620

gcctccaccg?cggtcttgac?gatcagccgc?tcggtcccgg?acctggcggc?gagcctcgca 1680

ctctcctcga?g 1691

Claims (9)

1, a kind of nikemycin biosynthetic controlling gene is one of following nucleotide sequences:

1) the SEQ ID № in the sequence table: 1;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding;

4) under the rigorous degree condition of height can with the SEQ ID № in the sequence table: 1 hybridization sequence.

2, gene according to claim 1 is characterized in that: described gene is the SEQ ID № in the sequence table: 1.

3, nikemycin biosynthetic controlling albumen is to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 is identical active by SEQ ID №: 2 deutero-protein.

4, albumen according to claim 3 is characterized in that: described albumen has SEQ ID № in the sequence table: 2 amino acid residue sequence.

5, contain described expression carrier of claim 1 or engineering bacteria.

6, expression vector according to claim 5 or engineering bacteria is characterized in that: described expression vector is recombinant plasmid pKC1139 ∷ sanG.

7, expression vector according to claim 5 or engineering bacteria is characterized in that: described engineering bacteria is streptomyces ansochromogenes (Streptomyces ansochromogenes) NK2 CGMCC № 1181.

8, the application of the described gene of claim 1 in nikemycin is produced.

9, the application of streptomyces ansochromogenes (Streptomyces ansochromogenes) NK2 CGMCC № 1181 in nikemycin is produced.

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