CN101962647B

CN101962647B - Biosynthesis gene cluster of Nocathiacins and application thereof

Info

Publication number: CN101962647B
Application number: CN 201010240885
Authority: CN
Inventors: 刘�文; 丁莹; 虞沂; 潘海学
Original assignee: Shanghai Institute of Organic Chemistry of CAS
Current assignee: Shanghai Institute of Organic Chemistry of CAS
Priority date: 2009-07-24
Filing date: 2010-07-23
Publication date: 2013-07-31
Anticipated expiration: 2030-07-23
Also published as: WO2011009416A1; CN101962647A; CN101684471A

Abstract

The invention relates to a biosynthesis gene cluster of Nocathiacins and an application thereof, in particular to providing the cloning, the sequencing, the analysis, the functional research of an antibiotic-Nocathiacins biosynthesis gene cluster with good antibiosis activity generated by Nocardia and an application thereof. The integral gene cluster contains 37 genes, wherein the 37genes comprise 8 genes relevant to the biosynthesis of big ring skeletons, 4 genes relevant to the side chain synthesis of indole acid, 6 genes relevant to glycosyl synthesis, 5 modified enzyme genes after the redox of P450, 2 methyl transferase genes, 2 resistance genes, 3 regulator genes, 3 genes with the unknown functions and 4 genes relevant to transcription and translation, and a series of new sulfur peptide antibiotics can be generated by utilizing the heterologous expression of the biosynthesis genes. The genes provided by the invention and proteins thereof can search and discover compounds or the genes and the proteins for medicine, industry and agriculture.

Description

The biological synthesis gene cluster and the application thereof of promise card thiazole rhzomorph

Technical field

The invention belongs to microbial gene resource and genetically engineered field, be specifically related to the clone of the biological synthesis gene cluster of sulphur peptide antibiotics promise card thiazole rhzomorph (Nocathiacins), sequential analysis, gene functional research and application thereof.

Technical background

Promise card thiazole rhzomorph (Nocathiacins) is that a class is rich in elementary sulfur, the cyclic peptide microbiotic that amino-acid residue is highly modified, they are as crucial member in the sulphur peptide family, be at streptococcus aureus (the Methicillin-Resistant Staphylococcus aureus of screening at first to the new penicillium resistance is arranged, MRSA) and multiple chemical sproof enterococcus faecalis (Multi-Drug Resistant Enterococcus faecium arranged, MREF) growth has in the inhibiting antibiotic process, [the J.Antibiot.1998 that from pedotheque, finds, 51,715].1998, the Tokushima research centre takes the lead in from amycolatosis (Amycolatopsis sp.MJ347-81F4) separation and purification to MJ347-81F4-A (Nocathiacin I) and B, and finished their fermentation, active testing and structure determination, but do not determine its absolute configuration.2003, Bristol-Myers Squibb institute of materia medica has found Nocathiacin I, II, III again from Nocardia bacteria (Nocardia sp.WW-12651), and it fermentation, separation and purification and active testing have been carried out, also utilize methods such as NMR, X-ray to determine its absolute configuration (Fig. 1) [J.Antibiot..2003 first, 56,226,232; Clough, B.; J.Org.Chem.2002,67,8699].

Promise card thiazole rhzomorph is the same with sulphur peptide family other microbiotic, and also to have one be core with trisubstituted pyridine, by a plurality of thiazoles and the dehydration cyclic peptide center that amino acid constituted.Wherein the Nocathiacin I is structurally extremely similar to another member's sugar sulphur hexides α of this family, difference is many one 2, the side chain of 3-dehydrogenation alanimamides, indolic acid unit link to each other with big ring the part ester bond substituted original thioester bond.

Studies show that promise card thiazole rhzomorph can suppress the growth of gram-positive microorganism well, particularly has extremely strong lethal effect to multiple chemical sproof conditioned pathogen.Wherein the Nocathiacin I is 0.003 μ g/mL to the MIC value of MRSA, to streptococcus pneumoniae (the penicillin-resistant Streptococcus pneumoniae that penicillin resistance is arranged, PRSP) MIC value reaches 0.001 μ g/mL, exceeds 10-20 doubly than vancomycin.Simultaneously, it also (vancomycin-resistant Enterococci faeccium VREF) has good resistance, and the MIC value is 0.015 μ g/mL to the enterococcus faecalis that vancomycin (Vancomycin) resistance is arranged of recent appearance.And compare with vancomycin, promise card thiazole rhzomorph has significant more curative effect, the PD of Nocathiacin I, II, III to the mouse that infects MRSA ₅₀Value is respectively 0.8,0.62,0.89mg/kg, and vancomycin only is 1.3mg/kg under similarity condition.Nocathiacin I and II are as two best compounds of sulphur peptide family anti-microbial activity, also having better water-solubility than other members under low pH, may be because contain the cause [Med.Chem.Lett.2004.14.171-175] of a dimethylamino hexose in the molecule.

Find that in the recent period antibiotic mechanism of action of this class and thiostrepton are similar, also be to combine with the 23S rRNA-L11 albumen composition of 50S subunit, by stoping or promote the variation of L11 conformation, influence the identification of aa-tRNA.Ef-Tu.GTP mixture and the GTPase activity of elongation factor, thereby [the J.Am.Chem.Soc.2008 that brings into play active function that synthesizes that suppresses the bacterial body internal protein, 130,12102-12110].Its concrete structure activity relationship also is not very clear at present, but their similar cyclic peptide centers may be that it has superior active key structure by inference.

Promise card thiazole rhzomorph has just caused the great interest of scientists as the lead compound of anti-infectives of new generation from finding certainly.Consider that their solubleness does not reach the requirement of intravenous injection medicament as yet, many chemists have carried out chemical semi-synthetic transformation to it.Bristol-Myers Squibb institute of materia medica is by having added some polarity water soluble groups on the Nocathiacin I, improve their bioavailability: as at alkoxide [Bioorganic ﹠ Medicinal Chemistry Letters.2004 on the hydroxyl of pyridine ring or on the nitrogen of indolic acid side chain, 14,3743-3746]; 2, add ammonia or mercaptan [Tetrahedron Letters.2004,5,1059-1063] by the Michael addition on the 3-dehydrogenation alanimamides side chain; Perhaps remove 2 with chemical method or enzyme process, 3-dehydrogenation alanimamides obtains the Nocathiacin IV, further again alkoxide or interpolation alkylamine [J.Org.Chem.2002,67,8789].But these methods often are difficult in and improve the water miscible primary formation good antibacterial activity that keeps simultaneously.And the antibiotic chemical structure of this class is extremely complicated, two people synthetic [Tetrahedron Lett.1984,25,2127 of only having finished part of module and acidic hydrolysis product during the last ten years; J.Org.Chem.1996,61,4623; J.Org.Lett.2003,5,4421; Tetrahedron Lett.1991,32,4263; Angewandte Chemie.2005,117,3802-3806; Chem.Commun.2008,591-593].Finished up to the talents such as organic synthesis great master Moody and Nicolaou in recent years and to have started the complete synthesis of thiophene star A, shallow lake sulfomycin D, thiostrepton, SIM-A, GE2270A/T, complete synthesis also nobody report [Angew.Chem.Int.Ed.2007 of promise card thiazole rhzomorph, 46,7930-7954].And because the polynuclear plane of such microbiotic complexity, numerous chiral centres makes that to utilize complete synthesis method to carry out adaptation step merely various, and real cost of production is too high.

And in recent years along with the further investigation of genomics and proteomics and the fast development of new bio technology, make us utilize microorganism as " cell factory ", synthesizing natural product and analogue thereof with good biological activity and novel mechanism of action in a large number by Genetic Control becomes possibility.This also provides a new thinking for we obtain needed novel sulphur peptide antibiotics in microbe.

Therefore we are target molecule with microbe-derived promise card thiazole rhzomorph, biological synthesis gene cluster from clone's promise card thiazole rhzomorph, the method that adopts microbiology, molecular biology, biological chemistry and organic chemistry to combine is studied its biosynthesizing, illustrate its biosynthetic pathway and regulation mechanism, use the principle of metabolic engineering on this basis, the biosynthetic pathway of rational modification promise card thiazole rhzomorph, explore bioavailability better, and can pass through the mass-produced newtype drug of microbial fermentation.

Summary of the invention

The present invention relates to clone, order-checking, analysis, functional study and the application thereof of a class by the biological synthesis gene cluster of the good antibiotic active microbiotic of having of Nocardia bacteria generation-Nuo Ka thiazole rhzomorph (Nocathiacins).

Whole gene cluster comprises the nucleotide sequence or the complementary sequence (sequence 1) (SEQ ID NO:1) of 37 genes altogether among the present invention, 8 gene noc28 is wherein arranged, noc20, noc21, noc22, noc23, noc9, noc24 and noc30 are responsible for the biosynthesizing of the big ring skeleton of Nocathiacin I; 4 gene noc25, noc26, noc27 and noc29 are responsible for the biosynthesizing of indolic acid side chain; 6 gene noc6, noc10, noc11, noc12, noc13 and noc14 are responsible for 4-N, N-dimethyl-2,4, the biosynthesizing of 6-deoxyhexamethylose; 5 Cytochrome P450 oxidoreductase gene noc7, noc15, noc16, noc18, noc19 modifies after the redox of responsible Nocathiacin I; 2 methyl transferase gene noc8, noc36, methylate the back modification and the resistance of self of responsible Nocathiacin I respectively; 2 resistant gene noc17, noc37; 3 regulatory gene noc5, noc33, noc34; 4 gene noc1s relevant with transcription and translation, noc2, noc3, noc4; And the gene noc31 of 3 unknown function, noc32, noc35.

The present invention also provides the nucleotide sequence of a coding promise card thiazole rhzomorph precursor peptide, is made up of the aminoacid sequence in the sequence 2, and called after noc28, the nucleotide sequence of its gene are arranged in sequence 1 38432-38581 base place.

The present invention also provides the nucleotide sequence of the cyclase of coding promise card thiazole rhzomorph thiazole ring formation, is made up of the aminoacid sequence in the sequence 3, and called after noc23, the nucleotide sequence of its gene are arranged in sequence 1 30980-32875 base place.

The present invention also provides the nucleotide sequence of the nadh dehydrogenase of coding promise card thiazole rhzomorph thiazole ring formation, is made up of the aminoacid sequence in the sequence 4, and called after noc22, the nucleotide sequence of its gene are arranged in sequence 1 29544-30983 base place.

The present invention also provides the nucleotide sequence of the dehydratase of the big ring skeleton formation of a coding promise card thiazole rhzomorph, is made up of the aminoacid sequence in the sequence 5, and called after noc21, the nucleotide sequence of its gene are arranged in sequence 1 26965-29523 base place.

