CN101962647B - Biosynthesis gene cluster of Nocathiacins and application thereof - Google Patents

Abstract

The present invention relates to the biosynthetic gene cluster of Nocathiacins and its application, and specifically provides a biosynthetic gene cluster of Nocathiacins, an antibiotic with good antibacterial activity produced by Nocardia Cloning, sequencing, analysis, functional studies and their applications. The entire gene cluster contains a total of 37 genes: 8 genes related to macrocyclic skeleton biosynthesis, 4 genes related to indolic acid side chain synthesis, 6 genes related to glycosyl synthesis, 5 genes related to P450 redox Modifier genes, 2 methyltransferase genes, 2 resistance genes, 3 regulatory genes, 3 genes with unknown functions and 4 genes related to transcription and translation. A series of novel thiopeptide antibiotics can be produced by heterologous expression of the above biosynthetic genes. The gene and protein thereof provided by the present invention can be used to find and discover compounds or genes and proteins used in medicine, industry or agriculture.

Description

Biosynthetic gene cluster of nocathiazole and its application

technical field

The invention belongs to the field of microbial gene resources and genetic engineering, and in particular relates to the cloning, sequence analysis, gene function research and application of the biosynthetic gene cluster of the thiopeptide antibiotic Nocathiacins. the

technical background

Nocathiacins are a class of cyclic peptide antibiotics rich in elemental sulfur and highly modified amino acid residues. As important members of the thiopeptide family, they were initially screened for new penicillin-resistant Staphylococcus aureus (Methicillin-Resistant Staphylococcus aureus, MRSA) and multidrug-resistant Enterococcus faecium (Multi-Drug Resistant Enterococcus faecium, MREF) were found in soil samples during the course of growth-inhibiting antibiotics [J. Antibiot. 1998, 51, 715]. In 1998, Tokushima Research Center took the lead in isolating and purifying MJ347-81F4-A (Nocathiacin Ⅰ) and B from Amycolatopsis sp.MJ347-81F4, and completed their fermentation, activity testing and structure determination. However, its absolute configuration has not been determined. In 2003, Bristol-Myers Squibb Institute of Pharmaceutical Sciences discovered Nocathiacin Ⅰ, Ⅱ, Ⅲ from Nocardia sp.WW-12651, and carried out fermentation, separation and purification and activity tests, and also used NMR , X-ray and other methods determined its absolute configuration for the first time (Figure 1) [J.Antibiot..2003, 56, 226, 232; Clough, B.; J.Org.Chem.2002, 67, 8699]. the

Nocathiazole, like other antibiotics in the thiopeptide family, also has a cyclic peptide center with a trisubstituted pyridine as the core and multiple thiazoles and dehydrated amino acids. Among them, Nocathiacin Ⅰ is very similar in structure to another member of the family, sugar thiohexose dianhydride α, the difference is that there is an additional side chain of 2,3-dehydroalaninamide, and the indole acid unit is connected with the macrocycle. The ester bond of the linking part replaces the original thioester bond. the

Studies have shown that nocathiazole can well inhibit the growth of Gram-positive bacteria, especially has a strong killing effect on multidrug-resistant opportunistic pathogens. Among them, the MIC value of Nocathiacin Ⅰ against MRSA is 0.003 μg/mL, and the MIC value against penicillin-resistant Streptococcus pneumoniae (PRSP) reaches 0.001 μg/mL, which is 10-20 times higher than that of vancomycin. times. At the same time, it also has good resistance to the recently emerged vancomycin (Vancomycin)-resistant Enterococci faeccium (VREF), with an MIC value of 0.015 μg/mL. Moreover, compared with vancomycin, nocathiacin has a more significant curative effect on mice infected with MRSA. The PD ₅₀ values of nocathiacin Ⅰ, Ⅱ, and Ⅲ were 0.8, 0.62, and 0.89 mg/kg, respectively. Under the same conditions, it is only 1.3mg/kg. Nocathiacin Ⅰ and Ⅱ, as the two compounds with the best antibacterial activity in the thiopeptide family, have better water solubility than other members at lower pH, probably because the molecule contains a dimethylhexosamine[Med .Chem.Lett.2004.14.171-175].

Recently, it was found that the mechanism of action of this class of antibiotics is similar to that of thiostrepton, which also binds to the 23S rRNA-L11 protein complex of the 50S subunit, and affects aa-tRNA.Ef-Tu.GTP by preventing or promoting the conformational change of L11 The recognition of the complex and the GTPase activity of the elongation factor, thereby inhibiting the synthesis of proteins in bacteria to play an active role [J.Am.Chem.Soc.2008, 130, 12102-12110]. At present, their specific structure-activity relationship is not very clear, but it is speculated that their similar cyclic peptide center may be the key structure for their superior activity. the

As the leading compound of a new generation of anti-infective drugs, nocathiazole has attracted great interest of scientists since its discovery. Considering that their solubility has not yet reached the requirements of intravenous injection, many chemists have carried out chemical semi-synthetic transformation on them. The Bristol-Myers Squibb Institute of Pharmacy has added some polar water-soluble groups to Nocathiacin Ⅰ to improve their bioavailability: such as alkoxylation on the hydroxyl group of the pyridine ring or on the nitrogen of the indole side chain[ Bioorganic & Medicinal Chemistry Letters.2004, 14, 3743-3746]; Addition of ammonia or thiols to 2,3-dehydroalaninamide side chains via Michael addition [Tetrahedron Letters.2004, 5, 1059-1063]; Or remove 2,3-dehydroalaninamide by chemical or enzymatic method to obtain Nocathiacin IV, and then further alkoxylate or add hydrocarbyl amine [J.Org.Chem.2002, 67, 8789]. However, these methods are often difficult to improve the water solubility while maintaining the good antibacterial activity of the original structure. And the chemical structure of this class of antibiotics is extremely complicated, and people have only completed the synthetic [Tetrahedron Lett.1984,25,2127 of partial module and acidic hydrolyzate; J.Org.Chem.1996,61,4623; J .Org. Lett.2003, 5, 4421; Tetrahedron Lett. 1991, 32, 4263; Angewandte Chemie. It was not until recent years that organic synthesis masters Moody and Nicolaou completed the total synthesis of thiazocin A, amythiamycin D, thiostrepton, siomycin A, GE2270A/T, and the synthesis of nocardiazolin Total synthesis has not yet been reported [Angew.Chem.Int.Ed.2007, 46, 7930-7954]. Moreover, due to the complex polycyclic structure and numerous chiral centers of this class of antibiotics, there are many steps for transformation by simply using the total synthesis method, and the actual production cost is too high. the

In recent years, with the in-depth research of genomics and proteomics and the rapid development of new biotechnology, we use microorganisms as "cell factories" to synthesize a large number of natural products with good biological activities and new mechanisms of action through genetic control. Its analogues are possible. This also provides a new way of thinking for us to obtain the new thiopeptide antibiotics we need in microorganisms. the

Therefore, we use nocathiazole derived from microorganisms as the target molecule, start from cloning the biosynthetic gene cluster of nocathiazole, and use a combination of microbiology, molecular biology, biochemistry and organic chemistry to study its biological Synthesis, elucidate its biosynthetic pathway and regulation mechanism, on this basis, use the principle of metabolic engineering to rationally modify the biosynthetic pathway of nocathiazole, and explore new drugs with better bioavailability and mass production through microbial fermentation . the

Contents of the invention

The invention relates to the cloning, sequencing, analysis, functional research and application of a biosynthetic gene cluster of Nocathiacins, an antibiotic with good antibacterial activity produced by Nocardia bacterium. the

In the present invention, the entire gene cluster comprises 37 gene nucleotide sequences or complementary sequences (sequence 1) (SEQ ID NO: 1), wherein there are 8 genes noc28, noc20, noc21, noc22, noc23, noc9, noc24 and noc30 is responsible for the biosynthesis of Nocathiacin I macrocyclic skeleton; 4 genes noc25, noc26, noc27 and noc29 are responsible for the biosynthesis of indole side chain; 6 genes noc6, noc10, noc11, noc12, noc13 and noc14 are responsible for 4-N,N - Biosynthesis of dimethyl-2,4,6-deoxyhexose; 5 cytochrome P450 oxidoreductase genes noc7, noc15, noc16, noc18, noc19 responsible for post-redox modification of Nocathiacin I; 2 methyltransfers Enzyme genes noc8 and noc36 are respectively responsible for the post-methylation modification of Nocathiacin I and its own resistance; 2 resistance genes noc17 and noc37; 3 regulatory genes noc5, noc33 and noc34; 4 genes related to transcription and translation noc1, noc2, noc3, noc4; and 3 genes of unknown function noc31, noc32, noc35. the

The present invention also provides a nucleotide sequence encoding nocardiazolin precursor peptide, which is composed of the amino acid sequence in sequence 2 and named noc28, and the nucleotide sequence of its gene is located at base 38432-38581 in sequence 1 base. the

The present invention also provides a nucleotide sequence encoding the cyclase that forms the thiazole ring of nocardiazolin, which consists of the amino acid sequence in sequence 3 and is named noc23. The nucleotide sequence of its gene is located in sequence 1. 30980-32875 bases. the

The present invention also provides a nucleotide sequence encoding the NADH dehydrogenase formed by the thiazole ring of nocardiazolin, which is composed of the amino acid sequence in sequence 4, named noc22, and the nucleotide sequence of its gene is located in sequence 1 At bases 29544-30983. the

The present invention also provides a nucleotide sequence encoding the dehydratase formed by the macrocyclic skeleton of nocathiazole, which is composed of the amino acid sequence in sequence 5, named noc21, and the nucleotide sequence of its gene is located at the first sequence in sequence 1 26965-29523 bases. the

