CN106434785A - Preparation method of 2-n-heptyl-4-hydroxyquinoline - Google Patents
Preparation method of 2-n-heptyl-4-hydroxyquinoline Download PDFInfo
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- CN106434785A CN106434785A CN201610402625.XA CN201610402625A CN106434785A CN 106434785 A CN106434785 A CN 106434785A CN 201610402625 A CN201610402625 A CN 201610402625A CN 106434785 A CN106434785 A CN 106434785A
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- heptyl
- hydroxyquinoline
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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Abstract
The invention discloses a genetic engineering preparation method of an active organic molecule 2-n-heptyl-4-hydroxyquinoline in the fields of biomedicines and pesticides. The preparation method includes the following steps: cloning and identifying a zunyimycins biosynthesis gene cluster of a Streptomyces Streptomycessp. FJS31-2 strain by using a genetic engineering technology; integrating the gene cluster into a prokaryotic expression plasmid vector pET32a to build a heterologous expression plasmid vector pET-abx of the gene cluster; and transforming the expression plasmid vector pET-abx into Escherichia coli cells BL21 (DE3) Rosetta, carrying out IPTG induction synthesis, and carrying out product purification and structure parsing on a fermentation product to obtain the 2-n-heptyl-4-hydroxyquinoline compound. The 2-n-heptyl-4-hydroxyquinoline is prepared through creative Escherichia coli heterologous expression of the antibiotic biosynthesis gene cluster of the Streptomyces, so genetic engineering bacteria and a new development direction are provided for synthesis of quinoline derivatives through microbial heterologous expression of the heterologous antibiotic biosynthesis gene cluster.
Description
Technical field
The present invention relates to biological medicine and pesticide field, and in particular to a kind of preparation side of 2-n- heptyl -4- hydroxyquinoline
Method.
Background technology
Quinoline derivatives are the important heterocyclic compounds of a class, are widely used in spice, dyestuff and medical industry etc.
Field, and quinolines have extensive biological activity, which has low toxicity, efficient, environmentally friendly, structure change in addition
Various the features such as, it has also become one of heterocycle structure of current novel pesticide research, therefore the preparation of quinoline derivatives is also had many
The method of kind, the predominantly biosynthesiss of chemosynthesis and non-genomic engineering means.
In biosynthesiss, 2-n- heptyl -4- hydroxyquinoline is mainly by the microorganism from non-Streptomyces such as
Pseudomonas methanica isolated strains are produced by fermentation, be there is no and are existed by gene or gene cluster heterogenous expression mode
The method for producing the compound in e. coli host bacteria body, escherichia coli itself do not produce 2-n- heptyl -4- hydroxyquinoline
(2-n-heptyl-4-hydroxyquinoline).
2-n-heptyl-4-hydroxyquinoline is produced for quinolines from now on hence with microorganism heterogenous expression
Derivatives class biological medicine and pesticide field development provide genetic engineering bacterium and new developing direction.
Content of the invention
The invention is intended to going out quinoline derivatives using microorganism heterogenous expression, a kind of 2-n- heptyl -4- hydroxyl quinoline is provided
The new preparation method of quinoline.
The preparation method of the 2-n- heptyl -4- hydroxyquinoline in this programme, first, with technique for gene engineering to streptomycete
The zunyimycins biological synthesis gene cluster of Streptomyces sp.FJS31-2 bacterial strain is cloned and is identified；Then, will
The gene cluster is integrated in prokaryotic expression plasmid vector pET32a, builds the heterogenous expression plasmid vector pET-abx of the gene cluster；
Again, expression plasmid carrier pET-abx is converted competent escherichia coli cell BL21 (DE3) Rosetta, finally, passes through
The bacterial strain of IPTG induction synthesis destination protein, obtains tunning, carries out product purification and structure solution to tunning after fermentation
Analysis, obtains 2-n- heptyl -4- hydroxyquinoline compounds.
Wherein, gene heterogenous expression technology refers to be analyzed genome sequence with the method for bioinformatics, finds
The conserved sequence of the gene cluster of some of which enzyme, the product structure of the function of predicted gene, biosynthesis pathway and gene cluster,
After the means such as gene knockout verify gene cluster function, gene cluster DNA sequence is integrated into corresponding expression plasmid vector, conversion
Host Strains, the Host Strains production purpose product for being.
Heterogenous expression is in non-body production bacterium the expression for analyzing gene, is operated by genetic engineering, will be original
The gene cluster of the secondary metabolite in bacterial strain is separated, and after modified transformation, whole gene cluster is imported in expressive host,
Fermentation isolation identification compound, analyzes the noval chemical compound for obtaining.