The present invention also provides the nucleotide sequence of the dehydratase of the big ring skeleton formation of a coding promise card thiazole rhzomorph, is made up of the aminoacid sequence in the sequence 6, and called after noc20, the nucleotide sequence of its gene are arranged in sequence 1 25968-26960 base place.

The present invention also provides the nucleotide sequence of a coding free radical SAM thiamines synthetic enzyme, is made up of the aminoacid sequence in the sequence 7, and called after noc27, the nucleotide sequence of its gene are arranged in sequence 1 36848-37948 base place.

The present invention also provides the nucleotide sequence of a coding acyl group-CoA synthetic enzyme, is made up of the aminoacid sequence in the sequence 8, and called after noc25, the nucleotide sequence of its gene are arranged in sequence 1 34680-35912 base place.

The present invention also provides the nucleotide sequence of a coding acyltransferase, is made up of the aminoacid sequence in the sequence 9, and called after noc26, the nucleotide sequence of its gene are arranged in sequence 1 35933-36820 base place.

The present invention also provides the oxydase of coding SAM dependence or the nucleotide sequence of methyltransgerase, is made up of the aminoacid sequence in the sequence 10, and called after noc29, the nucleotide sequence of its gene are arranged in sequence 1 38714-39976 base place.

The present invention also provides the nucleotide sequence of the two methyltransgerases of coding N-, is made up of the aminoacid sequence in the sequence 11, and called after noc10, the nucleotide sequence of its gene are arranged in sequence 1 14990-15703 base place.

The present invention also provides the nucleotide sequence of the methyltransgerase on 3 C of a coding NDP-hexose, is made up of the aminoacid sequence in the sequence 12, and called after noc11, the nucleotide sequence of its gene are arranged in sequence 1 15728-16972 base place.

The present invention also provides the nucleotide sequence of a coding NDP-hexose-3-ketoreductase, is made up of the aminoacid sequence in the sequence 13, and called after noc12, the nucleotide sequence of its gene are arranged in sequence 1 16984-17970 base place.

The present invention also provides a coding dTDP-4-ketone-6-deoxyglucose 2, and the nucleotide sequence of 3-dehydratase is made up of the aminoacid sequence in the sequence 14, and called after noc13, the nucleotide sequence of its gene are arranged in sequence 1 17988-19418 base place.

The present invention also provides the nucleotide sequence of the transaminase on 4 C of a coding NDP-6-DDG, form by the aminoacid sequence in the sequence 15, called after noc14, the nucleotide sequence of its gene are arranged in sequence 1 19424-20560 base place.

The present invention also provides the nucleotide sequence of the transaminase of an encoding glycosyl transferring enzyme, is made up of the aminoacid sequence in the sequence 16, and called after noc6, the nucleotide sequence of its gene are arranged in sequence 1 11505-12683 base place.

The present invention also provides the nucleotide sequence of a Codocyte cytochrome p 450 oxydo-reductase, is made up of the aminoacid sequence in the sequence 17, and called after noc7, the nucleotide sequence of its gene are arranged in sequence 1 12704-13912 base place.

The present invention also provides the nucleotide sequence of a Codocyte cytochrome p 450 oxydo-reductase, is made up of the aminoacid sequence in the sequence 18, and called after noc15, the nucleotide sequence of its gene are arranged in sequence 1 20591-21703 base place.

The present invention also provides the nucleotide sequence of a Codocyte cytochrome p 450 oxydo-reductase, is made up of the aminoacid sequence in the sequence 19, and called after noc16, the nucleotide sequence of its gene are arranged in sequence 1 21696-22934 base place.

The present invention also provides the nucleotide sequence of a Codocyte cytochrome p 450 oxydo-reductase, is made up of the aminoacid sequence in the sequence 20, and called after noc18, the nucleotide sequence of its gene are arranged in sequence 1 23507-24649 base place.

The present invention also provides the nucleotide sequence of a Codocyte cytochrome p 450 oxydo-reductase, is made up of the aminoacid sequence in the sequence 21, and called after noc19, the nucleotide sequence of its gene are arranged in sequence 1 24646-25926 base place.

The present invention also provides the nucleotide sequence of a coding methyltransgerase, is made up of the aminoacid sequence in the sequence 22, and called after noc8, the nucleotide sequence of its gene are arranged in sequence 1 13909-14532 base place.

The present invention also provides the nucleotide sequence of a coding methyltransgerase, is made up of the aminoacid sequence in the sequence 23, and called after noc36, the nucleotide sequence of its gene are arranged in sequence 1 43983-44768 base place.

The present invention also provides the nucleotide sequence of a coding bleomycin resistance protein, is made up of the aminoacid sequence in the sequence 24, and called after noc17, the nucleotide sequence of its gene are arranged in sequence 1 22956-23480 base place.

The present invention also provides the nucleotide sequence of a coding phosphor hydrochlorate ABC transporter, is made up of the aminoacid sequence in the sequence 25, and called after noc37, the nucleotide sequence of its gene are arranged in sequence 1 44914-45423 base place.

The present invention also provides the nucleotide sequence of the transcription regulatory protein of a coding SARP family, is made up of the aminoacid sequence in the sequence 26, and called after noc5, the nucleotide sequence of its gene are arranged in sequence 1 10404-11387 base place.

The present invention also provides the nucleotide sequence of a coding heat shock protein(HSP), is made up of the aminoacid sequence in the sequence 27, and called after noc33, the nucleotide sequence of its gene are arranged in sequence 1 42031-42456 base place.

The present invention also provides the nucleotide sequence of a coding hsp18 transcription regulaton factor, is made up of the aminoacid sequence in the sequence 28, and called after noc34, the nucleotide sequence of its gene are arranged in sequence 1 42565-43173 base place.

The present invention also provides the nucleotide sequence of the signal factor of a coding RNA polysaccharase ECF-subtribe, is made up of the aminoacid sequence in the sequence 29, and called after noc3, the nucleotide sequence of its gene are arranged in sequence 1 8731-9867 base place.

The present invention also provides the nucleotide sequence of the signal factor of a coding ATP enzyme, is made up of the aminoacid sequence in the sequence 30, and called after noc4, the nucleotide sequence of its gene are arranged in sequence 1 9968-10387 base place.

The present invention also provides the nucleotide sequence of an encoding D GPFAETKE family protein, is made up of the aminoacid sequence in the sequence 31, and called after noc2, the nucleotide sequence of its gene are arranged in sequence 1 8282-8725 base place.

The present invention also provides the nucleotide sequence of a coding 50S ribosomal protein L 18, is made up of the aminoacid sequence in the sequence 32, and called after noc1, the nucleotide sequence of its gene are arranged in sequence 1 7731-8084 base place.

The present invention also provides a proteic nucleotide sequence of coding unknown function, is made up of the aminoacid sequence in the sequence 33, and called after noc9, the nucleotide sequence of its gene are arranged in sequence 1 14529-14984 base place.

The present invention also provides a proteic nucleotide sequence of coding unknown function, is made up of the aminoacid sequence in the sequence 34, and called after noc24, the nucleotide sequence of its gene are arranged in sequence 1 32902-34683 base place.

The present invention also provides a proteic nucleotide sequence of coding unknown function, is made up of the aminoacid sequence in the sequence 35, and called after noc30, the nucleotide sequence of its gene are arranged in sequence 1 40174-40914 base place.

The present invention also provides a proteic nucleotide sequence of coding unknown function, is made up of the aminoacid sequence in the sequence 36, and called after noc31, the nucleotide sequence of its gene are arranged in sequence 1 41001-41408 base place.

The present invention also provides a proteic nucleotide sequence of coding unknown function, is made up of the aminoacid sequence in the sequence 37, and called after noc32, the nucleotide sequence of its gene are arranged in sequence 1 41492-41953 base place.

The present invention also provides a proteic nucleotide sequence of coding unknown function, is made up of the aminoacid sequence in the sequence 38, and called after noc35, the nucleotide sequence of its gene are arranged in sequence 1 43228-43758 base place.

In another preference, described gene cluster comprises the structure gene that is made of noc6 to noc30; Or comprise structure gene, resistant gene and the regulatory gene that constitutes by noc1 to noc37.

In another preference, the nucleotide sequence of described gene cluster is shown in 1-44560 position among 11505-40916 position or the SEQ ID NO:1 among 7731-45423 position, the SEQ ID NO:1 among the SEQ ID NO:1.

The present invention also provides the proteins encoded of the biological synthesis gene cluster of the above-mentioned promise card thiazole rhzomorph of the present invention.Preferably, the aminoacid sequence of described proteins encoded is shown in SEQ ID NO:2-38.

The present invention also provides the expression vector of the biological synthesis gene cluster that contains above-mentioned promise card thiazole rhzomorph.Preferably, described expression vector is a clay.

The present invention also provides the host cell that contains the biological synthesis gene cluster that is integrated with above-mentioned promise card thiazole rhzomorph on above-mentioned expression vector or the karyomit(e) of reorganization.Preferably, described host cell is a streptomycete.

The present invention also provides a kind of method that produces promise card thiazole rhzomorph, comprises step: express promise card thiazole rhzomorph thereby cultivate above-mentioned host cell, and separate promise card thiazole rhzomorph from nutrient solution.

The present invention also provides the biological synthesis gene cluster of above-mentioned promise card thiazole rhzomorph or the purposes of its portion gene, and they are used to carry out the allos complementation in the mutant strain of S.actuosus ATCC 25421 genes involveds disappearance, thereby recovers to produce nosiheptide; Perhaps the back modifying factor is carried out the heterogenous expression generation in S.actuosus ATCC 25421, thereby produce the analog of nosiheptide.

The complementary sequence of sequence 1 can obtain according to DNA base complementrity principle.The nucleotide sequence of sequence 1 or partial nucleotide sequence can be by polymerase chain reaction (PCR) or with suitable digestion with restriction enzyme corresponding D NA fragment or utilize other suitable technique to obtain.The present invention also provides the approach that obtains to comprise at least the recombinant plasmid of dna fragmentation in the partial sequence 1.

The present invention also provides promise card thiazole rhzomorph biosynthetic microbe approach, and the gene of one of them includes the nucleotide sequence in the sequence 1 at least.

Nucleotide sequence provided by the present invention or partial nucleotide sequence, the DNA that can utilize the method for polymerase chain reaction (PCR) or comprise sequence of the present invention obtains from the other biological body and the similar gene of promise card thiazole rhzomorph biosynthesis gene with methods such as Southern hybridization as probe.