The present invention also provides a nucleotide sequence encoding the dehydratase formed by the macrocyclic skeleton of nocathiazole, which is composed of the amino acid sequence in sequence 6, named noc20, and the nucleotide sequence of its gene is located in sequence 1 25968-26960 bases. the

The present invention also provides a nucleotide sequence encoding free radical SAM thiamine synthetase, which is composed of the amino acid sequence in sequence 7, named noc27, and the nucleotide sequence of its gene is located at base 36848-37948 in sequence 1 place. the

The present invention also provides a nucleotide sequence encoding acyl-CoA synthetase, which is composed of the amino acid sequence in sequence 8, named noc25, and the nucleotide sequence of its gene is located at base 34680-35912 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an acyltransferase, which is composed of the amino acid sequence in sequence 9, named noc26, and the nucleotide sequence of its gene is located at base 35933-36820 in sequence 1. the

The present invention also provides a nucleotide sequence encoding a SAM-dependent oxidase or methyltransferase, which consists of the amino acid sequence in sequence 10 and is named noc29. The nucleotide sequence of its gene is located at No. 38714- in sequence 1 39976 bases. the

The present invention also provides a nucleotide sequence encoding N-dimethyltransferase, which is composed of the amino acid sequence in sequence 11, named noc10, and the nucleotide sequence of its gene is located at base 14990-15703 in sequence 1 place. the

The present invention also provides a nucleotide sequence encoding the methyltransferase at the 3-position C of NDP-hexose sugar, which is composed of the amino acid sequence in sequence 12, named noc11, and the nucleotide sequence of its gene is located in sequence 1 At bases 15728-16972. the

The present invention also provides a nucleotide sequence encoding NDP-hexose-3-ketone reductase, which consists of the amino acid sequence in sequence 13 and is named noc12. The nucleotide sequence of its gene is located at 16984- 17970 bases. the

The present invention also provides a nucleotide sequence encoding dTDP-4-keto-6-deoxyglucose 2,3-dehydratase, which consists of the amino acid sequence in sequence 14 and is named noc13. The nucleotide sequence of its gene is located at 17988-19418 bases in sequence 1. the

The present invention also provides a nucleotide sequence encoding the aminotransferase at the 4-position C of NDP-6-deoxy-D-glucose, which is composed of the amino acid sequence in sequence 15, named noc14, and the nucleotide sequence of its gene Located at bases 19424-20560 in sequence 1. the

The present invention also provides a nucleotide sequence encoding aminotransferase of glycosyltransferase, which is composed of the amino acid sequence in sequence 16, named noc6, and the nucleotide sequence of its gene is located at base 11505-12683 in sequence 1 base. the

The present invention also provides a nucleotide sequence encoding cytochrome P450 oxidoreductase, which is composed of the amino acid sequence in sequence 17, named noc7, and the nucleotide sequence of its gene is located at base 12704-13912 in sequence 1 . the

The present invention also provides a nucleotide sequence encoding cytochrome P450 oxidoreductase, which is composed of the amino acid sequence in sequence 18, named noc15, and the nucleotide sequence of its gene is located at base 20591-21703 in sequence 1 . the

The present invention also provides a nucleotide sequence encoding cytochrome P450 oxidoreductase, which is composed of the amino acid sequence in sequence 19, named noc16, and the nucleotide sequence of its gene is located at base 21696-22934 in sequence 1 . the

The present invention also provides a nucleotide sequence encoding cytochrome P450 oxidoreductase, which is composed of the amino acid sequence in sequence 20, named noc18, and the nucleotide sequence of its gene is located at base 23507-24649 in sequence 1 . the

The present invention also provides a nucleotide sequence encoding cytochrome P450 oxidoreductase, which is composed of the amino acid sequence in sequence 21, named noc19, and the nucleotide sequence of its gene is located at the 24646-25926th base in sequence 1 . the

The present invention also provides a nucleotide sequence encoding a methyltransferase, which is composed of the amino acid sequence in sequence 22 and is named noc8. The nucleotide sequence of its gene is located at base 13909-14532 in sequence 1. the

The present invention also provides a nucleotide sequence encoding methyltransferase, which is composed of the amino acid sequence in sequence 23 and is named noc36. The nucleotide sequence of its gene is located at base 43983-44768 in sequence 1. the

The present invention also provides a nucleotide sequence encoding bleomycin resistance protein, which is composed of the amino acid sequence in sequence 24 and named noc17, and the nucleotide sequence of its gene is located at base 22956-23480 in sequence 1 place. the

The present invention also provides a nucleotide sequence encoding phosphate ABC transporter, which is composed of the amino acid sequence in sequence 25 and named noc37, and the nucleotide sequence of its gene is located at the 44914-45423 base in sequence 1. the

The present invention also provides a nucleotide sequence encoding a transcriptional regulatory protein of the SARP family, which consists of the amino acid sequence in sequence 26 and is named noc5, and the nucleotide sequence of its gene is located at base 10404-11387 in sequence 1 . the

The present invention also provides a nucleotide sequence encoding a heat shock protein, which consists of the amino acid sequence in sequence 27 and is named noc33. The nucleotide sequence of its gene is located at base 42031-42456 in sequence 1. the

The present invention also provides a nucleotide sequence encoding hsp18 transcription regulator, which is composed of the amino acid sequence in sequence 28 and is named noc34. The nucleotide sequence of its gene is located at base 42565-43173 in sequence 1. the

The present invention also provides a nucleotide sequence encoding a signal factor of the ECF-subfamily of RNA polymerase, which consists of the amino acid sequence in sequence 29 and is named noc3, and the nucleotide sequence of its gene is located at the 8731-th sequence in sequence 1. 9867 bases. the

The present invention also provides a nucleotide sequence encoding an ATPase signal factor, which is composed of the amino acid sequence in sequence 30 and is named noc4, and the nucleotide sequence of its gene is located at base 9968-10387 in sequence 1. the

The present invention also provides a nucleotide sequence encoding DGPFAETKE family protein, composed of the amino acid sequence in sequence 31, named noc2, the nucleotide sequence of its gene is located at base 8282-8725 in sequence 1. the

The present invention also provides a nucleotide sequence encoding 50S ribosomal protein L18, which consists of the amino acid sequence in sequence 32 and is named noc1. The nucleotide sequence of its gene is located at base 7731-8084 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which is composed of the amino acid sequence in sequence 33 and is named noc9. The nucleotide sequence of its gene is located at base 14529-14984 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which consists of the amino acid sequence in sequence 34 and is named noc24. The nucleotide sequence of its gene is located at base 32902-34683 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which consists of the amino acid sequence in sequence 35 and is named noc30. The nucleotide sequence of its gene is located at base 40174-40914 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which consists of the amino acid sequence in sequence 36 and is named noc31. The nucleotide sequence of its gene is located at base 41001-41408 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which consists of the amino acid sequence in sequence 37 and is named noc32. The nucleotide sequence of its gene is located at base 41492-41953 in sequence 1. the

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which consists of the amino acid sequence in sequence 38 and is named noc35. The nucleotide sequence of its gene is located at base 43228-43758 in sequence 1. the

In another preferred example, the gene cluster includes structural genes consisting of noc6 to noc30; or includes structural genes, resistance genes and regulatory genes consisting of noc1 to noc37. the

In another preferred example, the nucleotide sequence of the gene cluster is as 7731-45423 in SEQ ID NO: 1, 11505-40916 in SEQ ID NO: 1, or 1 in SEQ ID NO: 1 1-44560 digits shown. the

The present invention also provides the encoded protein of the above-mentioned nocathiazole biosynthetic gene cluster of the present invention. Preferably, the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2-38. the

The present invention also provides an expression vector containing the above-mentioned nocathiazole biosynthetic gene cluster. Preferably, the expression vector is a cosmid. the

The present invention also provides a recombinant host cell containing the above-mentioned expression vector or the above-mentioned nocathiazole biosynthetic gene cluster integrated on the chromosome. Preferably, the host cell is Streptomyces. the

The present invention also provides a method for producing nocardiazolin, comprising the steps of: cultivating the above-mentioned host cells to express nocardiazolin, and isolating nocardiazolin from the culture solution. the

The present invention also provides the use of the biosynthetic gene cluster of nocardiazolin or some genes thereof, which are used to carry out heterologous complementation in the mutant strain of S.actuosus ATCC 25421 related gene deletion, thereby restoring the production of nocathiazole Peptidectin; or heterologous expression of the post-modified gene in S.actuosus ATCC 25421 to produce a structural analog of nopeptidemectin. the

The complementary sequence of sequence 1 can be obtained according to the principle of DNA base complementarity. The nucleotide sequence or partial nucleotide sequence of Sequence 1 can be obtained by polymerase chain reaction (PCR) or by digesting the corresponding DNA fragment with a suitable restriction endonuclease or using other suitable techniques. The present invention also provides a method for obtaining a recombinant plasmid comprising at least a partial sequence 1 of the DNA fragment. the

The present invention also provides microbial pathways for the biosynthesis of nocathiazole, at least one of the genes including the nucleotide sequence in sequence 1. the

The nucleotide sequence or partial nucleotide sequence provided by the present invention can be obtained from other organisms by methods such as Southern hybridization using the method of polymerase chain reaction (PCR) or DNA comprising the sequence of the present invention as a probe. Genes similar to nocathiazole biosynthetic genes. the

The cloned DNA comprising the nucleotide sequence provided by the present invention or at least a part of the nucleotide sequence can be used to locate more library plasmids from the Nocardia sp.WW-12651 genome library. These library plasmids contain at least part of the sequence of the present invention and also contain DNA from previously uncloned adjacent regions of the genome of Nocardia sp. WW-12651. the