Wherein, to streptomycete Streptomyces sp.FJS31-2 strain fermentation and isolated halogenation type polyketide
Thing zunyimycins, therefore zunyimycins biological synthesis gene cluster be streptomycete Streptomyces sp.FJS31-2
Bacterial strain can express the gene of the halogenation type polyketide that ferments.
Beneficial effects of the present invention are：By streptomycete Streptomyces sp.FJS31-2 bacterium source
Zunyimycin biological synthesis gene cluster heterogenous expression in the escherichia coli body, prepares 2-n- heptyl -4- hydroxyquinoline first,
It is to provide new developing direction by genetic engineering means micro-organisms quinoline derivatives.
Optimize further,：The preserving number of streptomycete Streptomyces sp.FJS31-2 bacterial strain is：CGMCC
4.7321, preserve in common DSMZ of China.
Optimize further, the NCBI of the zunyimycins biological synthesis gene cluster submits Serial No. to
No.KU243130.
Optimize further, the temperature of the IPTG induction synthesis is 18～28 DEG C, and the optimum temperature of expression is 18 DEG C.
Description of the drawings
Fig. 1 is 2-n- heptyl -4- hydroxyquinoline mass spectral analyses figure of the present invention；
Fig. 2 is 2-n- heptyl -4- hydroxyquinoline hydrogen analysis of spectrum figure of the present invention；
Fig. 3 is 2-n- heptyl -4- hydroxyquinoline carbon analysis of spectrum figure of the present invention；
Fig. 4 is 2-n- heptyl -4- hydroxyquinoline HMBC spectrum analysis figure of the present invention；
Fig. 5 is 2-n- heptyl -4- hydroxyquinoline hsqc spectrum map analysis figure of the present invention；
Fig. 6 is 2-n- heptyl -4- hydroxyquinoline ROESY spectrum analysis figure of the present invention；
Fig. 7 is 2-n- heptyl -4- hydroxyquinoline COSY spectrum analysis figure of the present invention.
Specific embodiment
Below by specific embodiment, the present invention is further detailed explanation：
Streptomycete Streptomyces sp.FJS31-2 material information
1. name biology：
Streptomycete Streptomyces sp.FJS31-2 (genome sequence row number：PRJNA320463)
2. Basic Biological Character：
Aerial hyphae prosperity, ecru bacterium colony, edge roughness, there is gauffer；Spore number is on the low side；Mol%G+C=73%.
3. isolation medium：
Solid GYM (ISP2)：Glucose 4g, Fructus Hordei Germinatus extract 4g, yeast powder 10g, Calcium Carbonate 2g, agar powder 18g, add water
To 1000 milliliters, sterilizing.
4. condition of culture is advised：
Solid GYM culture medium：Most 28 DEG C of suitable cultivation temperature, it is proposed that incubation time：4 days.
5. separately point：
Fanjing Mountain Nature Reserve in Guizhou Province (height above sea level 800m, 108 ° 47 ' 50 of east longitude ", 27 ° 56 ' 32 of north latitude ").
6. preservation place：
China of Institute of Micro-biology of the Chinese Academy of Sciences common Culture Collection (bacterium guarantor number：CGMCC 4.7321).Address：Court of Beijing
The institute 3 of positive area's North Star West Road 1：Institute of Microorganism, Academia Sinica；Postcode：100101.
7. preservation time：
The preservation time is on June 2nd, 2016.
8. classification foundation：
Primer (27f/1492r) amplification is guarded through 16S rRNA, sequencing simultaneously compares analysis and reference strain online
The homology of Streptomyces sparsogenes strain NBRC 13086 is 99%, and preliminary classification is streptomycete.
Streptomycete Streptomyces sp.FJS3-2 16s RNA sequence：
TGCAGTCGAACGATGAAGCCGCTTCGGTGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGC
ACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGACCTGGTGAGGCATCTTACTGGGTGGAAAGCTCC
GGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGG
CCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGC
ACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGG
AAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCG
CAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTA
ACCCCGGGTCTGCATTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATG
CGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGG
GAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTGGGCGACATTCCACGTTGT
CCGtGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAaGgAATTGACGGG
GGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACATCGG
AAACATCCAGAGATGGGTGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGAT
GTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACA
GGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACA
CGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGA
TTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTC
CCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGAG
GGAGTC
Two type polyketide zunyimycins biological synthesis gene cluster information of halogenation
To streptomycete Streptomyces sp.FJS31-2 strain fermentation and isolated halogenation type polyketide
zunyimycins.