Comprise nucleotide sequence provided by the present invention or at least the cloned DNA of partial nucleotide sequence can be used for from Nocardia bacteria Nocardia sp.WW-12651 genomic library more library, location plasmid.These library plasmids comprise the partial sequence among the present invention at least, also include the DNA that former adjacent domain is not cloned in the Nocardia sp.WW-12651 genome.

Nucleotide sequence provided by the present invention or at least partial nucleotide sequence can be modified or be suddenlyd change.These approach comprise insertion, displacement or disappearance, the polymerase chain reaction, mistake mediation polymerase chain reaction, the locus specificity sudden change, not homotactic reconnecting, the different piece of sequence or carry out orthogenesis (DNAshuffling) with the homologous sequence in other sources, or by ultraviolet ray or chemical reagent mutagenesis etc.

Comprise nucleotide sequence provided by the present invention or at least the clone gene of partial nucleotide sequence can in foreign host, express to obtain the meta-bolites of corresponding enzyme or other higher biological activitys or output by suitable expression system.These foreign host comprise streptomycete, pseudomonas, intestinal bacteria, genus bacillus, yeast, plant and animal etc.

Aminoacid sequence provided by the present invention can be used for separating needed albumen and can be used for the preparation of antibody.

Comprise aminoacid sequence provided by the present invention or at least the polypeptide of partial sequence may after remove or substituting some amino acid, still have biological activity even new biologic activity is arranged, perhaps improved output or optimized the albumen dynamic characteristic or other character of being devoted to obtain.

Comprise nucleotide sequence provided by the present invention or at least partial nucleotide sequence gene or gene cluster can be expressed in heterologous host and understand their functions in host's metabolic chain by the DNA chip technology.

Comprise nucleotide sequence provided by the present invention or at least the gene or the gene cluster of partial nucleotide sequence can come construction recombination plasmid to obtain its biosynthetic pathway by genetic recombination, also can and then obtain new biosynthetic pathway by insertion, displacement, disappearance or inactivation.

Comprise nucleotide sequence provided by the present invention or the clone gene of partial nucleotide sequence or dna fragmentation can obtain new promise card thiazole rhzomorph analog by interrupting biosynthetic one or several step of promise card thiazole rhzomorph at least.Comprise the output that dna fragmentation or gene can be used for improving promise card thiazole rhzomorph or derivatives thereof, the invention provides the approach that in genetically engineered microorganism, improves output.

The biosynthetic precursor peptide of nucleotide sequence coded rrna mechanism promise card thiazole rhzomorph provided by the present invention can be by inserting, replace or disappearance, the polymerase chain reaction, mistake mediation polymerase chain reaction, the locus specificity sudden change, not homotactic reconnecting, the different piece of sequence or carry out orthogenesis (DNAshuffling) with the homologous sequence in other sources, methods such as ultraviolet ray or chemical reagent mutagenesis produce new sulphur peptide antibiotics or other polypeptide class meta-bolitess.

Structural units such as comprising nucleotide sequence coded albumen provided by the present invention can catalysis synthetizing thiazolium ring, hydroxylation pyridine ring, indolic acid, and can be by recombinating with the biosynthetic pathway or the part biological route of synthesis of other natural products, obtaining to comprise has these structural units and has better bioactive meta-bolites.

Comprise big ring skeleton and its analog that nucleotide sequence coded albumen provided by the present invention can the synthetic promise card thiazole rhzomorph of catalysis.

Comprise nucleotide sequence coded albumen provided by the present invention and can catalysis synthesize 4-N, N-dimethyl-2,4, the 6-deoxyhexamethylose, and can obtain new glycation product by recombinating with the biosynthetic pathway or the part biological route of synthesis of other natural products.

The back modifying factor of promise card thiazole rhzomorph provided by the present invention provides the approach that obtains analogue by genetic modification, and the redox reaction that is comprised also can have other application.

In a word, all relevant genes of promise card thiazole rhzomorph biosynthesizing and the albumen information of comprising provided by the present invention can help people to understand the biosynthesizing mechanism of sulphur peptide antibiotics, for further genetic modification provides material and knowledge.Gene provided by the present invention and protein thereof also can be used for seeking and find can be used for medicine, industry or agriculture compound or gene, albumen.

Description of drawings

Fig. 1: the chemical structure of promise card thiazole rhzomorph.

Fig. 2: the gene structure and the restriction mapping of promise card thiazole rhzomorph biological synthesis gene cluster.(A) the glutinous grain of 6 overlappings has been represented the DNA zone of Nocardia bacteria Nocardia sp.WW-12651 genome 45kb, and S represents Restriction enzyme Sma I, and entity is represented by the part of dna sequencing, the probe portion of the thick vertical line expressive notation of black; (B) genomic constitution of promise card thiazole rhzomorph biological synthesis gene cluster.

Fig. 3: the biosynthetic pathway of the big ring skeleton of promise card thiazole rhzomorph of proposition.

Fig. 4: the biosynthetic pathway of the promise card thiazole rhzomorph indolic acid side chain of proposition.

Fig. 5: the promise card thiazole rhzomorph 4-N of proposition, N-dimethyl-2,4, the biosynthetic pathway of 6-deoxyhexamethylose.

Fig. 6: the high performance liquid chromatography-mass spectrum (HPLC-MS) of tunning Nocathiacin I is analyzed.

(A) high performance liquid chromatography; (B) mass spectrum

Fig. 7: the portion gene relevant with the biosynthesizing of the big ring skeleton of promise card thiazole rhzomorph is to the allos complementation of similar gene in the nosiheptide gene cluster with frame deletion mutantion strain.

(A) nosiheptide produces the HPLC-MS detection of bacterium Streptomyces actuosus ATCC25421 wild-type tunning

(B) nosiheptide nosE detects with the HPLC-MS of frame deletion mutantion strain tunning

(C) HPLC-MS of promise card thiazole rhzomorph noc21 allos covering mutant strain tunning detects

(D) nosiheptide nosD detects with the HPLC-MS of frame deletion mutantion strain tunning

(E) HPLC-MS of promise card thiazole rhzomorph noc20 allos covering mutant strain tunning detects

Fig. 8: the portion gene relevant with the biosynthesizing of promise card thiazole rhzomorph indolic acid side chain is to the allos complementation of similar gene in the nosiheptide gene cluster with frame deletion mutantion strain.

(B) nosiheptide nosI detects with the HPLC-MS of frame deletion mutantion strain tunning

(C) HPLC-MS of promise card thiazole rhzomorph noc25 allos covering mutant strain tunning detects

(D) nosiheptide nosL detects with the HPLC-MS of frame deletion mutantion strain tunning

(E) HPLC-MS of promise card thiazole rhzomorph noc27 allos covering mutant strain tunning detects

Fig. 9: in the promise card thiazole rhzomorph biological synthesis gene cluster after the part P450 redox modifying factor to the allos complementation of similar gene in the nosiheptide gene cluster with frame deletion mutantion strain.

(B) nosiheptide nosC detects with the HPLC-MS of frame deletion mutantion strain tunning

(C) HPLC-MS of promise card thiazole rhzomorph noc19 allos covering mutant strain tunning detects

(D) nosiheptide nosB detects with the HPLC-MS of frame deletion mutantion strain tunning

(E) HPLC-MS of promise card thiazole rhzomorph noc7 allos covering mutant strain tunning detects

Figure 10: the heterogenous expression of modifying factor in nosiheptide generation bacterium Streptomyces actuosus ATCC25421 after the part P450 redox in the promise card thiazole rhzomorph biological synthesis gene cluster.

(B) HPLC-MS of promise card thiazole rhzomorph noc16 heterogenous expression mutant strain tunning detects

(C) HPLC-MS of promise card thiazole rhzomorph noc18 heterogenous expression mutant strain tunning detects

The chemical structure of nosiheptide analog A

The chemical structure of nosiheptide analog B.

Figure 11: shear in the relevant gene pairs nosiheptide gene cluster similar gene with the allos complementation of frame deletion mutantion strain with the Serine side chain in the promise card thiazole rhzomorph biological synthesis gene cluster.

(B) nosiheptide nosA detects with the HPLC-MS of frame deletion mutantion strain tunning

(C) HPLC-MS of promise card thiazole rhzomorph noc9 allos covering mutant strain tunning detects

Nomenclature

Fig. 1 Nocathiacin I: promise card thiazole rhzomorph I; Nocathiacin II: promise card thiazole rhzomorph II; Nocathiacin III: promise card thiazole rhzomorph III; MJ347-81F4-B: the demethylation product of promise card thiazole rhzomorph I.

Fig. 2 (A) B ₃: glutinous grain pDY2-61-B ₃, D ₂: glutinous grain pDY2-61-D ₂, C ₂: glutinous grain pDY2-61-C ₂, A ₁: glutinous grain pDY2-61-A ₁, C ₄: glutinous grain pDY2-61-C ₄Letter S represents the SmaI restriction enzyme site; (B) suger 4-N, N-dimethyl-2,4, modifying factor Indole acid promise card thiazole rhzomorph indolic acid side chain biosynthesis gene resistance resistant gene regulation regulatory gene Unknown unknown gene after the big ring skeleton of the 6-deoxyhexamethylose biosynthesis gene cyclopeptide promise card thiazole rhzomorph biosynthesis gene oxidation promise card thiazole rhzomorph P450 redox.

Gene noc20,21,22 in the promise card thiazole rhzomorph gene cluster of describing in corresponding respectively this specification sheets of Fig. 3 Noc20,21,22,23,23 coded albumen, LP represents the signal peptide of promise card thiazole rhzomorph.

Gene noc25,26,27 in the promise card thiazole rhzomorph gene cluster of describing in corresponding respectively this specification sheets of Fig. 4 Noc25,26,27,29,29 coded albumen, Ado represents free radical.

Gene noc6,10,12,13 in the promise card thiazole rhzomorph gene cluster of describing in corresponding respectively this specification sheets of Fig. 5 Noc6,10,12,13,14,14 coded albumen.

Fig. 6 (A) 15min is that retention time (B) 1437 of Nocathiacin I is the molecular ion peak of Nocathiacin I.

Embodiment:

The present invention is described in more detail below in conjunction with accompanying drawing.