The nucleotide sequences provided by the present invention or at least part of the nucleotide sequences may be modified or mutated. These pathways include insertions, substitutions, or deletions, polymerase chain reactions, error-mediated polymerase chain reactions, site-specific mutations, rejoining of different sequences, different parts of a sequence, or alignment with homologous sequences from other sources Evolution (DNAshuffling), or mutagenesis by ultraviolet light or chemical reagents, etc. the

The cloned gene comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be expressed in a foreign host through a suitable expression system to obtain corresponding enzymes or other metabolites with higher biological activity or yield. These exogenous hosts include Streptomyces, Pseudomonas, Escherichia coli, Bacillus, yeast, plants and animals, etc. the

The amino acid sequence provided by the present invention can be used to isolate the desired protein and can be used for antibody preparation. the

The polypeptide comprising the amino acid sequence or at least part of the sequence provided by the present invention may still have biological activity or even have new biological activity after removing or substituting certain amino acids, or increase the yield or optimize protein dynamics characteristics or other efforts to obtained properties. the

Genes or gene clusters comprising the nucleotide sequences provided by the present invention or at least part of the nucleotide sequences can be expressed in heterologous hosts, and their functions in the host metabolic chain can be understood by DNA chip technology. the

The gene or gene cluster comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be constructed by genetic recombination to obtain a recombinant plasmid to obtain its biosynthetic pathway, and can also be obtained by insertion, substitution, deletion or inactivation new biosynthetic pathways. the

The cloned gene or DNA fragment comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can obtain a new nocathiazole with similar structure by interrupting one or several steps of nocathiazole biosynthesis thing. The inclusion of DNA fragments or genes can be used to increase the yield of nocardiazolin or its derivatives, and the present invention provides a way to increase the yield in genetically engineered microorganisms. the

The nucleotide sequence coded by the present invention encodes a precursor peptide for the biosynthesis of nocathiazole by the ribosome mechanism, which can be inserted, replaced or deleted, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific Sexual mutation, rejoining of different sequences, different parts of the sequence or directed evolution (DNAshuffling) with homologous sequences from other sources, ultraviolet or chemical reagent mutagenesis, etc. to produce new thiopeptide antibiotics or other polypeptide metabolites . the

The protein encoded by the nucleotide sequence provided by the present invention can catalyze the synthesis of structural units such as thiazole ring, hydroxylated pyridine ring, indole acid, etc., and can be recombined with the biosynthesis pathway or part of the biosynthesis pathway of other natural products. Metabolites containing these structural units and having better biological activity are obtained. the

The protein encoded by the nucleotide sequence provided by the present invention can catalyze the synthesis of the macrocyclic skeleton of nocardiazolin and its structural analogues. the

The protein encoded by the nucleotide sequence provided by the present invention can catalyze the synthesis of 4-N,N-dimethyl-2,4,6-deoxyhexose, and can be biosynthetic with other natural products or part of the biological Synthetic pathways are reorganized to obtain novel glycosylation products. the

The post-modification gene of nocathiazole provided by the present invention provides a way to obtain analogs through genetic modification, and the included redox reaction can also have other applications. the

In a word, the information provided by the present invention includes all the genes and proteins related to the biosynthesis of nocardiazolin, which can help people understand the biosynthesis mechanism of thiopeptide antibiotics, and provide materials and knowledge for further genetic modification. The gene and protein thereof provided by the present invention can also be used to find and discover compounds or genes and proteins that can be used in medicine, industry or agriculture. the

Description of drawings

Figure 1: Chemical structure of nocardiazolin. the

Figure 2: Gene structure and restriction enzyme map of the nocardiazolin biosynthesis gene cluster. (A) 6 overlapping cosmids represent the 45kb DNA region of Nocardia sp.WW-12651 genome, S represents the restriction endonuclease SmaI, the entity represents the part that has been sequenced by DNA, black thick vertical lines Labeled probe fraction is indicated; (B) Gene composition of the nocardiazolin biosynthetic gene cluster. the

Figure 3: Proposed biosynthetic pathway of the macrocyclic skeleton of nocathiazole. the

Figure 4: Proposed biosynthetic pathway for the indole side chain of nocathiazole. the

Figure 5: Proposed biosynthetic pathway of nocardiazolin 4-N,N-dimethyl-2,4,6-deoxyhexose. the

Figure 6: High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the fermentation product Nocathiacin Ⅰ. the

(A) High performance liquid chromatography; (B) Mass spectrometry

Figure 7: Heterologous complementation of some genes related to the biosynthesis of the macrocyclic skeleton of nocapetiazolin to the in-frame deletion mutant of similar genes in the nocapethiazole gene cluster. the

(A) HPLC-MS detection of the wild-type fermentation product of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421

(B) HPLC-MS detection of the fermentation product of the nosE nosE in-frame deletion mutant

(C) HPLC-MS detection of the fermentation product of nocathiazole noc21 heterologous anaplerotic mutant strain

(D) HPLC-MS detection of the fermentation product of nosD in-frame deletion mutant strain

(E) HPLC-MS detection of the fermentation product of nocathiazole noc20 heterologous anaplerotic mutant strain

Figure 8: Heterologous complementation of some genes related to the biosynthesis of the indolic acid side chain of nocathiazole to the in-frame deletion mutant of similar genes in the nocaphizolin gene cluster. the

(A) HPLC-MS detection of the wild-type fermentation product of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421

(B) HPLC-MS detection of the fermentation product of the nosI nosI in-frame deletion mutant strain

(C) HPLC-MS detection of the fermentation product of nocardiazolin noc25 heterologous anaplerotic mutant strain

(D) HPLC-MS detection of the fermentation product of nosL in-frame deletion mutant strain of nopeptidemectin

(E) HPLC-MS detection of the fermentation product of nocardiazolin noc27 heterologous anaplerotic mutant strain

Figure 9: Heterologous complementation of partial P450 post-redox modification genes in the nocathiazole biosynthetic gene cluster to the in-frame deletion mutant of similar genes in the nocathiazole gene cluster. the

(A) HPLC-MS detection of the wild-type fermentation product of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421

(B) HPLC-MS detection of the fermentation product of the nosC in-frame deletion mutant strain of nopeptidemectin

(C) HPLC-MS detection of the fermentation product of nocardiazolin noc19 heterologous anaplerotic mutant strain

(D) HPLC-MS detection of the fermentation product of the nosB in-frame deletion mutant of nopeptidemectin

(E) HPLC-MS detection of the fermentation product of nocardiazolin noc7 heterologous anaplerotic mutant strain

Figure 10: Heterologous expression of some P450 post-redox modification genes in the nocathiazole biosynthetic gene cluster in the nocaptomyces acting strain Streptomyces actuosus ATCC25421. the

(A) HPLC-MS detection of the wild-type fermentation product of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421

(B) HPLC-MS detection of fermentation products of nocardiazolin noc16 heterologous expression mutant strain

(C) HPLC-MS detection of fermentation products of nocardiazolin noc18 heterologous expression mutant strain

Chemical structure of nopeptidemectin structural analogue A

Chemical structure of nopeptidemectin structural analog B. the

Figure 11: Heterologous complementation of genes related to serine side chain cleavage in the nocathiazole biosynthetic gene cluster to the in-frame deletion mutant of similar genes in the nocathiazole gene cluster. the

(A) HPLC-MS detection of the wild-type fermentation product of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421

(B) HPLC-MS detection of the fermentation product of the nosA nosA in-frame deletion mutant strain

(C) HPLC-MS detection of the fermentation product of nocardiazolin noc9 heterologous anaplerotic mutant strain

Symbol Description

Figure 1 Nocathiacin Ⅰ: Nocathiazole Ⅰ; Nocathiacin Ⅱ: Nocathiazole Ⅱ; Nocathiacin Ⅲ: Nocathiazole Ⅲ; MJ347-81F4-B: Nocathiacin Ⅰ demethylation product. the

Fig. 2(A) B ₃ : cosmid pDY2-61-B ₃ , D ₂ : cosmid pDY2-61-D ₂ , C ₂ : cosmid pDY2-61-C ₂ , A ₁ : cosmid pDY2-61- A ₁ , C ₄ : cosmid pDY2-61-C ₄ ; letter S stands for SmaI restriction site; (B) suger 4-N,N-dimethyl-2,4,6-deoxyhexose biological Synthetic gene cyclopeptide nocardiazolin macrocyclic skeleton biosynthesis gene oxidation nocardiazolin P450 post-redox modification gene Indole acid nocardiazolin indole acid side chain biosynthesis gene resistance resistance gene regulation regulatory gene Unknown Gene.

Fig. 3 Noc20, 21, 22, 23 respectively correspond to the proteins encoded by the genes noc20, 21, 22, 23 in the nocardiazolin gene cluster described in this specification, and LP represents the signal peptide of nocardiazolin. the

Fig. 4 Noc25, 26, 27, 29 respectively correspond to the proteins encoded by genes noc25, 26, 27, 29 in the nocardiazolin gene cluster described in this specification, and Ado· represents a free radical. the

Noc6, 10, 12, 13 and 14 in Fig. 5 correspond to the proteins encoded by genes noc6, 10, 12, 13 and 14 in the nocathiazole gene cluster described in this specification, respectively. the

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

Detailed ways:

The present invention will be described in further detail below in conjunction with the accompanying drawings. the

1. Cloning of the N, N-dimethylase gene fragment in the nocardiazolin biosynthetic gene cluster:

Although the research on the biosynthesis of thiopeptide antibiotics started as early as the late 1970s, so far the research on their metabolic pathways in microorganisms has not been effectively broken through. Early researchers mainly completed Thiostrepton, Nosiheptide, Thiopeptide I (sulfomycin I), GE2270A, A10255G/B/E, Nocathiacins (Nocathiacins) Amino acid precursor labeling experiments (C ¹³ , C ¹⁴ , H ² , H ³ , N ^{15 ,} etc.), and thus speculated on the possible biosynthetic pathways of some of their main structures: such as the source of the thiazole or thiazoline ring Cysteine and serine; while indole acid or quinac acid structure is obtained from tryptophan through multi-step transformation; it is also believed that the aza six-membered ring of their core structure is obtained by [4+2] ring addition [J.Am.Chem.Soc.1979,101,5069; 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.].