Wherein, R1, R2, R3 are independently selected from H or Cl.
Table 1：Zunyimycins biological synthesis gene cluster information：
>gi|1022946594|gb|KU243130.1|UNVERIFIED:Streptomyces sp.FJS31-
2anthrabenzoxocinones biosynthetic gene cluster,complete sequence
CGACGCCGGGTCAGGAGCGCTTGAGGGGCGCCGGGGAGGTGGCGGGCCGGGAGGCGGAGCGGACCCGCTCGGGCCAG
GGCGGGCAGTTCACCAGCTCGGCCCACTCCCGGTCGTAGCGCCCCGGCACGGCCCGGCCCGCGGAGGCCCAGCGCTC
GACCAGGGCGGCGTAGATGGGCACACAGGTGCTTTGATGGAGCGTTTCGCCGCCCGCGGTGCTCCGGTCCAGCGCTC
TGCTCGGCTGCGAGAGAGAACGGCGTCGCATCGTCGTCGTCATGTCAGGCCAACGCGCCGCGTCCGGTGCGGTCACC
GGCTTCCCGCGGAGCTCACCCGGACCGGGGTGGTCTCGCACGTATCATCGACCGCGCCCGAAAGTGAAAAGCACTGC
CGAATTCAGAAAGTATTCCGTATCGCTGCAGGCCCTGGTTCACAAACTCCGATTCACAAATGGATACGTCGAACTGA
TCAGCGATTTGCGGCGGGTAGGGGCGGTCTCGGTTCCGGTCTCCTGGGTGCGTGCTGCTCATGAATCGATTCGCGTA
TCAGCAGGCACAGATGCGATTGAGCCGAAATATGGTGCGGATCTACTGATCATGCAGGCCGGAGGTATGCTCGGCTG
CGACCTGAATACGGGGGAGCGGGCGGGGCCTTGTGGCACCACAACGAGCCGGGGGAGTTCGCGAATGAGGTTTCAGG
TTCTGGGAAACTTCGAAGTCCTGAGCGACACCCGCGCCCGGACGCCGAGCGCTCCCAAGCTCCGCCGGGCCCTGGCC
CTGCTGATACTGCGGCACAACGAGGTCGTGCCCACCAAGGCCCTCATCGACGAACTCTGGGGAAGCCGGCCCCCGGA
CAAAGCCATTCGGGCTGTACACACCTACATCTATGAACTCCGCCGCAGCCTGGCCCGCCCGGGCGGTGGCGGAGAGC
TGTTCCTCCAGACCCGGCCGAGCGGCTATACCGTTCGCGTACCGGAAAGCGCGATCGACCTCAATTCCTTTCGAGTC
CTGGTCAAAGAGGGCAGGGAGGCGCTTGCTGCGGGGGACCCGGGACACGCCCGGGAAGTGCTGAACCGGGCCCTGGG
GCTGTGGCAGGGCAGCGCCCTGGCCAATGTGGACTGCGGGGAACTGCTCGAAGCGCATGCGACCGAGCTGGAGGAGA
GCCGGCTGCGTGCCCTGGAGATGCGCATCGAAGCCGATTTCCAACTCAGCCGGCACCATGAATTGACCGGTGAGCTC
AAGGCGCTGGCCGCCGCCAGGCCCCTGCACGAGGGCATCCACGCCAAACTGATGCTCGCGCTCTACCGTTCGGGGCG
GCGCGGCGAGGCGCTCAAGGTCTTCCACGACCTGCGCCGCCATCTGGTCGACGAGCTGGGCCTGGAGCCGGGCCCCG
AGCTCCAGCGGCTGCAGCGCTCCATGCTCGCCGGGGACCCCTCGCTCGACCCGCCGACCGCGCCACCGCTCCCGCCG
CCCCGCCGCGCGCAGCCCCCGGCACCGCCCGCCCAGCTCCCCCGGGACACCGTCGACTTCACCGGGCGGCAGGCCGT
GCTCGACGAGGTCGGCGCCCTGCTCGCGCCCCACGGCGACGGCACGGGCCTGCCCGTGGTGTCGCTGGTCGGCATGC