1. N in the promise card thiazole rhzomorph biological synthesis gene cluster, the clone of N-dimethyl enzyme gene fragment:

Although the research of relevant sulphur peptide antibiotics biosynthesizing aspect just began as far back as the end of the seventies in last century, all fail effectively to break through in the research of microbe intracellular metabolite approach about them up to now.Early stage investigator has mainly finished the amino acid precursor labelling experiment (C of thiostrepton (Thiostrepton), nosiheptide (Nosiheptide), sulphur peptimycin I (sulfomycin I), GE2270A, A10255G/B/E, promise card thiazole rhzomorph (Nocathiacins) etc. ¹³, C ¹⁴, H ², H ³, N ¹⁵Deng), and inferred the possible biosynthetic pathways of they part primary structures thus: as think that wherein thiazole or thiazoline ring derive from halfcystine and Serine; Indolic acid or quinaldinic acid structure are then transformed through multistep by tryptophane and obtain; Think that also the azepine six-ring of their core textures forms [J.Am.Chem.Soc.1979,101,5069 by [4+2] cycloaddition reaction; 1988,110,5800; 1993,115,7557; 1993,115,7992; 1995,117,7606; 1996,118,11363; Biorg.Med.Chem.1996,4,1135; J.Antibiot.1992,45,1499; 2001,54,1066; J.Chem.Soc.Chem.Commun.1993,1612; Acc.Chem.Res.1993,26,116; Tetrahedron Letters.2008,49,6265-6268.].

Labelling experiment shows that equally also this class microbiotic has similar amino acid precursor mostly, and this illustrates that in a sense they may be to adopt similar biosynthetic pathway to obtain in microbe.And the biosynthetic pathway of typical poly-peptides natural product can be divided into two classes substantially in the microbe: rrna approach and non-ribosomal approach.Floss group once suppresses result of experiment according to paraxin and infers that this class microbiotic may be to adopt non-ribosomal approach synthetic.Investigators have attempted a large amount of methods and have cloned the antibiotic biological synthesis gene cluster of this class subsequently, as mutant strain complementation, reverse genetics, resistant gene is that the gene fragment of probe, coding rrna approach precursor peptide is that the conserved sequence of probe, non-ribosomal approach biosynthesis gene is methods such as primer, transposon interrupt at random, but does not all have successfully.This just points out us need seek a brand-brand-new way and clones the antibiotic biological synthesis gene cluster of this class.

Be distributed in these characteristics on chromosomal the same area according to the biosynthesis gene of the glycosyl of glycopeptide antibiotics and precursor skeleton is mostly chain, the inventor determines the biosynthesis gene of deoxidation aminosugar from clone Nocathiacin I, clones the biological synthesis gene cluster of whole molecule.We are at first according to biosynthetic pathway [Antimicrobial Agents And Chemotherapy, 1999,43, the 1565-1573 of a large amount of microbe-derived natural product deoxidation glycosyls; Chemistry﹠ Biology, 2004,11,959-969; Molecular Microbiology, 2000,37,752-762.] inferred 4-N in the Nocathiacin I, N-dimethyl-2,4, the biosynthetic pathway that the 6-deoxyhexamethylose is possible, and choose the N that may be present in this approach catalysis later stage, the conserved sequence of N-dimethyl transferring enzyme, PCR primer Dimeth-For1:5 '-GCTGAC GTCGCCTGCGGSAC (C/G) GG (A/T/C/G) that has designed degeneracy is (A/T/C/G) (A/T/C/G) CA-3 and Dimeth-Rev2:5 '-CGCG AACGT (G/C) TC (G/C) GG (A/G) AA CCACCA (A/T/G/C) GG (A/T/G/C) TC-3 ' (G/A/T), total DNA with Nocardia sp.WW-12651 is that template is carried out pcr amplification then, obtain the PCR product of about 0.3kb, be cloned into the pSP72 carrier, find and known N that through sequencing analysis N-dimethyl transferase gene has very high homology.

2. the clone of promise card thiazole rhzomorph biological synthesis gene cluster, sequential analysis and functional analysis:

With above-mentioned N of being cloned into, N-dimethyl enzyme gene fragment is labeled as probe with digoxin, and the genomic library of the Nocardiasp.WW-12651 that builds is screened, and obtains 6 altogether and inserts the glutinous grain that fragments overlap each other, and is respectively: cDY446-2-47-A ₁, B ₃, C ₂, C ₄, D ₂, D ₄, contained the DNA zone (Fig. 2 A and 2B) (method for making is seen embodiment 2) of the about 50kb of karyomit(e).Choose the glutinous grain cDY446-2-47-B of high order end and low order end in the restriction enzyme digestion physical map ₃, C ₂Carry out the subclone order-checking, find the DNA zone of the 45.560kb of mensuration by analysis of biological information, GC content is 73.3%, (open reading frame, ORF), wherein relevant with the biosynthesizing of promise card thiazole rhzomorph has 37 to have comprised 44 open reading frames altogether.The functional analysis of each gene sees Table 1.

The functional analysis of each gene and proteins encoded in the table 1 promise card thiazole rhzomorph biological synthesis gene cluster

Annotate: 1.noc1 to noc5 is biosynthetic regulatory gene of promise card thiazole rhzomorph and resistant gene; Noc6 to noc30 is a structure gene; Noc31 to noc37 is regulatory gene and resistant gene.

2.orf (7) are the biosynthesis gene of methoxy propyl diacid (not belonging to promise card thiazole rhzomorph gene cluster) to orf (1).

3. promise card thiazole rhzomorph biological synthesis gene cluster border is determined

According to the functional analysis of gene coded protein, the biological synthesis gene cluster of our preliminary judgement promise card thiazole rhzomorph be from gene noc1 to noc37, totally 37 open reading frames.Wherein 1 gene (noc28) is responsible for the precursor peptide of coding promise card thiazole rhzomorph, and 7 genes (noc28, noc20, noc21, noc22, noc23, noc9, noc24 and noc30) are responsible for the precursor peptide of noc28 coding is modified, and form whole big ring skeleton; It is precursor that 4 genes (noc25, noc26, noc27 and noc29) are responsible for the tryptophane, synthesis of indole acid side chain; 6 genes (noc6, noc10, noc11, noc12, noc13 and noc14) are responsible for 4-N, N-dimethyl-2,4, the biosynthesizing of 6-deoxyhexamethylose; 5 Cytochrome P450 oxidoreductase genes (noc7, noc15, noc16, noc18 noc19) is responsible for modifying after the redox of Nocathiacin I; (noc8 noc36) is responsible for back modification of methylating of Nocathiacin I and the resistance of self respectively to 2 methyl transferase genes; Also have in addition 2 resistant genes (noc17, noc37); 3 regulatory gene (noc5, noc33, noc34); 4 genes relevant with transcription and translation (noc1, noc2, noc3, noc4); And the gene of 3 unknown function (noc31, noc32, noc35).Because orf (1)-orf (8) is and the relevant gene of methoxy propyl diacid biosynthesizing, gene noc1-noc3 then encode respectively 50S rrna L18 albumen, DGPFAETKE family protein and ECF subfamily RNA polymerase signal factor, these genes may be relevant with the method for the transcribing translation of promise card thiazole rhzomorph precursor peptide.So the right side boundary of promise card thiazole rhzomorph biological synthesis gene cluster should be between orf (1)-noc1.The gene noc31-37 on this sequence right side is some regulatory gene and resistant gene, may be relevant with regulation and control with the resistance of promise card thiazole rhzomorph generation bacterium self, so promise card thiazole rhzomorph biological synthesis gene cluster left border should be positioned at the noc37 place.The heterogenous expression of the plasmid by comprising promise card thiazole rhzomorph biological synthesis gene cluster can be further confirms the border of promise card thiazole rhzomorph biological synthesis gene cluster in the body.

4. the biosynthesizing of the big ring skeleton of promise card thiazole rhzomorph

It is relevant with the biosynthesizing of its big ring skeleton to have 8 genes in the biological synthesis gene cluster of promise card thiazole rhzomorph.Noc28 wherein, the precursor peptide of coding promise card thiazole rhzomorph, the amino acid precursor of its C end structure peptide sequence SCTTCECSCSCSS and the big ring skeleton of promise card thiazole rhzomorph fits like a glove in proper order, and the N end is rich in the signal peptide part of hydrophobic amino acid, then mainly be responsible for the identification of precursor peptide, and mediate it at intracellular transport.This has also confirmed that from gene level the big ring skeleton of promise card thiazole rhzomorph should be to come synthetic according to rrna mechanism.This process is similar to cell peptide and proteinic synthetic, at first progressively forms the poly-peptide chain precursor of promise card thiazole rhzomorph in rrna through transcription and translation.Then the hydrolysis from the rrna of this precursor discharges, again by the cyclodehydration enzyme catalysis of noc23 coding wherein the sulfydryl attack on the halfcystine face carbonyl on the Serine, forms 5 yuan of N heterocycles, then slough a part water, the formation thiazoline.A part hydrogen is sloughed in the oxydo-reductase effect that is relied on by the NADH of noc22 coding then, changes thiazoline into thiazole.Next under the catalysis of the dehydratase of noc20 or noc21 coding, slough the hydroxyl on the serine residue, form 2, the 3-dehydroalanine further forms hydroxylation dehydropiperidine six-ring by intramolecularly Diels-Alder reaction.And then under the effect of the agnoprotein of noc30 coding and a series of dehydratase and oxydo-reductase, form hydroxylation pyridine cyclic peptide center, thereby made up maximum in a promise card thiazole rhzomorph cyclic peptide structures through polystep reaction.Whole process as shown in Figure 3.

5. the biosynthesizing of skatole acid structural unit

The biosynthetic pathway of skatole acid structural unit as shown in Figure 4.At first be precursor with the tryptophane, through the proteic catalysis of radical S-adenosylmethionine (AdoMet) type of noc27 coding a series of intramolecular rearrangements take place, hydrolysis decarboxylation forms 3-skatole-2-formic acid structure again.Then activate the carboxyl of indolic acid, be converted into the CoA-thioester bond by the acyl group-CoA synthetic enzyme of noc25 coding.Then under the effect of the acyltransferase of noc26 coding, form the indolic acid side chain and be connected with thioester bond between the big ring skeleton of promise card thiazole rhzomorph.Further shift methyl on the methionine(Met) to the carbon of 4 of indolic acid by the methyltransgerase of noc29 coding, act on a last hydroxyl on 4 carbon by the oxydo-reductase in the gene cluster again, realizing under the effect of a series of relevant enzyme that the indolic acid side chain is connected with the big ester bond that encircles between skeleton at last.Wherein 4 of indolic acid methylate may occur in side chain and big ring skeleton thioester bond and is connected before or after the connection.