Labeling experiments also showed that most of these antibiotics have similar amino acid precursors, which in a sense indicates that they may be obtained through similar biosynthetic pathways in microorganisms. The biosynthetic pathways of typical polypeptide natural products in microorganisms can be roughly divided into two categories: ribosomal pathway and non-ribosomal pathway. According to the results of chloramphenicol inhibition experiments, the Floss group speculated that these antibiotics may be synthesized by non-ribosomal pathways. Subsequently, researchers tried a large number of methods to clone the biosynthetic gene clusters of such antibiotics, such as complementation of mutants, reverse genetics, resistance genes as probes, and gene fragments encoding ribosomal pathway precursor peptides as probes. The conserved sequences of biosynthetic genes in the non-ribosomal pathway are primers, random interruption of transposons and other methods, but they are all unsuccessful. This suggests that we need to find a new way to clone the biosynthetic gene clusters of these antibiotics. the

According to the fact that most of the biosynthetic genes of the glycosyl and the parent skeleton of glycopeptide antibiotics are concatenated and distributed on the same region of the chromosome, the inventors decided to clone the whole molecule by cloning the biosynthetic genes of deoxyaminosugars in Nocathiacin Ⅰ biosynthetic gene clusters. We firstly based on the biosynthetic pathway of deoxyglycosylation of natural products derived from a large number of microorganisms [Antimicrobial Agents And Chemotherapy, 1999, 43, 1565-1573; Chemistry & Biology, 2004, 11, 959-969; Molecular Microbiology, 2000, 37, 752- 762.] Inferred the possible biosynthetic pathway of 4-N,N-dimethyl-2,4,6-deoxyhexose in Nocathiacin Ⅰ, and selected N,N-dimethyl For the conserved sequence of transferase, a degenerate PCR primer Dimeth-For1 was designed: 5′-GCTGAC GTCGCCTGCGGSAC(C/G)GG(A/T/C/G)(G/A/T)(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', then use the total DNA of Nocardia sp.WW-12651 as a template for PCR amplification to obtain a PCR product of about 0.3kb, which is cloned into the pSP72 vector, Sequencing analysis found that it has a high homology with known N, N-dimethyltransferase genes. the

2. Cloning, sequence analysis and functional analysis of nocardiazolin biosynthesis gene cluster: 

The N,N-dimethylase gene fragment cloned above was labeled with digoxin as a probe, and the constructed genome library of Nocardiasp.WW-12651 was screened, and a total of 6 cosmids with overlapping inserts were obtained. They are: cDY446-2-47-A ₁ , B ₃ , C ₂ , C ₄ , D ₂ , D ₄ , covering a DNA region of about 50 kb in the chromosome (Fig. 2A and 2B) (see Example 2 for the preparation method). The leftmost and rightmost cosmids cDY446-2-47-B ₃ and C ₂ in the physical map of restriction enzyme digestion were selected for subcloning and sequencing. Through bioinformatics analysis, it was found that the 45.560kb DNA region determined had a GC content of 73.3 %, a total of 44 open reading frames (open reading frame, ORF) were included, of which 37 were related to nocardiazolin biosynthesis. The functional analysis of each gene is shown in Table 1.

Table 1 Functional analysis of genes and encoded proteins in the nocardiazolin biosynthesis gene cluster

Note: 1. noc1 to noc5 are regulatory genes and resistance genes of nocathiazole biosynthesis; noc6 to noc30 are structural genes; noc31 to noc37 are regulatory genes and resistance genes. the

2. orf(-7) to orf(-1) are the biosynthetic genes of methoxymalonate (not belonging to the nocardiazolin gene cluster). the

3. Determination of Nocardiazolin Biosynthetic Gene Cluster Boundary

According to the functional analysis of the gene-encoded protein, we preliminarily determined that the nocardiazolin biosynthetic gene cluster is from gene noc1 to noc37, a total of 37 open reading frames. One gene (noc28) is responsible for encoding the precursor peptide of nocathiazole, and seven genes (noc28, noc20, noc21, noc22, noc23, noc9, noc24 and noc30) are responsible for modifying the precursor peptide encoded by noc28, Forms the entire macrocyclic backbone; 4 genes (noc25, noc26, noc27 and noc29) are responsible for the synthesis of indole side chains from tryptophan as a precursor; 6 genes (noc6, noc10, noc11, noc12, noc13 and noc14) Responsible for the biosynthesis of 4-N,N-dimethyl-2,4,6-deoxyhexose; 5 cytochrome P450 oxidoreductase genes (noc7, noc15, noc16, noc18, noc19) are responsible for the redox of Nocathiacin Ⅰ post-modification; 2 methyltransferase genes (noc8, noc36) are responsible for the post-methylation modification of Nocathiacin Ⅰ and its own resistance; in addition, there are 2 resistance genes (noc17, noc37); 3 regulatory genes ( noc5, noc33, noc34); 4 genes related to transcription and translation (noc1, noc2, noc3, noc4); and 3 genes of unknown function (noc31, noc32, noc35). Because orf(-1)-orf(-8) are genes related to the biosynthesis of methoxymalonate, and the genes noc1-noc3 encode 50S ribosomal L18 protein, DGPFAETKE family protein and ECF subfamily RNA polymerase respectively Signaling factors, these genes may be related to the transcriptional translation of the nocardiazolin precursor peptide. So the right boundary of the nocathiazole biosynthesis gene cluster should be between orf(-1)-noc1. The genes noc31-37 on the right side of the sequence are some regulatory genes and resistance genes, which may be related to the resistance and regulation of nocathiazole-producing bacteria themselves, so the left boundary of the nocathiazole biosynthesis gene cluster should be located at at noc37. The boundaries of the nocardiazolin biosynthetic gene cluster were further confirmed in vivo by heterologous expression of plasmids containing the nocardiazolin biosynthetic gene cluster. the

4. Biosynthesis of macrocyclic skeleton of nocathiazole

A total of 8 genes in the biosynthetic gene cluster of nocathiazole were related to the biosynthesis of its macrocyclic skeleton. Among them, noc28 encodes the precursor peptide of nocathiazole, and its C-terminal structural peptide sequence SCTTCECSCSSSS is completely consistent with the amino acid precursor sequence of the macrocyclic skeleton of nocathiazole, while the signal peptide part of the N-terminal rich in hydrophobic amino acids, It is mainly responsible for the recognition of precursor peptides and mediates their intracellular transport. This also confirmed from the genetic level that the macrocyclic skeleton of nocathiazole should be synthesized according to the ribosome mechanism. This process is similar to the synthesis of cellular peptides and proteins. First, the polypeptide chain precursor of nocathiazole is gradually formed in ribosomes through transcription and translation. Then the precursor is hydrolyzed and released from the ribosome, and the cyclodehydratase encoded by noc23 catalyzes the sulfhydryl group on the cysteine to attack the carbonyl group on the adjacent serine to form a 5-membered N heterocycle, and then remove a molecule of water , forming thiazolines. Then the NADH-dependent oxidoreductase encoded by noc22 removes a molecule of hydrogen and converts thiazoline to thiazole. Next, under the catalysis of the dehydratase encoded by noc20 or noc21, the hydroxyl group on the serine residue is removed to form 2,3-dehydroalanine, which is further formed by intramolecular Diels-Alder reaction to form hydroxylated dehydropiperidine six Yuan ring. Then, under the action of an unknown protein encoded by noc30 and a series of dehydratases and oxidoreductases, a hydroxylated pyridine cyclic peptide center was formed through a multi-step reaction, thereby constructing the largest cyclic peptide structure in nocathiazole. The whole process is shown in Figure 3. the

5. Biosynthesis of methyl indole acid building blocks

The biosynthetic pathway of the methyl indole acid building block is shown in Figure 4. First, using tryptophan as a precursor, a series of intramolecular rearrangements are catalyzed by the radical S-adenosylmethionine (AdoMet) type protein encoded by noc27, and then hydrolyzed and decarboxylated to form a 3-methylindole-2-carboxylic acid structure. The carboxyl group of indole acid is then activated by the acyl-CoA synthetase encoded by noc25, converting it to a CoA-thioester bond. Then, under the action of the acyltransferase encoded by noc26, a thioester linkage between the side chain of indole acid and the macrocyclic backbone of nocathiazole is formed. Further, the methyltransferase encoded by noc29 transfers the methyl group on the methionine to the carbon at the 4-position of indolic acid, and then the oxidoreductase in the gene cluster acts on a hydroxyl group on the carbon at the 4-position, and finally in a Under the action of a series of related enzymes, the ester linkage between the indole side chain and the macrocyclic backbone is realized. The methylation at the 4-position of indolic acid may occur before or after the side chain is linked to the thioester bond of the macrocyclic skeleton. the

6. Biosynthesis of 4-N,N-dimethyl-2,4,6-deoxyhexose units

There are 6 genes in the biosynthesis gene cluster of nocathiazole related to the biosynthesis of its sugar moiety. First, a molecule of phosphorylated D-glucose is activated under the action of dNDP-D-glucose synthase, followed by dehydration catalyzed by dNDP-D-glucose-4,6-dehydratase to form a ketone group at the 4-position, and then encoded by noc13 The 2,3-dehydratase removes another molecule of water to form a 3,4-keto-6-deoxyhexose intermediate, and then the 3-position is reduced to a hydroxyl group under the catalysis of the 3-keto-reductase encoded by noc12, After epimerization by the action of 3,5 isomerase, a methyl group is placed on the 3-position under the action of the methyltransferase encoded by noc11, and then catalyzed by the aminotransferase encoded by noc14 on the 4-position An amino group, then two methyl groups are added to the amino group under the action of N,N-dimethyltransferase encoded by noc10, and finally the glycosyltransferase encoded by noc6 transfers the sugar group to the large On the ring skeleton, the whole process is shown in Figure 5. the