CGGGCGTCGGCAAGACGGCCACCGCCATCCACCTCGCCCACGCGGTCCGCGCCCGCTACCCCGACGGGCAGCTGTAC
GTACCGCTGGGCGGATCGCAGCCCACTCCGGCGACCGCGGCGGAGGGGATGGAACACATTCTGCGGGGGATCGGTGT
GGCGCCGCGCGACATCCCGAGCACGCTGGGCGGGCGGATGGCCCTGTTCCGCACCTGGAGCTCCGACCGCCGGGTGC
TGCTGGTACTGGACGACGCCGACTCGCCGCAGCAGGTCGAACCGCTGCTCCCGGGCGGCACCGGCTGCGCCGTGCTG
ATCACCGGCCGCTCCCTGCTGTACGGGCTGCGCGGGGCGCGGACCATCGCGCTGAGCTGCCTGTCCACCGCGGAGGG
CGGGCAGCTGCTCACCCGGCTGATCGGGCGGGAGCGGACGGACGCCGAGCCCGAGGCCGTCGCCGATGTGGTGCGGC
TGGCGGACGGTCTGCCGCTGGCCATCACCTTCCTCGGCGAGCGGCTGATGGCGTTGCGGCCGGTCAGTATCGCCTGC
GTGCTGGCGAAGATACGATCCGCCAAGGGGCAGCACCGGCTCTCCGAGCTCTCGGCGCTGGGGCTCGACCTGTACGA
CCGGCTGGACAGCTGCTTCCGCAAGCTCGACGAGAACACGCAGCAGGCCTTTCTGCGGCTGGCCCTGATGCCGCGCC
GGCTGTTCACCGCCGGGCAGGGCGCCCGTGCGCTGGGCACCGACACCACCTCGGCCGACGTGGTGCTCATGCGGCTG
GTCGACGCCTCACTGGTGGAGGTCGCGGGGGAGCGGGAGGTGGGCGCGAGACACTACCGCTTC
CGGGAACTGGTCCGCGAGTACGCGCTGGAGCGGATGGCGTGACCCGTACGGCGCCCGCGGCGCGCCACTTCACGGGC
TTGTGCGCCCGCTGCGCCCGGATGGCCGTGGGCCGCGCGTTCAGGTGGTGTCCGCGGCCCGGTGTCCGGCCCGCAGC
GCGGGTAGCACCACCTGGTCGACCATGGCCTCGATGGCCCGGTCGTCCGGCGGGGTGCCGTTGAGCAGATAGTGCAT
CATCACCTGCGCGGGCCCGGTCCGCGCGGCGAACGGGCCGGCCGACTCCCCGGGGATCTCGCCGCGCTCGGCCGCCC
GCCGCAGGATCTCCGCGATCGCCTCCAGCCGCGGCTCGATGAACCGGGTGCGGAACACCGCGATCAGCTCGGGGTGG
CGCAGCAGATCCCCCAGCAGCACCATCGCGGGCGGGCCGGTGCGGCCGGTGAGCGAGTCGGCCATCCGCCGCAGCGC
CGCCACCAGTTCCTCGCGCAGATCGCCCGTCTCGGGCAGTTCCTCGCTGGGCAGCAGGACATGTTCGAAGGCGTCGA
GCAGCAGGTCCTGCGGGGTGCTCCAGCGGCGGTACAGCGCCGCCTTACCGGTGCGCGCCCGTTCCGCGACCCCCTTG
AGCGTCAGCCTGCCGTAGCCCAGTTCGGCGCACTCGGCGATCGTCGCCTTGTAGATGGCCCGCTCCAGGGCCGCGCC
CCTGCGACGGGTCCGTACCGGCGGCGCCGGTGCTTCCTCCGCGCCCATCCGGCCGCCTTCCTCCCGGCAAGCATTGC
CCCCAAAAGCCCCGTCAAGCACCTTCGAGCCCCTTTGCGGACTCTATCAGCGGGGTCTACCCTGCCGAATAAGGAAC
AGATTAGTCTACTATCCGGCCGGCGGCGCATCGCTGCGCCCCCTCCTCACGATGGGCGAAAGAGGGGCGAACGACCA
TGGACGTCACCCAGGACCCCGCCCCGCCGCCCCGGTTCTGGTGGGCCATTCTGGTGATCGCGGGAGCGCAGCTGATG
GTGGTGCTGGACATCAGCATCGTGAACATCGCGCTGCCCAGCATCCGCGACGACCTCGGCTTCACCCGGTCCGGGCT