6.4-N, N-dimethyl-2,4, the unitary biosynthesizing of 6-deoxyhexamethylose

It is relevant with the biosynthesizing of its glycosyl to have 6 genes in the biological synthesis gene cluster of promise card thiazole rhzomorph.At first the D-glucose of a part phosphorylation is activated under the effect of dNDP-D-glucose synthetic enzyme, then by dNDP-D-glucose-4,6-dehydratase catalytic dehydration forms ketone group at 4, again by 2 of noc13 coding, the effect of 3-dehydratase is sloughed another molecular water and is formed 3,4-ketone-6-deoxyhexamethylose intermediate, then under the catalysis of the 3-ketone-reductase enzyme of noc12 coding 3 be reduced into hydroxyl, through 3, behind the effect generation epimerization of 5 isomerases, again under the effect of the methyltransgerase of noc11 coding on 3 a methyl, again by transamination enzyme catalysis amino on 4 of noc14 coding, then at the N of noc10 coding, on amino, add two methyl under the effect of N-dimethyl transferring enzyme, glycosyltransferase by the noc6 coding shifts this glycosyl to the big ring skeleton of promise card thiazole rhzomorph at last, and whole process as shown in Figure 5.

7. the back modification of promise card thiazole rhzomorph

Have 5 Cytochrome P450 oxidoreductase gene noc7 in the biological synthesis gene cluster of promise card thiazole rhzomorph, noc15, noc16, noc18, noc19, may with a series of redox in the promise card thiazole rhzomorph biosynthetic pathway after modify relevant.The respectively hydroxylation and the hydroxylation on the pyridine ring (Fig. 9) of catalysis promise card thiazole rhzomorph glutamic acid gamma position of noc7 and noc19 wherein, noc16 and noc18 be the hydroxylation (Figure 10) on the hydroxylation on the catalyzing indole acid N and 3 methyl respectively.Also having an agnoprotein noc9 in this gene cluster, is (Figure 11) that is responsible for the shearing formation amide structure of Serine side chain.

8. the application of promise card thiazole rhzomorph biological synthesis gene cluster

On the basis of clone, analysis promise card thiazole rhzomorph biological synthesis gene cluster, we have also developed the universal method of other sulphur peptide antibiotics biological synthesis gene clusters of cover quick clone.As utilizing the amino acid conserved sequence that wherein forms relevant cyclo(de)hydrase Noc23 with thiazole ring, design PCR primer has been cloned the biological synthesis gene cluster of thiostrepton, nosiheptide, siomycin etc., also utilizes the gene order relevant with the biosynthesizing of the big ring skeleton of promise card thiazole rhzomorph to clone the biological synthesis gene cluster of theiomycetin by the method for genome scanning simultaneously.

On this basis, we further analyze the similarity that has compared these gene clusters and biosynthetic pathway.As one having 15 intimate genes (table 2) in the biological synthesis gene cluster (CN200910053427.7) of promise card thiazole rhzomorph and nosiheptide (nosiheptide), wherein comprised 8 with the relevant gene of big ring skeleton biosynthesizing, 4 with the synthetic relevant gene of indolic acid side chain, 2 P450 oxidoreductase genes and 1 regulatory gene, and these genes put in order also similar substantially with transcriptional orientation.We at first by in the system that produces bacterium at nosiheptide to the same frame disappearance tentative confirmation of the similar gene of partial function these genes and the biosynthetic dependency of nosiheptide and its possible function, we import to these with carrying out the allos complementation in the frame deletion mutantion strain with the similar gene in the promise card thiazole rhzomorph gene cluster again on this basis, detect through fermentation and LC-MS, recovery has produced nosiheptide, has further confirmed these similar genes and big ring skeleton, the synthetic dependency of modifying after the redox that reaches of indolic acid side chain.Identify that with the tunning of frame deletion mutantion strain confirmation Nos B is responsible for the hydroxylation of seven glutamic acid gamma positions of promise silk as no B, the tunning of the complementary mutant strain of noc7 allos is identified and is confirmed that it is consistent with no B function.We have confirmed that also noc19 is responsible for the hydroxylation on the catalysis pyridine ring with quadrat method, and the shearing that noc9 is responsible for the catalytic serine side chain forms acid amides.Simultaneously we also produce heterogenous expression in the thalline system by 2 P450 oxidoreductase gene noc16 and noc18 at nosiheptide, tentative confirmation their possible functions be respectively to be responsible for hydroxylation on the indolic acid N and the hydroxylation on 3 methyl.

The homology of functional similarity gene relatively in table 2 promise card thiazole rhzomorph and the nosiheptide biological synthesis gene cluster

Nos	Noc	Identity (Identities)	Similarity (Positives)
				NosD	Noc20	183/328(55％)	215/328(65％)
NosE	Noc21	506/885(57％)	586/885(66％)
				NosF	Noc22	222/474(46％)	253/474(53％)

[0142]

NosG	Noc23	411/642(64％)	459/642(71％)
				NosH	Noc24	304/608(50％)	354/608(58％)
NosI	Noc25	55/91(60％)	64/91(70％)
				NosJ	Noc26	120/229(52％)	151/229(65％)
NosL	Noc27	280/357(78％)	315/357(88％)
				NosM	Noc28	41/48(85％)	45/48(93％)
NosN	Noc29	296/391(75％)	336/391(85％)
				NosO	Noc30	108/261(41％)	145/261(55％)
NosP	Noc5	144/266(54％)	187/266(70％)
				NosC	Noc19	289/406(71％)	336/406(82％)
NosA	Noc9		88/136(64％)					107/136(78％)
				NosB	Noc7	221/450(49％)	266/450(59％)

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) or the condition described in the streptomycete handbook (Practical Streptomyces Genetics), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber by weight.

Embodiment 1

Promise card thiazole rhzomorph produces the extraction of the total DNA of bacterium Nocardia bacteria Nocardia sp.WW-12651:

With 100 μ L Nocardia sp.WW-12651 (ATCC 202029) mycelia suspension inoculations in 3mL TSB liquid nutrient medium, 30 ℃, 250rpm, the about 36hr of shaking culture reaches the logarithmic phase later stage.Getting 3mL is inoculated among the 50mL TSB and (contains 5mM MgCl ₂, 0.5% glycine), 30 ℃, 250rpm reaches early stage stable growth phase behind the about 25hr of shaking culture, the muddiness that is creamy white, and a large amount of mycelia suspended substances are arranged.With bacterium liquid in 4 ℃, 3500rpm, centrifugal 15min collects mycelia, uses the lysis buffer washed twice, obtains the about 2.5mL of mycelia.Add 10mL lysis buffer (lysozyme 5mg/mL) in the 2.5mL mycelia, vortex adds achromopeptidase (Achromopeptidase) to 3mg/ml, mixing again to homogeneous.37 ℃ of water-bath 30min.Add the SDS of 0.1mL Proteinase K (20mg/mL is with the fresh preparation of lysis buffer), 1mL 10%, put into 70 ℃ of water-baths behind the mixing rapidly, 15min to basic clarification, puts cooled on ice.The KAc solution that adds 2.5mL 5M, cooled on ice 15min.Add the saturated phenol of 15mL Tris, mixing adds the 15mL chloroform more gently, mixing gently, 20000rpm, 4 ℃, centrifugal 20min.Rifle head with cut places new centrifuge tube with the water sucking-off, adds isopyknic chloroform: primary isoamyl alcohol=mixed solution extracting in 24: 1, mixing gently, 12000rpm, 4 ℃, centrifugal 10min.Rifle head with cut places new centrifuge tube with the water sucking-off again, adds the dehydrated alcohol of 2 times of volumes, and mixing has the DNA of agglomerate to occur.Its hook is gone out, place new centrifuge tube, add the washing with alcohol of 5mL70%, liquid is inclined to, blot with rifle.Add 5mL TE dissolving (add the active RNase A of no DNase to final concentration be 50 μ g/mL) again, 37 ℃ of water-bath 0.5hr.With isopyknic saturated phenol extracting twice, use isopyknic chloroform again: the mixed solution extracting twice of primary isoamyl alcohol=24: 1, add the NaAc solution of 0.1 times of volume 3M to aqueous phase, the dehydrated alcohol of 2 times of volumes, mixing has cotton-shaped DNA to occur gently.With 70% washing with alcohol DNA precipitation, sucking-off liquid.Add the 1mL absolute ethanol washing again, sucking-off liquid dries up in the super clean bench, is dissolved among the TE (pH 8.0) of proper volume.If be difficult to dissolving, can be stored in 4 ℃ at last at 55 ℃ of water-bath 2hr.

Embodiment 2

Promise card thiazole rhzomorph produces the structure of bacterium Nocardia bacteria Nocardia sp.WW-12651 genomic library:

At first determine the consumption of Sau3AI by a series of dilution experiment, a large amount of on this basis enzymes are cut the dna fragmentation that obtains and are slightly larger than 40kb, dephosphorization.(Gene 1992,116:43-49 for carrier pOJ446; US 7,109, and 019) cut and dephosphorization in the middle of two cos sequences with HpaI earlier, and then cut with BamHI from multiple clone site, obtain two connecting arms, be connected with the dna fragmentation that is about 40kb for preparing and spend the night.Take out package kit (Promega PackageneExtract) from-80 ℃ and be put on ice, treat its lucky thawing, add the above-mentioned connection liquid of 5uL immediately, flick mixing, note not producing bubble, room temperature (about 22 ℃) is placed 3hr.Add 445uL Phage damping fluid, mixing turns upside down.Add the 25uL chloroform again, the mixing that turns upside down makes it slowly streak whole liquid with termination reaction, gently gets rid of with whizzer, makes chloroform be sunken to the bottom, is stored in 4 ℃.With test kit with the streak inoculation on the LB flat board of E.coli LE392 bacterial classification cultivate.Choose single bacterium colony and in 3mL LB, (add the MgSO of 30 μ L 1M ₄The maltose solution of solution and 30 μ L 20%), 37 ℃, 250rpm, shaking culture is spent the night.The 500 μ L bacterium liquid of transferring again (add the MgSO of 500 μ L in the LB of 50mL ₄The maltose solution of solution and 500 μ L 20%), 37 ℃, 250rpm, shaking culture is to OD ₆₀₀=0.6-0.8.Get 2.5 μ L packing liquid, add 97.5 μ L phage damping fluids and 100 μ L E.Coli LE392 (OD ₆₀₀=0.67), mixing gently, 22 ℃ of water-bath 0.5hr.The LB mixing that adds 100 μ L again, 37 ℃, water-bath 75min is applied on the LB flat board and (contains Am 100 μ g/mL), and 37 ℃ of overnight incubation are to there being bacterium colony to grow.