7. Post-modification process of nocathiazole

There are five cytochrome P450 oxidoreductase genes noc7, noc15, noc16, noc18, noc19 in the biosynthetic gene cluster of nocardiazolin, which may be related to a series of post-redox modifications in the nocardiazolin biosynthetic pathway . Among them, noc7 and noc19 catalyze the hydroxylation of the γ-position of glutamic acid and the hydroxylation of the pyridine ring of nocardiazolin, respectively (Figure 9), and noc16 and noc18 catalyze the hydroxylation of the N of indolic acid and the hydroxylation of the 3-methyl group, respectively. Hydroxylation (Figure 10). There is also an unknown protein noc9 in this gene cluster, which is responsible for the cleavage of serine side chains to form amide structures (Fig. 11). the

8. Application of nocardiazolin biosynthetic gene cluster

Based on the cloning and analysis of the nocathiazole biosynthetic gene cluster, we also developed a general method for rapidly cloning the biosynthetic gene cluster of other thiopeptide antibiotics. For example, using the conserved amino acid sequence of the cyclodehydratase Noc23 related to the formation of the thiazole ring, PCR primers were designed to clone the biosynthetic gene clusters of thiostrepton, noepidectin, and siomycin, etc. The gene sequence related to the biosynthesis of nocathiazole macrocycle skeleton The biosynthesis gene cluster of thiomycin was cloned by genome scanning method. the

On this basis, we further analyzed and compared the similarity of these gene clusters and biosynthetic pathways. For example, there are 15 genes with similar functions in the biosynthesis gene cluster of nocathiazole and nosiheptide (CN200910053427.7) (Table 2), including 8 genes related to the biosynthesis of the macrocyclic skeleton There are 4 genes related to indole side chain synthesis, 2 P450 oxidoreductase genes and 1 regulatory gene, and the sequence and transcription direction of these genes are basically similar. First, we preliminarily confirmed the correlation of these genes with the biosynthesis of nopeptides and their possible functions by in-frame deletion of some functionally similar genes in the noepidectin-producing bacteria system. Similar genes in the nocathiazole gene cluster were introduced into these in-frame deletion mutants for heterologous complementation. After fermentation and LC-MS detection, the production of nocaptocin was restored, further confirming that these similar genes are closely related to the macrocyclic Correlation of backbone, indole side chain synthesis, and post-redox modification. For example, the identification of the fermentation product of the nos B in-frame deletion mutant confirmed that Nos B was responsible for the hydroxylation of the γ-position of glutamic acid in Nos7, and the identification of the fermentation product of the nos B heterologous complementation mutant confirmed that it had the same function as nos B. In the same way, we also confirmed that noc19 is responsible for catalyzing the hydroxylation on the pyridine ring, and noc9 is responsible for catalyzing the cleavage of serine side chains to form amides. At the same time, through the heterologous expression of two P450 oxidoreductase genes, noc16 and noc18, in the noepidectin-producing bacteria system, we preliminarily confirmed that their possible functions are responsible for the hydroxylation of indolic acid N and the 3-position formazan, respectively. hydroxylation on the base. the

Table 2 Homology comparison of functionally similar genes in the biosynthetic gene clusters of nocathiazole and nocapetide

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 illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions such as Sambrook et al., molecular cloning: laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) or Streptomyces manual (Practical Streptomyces Genetics) conditions described above, or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. the

Example 1

Extraction of total DNA from Nocardia sp.WW-12651, a nocardiazolin-producing bacterium:

Inoculate 100 μL of Nocardia sp.WW-12651 (ATCC 202029) mycelia suspension into 3 mL of TSB liquid medium, culture at 30° C., 250 rpm, and shake for about 36 hrs to reach the late logarithmic growth phase. Take 3mL and inoculate into 50mL TSB (containing 5mM MgCl ₂ , 0.5% glycine), culture at 30°C, 250rpm with shaking for about 25hrs, and then reach the early stage of stable growth phase, which is milky white and turbid with a large amount of suspended mycelia. Centrifuge the bacterial solution at 4°C, 3500 rpm for 15 minutes to collect mycelia, wash twice with lysis buffer to obtain about 2.5 mL of mycelia. Add 10 mL of lysis buffer (containing 5 mg/mL of lysozyme) to 2.5 mL of mycelium, vortex until uniform, then add Achromopeptidase (Achromopeptidase) to 3 mg/ml, and mix well. 37°C water bath for 30min. Add 0.1mL proteinase K (20mg/mL, freshly prepared with lysis buffer), 1mL 10% SDS, mix well, put it in a 70°C water bath for 15min, until it is basically clear, and cool it on ice. Add 2.5mL of 5M KAc solution and cool on ice for 15min. Add 15mL Tris-saturated phenol, mix gently, then add 15mL chloroform, mix gently, centrifuge at 20000rpm, 4°C for 20min. Use a broken pipette tip to suck out the aqueous phase and place it in a new centrifuge tube, add an equal volume of chloroform: isoamyl alcohol = 24:1 mixture for extraction, mix gently, and centrifuge at 12000 rpm at 4°C for 10 min. Then use a broken pipette tip to suck out the aqueous phase and place it in a new centrifuge tube, add 2 times the volume of absolute ethanol, mix well, and large groups of DNA appear. Hook it out, place it in a new centrifuge tube, add 5mL of 70% ethanol to wash, pour out the liquid, and dry it with a gun. Then add 5 mL of TE to dissolve (add RNase A without DNase activity to a final concentration of 50 μg/mL), and bathe in water at 37°C for 0.5 hr. Extract twice with an equal volume of saturated phenol, then extract twice with an equal volume of chloroform:isoamyl alcohol=24:1 mixture, add 0.1 volume of 3M NaAc solution to the aqueous phase, and 2 volumes of Absolute ethanol, mix gently, flocculent DNA appears. Wash the DNA pellet with 70% ethanol and aspirate the liquid. Then wash with 1 mL of absolute ethanol, suck out the liquid, dry it in an ultra-clean table, and dissolve it in an appropriate volume of TE (pH 8.0). If it is difficult to dissolve, it can be stored in a water bath at 55°C for 2 hours, and finally stored at 4°C.

Example 2

Construction of Genomic Library of Nocardia sp.WW-12651 Nocardia sp.WW-12651 Producer of Nocardiazolin:

First, through a series of dilution experiments to determine the amount of Sau3AI, on this basis, a large number of enzyme-digested DNA fragments slightly larger than 40kb, dephosphorylated. The vector pOJ446 (Gene 1992, 116: 43-49; US 7,109,019) was first cut and dephosphorylated from the middle of the two cos sequences with HpaI, and then cut with BamHI from the multiple cloning site to obtain two tethered arms, and The prepared DNA fragments with a length of about 40kb were ligated overnight. Take out the packaging kit (Promega PackageneExtract) from -80°C and put it on ice. After it just melts, add 5uL of the above connection solution immediately, flick and mix well, be careful not to generate air bubbles, and place at room temperature (about 22°C) for 3 hours. Add 445uL Phage buffer and mix by inverting up and down. Then add 25uL of chloroform, mix well by inverting up and down, make it pass through the whole liquid slowly to terminate the reaction, shake gently with a centrifuge, make the chloroform sink to the bottom, and store at 4°C. The E.coli LE392 strain included in the kit was streaked and inoculated on the LB plate for culture. Pick a single colony into 3 mL LB (add 30 μL 1M MgSO ₄ solution and 30 μL 20% maltose solution), 37° C., 250 rpm, shake culture overnight. Then transfer 500 μL of the bacterial solution to 50 mL of LB (add 500 μL of MgSO ₄ solution and 500 μL of 20% maltose solution), culture at 37° C. with shaking at 250 rpm until OD ₆₀₀ =0.6-0.8. Take 2.5 μL of packaging solution, add 97.5 μL of phage buffer and 100 μL of E.Coli LE392 (OD ₆₀₀ =0.67), mix gently, and bathe in 22° C. water for 0.5 hr. Then add 100 μL of LB and mix well, 37°C, water bath for 75min, spread on LB plates (containing Am 100μg/mL), and culture overnight at 37°C until colonies grow.