GCAGTGGGTGGTCAACGGCTACACCCTCGCCTTCGGCGGGCTGCTGCTGTTCGGCGGCCGGGTGGGCGATCTCCTGG
GCCGCCGCCGGGTGTTCGCCTGCGGAGTCTGCGCGTTCGCACTGATCTCCCTGCTCGGCGGCTTCGCCGACGGGCCC
GGCCGGCTCATCGCCGCCCGCGCCGCCCAGGGCGTGGCCGGCGCCCTCGTCGCGCCCACCGCCCTGGCGCTGATCAG
CACGACCTTCCCGGAAGGGCGGCAGCGCCGCGCCGCGTTCGGCGTCTACGGGGCGGTGTCCGCGGGCGGCGGGGCTA
TCGGGCTGCTGCTGGGCGGGCTGCTCACCGACACCCTCTCCTGGCGCTGGGTGCTCTGGGTCAATGTGCCCATCGGC
GCCGCGCTCGCCTGTGGCGCCCTGCTGGTGCTGCCCTCCTCCGCGCGCCGGGGCGGCCGGTTCGACCTGCCCGGGGC
GCTCTCGGTCACCGGCGGGCTGACCGCGCTGGTGTACGGGCTGGTCCGCGCCCCCCAGCACGGCTGGGCCGACCGCT
GGACGCTGACCTCGTTCGCCGCGGCGGCCGGGCTCCTGGTCCTGTTCGTGTTGATCGAGGTGCGCGGCCGGCAGCCG
CTGATGCCGTTGCGCGTCTTCGCCGACCGCGACCGCTCCGGTGCCTACGCGGTGACCCTCCTGACCGGCGCGGCGCT
GCTGAGCACGCTCTTCTTCCTCGCCCAGTTCGCGCAGGACGTGATGGGCTACTCACCGCTGCGCAACGGCGTGGCGT
TCCTGCCCAACGCCGCCTGCGTGCTGCTCTTCTCCGGGCTCGCCGCACGGCTGACCGCCCGCGTCGGCGCCATGCCG
CTGATGGCCGCGGGCGCCGCCTCGATGGCCCTCGGCCTGGCCTGGCTGTCGCGGCTCGACGTCGGCGCGAGGTACGC
CGCCGACCTGCTGCCGGGCCTGCTGCTGTACGGGTCCGGCCTCGGCCTGCTGTTCGTACCGCTGTCCATGACCGCGG
TCGCGACCGTCGCGGACGAGGACTCCGGCCTCGCCTCCGGGCTGCTCAACACCGCCCAGCAGGTGGGCGGTTCGCTG
GGCCTGGCCACGCTGAGCACCGTGGCGGCCGACTCCACCCGGGACGCCCTGCCCGCCGGCCGCCCGCCGACCCGCCC
GCAGCTCGCCCACGCGCTGGCCGAGGGCTGGGCCACGGCCCTGTCCGTCAGCGTGGCCTTCGTCGGCTGCGCGCTGG
TCCTCGTCGTGGCCGTGATCCGCGCCCGGCCGCGCGCCGCCGAGCCCGCCACACGGCAGGAGGCGGTCACCCGCACC
CCGGGCTGACCCGGGCCGATGCCCGCCCTTGTGAGAAACCTGTCCGCTTCCTGAGCGTCGATGTGCAGGCTCGCCGC
ATCGGCTCTTTCCGGTTCCCCGGACGCGACCCGGCCCCGGGCCGCGATGAGCCGAGCGCCGCACACACAGCCGCACA
CACAGGGGGAGTGGTACGGATGGAGCGGGTCGCGCTGGGACTTCCGGTCGGACCGGGCAACGAAACGCGGGTGGCAC
GGCTGCTGCGCCGCCTCGCCCCGGCCCAGGCCGGCGAGGTGCGCACCGAGGCGGTGGCGACGTTCACCGCGCCGGGC
CGGGTGCTGCACATCGCCGACACCACGGGCGGTCTCGACGGCCTCGTCGCCGACCTCACCGCGCACCCGGACGTCGC
GGCCGGGCTGCGCGAGCTGTGCGGCATGCCCCACCGGCTGACGGTGGGCTCGTATCTCATCCGGGCCAGGCTCACCC
GCTGGGCCCACTACGCCCGGCCGTACCCCGCGCGCCTCCCGGTGCACCGCCGCGCCCTGCTCTACCCGGTACGGCCG
GGCAAGGGCGAGGAACTGCGCCGTCTGCTGGCCGCAGGAATCGCCCCCGGCACCTCCCAGAATCTCGCGTCCGCCCT