Dull and stereotyped long 50000 clones of surpassing that have scrape with LB, add glycerine (final concentration 18%) and Apramycin (final concentration 50ug/ml), by every pipe 200uL packing, in-80 ℃ of preservations.From flat board, transfer 10 clones at random, be inoculated in the LB substratum and cultivate, prepare the extracting recombinant cosmid in a small amount by the alkaline process of escherichia coli plasmid.Hae III enzyme is cut evaluation, and detects with 0.7% agarose gel electrophoresis.According to the length of each dna fragmentation of electrophoretogram adduction, estimate that each glutinous grain inserts a segmental size and is about 35-40kb, and calculate tiring of library with this and be about 30000cfu/ μ g DNA.Because the size of most of actinomycetes chromosomal DNAs is about 8Mb, for inserting the library that fragment is 20kb, it is tired is that 2000～5000cfu/ μ g DNA just is enough to represent whole genome.According to above experiment, tire and reach 30000cfu/ μ g DNA in the library that we set up, and inserts fragment and be about about 40kb, and this library that shows that we set up has good quality, can satisfy the needs of library screening.

Embodiment 3

Promise card thiazole rhzomorph produces fermentation, product separation and purification and the evaluation of bacterium Nocardia bacteria Nocardia sp.WW-12651:

At first with 300 μ L-80 ℃ of frozen Nocardia sp.WW-12651 mycelia suspensions, be inoculated in (Zulkovsky starch 2%, glucose 0.5%, N-Z Case 0.3%, yeast extract 0.2%, flesh of fish extract 0.5%, lime carbonate 0.3%) in the seed culture medium of 25mL, 32 ℃, 250rpm cultivated 3 days.Therefrom get in the fermention medium that 2mL is transferred to 50mL (glucose 2%, HY-yeast 4121%, nutritious soy bean 1%), 30 ℃, 250rpm cultivated 4-5 days.Then fermented liquid is merged, transfer in the centrifuge tube, 3800rpm, centrifugal 15min discards the mycelia precipitation.Supernatant liquid merges organic phase with equal volume of ethyl acetate twice.Use anhydrous MgSO ₄Or anhydrous Na ₂SO ₄Drying is filtered, and 37 ℃ are evaporated to dried.Sample is dissolved in chloroform: in the mixed solvent of methyl alcohol=9: 1, whether there is Nocathiacins to have that (Nocathiacin I, II, III can be sent yellow-green fluorescence under long wave ultraviolet in the preliminary extract with TLC plate detection fermented liquid, TLC developping agent chloroform: methyl alcohol: formic acid=90: 10: 0.1), further can carry out biological activity assay (Nocathiacins can suppress the growth of S.ceolicolor M110, forms inhibition zone) with S.ceolicolor M110.

With the ethyl acetate crude extract of 2L fermented liquid, be dissolved in the mixed solvent of chloroform/methanol on this basis, behind the 100-200 order silica gel mixed sample, carry out column chromatography, elution requirement with 300-400 purpose silica gel: hexane/chloroform=1: 1,100ml; Chloroform 100ml; Chloroform/methanol=99: 1,100ml; Chloroform/methanol=98: 2,300ml; Chloroform/methanol=97: 3,200ml; Chloroform/methanol=95: 5,300ml; Chloroform/methanol=90: 10,200ml.Wherein in the component of chloroform/methanol=95: 5, detected the point of yellow-green fluorescence.Collect this component, 30 ℃ are evaporated to driedly, carry out the separation and purification of HPLC semipreparative column.

Detect wavelength: UV=220nm;

Pillar: Agilent ZORAX SB-C185 μ m 4.6 * 250mm, PN 880975-902, SN USCL024998, LN B07051;

Moving phase condition: v=1mL/min; A=H ₂O (0.1%TFA); B=CH ₃CN;

Time (min)	0	3	6	25	27	30	32
								B(％)	0	15	40	70	90	90	15

Collecting the HPLC retention time is the component of 15min, carries out HPLC-MS (ESI) and identifies, is the Nocathiacin I, corresponding [M+H] ⁺Molecular ion peak is m/z=1437.3.Simultaneously we also and have carried out HPLC-MS (ESI) and have identified, corresponding [M+H] to components such as Nocathiacin II, III, MJ347-81F4-B with similar approach separation and purification from the fermented liquid of Nocardia sp.WW-12651 ⁺Molecular ion peak is followed successively by m/z=1421.3 (Fig. 6), 1266.2,1368.3.

Embodiment 4

The biosynthesis gene of PCR clone promise card thiazole rhzomorph:

The PCR system comprises: and DMSO (8%, v/v), MgCl ₂(1.5mM), dNTP (0.2mM), merger property primer (0.2 μ M), Taq archaeal dna polymerase (2u) and the total DNA of an amount of template Nocardia sp.WW-12651.At first 95 ℃, 5min, 1 takes turns; 94 ℃ then, 1min, 63 ℃, 1min, 72 ℃, 1min, 10 take turns; 94 ℃, 1min, 55 ℃, 1min, 72 ℃, 1min, 20 take turns; Last 72 ℃, 10min, 1 takes turns.After PCR finishes, 1.5% agarose electrophoresis check result.Low melting point glue reclaims the dna fragmentation of expection size, with the EcoR V digestion of carrier pSP72, the 2.4kbDNA fragment of CIAP dephosphorization connects, transformed into escherichia coli DH5 α competent cell, be coated on and contain on the suitable antibiotic LB flat board, 37 ℃ are cultured to transformant and grow.Picking list bacterium colony overnight incubation in the liquid LB, extracting plasmid, Bgl II and EcoR V double digestion identify that the DNA that whether contains the expection size inserts fragment.And the plasmid that will be inserted with the big or small dna fragmentation of expection checks order.

Embodiment 5

Making nucleic acid molecular hybridization:

1) DIG dna marker: DNA to be marked is diluted to cumulative volume 15 μ L with sterilized water, and heat denatured is 10 minutes in the boiling water bath, places cryosel to bathe cooling immediately.Then add mixture of ribonucleotides (Hexanucleotide Mix) (10 *) 2 μ L, dNTP mark mixture (Labeling Mix) 2 μ L, Klenow enzyme labeling grade 1 μ L (Promega), after mixing, about 16 hours of 37 ℃ of water-baths.Add 0.8 μ L0.8M EDTA (pH8.0) with termination reaction, add 2.5 μ L4M LiCl, mix, add the DNA behind the dehydrated alcohol precipitation mark of 75 μ L precoolings again, place-80 ℃ of sedimentations 40 minutes.4 ℃, 12000rpm collected DNA in centrifugal 20 minutes, and the 70% washing with alcohol DNA precipitation with precooling is dissolved in 50 μ L TE (in (pH 8.0) again after the vacuum-drying.

2) quality examination behind the DIG dna probe mark: the dna probe of dilution mark is to following six gradients, 1,10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5The contrast DNA of dilution mark is respectively to following concentration 1 μ g/mL, 100ng/mL, 10ng/mL, 1ng/mL, 0.1ng/mL, 0.01ng/mL.The DNA sample spot of getting the above-mentioned concentration of 1 μ L respectively is on the nylon membrane of hybridization usefulness, according to 7) described step carries out color reaction, and the colored intensity of the contrast DNA of the dna probe of contrast marker and DIG mark is with the dna probe concentration of decision mark.

3) film of colony hybridization (library screening) shifts: therefrom get 50 μ L, add 450 μ L LB dilution and obtain 10 ^-1Extension rate, doubling dilution obtains 10 again ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Therefrom get 300 μ L respectively and be coated with the LB flat board that a block specifications is 15cm * 15cm (containing apramycin 50 μ g/mL).37 ℃ of overnight incubation are to there being bacterium colony to grow.Choose suitable Dilution ratio, make every dull and stereotyped about 1200-1500 clone.According to selected dilution proportion library, evenly be coated with four flat boards, 37 ℃ of overnight incubation with LB.Big or small clip nylon membrane according to flat board is covered in planar surface carefully and does not produce bubble, carries out position mark, takes off nylon membrane after 1 minute and places on the dry filter paper, is combined on the nylon membrane until bacterium colony in dry 10 minutes.The primary flat board places incubator 4-5hr, and the clone is regrowed as former flat board.Nylon membrane is placed sex change liquid (0.25M NaOH, 1.5M NaCl) saturated last 15 minute of filter paper (not soaking film), be transferred to neutralizer (pH 7.5 for 1.0MTris.HCl, 1.5M NaCl) saturated last 5 minute of filter paper.Be transferred to 2 * SSC (20xSSC storing solution (L ^-1): NaCl, 175.3g, Trisodium Citrate, 88.2g, pH=7.0) natural air drying on the saturated filter paper.Take off nylon membrane and place baking oven, fix 45 minutes for 120 ℃.In 3 * SSC/0.1%SDS solution, vibrate under the normal temperature and washed 3 hours, to remove cell debris.

4) film of Southern hybridization shifts: DNA sample electrophoresis on the sepharose of proper concn is carried out mark to suitable distance.Be soaked in depurination 20min among the HCl of 400mL 0.25M, make the tetrabromophenol sulfonphthalein flavescence, wash for several times with deionized water.Immerse (NaOH 0.5M, NaCl 1M) 15min in the ealkaline buffer under the room temperature, and vibration gently.Continue to soak gel 20min after changing ealkaline buffer, and vibration gently, it is inferior to give a baby a bath on the third day after its birth with deionized water.Get every limit all than the nylon membrane of the big 1mm of gel, soak fully, carry out mark with deionized water.Adopt upwards capillary transfer method, with the transfering buffering liquid transfer 8-24hr of 10 * SSC.Wash film slightly with 2 * SSC, 120 ℃ of baking 30min.

5) prehybridization and hybridization: preheating hybridization solution (20mL/100cm ²) to 68 ℃ of hybridization temperatures, put into the hybridization nylon membrane, vibrate gently and be incubated 30 minutes.With the sex change 5 minutes in boiling water bath of the dna probe of DIG mark, place cryosel to bathe cooling immediately.After the cooling, with the DIG hybridization solution (2.5mL/100cm of dna probe and suitable volumes ²) mix.Remove prehybridization solution and immediately dna probe/DIG hybridization solution is added, vibration keeps 64 ℃ of hybridization temperatures or 68 ℃ about 16 hours gently.

6) the tight wash-out in hybridization back: under the room temperature with 2 * SSC/0.1%SDS rinsing twice, each 5 minutes.68 ℃, with 0.1 * SSC/0.1%SDS vibration rinsing twice, each 15 minutes.