There are more than 50,000 colonies on the plate, scrape off with LB, add glycerol (final concentration 18%) and Apramycin (final concentration 50ug/ml), dispense 200uL per tube, and store at -80°C. Ten clones were randomly selected from the plate, inoculated in LB medium for culture, and the recombinant cosmids were extracted according to the alkaline method of Escherichia coli plasmids. It was identified by HaeⅢ enzyme digestion and detected by 0.7% agarose gel electrophoresis. According to the length of each DNA fragment added to the electrophoresis pattern, the size of each cosmid insert fragment is estimated to be about 35-40kb, and the titer of the library is calculated to be about 30000cfu/μg DNA. Because the size of chromosomal DNA of most actinomycetes is about 8Mb, for a library with an insert size of 20kb, a titer of 2000-5000cfu/μg DNA is sufficient to represent the entire genome. According to the above experiments, the titer of the library we established reached 30000cfu/μg DNA, and the insert fragment was about 40kb, which indicated that the library we established had good quality and could meet the needs of library screening. the

Example 3

Fermentation, product isolation, purification and identification of Nocardia sp.WW-12651, a nocardiazolin-producing bacterium:

First, inoculate 300 μL of Nocardia sp.WW-12651 mycelial suspension frozen at -80°C into 25 mL of seed medium (soluble starch 2%, glucose 0.5%, NZ Case 0.3%, yeast extract 0.2%, fish meat Extract 0.5%, calcium carbonate 0.3%), 32 ℃, 250rpm cultivated for 3 days. Get 2mL therefrom and transfer to 50mL fermentation medium (glucose 2%, HY-yeast 4121%, nutritional soybean 1%), 30 ℃, 250rpm culture 4-5 days. Then the fermentation broth was combined, transferred to a centrifuge tube, centrifuged at 3800rpm for 15min, and the mycelia precipitate was discarded. The supernatant was extracted twice with an equal volume of ethyl acetate, and the organic phases were combined. Dry over anhydrous MgSO ₄ or anhydrous Na ₂ SO ₄ , filter, and concentrate to dryness under reduced pressure at 37 °C. The sample was dissolved in a mixed solvent of chloroform:methanol=9:1, and the presence of Nocathiacins in the extract of the fermentation broth was initially detected with a TLC plate (Nocathiacin I, II, and III would emit yellow-green fluorescence under long-wave ultraviolet light, and TLC developed Chloroform:methanol:formic acid=90:10:0.1), and S.ceolicolor M110 can be used to detect biological activity (Nocathiacins can inhibit the growth of S.ceolicolor M110 and form an inhibition zone).

On this basis, dissolve the ethyl acetate crude extract of 2L fermentation broth in a mixed solvent of chloroform/methanol, mix the sample with 100-200 mesh silica gel, and perform column chromatography with 300-400 mesh silica gel. The elution conditions are: 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. Among them, in the composition of chloroform/methanol=95:5, a spot of yellow-green fluorescence was detected. The fraction was collected, concentrated to dryness under reduced pressure at 30°C, and subjected to HPLC semi-preparative column separation and purification. the

Detection wavelength: UV=220nm;

Column: Agilent ZORAX SB-C185μm 4.6×250mm, PN 880975-902, SN USCL024998, LN B07051;

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

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

The fraction with a HPLC retention time of 15 min was collected and identified by HPLC-MS (ESI). It was Nocathiacin I, and the corresponding [M+H] ⁺ molecular ion peak was m/z=1437.3. At the same time, we also separated and purified Nocathiacin II, III, MJ347-81F4-B and other components from the fermentation broth of Nocardia sp.WW-12651 by a similar method, and carried out HPLC-MS (ESI) identification, the corresponding [M+H ] ⁺ Molecular ion peaks are m/z=1421.3 (FIG. 6), 1266.2, 1368.3 in sequence.

Example 4

PCR cloning of the biosynthetic gene of nocardiazolin:

The PCR system includes: DMSO (8%, v/v), MgCl ₂ (1.5mM), dNTP (0.2mM), degenerate primer (0.2μM), Taq DNA polymerase (2u) and an appropriate amount of template Nocardia sp.WW- 12651 total DNA. First 95°C, 5min, 1 round; then 94°C, 1min, 63°C, 1min, 72°C, 1min, 10 rounds; 94°C, 1min, 55°C, 1min, 72°C, 1min, 20 rounds; finally 72°C, 10 minutes, 1 round. After the PCR was finished, the results were checked by 1.5% agarose electrophoresis. The DNA fragment of the expected size was recovered by low-melting point gel, ligated with the 2.4kb DNA fragment digested by EcoRⅤ of vector pSP72, and dephosphorylated by CIAP, transformed into E. coli DH5α competent cells, spread on LB plates containing appropriate antibiotics, and cultured at 37°C until transformation The child grows. Pick a single colony and culture it overnight in liquid LB, extract the plasmid, and double-enzyme digest with BglII and EcoRⅤ to identify whether it contains a DNA insert of the expected size. Plasmids inserted with DNA fragments of the expected size were sequenced.

Example 5

Nucleic acid molecular hybridization:

1) DIG DNA labeling: Dilute the DNA to be labeled with sterile water to a total volume of 15 μL, heat and denature in a boiling water bath for 10 minutes, and immediately place it in an ice-salt bath to cool. Then add 2 μL of Hexanucleotide Mix (10×), 2 μL of dNTP labeling mix (Labeling Mix), 1 μL of Klenow enzyme labeling grade (Promega), mix well, and place in a 37°C water bath for about 16 hours. Add 0.8 μL of 0.8M EDTA (pH8.0) to terminate the reaction, add 2.5 μL of 4M LiCl, mix well, then add 75 μL of pre-cooled absolute ethanol to precipitate the labeled DNA, and place it at -80°C for 40 minutes. Collect DNA by centrifugation at 12000rpm at 4°C for 20 minutes, wash the DNA precipitate with pre-cooled 70% ethanol, dry it in vacuum and redissolve it in 50 μL TE ((pH 8.0).

2) Quality detection after DIG DNA probe labeling: Dilute the labeled DNA probe to the following six gradients, 1, ^{10 -1} , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 . Dilute the labeled control DNA to the following concentrations 1 μg/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, respectively. Take 1 μL of DNA samples of the above concentrations and spot them on the nylon membrane for hybridization, carry out the color reaction according to the steps described in 7), and compare the color intensity of the labeled DNA probe and the DIG-labeled control DNA to determine the color of the labeled DNA probe. needle concentration.

3) Membrane transfer of colony hybridization (library screening): take 50 μL from it, add 450 μL LB to dilute to obtain a dilution factor of 10 ^-1 , and further dilute to obtain 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 . Take 300 μL of it and apply it on an LB plate (containing 50 μg/mL apramycin) with a size of 15 cm×15 cm. Incubate overnight at 37°C until colonies grow. Choose an appropriate dilution ratio to make about 1200-1500 clones per plate. Dilute the library with LB according to the selected ratio, spread evenly on four plates, and incubate overnight at 37°C. Cut the nylon membrane according to the size of the plate, carefully cover the surface of the plate to avoid air bubbles, and mark the position. After 1 minute, remove the nylon membrane and place it on dry filter paper, and dry it for 10 minutes until the colonies are combined on the nylon membrane. The original plate was placed in the incubator for 4-5 hr to allow the clones to re-grow as the original plate. Place the nylon membrane on filter paper saturated with denaturing solution (0.25M NaOH, 1.5M NaCl) for 15 minutes (do not soak the membrane), transfer to filter paper saturated with neutralizing solution (1.0MTris.HCl, 1.5M NaCl, pH 7.5) for 5 minutes. Transfer to filter paper saturated with 2×SSC (20×SSC stock solution (L ⁻¹ ): NaCl, 175.3 g, sodium citrate, 88.2 g, pH=7.0) and air-dry naturally. Remove the nylon membrane and place it in an oven, and fix it at 120°C for 45 minutes. Shake and wash in 3×SSC/0.1% SDS solution at room temperature for 3 hours to remove cell debris.

4) Membrane transfer of Southern hybridization: DNA samples are electrophoresed to an appropriate distance on an agarose gel of appropriate concentration, and marked. Soak in 400mL of 0.25M HCl for 20min to depurinate the bromophenol blue, and wash it several times with deionized water. Immerse in alkaline buffer solution (NaOH 0.5M, NaCl 1M) for 15min at room temperature, and shake gently. After replacing the alkaline buffer solution, continue to soak the gel for 20 minutes, shake it gently, and wash it three times with deionized water. Take a nylon membrane that is 1 mm larger than the gel on each side, soak it completely with deionized water, and mark it. Using the upward capillary transfer method, transfer with 10×SSC transfer buffer for 8-24 hr. Wash the membrane slightly with 2×SSC, and bake at 120°C for 30min. the

5) Pre-hybridization and hybridization: preheat the hybridization solution (20mL/100cm ² ) to a hybridization temperature of 68°C, put it into a hybridized nylon membrane, shake gently and incubate for 30 minutes. Denature the DIG-labeled DNA probes in a boiling water bath for 5 minutes, and immediately place them in an ice-salt bath to cool. After cooling, the DNA probe was mixed evenly with an appropriate volume of DIG hybridization solution (2.5 mL/100 cm ² ). Remove the pre-hybridization solution and immediately add the DNA probe/DIG hybridization solution, shake gently and keep the hybridization temperature at 64°C or 68°C for about 16 hours.

6) Stringent elution after hybridization: wash twice with 2×SSC/0.1% SDS at room temperature, 5 minutes each time. At 68°C, shake and rinse twice with 0.1×SSC/0.1% SDS, each time for 15 minutes. the

7) Color reaction and detection: The nylon membrane after stringent elution is equilibrated in washing buffer (0.1M maleic acid, 0.15M NaCl, pH=7.5, 0.3% (v/v) Tween 20) for 1-5 minutes , followed by blocking for 30 minutes in blocking buffer (blocking reagent dissolved in 0.1M maleic acid at a concentration of 10%, 0.15M NaCl, pH=7.5), and then soaked in the antibody for 30 minutes. Rinse the nylon membrane twice with washing buffer, equilibrate with detection buffer (0.1M Tris-HCl, 0.1M NaCl, pH=9.5) for 2-5 minutes, and finally place the nylon membrane in 10 mL of newly prepared chromogenic solution In [NBT (nitroblue tetrazoliumchloride) dissolved in 70% DMF, the concentration is 70mg/mL, BCIP (5-bromo-4-chloro-3-indolyl-phosphate) dissolved in water, the concentration is 50mg/mL. Add 45μL NBT, 35μL BCIP] to 10mL chromogenic solution, and place in the dark for color development. After proper color development, rinse with deionized water to terminate the reaction. the

Example 6

Obtaining of heterologous complementation mutant strains and analysis of fermentation products:

Firstly, a mutant strain with in-frame deletion of the target gene was constructed in the system of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421. The vector used is the plasmid pKC1139 (Gene 1992, 116: 43-49; US 7,109,019) containing a temperature-sensitive replicon, and the constructed plasmid for in-frame deletion is passed through E.coli S17-1 (US 5,268,276) and Streptomyces actuosus ATCC25421 genera indirect conjugative transfer method (see below) was introduced into nopeptidemectin-producing bacteria, cultured at 30°C for 4-5 days, and conjugants with Am resistance were screened. Then the obtained zygotes were inoculated into TSB liquid medium (apramycin (Am) 50 μg/ml) and cultured with shaking at 30° C. for 2-3 days. Then spread on MS plates containing 100 μg/ml apramycin, and integrate at 42° C. to allow single exchange. The obtained single-crossover mutant strains were subjected to 10-20 rounds of relaxation culture (without adding apramycin) at 30° C., and then a mutant strain sensitive to apramycin was obtained through resistance screening. The total DNA was extracted and screened by PCR to obtain mutants with double crossover. The obtained double crossover mutant was verified by PCR sequencing on the genotype, and the in-frame deletion did occur, and there was no mutation in the internal gene. Then, through fermentation and LC-MS detection, it was verified phenotypically that nopeptidemectin was no longer produced, and a mutant strain with in-frame deletion of the target gene was obtained. the

On this basis, genes with similar functions in the nocardiazolin biosynthetic gene cluster and the erythromycin The gene promoter ErmE ^* was cloned into the corresponding site of the integrative vector pSET152 (Gene 1992, 116: 43-49) to obtain a plasmid for heterologous complementation. The constructed plasmid for heterologous complementation was introduced into the mutant strain with in-frame deletion of the corresponding gene of nopeptidemectin by the method of intergenus conjugation transfer, and the mutant strain of heterologous complementation was obtained by screening for apramycin resistance . After the obtained mutant strains were verified on the genotype, they were fermented and detected by LC-MS, and nopeptidemectin was recovered, indicating that the functions of these genes were consistent ( FIGS. 7 , 8 , 9 and 11 ).

Among them, Figure 7 illustrates that noc21 and noc20 complement the dehydratase functions of NosE and NosD, respectively. the

Figure 8 illustrates that noc25 complements the acyl-CoA synthetase function of NosI and noc27 complements the free radical SAM thiamine synthase function of NosL. the

Figure 9 illustrates that noc19 and noc7 complement the P450 oxidoreductase function of NosC and NosB respectively (noc7 and noc19 catalyze the hydroxylation of the γ-position of glutamic acid and the hydroxylation of the pyridine ring of nocathiazole, respectively). the

Figure 11 illustrates that noc9 complements the serine side chain cleavage function of NosA-encoded protein. the

E.coli S17-1 and nopeptidemectin-producing strain Streptomyces actuosus ATCC25421 and in-frame deletion mutant strains are conjugatively transferred as follows:

Pick a single colony from the E. coli culture plate containing the appropriate plasmid and inoculate it into a test tube for overnight culture. Pipette 200-300 μl of bacterial liquid and transfer it to 20ml LB, and place it in a shaker at 37°C until the OD ₆₀₀ is 0.3-0.4. Centrifuge the bacterial solution, wash twice with an equal volume of LB, and then resuspend with 2ml of LB to serve as E. coli donor cells. Take 50 μl of the spore suspension (2-3x10 ⁹ cells/ml) of Streptomyces actuosus ATCC25421 or the in-frame deletion mutant strain frozen at -80°C and stored in 20% glycerol, dilute 10 times with H ₂ O to 500 μl, and then use Wash twice with a volume of TES buffer (0.05M, pH 8.0), resuspend in an equal volume of TES buffer, and heat shock at 50°C for 10 minutes to germinate the spores. Then add an equal volume of TSB medium, incubate at 37°C for 3-4 hours, centrifuge and resuspend in 500 μl LB as Streptomyces recipient cells. Mix 100 μL of recipient cells of different concentrations with an equal volume of donor cells, and spread directly on IWL-4 containing 10 mM MgCl ₂ (Soluble Starch 10 g/L, K ₂ HPO ₄ 1 g/L, MgSO ₄ 1 g/L, NaCl 1g/L, (NH ₄ )SO ₄ 2g/L, CaCO ₃ 2g/L, Yeast extract 0.5g/L, tryptone 1g/L, FeSO ₄ 7H ₂ O 0.001g/L, MnCl ₂ 7H ₂ O 0.001 g/L, ZnSO ₄ 7H ₂ O 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 L, Soluble Starch 5g/L, NaCl 2.5g/L, Na ₂ SO ₄ 10g/L, agar powder 20g/L, pH=7.5) plate, cultured at 30°C for 12-16 hours. The surface of the plate was gently washed with sterile water to wash away most E. coli, and then 1 mL of sterile water containing nalidixic acid (final concentration of 50 ng/μL) and corresponding antibiotics was covered on the surface of each plate. Culture at 30°C for more than 5 days to pick zygotes.

Analysis and identification methods for the fermentation and fermentation products of the nopeptidemectin-producing strain Streptomyces actuosus ATCC25421 and its mutant strains:

Take 100ul of the spore suspension of Streptomyces actuosus ATCC25421 or the mutant strain and inoculate it into a 250ml cone containing 50ml of seed medium (Sucrose 20g/L, corn steep liquor 30ml/L, peptone 5g/L, CaCO ₃ 5g/L; pH 7.0) Cultivate in the bottle at 30°C, 270rpm for 24-48 hours, transfer 10ml mycelia suspension to 50ml fermentation medium (Glucose 30g/L, Cotton meal 10g/L, NaCl 3g/L, 2x trace element 5ml/L , CaCO ₃ 3g/L; PH 7.0) Erlenmeyer flask, 28°C, 270rpm for 96-120 hours.

Centrifuge the fermented bacterial liquid, discard the supernatant, add an appropriate volume of THF to the mycelium precipitate, stir for 30 minutes, filter with suction, remove the solid precipitate, collect the organic phase, spin dry to obtain a solid powder, and dissolve it in an appropriate volume of THF . the

Take 20μl for HPLC-MS detection, detection wavelength: UV=220nm; column: Agilent ZORAX SB-C185μm 4.6×250mm, PN 880975-902, SN USCL024998, LN B07051; mobile phase conditions: v=1mL/min; A=H ₂ O (containing 0.1% HCOOH); B=CH ₃ CN (containing 0.1% HCOOH);

time (min) 0 3 6 12 19 twenty two 28 30 B(%) 0 15 40 40 55 85 85 15

Example 7

Obtaining of heterologous expression mutant strains and analysis of fermentation products:

The target gene in the nocardiazolin biosynthetic gene cluster and the erythromycin promoter ErmE ^* from pLL6212 were cloned into the corresponding sites of the integrated vector pSET152 to obtain a plasmid for heterologous expression. The constructed plasmid was introduced into nopeptidemectin-producing strain Streptomycesactuosus ATCC25421 by the method of intergenus conjugation transfer in Example 6, and a heterologous expression mutant strain was obtained by screening for apramycin resistance. After genotype verification is correct, phenotypic analysis is performed with reference to the fermentation and detection methods in Example 6, and the function of the target gene is confirmed by LC-MS detection of the newly produced structural analogs of nopeptidemectin.

The results are shown in FIG. 10 , the encoded proteins of noc16 and noc18 have the function of P450 oxidoreductase (noc16 and noc18 catalyze the hydroxylation on the N of indolic acid and the hydroxylation on the 3-methyl group, respectively). the

Example 8

Heterologous expression of the nocardiazolin biosynthetic gene cluster:

In order to confirm the function of the nocardiazolin biosynthetic gene cluster we cloned, we screened the positive library cosmid CDY446-2-47-C2 ( Including the structural gene noc6-noc30 of the gene cluster, and the downstream resistance gene and regulatory gene noc31-37), CDY446-2-47-E2 (including all genes noc1-noc37 of the nocathiazole synthesis gene cluster) (each The preparation method of the cosmid is shown in Example 2), and it is introduced into Streptomyces albus for heterologous expression by the method of combined transfer. Firstly, the cosmid was transformed into Escherichia coli ET12567/PUZ8002 (US 7,595,187), and the transformants were screened with abramycin, kanamycin and chloramphenicol. Selected transformants were inoculated in LB for overnight culture at 37°C (kanamycin = 25 μg/mL, abramycin = 100 μg/mL, chloramphenicol = 34 μg/mL), and transferred 0.2 mL to 20 mL LB at 37 °C Cultivate until the OD ₆₀₀ is 0.3-0.4, collect the cells by centrifugation, wash twice with an equal volume of LB, resuspend in 2 mL LB, and use them as E. coli donor cells. At the same time, collect Streptomyces albus spores, dilute to 500 μL with sterile water, centrifuge at 8000 rpm for 3 minutes to remove the supernatant, wash twice with an equal volume of TES buffer, resuspend in an equal volume of TES buffer, and heat shock at 50°C for 10 minutes. The spores germinate. Add an equal volume of TSB and incubate at 37°C for 2-5hr. Spores were collected by centrifugation and resuspended in 0.5-1 mL LB as Streptomyces recipient cells. Mix 100 μL of recipient cells of different concentrations with an equal volume of donor cells and spread them directly on MS plates containing 10 mM MgCl ₂ . After incubating at 30°C for 16-20 hrs, gently wash the surface of the plates with sterile water to remove large amounts of For some Escherichia coli, 1 mL of sterile water containing nalidixic acid (final concentration 50 μg/mL) and abramycin (final concentration 100 μg/mL) was covered on the surface of each plate. Culture at 30°C for more than 5 days to pick zygotes. After the surface is dry, it is placed in a 30-degree incubator to continue culturing for 3-5 days until zygotes appear on the surface of the medium, and the zygotes are transferred into cosmids containing the nocardiazolin biosynthesis gene cluster.