7) color reaction and detection: the nylon membrane behind the tight wash-out is at lavation buffer solution (0.1M toxilic acid, 0.15M NaCl, pH=7.5,0.3% (v/v) Tween 20) middle balance 1-5 minute, then (closed reagent is dissolved in the 0.1M toxilic acid with 10% concentration at the sealing damping fluid, 0.15M NaCl, pH=7.5) middle sealing is 30 minutes, soaks 30 minutes in antibody then.Behind twice of lavation buffer solution rinsing nylon membrane, with detecting damping fluid (0.1M Tris-HCl, 0.1M NaCl, pH=9.5) middle balance 2-5 minute, [NBT (nitroblue tetrazoliumchloride) is dissolved in 70%DMF at last nylon membrane to be placed the new chromophoric solution of preparing of 10mL, concentration is 70mg/mL, and BCIP (5-bromo-4-chloro-3-indolyl-phosphate) is water-soluble, and concentration is 50mg/mL.Add 45 μ L NBT in the time spent 10mL chromophoric solution, 35 μ L BCIP], place dark to develop the color.Develop the color the suitable back rinsed with deionized water of using with termination reaction.

Embodiment 6

Acquisition and the tunning analysis of the complementary mutant strain of allos:

At first produce in the system of bacterium Streptomyces actuosus ATCC25421 the establishing target gene with the mutant strain of frame disappearance at nosiheptide.Used carrier is to contain the plasmid pKC1139 of temperature sensitive type replicon (Gene 1992,116:43-49; US 7,109,019), with being used for of building plasmid with the frame disappearance, import in the generation bacterium of nosiheptide by engaging the method (as follows) that shifts between E.coli S17-1 (US 5,268,276) and Streptomyces actuosus ATCC25421 genus, cultivated 4-5 days the zygote that screening has the Am resistance for 30 ℃.Then the zygote that obtains is inoculated in the TSB liquid nutrient medium (apramycin (Am) 50 μ g/ml) 30 ℃ shaking culture 2-3 days.Coat again on the MS flat board that contains apramycin 100 μ g/ml, make it to take place single cross 42 ℃ of integration and change.The single cross that obtains is changed mutant strain 30 ℃ of lax cultivations (not adding apramycin) of carrying out the 10-20 wheel, obtain mutant strain by resistance screening again the apramycin sensitivity.The total DNA of extracting carries out the PCR screening, obtains to take place the mutant strain of double exchange.And the double exchange mutant strain that obtains is carried out the PCR order-checking on genotype, verified taken place really with the frame disappearance, and internal gene not to have sudden change.Detect by fermentation and LC-MS then, on phenotype, verified, no longer produce nosiheptide, just obtained the mutant strain of target gene with the frame disappearance.

On this basis, with intimate gene in the promise card thiazole rhzomorph biological synthesis gene cluster and the erythromycin promotor ErmE that comes from pLL6212 (the erythromycin promoter sequence is cloned into the plasmid of deriving that plasmid 7zf (available from Promega) is obtained) ^*(Gene 1992, in corresponding site 116:43-49), obtain being used for allos complementary plasmid to be cloned into integrating vector pSET152.The allos complementary plasmid that is used for that builds is imported to the mutant strain of nosiheptide corresponding gene with the frame disappearance by engaging the method that shifts between belonging to,, obtain the complementary mutant strain of allos through the apramycin resistance screening.The mutant strain of gained ferments and the LC-MS detection after verifying on the genotype, recovers to have produced nosiheptide, and the function that these genes are described is consistent (Fig. 7,8,9 and 11).

Wherein, Fig. 7 has illustrated that noc21 and noc20 have covered the dehydratase function of NosE and NosD respectively.

Fig. 8 has illustrated that noc25 has covered the acyl group of NosI-CoA synthetic enzyme function, and noc27 has covered the free radical SAM thiamines synthetic enzyme function of NosL.

Fig. 9 has illustrated that noc19 and noc7 have covered the P450 oxydo-reductase function of NosC and NosB (hydroxylation of noc7 and noc19 difference catalysis promise card thiazole rhzomorph glutamic acid gamma position and the hydroxylation on the pyridine ring) respectively.

Figure 11 has illustrated that noc9 has covered the Serine side chain shearing function of NosA proteins encoded.

E.coli S17-1 and nosiheptide produce bacterium Streptomyces actuosus ATCC25421 and as follows with engaging the method that shifts between frame deletion mutantion strain genus:

Picking list colony inoculation overnight incubation to the test tube from the intestinal bacteria culture plate that contains suitable plasmid is drawn 200-300 μ l bacterium liquid and is transferred among the 20ml LB, places 37 ℃ of shaking tables to be cultured to OD ₆₀₀Be 0.3-0.4.With the centrifugation of bacterium liquid,, use the LB of 2ml resuspended then, as the intestinal bacteria donorcells with isopyknic LB washed twice.Get the frozen Streptomyces actuosus ATCC25421 that preserves in-80 ℃, 20% glycerine of 50 μ l or with the spore suspension (2-3x10 of frame deletion mutantion strain ⁹Individual/as ml), to use H ₂O dilutes 10 times to 500 μ l, uses isopyknic TES damping fluid (0.05M, pH 8.0) washed twice then, is resuspended in isopyknic TES damping fluid, and 50 ℃ of heat shock 10min make spore germination.Add isopyknic TSB substratum then, 37 ℃ of temperature were bathed centrifugal being resuspended among the 500 μ l LB as the streptomycete recipient cell 3-4 hour.The recipient cell 100 μ L of different concns are mixed with isopyknic donorcells, directly be coated on and contain 10mM MgCl ₂IWL-4 (Soluble Starch 10g/L, K ₂HPO ₄1g/L, MgSO ₄1g/L, NaCl 1g/L, (NH ₄) SO ₄2g/L, CaCO ₃2g/L, Yeast extract 0.5g/L, tryptone1g/L, FeSO ₄7H ₂O0.001g/L, MnCl ₂7H ₂O 0.001g/L, ZnSO ₄7H ₂O 0.001g/L, agar powder 20g/L, pH=7.2) or AS-1 (Yeast extract 1g/L, L-alanine 0.2g/L, L-arginine 0.5g/L, Soluble Starch 5g/L, NaCl2.5g/L, Na ₂SO ₄10g/L, agar powder 20g/L pH=7.5) on the flat board, cultivated 12-16 hour for 30 ℃.Wash planar surface gently with the most of intestinal bacteria of flush away with sterilized water, contain nalidixic acid (final concentration is 50ng/ μ L) and corresponding antibiotic sterilized water at each dull and stereotyped surface coverage 1mL then.Cultivate picking zygote more than 5 days for 30 ℃.

Nosiheptide produces bacterium Streptomyces actuosus ATCC25421 and the fermentation of mutant strain and the Analysis and Identification method of tunning:

The spore suspension of getting 100ul Streptomyces actuosus ATCC25421 or mutant strain is inoculated into 50ml seed culture medium (Sucrose 20g/L, corn steep liquor 30ml/L, peptone 5g/L, CaCO is housed ₃5g/L; PH 7.0) the 250ml conical flask in, 30 ℃, 270rpm cultivated 24-48 hour, switching 10ml mycelia suspension is to 50ml fermention medium (Glucose 30g/L, Cotton meal 10g/L, NaCl 3g/L, 2x trace element 5ml/L, CaCO are housed ₃3g/L; PH 7.0) Erlenmeyer flask in, 28 ℃, 270rpm cultivated 96-120 hour.

The bacterium liquid that ferments is centrifugal, and supernatant discarded adds the THF of proper volume in mycelia precipitation, stir 30min, and suction filtration is removed solid precipitation, collects organic phase, be spin-dried for pressed powder, be dissolved among the THF of proper volume.

Get 20 μ l and carry out the HPLC-MS detection, detect wavelength: UV=220nm; Pillar: Agilent ZORAX SB-C185 μ m 4.6 * 250mm, PN 880975-902, SN USCL024998, LN B07051; Moving phase condition: v=1mL/min; A=H ₂O (containing 0.1%HCOOH); B=CH ₃CN (containing 0.1%HCOOH);

Time (min)	0	3	6	12	19	22	28	30
									B(％)	0	15	40	40	55	85	85	15

Embodiment 7

Acquisition of heterogenous expression mutant strain and tunning analysis:

With target gene in the promise card thiazole rhzomorph biological synthesis gene cluster and the erythromycin promotor ErmE that comes from pLL6212 ^*Be cloned in the corresponding site of integrating vector pSET152, obtain being used for the plasmid of heterogenous expression.The plasmid that builds is imported among the generation bacterium Streptomycesactuosus ATCC25421 of nosiheptide by engaging the method that shifts between belonging among the embodiment 6,, obtain the heterogenous expression mutant strain through the apramycin resistance screening.By genotype checking correct after, refer again to fermentation among the embodiment 6 and detection method and carry out analysis on the phenotype, the analog that detects the new nosiheptide that produces by LC-MS confirms the function of target gene.

The result as shown in figure 10, the proteins encoded of noc16 and noc18 has P450 oxydo-reductase function (noc16 and noc18 be the hydroxylation on the hydroxylation on the catalyzing indole acid N and 3 methyl respectively).

Embodiment 8

The heterogenous expression of promise card thiazole rhzomorph biological synthesis gene cluster:

In order to confirm the function of the promise card thiazole rhzomorph biological synthesis gene cluster that we are cloned into, the positive library that comprises promise card thiazole rhzomorph biological synthesis gene cluster glutinous grain CDY446-2-47-C2 (the structure gene noc6-noc30 that comprises gene cluster that we obtain screening among the embodiment 5, and the resistant gene in downstream and regulatory gene noc31-37), CDY446-2-47-E2 (comprise promise card thiazole rhzomorph synthetic gene bunch full gene noc1-noc37) (method for making of each clay is seen embodiment 2) carries out heterogenous expression by importing to streptomycete Streptomyces albus in conjunction with the method that shifts.At first will stick grain and be transformed among the intestinal bacteria ET12567/PUZ8002 (US 7,595,187), and utilize A Baila mycin, kantlex and paraxin that transformant is screened.Select transformant and be inoculated in 37 ℃ of incubated overnight (kantlex=25 μ g/mL, A Baila mycin=100 μ g/mL, paraxin=34 μ g/mL) among the LB, switching 0.2mL in the 20mL LB 37 ℃ be cultured to OD ₆₀₀Be 0.3-0.4, centrifugal collection thalline washes twice with isopyknic LB, is resuspended among the 2mL LB, as the intestinal bacteria donorcells.Collect streptomycete Streptomyces albus spore simultaneously, be diluted to 500 μ L with sterilized water, 8000rpml is centrifugal, and 3min removes supernatant, and isopyknic TES damping fluid washes twice, is resuspended in isopyknic TES damping fluid, and 50 ℃ of heat shock 10min make spore germination.Add isopyknic TSB again, 37 ℃ of incubation 2-5hr.Centrifugal collection spore is resuspended among the 0.5-1mL LB as the streptomycete recipient cell.The recipient cell 100 μ L of different concns are mixed with isopyknic donorcells directly to be coated on contain 10mM MgCl ₂The MS flat board on, behind 30 ℃ of cultivation 16-20hr, wash planar surface gently with the most of intestinal bacteria of flush away with sterilized water, contain the sterilized water of nalidixic acid (final concentration is 50 μ g/mL) and A Baila mycin (final concentration is 100 μ g/mL) at each dull and stereotyped surface coverage 1mL.Cultivate picking zygote more than 5 days for 30 ℃.Treat that surface drying is placed on 30 degree incubators and continues to cultivate 3-5 days, zygote occurs, changed the clay that contains promise card thiazole rhzomorph biological synthesis gene cluster in the described zygote over to until media surface.

Zygote of picking (is added 50ug/mL A Baila mycin) in TSB substratum enlarged culturing, and coat and give birth to spore on the MS substratum.Inoculate an amount of spore in the R5A liquid nutrient medium of the routine that is fit to thalli growth, 30 ℃, 250rpm cultivated 5 days.Use ethyl acetate extraction mycelia and bacterium liquid respectively, be dissolved in an amount of methyl alcohol after concentrating, HPLC-MS detects.Separation and Extraction and detection method are referring to embodiment 3.

The result shows, can detect promise card thiazole rhzomorph in the bacterium liquid, the zygote that has wherein changed glutinous grain CDY446-2-47-C2 over to has only produced micro-promise card thiazole rhzomorph, and the zygote that changes clay CDY446-2-47-E2 over to can produce promise card thiazole rhzomorph (about 0.1mg/L).

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

Claims

1. the biological synthesis gene cluster of microbiotic-promise card thiazole rhzomorph is characterized in that the nucleotide sequence of described gene cluster is selected from down group:

(a) from the nucleotide sequence of noc1 to noc37, described sequence is shown in 7731-45423 position among the SEQ ID NO:1;

(b) from the nucleotide sequence of noc6 to noc37, described sequence is shown in 11505-45423 position among the SEQ ID NO:1;

And the included coding promise card thiazole rhzomorph biosynthesis related genes of described gene cluster is specially:

1) be responsible for the biosynthetic gene of the big ring skeleton of promise card thiazole rhzomorph I, i.e. noc28, noc20, noc21, noc22, noc23, noc9, noc24, noc30 be totally 8 genes:

Noc28 is positioned at gene cluster nucleotide sequence 38432-38581 bit base place, and length is 150 base pairs, the precursor peptide of coding promise card thiazole rhzomorph, and length is 49 amino acid;

Noc23 is positioned at gene cluster nucleotide sequence 30980-32875 bit base place, and length is 1896 base pairs, coding heterocyclization albumen, and length is 631 amino acid;

Noc22 is positioned at gene cluster nucleotide sequence 29544-30983 bit base place, and length is 1440 base pairs, the coding nadh dehydrogenase, and length is 479 amino acid;

Noc21 is positioned at gene cluster nucleotide sequence 26965-29523 bit base place, and length is 2559 base pairs, the coding dehydratase, and length is 852 amino acid;

Noc20 is positioned at gene cluster nucleotide sequence 25968-26960 bit base place, and length is 993 base pairs, the coding dehydratase, and length is 330 amino acid;

Noc9 is positioned at gene cluster nucleotide sequence 14529-14984 bit base place, and length is 456 base pairs, coding unknown function albumen, and length is 151 amino acid;

Noc24 is positioned at gene cluster nucleotide sequence 32902-34683 bit base place, and length is 1782 base pairs, coding unknown function albumen, and length is 593 amino acid;

Noc30 is positioned at gene cluster nucleotide sequence 40174-40914 bit base place, and length is 741 base pairs, coding unknown function albumen, and length is 246 amino acid;

2) be responsible for the biosynthetic gene of promise card thiazole rhzomorph I indolic acid side chain, i.e. noc25, noc26, noc27, noc29, totally 4 genes:

Noc27 is positioned at gene cluster nucleotide sequence 36848-37948 bit base place, and length is 1101 base pairs, coding free radical SAM thiamines synthetic enzyme, and length is 366 amino acid;

Noc25 is positioned at gene cluster nucleotide sequence 34680-35912 bit base place, and length is 1233 base pairs, coding acyl group-CoA synthetic enzyme, and length is 410 amino acid;

Noc26 is positioned at gene cluster nucleotide sequence 35933-36820 bit base place, and length is 888 base pairs, the coding acyltransferase, and length is 295 amino acid;

Noc29 is positioned at gene cluster nucleotide sequence 38714-39976 bit base place, and length is 1263 base pairs, the methyltransgerase that coding SAM relies on, and length is 420 amino acid;

3) be responsible for the biosynthetic gene of promise card thiazole rhzomorph I deoxidation glycosyl, i.e. noc6, noc10, noc11, noc12, noc13, noc14, totally 6 genes:

Noc10 is positioned at gene cluster nucleotide sequence 14990-15703 bit base place, and length is 714 base pairs, the two methyltransgerases of coding N-, and length is 237 amino acid;

Noc11 is positioned at gene cluster nucleotide sequence 15728-16972 bit base place, and length is 1245 base pairs, the methyltransgerase on 3 C of coding NDP-hexose, and length is 414 amino acid;

Noc12 is positioned at gene cluster nucleotide sequence 16984-17970 bit base place, and length is 987 base pairs, coding NDP-hexose-3-ketoreductase, and length is 328 amino acid;

Noc13 is positioned at gene cluster nucleotide sequence 17988-19418 bit base place, and length is 1431 base pairs, coding dTDP-4-ketone-6-deoxyglucose 2, and the 3-dehydratase, length is 476 amino acid;

Noc14 is positioned at gene cluster nucleotide sequence 19424-20560 bit base place, and length is 1137 base pairs, the transaminase on 4 C of coding NDP-6-DDG, and length is 378 amino acid;

Noc6 is positioned at gene cluster nucleotide sequence 11505-12683 bit base place, and length is 1179 base pairs, the encoding glycosyl transferring enzyme, and length is 392 amino acid;

4) Cytochrome P450 oxidoreductase gene, i.e. noc7, noc15, noc16, noc18, noc19, totally 5 genes:

Noc7 is positioned at gene cluster nucleotide sequence 12704-13912 bit base place, and length is 1209 base pairs, Codocyte cytochrome p 450 oxydo-reductase, and length is 402 amino acid;

Noc15 is positioned at gene cluster nucleotide sequence 20591-21703 bit base place, and length is 1113 base pairs, Codocyte cytochrome p 450 oxydo-reductase, and length is 370 amino acid;

Noc16 is positioned at gene cluster nucleotide sequence 21696-22934 bit base place, and length is 1239 base pairs, Codocyte cytochrome p 450 oxydo-reductase, and length is 412 amino acid;

Noc18 is positioned at gene cluster nucleotide sequence 23507-24649 bit base place, and length is 1143 base pairs, Codocyte cytochrome p 450 oxydo-reductase, and length is 380 amino acid;

Noc19 is positioned at gene cluster nucleotide sequence 24646-25926 bit base place, and length is 1281 base pairs, Codocyte cytochrome p 450 oxydo-reductase, and length is 426 amino acid;

5) methyl transferase gene, i.e. noc8, noc36, totally 2 genes:

Noc8 is positioned at gene cluster nucleotide sequence 13909-14532 bit base place, and length is 624 base pairs, the coding methyltransgerase, and length is 207 amino acid;

Noc36 is positioned at gene cluster nucleotide sequence 43983-44768 bit base place, and length is 786 base pairs, the coding methyltransgerase, and length is 261 amino acid;

6) resistant gene, i.e. noc17, noc37, totally 2 genes:

Noc17 is positioned at gene cluster nucleotide sequence 22956-23480 bit base place, and length is 525 base pairs, coding bleomycin resistance protein, and length is 174 amino acid;

Noc37 is positioned at gene cluster nucleotide sequence 44914-45423 bit base place, and length is 510 base pairs, coding phosphor hydrochlorate ABC transporter, and length is 169 amino acid;

7) regulatory gene, i.e. noc5, noc33, noc34:

Noc5 is positioned at gene cluster nucleotide sequence 10404-11387 bit base place, and length is 984 base pairs, the transcription regulatory protein of coding SARP family, and length is 327 amino acid;

Noc33 is positioned at gene cluster nucleotide sequence 42031-42456 bit base place, and length is 426 base pairs, the coding heat shock protein(HSP), and length is 141 amino acid;

Noc34 is positioned at gene cluster nucleotide sequence 42565-43173 bit base place, and length is 609 base pairs, coding hsp18 transcription regulaton factor, and length is 202 amino acid;

8) be responsible for the gene of transcription and translation, i.e. noc1, noc2, noc3, noc4:

Noc1 is positioned at gene cluster nucleotide sequence 7731-8084 bit base place, and length is 354 base pairs, coding 50S ribosomal protein L 18, and length is 117 amino acid;

Noc2 is positioned at gene cluster nucleotide sequence 8282-8725 bit base place, and length is 444 base pairs, encoding D GPFAETKE family protein, and length is 147 amino acid;

Noc3 is positioned at gene cluster nucleotide sequence 8731-9867 bit base place, and length is 1137 base pairs, the signal factor of coding RNA polysaccharase ECF-subtribe, and length is 378 amino acid;

Noc4 is positioned at gene cluster nucleotide sequence 9968-10387 bit base place, and length is 420 base pairs, coding ATP enzyme, and length is 139 amino acid;

9) unknown function gene, i.e. noc31, noc32, noc35, totally 3 genes:

Noc31 is positioned at gene cluster nucleotide sequence 41001-41408 bit base place, and length is 408 base pairs, coding unknown function albumen, and length is 135 amino acid;

Noc32 is positioned at gene cluster nucleotide sequence 41492-41953 bit base place, and length is 462 base pairs, coding unknown function albumen, and length is 153 amino acid;

Noc35 is positioned at gene cluster nucleotide sequence 43228-43758 bit base place, and length is 531 base pairs, coding unknown function albumen, and length is 176 amino acid.

2. gene cluster according to claim 1 is characterized in that, the nucleotide sequence of described gene cluster is shown in 7731-45423 position among the SEQ ID NO:1.

3. expression vector that contains the biological synthesis gene cluster of the described promise card of claim 1 thiazole rhzomorph.

4. the host cell that contains the biological synthesis gene cluster that is integrated with the described promise card of claim 1 thiazole rhzomorph on described expression vector of claim 3 or the karyomit(e) of a reorganization.

5. a method that produces promise card thiazole rhzomorph is characterized in that, comprises step: express promise card thiazole rhzomorph thereby cultivate the described host cell of claim 4, and separate promise card thiazole rhzomorph from nutrient solution.