One zygote was picked and cultured in TSB medium (supplemented with 50ug/mL abramycin), and spread on MS medium to produce spores. Inoculate an appropriate amount of spores into a conventional R5A liquid medium suitable for cell growth, and culture for 5 days at 30° C. and 250 rpm. The mycelium and bacterial liquid were extracted with ethyl acetate respectively, concentrated and dissolved in an appropriate amount of methanol, and detected by HPLC-MS. See Example 3 for separation, extraction and detection methods. the

The results showed that nocardiazolin could be detected in the bacterial fluid, and the zygote transferred into the cosmid CDY446-2-47-C2 produced only a trace amount of nocardiazolin, while the zygote transferred into the cosmid CDY446-2 The zygote of -47-E2 can produce nocardiazolin (about 0.1mg/L). the

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. the

sequence listing

Claims (5)

1. A biosynthetic gene cluster of antibiotic-nocardiazolin, characterized in that, the nucleotide sequence of the gene cluster is selected from the group: (a) from the nucleotide sequence of noc1 to noc37, described sequence is as shown in No. 7731-45423 in SEQ ID NO:1; (b) from the nucleotide sequence of noc6 to noc37, described sequence is as shown in No. 11505-45423 in SEQ ID NO:1; In addition, the genes encoding nocardiazolin biosynthesis included in the gene cluster are specifically: 1) The genes responsible for the biosynthesis of the macrocyclic skeleton of nocathiazole I, namely noc28, noc20, noc21, noc22, noc23, noc9, noc24, noc30, a total of 8 genes: noc28 is located at base 38432-38581 in the nucleotide sequence of the gene cluster, with a length of 150 base pairs, encoding the precursor peptide of nocathiazole, with a length of 49 amino acids; noc23 is located at base 30980-32875 in the nucleotide sequence of the gene cluster, with a length of 1896 base pairs, encoding a heterocyclic protein with a length of 631 amino acids; noc22 is located at base 29544-30983 in the nucleotide sequence of the gene cluster, with a length of 1440 base pairs, encoding NADH dehydrogenase, and a length of 479 amino acids; noc21 is located at base 26965-29523 in the nucleotide sequence of the gene cluster, with a length of 2559 base pairs, encoding a dehydratase with a length of 852 amino acids; noc20 is located at base 25968-26960 in the nucleotide sequence of the gene cluster, with a length of 993 base pairs, encoding a dehydratase with a length of 330 amino acids; noc9 is located at base 14529-14984 in the nucleotide sequence of the gene cluster, with a length of 456 base pairs, encoding an unknown functional protein, and a length of 151 amino acids; noc24 is located at base 32902-34683 in the nucleotide sequence of the gene cluster, with a length of 1782 base pairs, encoding an unknown functional protein, and a length of 593 amino acids; noc30 is located at base 40174-40914 in the nucleotide sequence of the gene cluster, with a length of 741 base pairs, encoding an unknown functional protein, and a length of 246 amino acids; 2) The genes responsible for the biosynthesis of the indole side chain of nocardiazolin I, namely noc25, noc26, noc27, noc29, a total of 4 genes: noc27 is located at base 36848-37948 in the nucleotide sequence of the gene cluster, with a length of 1101 base pairs, encoding free radical SAM thiamine synthetase, with a length of 366 amino acids; noc25 is located at base 34680-35912 in the nucleotide sequence of the gene cluster, with a length of 1233 base pairs, encoding an acyl-CoA synthetase, with a length of 410 amino acids; noc26 is located at base 35933-36820 in the nucleotide sequence of the gene cluster, with a length of 888 base pairs, encoding an acyltransferase, and a length of 295 amino acids; noc29 is located at base 38714-39976 in the nucleotide sequence of the gene cluster, with a length of 1263 base pairs, encoding a SAM-dependent methyltransferase, with a length of 420 amino acids; 3) The genes responsible for the biosynthesis of nocardiazolin I deoxyglycosyl groups, namely noc6, noc10, noc11, noc12, noc13, noc14, a total of 6 genes: noc10 is located at base 14990-15703 in the nucleotide sequence of the gene cluster, with a length of 714 base pairs, encoding N-dimethyltransferase, with a length of 237 amino acids; noc11 is located at base 15728-16972 in the nucleotide sequence of the gene cluster, with a length of 1245 base pairs, encoding a methyltransferase on the 3-position C of NDP-hexose sugar, with a length of 414 amino acids; noc12 is located at base 16984-17970 in the nucleotide sequence of the gene cluster, with a length of 987 base pairs, encoding NDP-hexose-3-ketone reductase, with a length of 328 amino acids; noc13 is located at base 17988-19418 in the nucleotide sequence of the gene cluster, with a length of 1431 base pairs, encoding dTDP-4-keto-6-deoxyglucose 2,3-dehydratase, with a length of 476 amino acids; noc14 is located at base 19424-20560 in the nucleotide sequence of the gene cluster, with a length of 1137 base pairs, encoding an aminotransferase at position 4 C of NDP-6-deoxy-D-glucose, with a length of 378 amino acids; noc6 is located at base 11505-12683 in the nucleotide sequence of the gene cluster, with a length of 1179 base pairs, encoding a glycosyltransferase, and a length of 392 amino acids; 4) Cytochrome P450 oxidoreductase genes, namely noc7, noc15, noc16, noc18, noc19, a total of 5 genes: noc7 is located at base 12704-13912 in the nucleotide sequence of the gene cluster, with a length of 1209 base pairs, encoding cytochrome P450 oxidoreductase, with a length of 402 amino acids; noc15 is located at base 20591-21703 in the nucleotide sequence of the gene cluster, with a length of 1113 base pairs, encoding cytochrome P450 oxidoreductase, and a length of 370 amino acids; noc16 is located at base 21696-22934 in the nucleotide sequence of the gene cluster, with a length of 1239 base pairs, encoding cytochrome P450 oxidoreductase, with a length of 412 amino acids; noc18 is located at base 23507-24649 in the nucleotide sequence of the gene cluster, with a length of 1143 base pairs, encoding cytochrome P450 oxidoreductase, and a length of 380 amino acids; noc19 is located at base 24646-25926 in the nucleotide sequence of the gene cluster, with a length of 1281 base pairs, encoding cytochrome P450 oxidoreductase, with a length of 426 amino acids; 5) Methyltransferase genes, namely noc8, noc36, a total of 2 genes: noc8 is located at base 13909-14532 in the nucleotide sequence of the gene cluster, with a length of 624 base pairs, encoding a methyltransferase with a length of 207 amino acids; noc36 is located at base 43983-44768 in the nucleotide sequence of the gene cluster, with a length of 786 base pairs, encoding a methyltransferase, with a length of 261 amino acids; 6) Resistance genes, namely noc17, noc37, a total of 2 genes: noc17 is located at base 22956-23480 in the nucleotide sequence of the gene cluster, with a length of 525 base pairs, encoding bleomycin resistance protein, and a length of 174 amino acids; noc37 is located at base 44914-45423 in the nucleotide sequence of the gene cluster, with a length of 510 base pairs, encoding a phosphate ABC transporter, with a length of 169 amino acids; 7) Regulatory genes, namely noc5, noc33, noc34: noc5 is located at base 10404-11387 in the nucleotide sequence of the gene cluster, with a length of 984 base pairs, encoding a transcriptional regulatory protein of the SARP family, with a length of 327 amino acids; noc33 is located at base 42031-42456 in the nucleotide sequence of the gene cluster, with a length of 426 base pairs, encoding a heat shock protein with a length of 141 amino acids; noc34 is located at base 42565-43173 in the nucleotide sequence of the gene cluster, with a length of 609 base pairs, encoding the hsp18 transcription regulator, and a length of 202 amino acids; 8) Genes responsible for transcription and translation, namely noc1, noc2, noc3, noc4: noc1 is located at base 7731-8084 in the nucleotide sequence of the gene cluster, with a length of 354 base pairs, encoding 50S ribosomal protein L18, with a length of 117 amino acids; noc2 is located at bases 8282-8725 in the nucleotide sequence of the gene cluster, with a length of 444 base pairs, encoding DGPFAETKE family proteins, with a length of 147 amino acids; noc3 is located at bases 8731-9867 in the nucleotide sequence of the gene cluster, with a length of 1137 base pairs, encoding a signal factor of the ECF-subfamily of RNA polymerase, with a length of 378 amino acids; noc4 is located at base 9968-10387 in the nucleotide sequence of the gene cluster, with a length of 420 base pairs, encoding ATPase, and a length of 139 amino acids; 9) Unknown functional genes, namely noc31, noc32, noc35, a total of 3 genes: noc31 is located at base 41001-41408 in the nucleotide sequence of the gene cluster, with a length of 408 base pairs, encoding an unknown functional protein, and a length of 135 amino acids; noc32 is located at base 41492-41953 in the nucleotide sequence of the gene cluster, with a length of 462 base pairs, encoding an unknown functional protein, and a length of 153 amino acids; noc35 is located at base 43228-43758 in the nucleotide sequence of the gene cluster, with a length of 531 base pairs, encoding a protein of unknown function, and a length of 176 amino acids. 2. The gene cluster according to claim 1, wherein the nucleotide sequence of the gene cluster is as shown in the 7731-45423rd position in SEQID NO:1. 3. An expression vector containing the biosynthetic gene cluster of nocathiazole according to claim 1. 4. A recombinant host cell containing the expression vector according to claim 3 or the biosynthetic gene cluster of nocathiazole according to claim 1 integrated on the chromosome. 5. A method for producing nocardiazolin, characterized in that it comprises the steps of: cultivating the host cell according to claim 4 to express nocardiazolin, and isolating nocardiazolin from the culture solution.

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