GATCAGCAGCACCGTCTTCGCCCGCCAGGACCTCGTCGTCCGGTTCTGGGAGGCCACCGCGGACCCGGCCGAGGAGC
TCGACAACATCGCCCGCGTCGTCCCGCGTTCCGGCCTGGGGGCCAAGCTCAACCGGCTCCTGGACATCGAGCAGGAC
CTGACCACCGACGCCGGATTCCGGGCGTTCTTCACGGGTTGCGCGATGTCCCCCGTCCCCGGC
GCGGGCGCCTGGGCCCGGCCGTAGCCGCGCCGGCACCACCCACCTCACCACCGTCACTTCCGAGGAGGAAGCGTGAC
CGCACAGCCTGTCCCCGTCGTCTCGCTGGACGACGCCTTGCCCAACCGCCGGCGCGGCGGGGATCTGCGCACCCTGC
TCAGCCCCAAGACGGTCGGCGCCACCTCCGGCTTCCTCGGCGTGGCGCTCATCGAGCCCGGCGAATACGTGGCCGAG
CACTACCACCCCTACTCCGAGGAGTTCCTGTACGTGGTGTGCGGCGACCTCGTGGTCGACCTCGACGGCACGCCGTG
CGAACTCCACGCCGACCAGGGGCTGATGGTCCCGGTCGGGATGCGCCACCGCGTGCGCAACACCGGAGCCGTGCAGG
CCAGGATCGTCTTCCACCTCTCCCCGCTGGCCCCGCGCCCGGAACTGGGCCATGTGGACACCGAGACGGTCCCCTCC
CCGGCCACAGGCACGCTCGCCCGGGAGGGGCGGTGAGTCGCACCGCCGTCATCACCGGGATCGGGGTGGTGGCCCCC
GGCGGAGTCGGCACCAAGGGCTTTTGGGAGCTGCTCATCTCCGGGCGCACCGCCACCCGGCGGATCTCCCTCTTCGA
CCCCACCGGATTCCGCTCCCAGGTGGCCGCCGAATGCGACTTCGACCCCGCCGCCGAAGGACTGAGCCCGCAGGAGA
TACGCCGGATGGACCGGGCCGCCCAGTTCGCGGTCGCCGCGGTGAGGGAAGCGCTCGCCGACAGCGGGCTGACCCCC
GCCGAGGCGCCCGCGCACCGCACCGGCGTGAGCCTGGGCAGCGCCGTCGGGTGCACCACGGGACTGGAGGAGGAGTA
TGTCGTCCTGAGCGACGGCGGCCGGCTCCACCTGGTCGACCACCGCTACAAAGTCCCCGAGCTGTACGGGTACATGG
TGCCCAGCACGCTTGCCGTCGAGACGGCGTGGGCGTGCGGCGCCGAGGGCCCGGTCGGCCTGATCTCCACCGGCTGC
ACCTCGGGTCTCGACGCCGTCGGCTACGGCGCCCAGCTCATCGCCGAGGGCTCGGCCGACGTGATGGTCGCGGGCGC
CACCGACGCGCCCATCTCGCCCATCACCTCGGCCTGCTTCGCCGCCATCAAGGCCACCTCGCCCAACAACGACGACC
CGGCCCACGCCTCCCGCCCCTTCGACCGCCTGCGGGACGGCTTCGTCCTTGGCGAGGGCGCGGCCGTGATGGTGCTG
GAGGAGCGCGGCCGAGCCGAGGCCCGGGGCGCTCATGTCTACGCCGAGCTCGCGGCCTTCGCGAGCCGCAGCAACGC
CTTCCACATGACCGGACTGCGCCCCGACGGCCGGGAGATGGCCGAGGCCATCCGGGTCGCCCTGGACCGGGCCCGCC
TCGCGCCGGGCGATGTCGACTACATCAACGCGCACGGCTCCGGCACCCGCCAGAACGACCGCCACGAAACCGCCGCC
TTCAAGCGCGCGCTGGGCGAGCGGGCCCGCGAGGTGCCCGTCAGCTCCATCAAGTCCATGGTCGGCCACTCGCTCGG
GGCGATCGGCGCGATCGAGGTCGCCGCCTGCGCGCTGGCGCTGGAACACCAGGTCGTGCCGCCCACCGCCAATCTGC
ACCACCCCGACCCCGAATGCGACTTGGACTACGTCCCGCTGACGGCCCGTGACCACACCATGGACGTGGTGCTCTCG
GTCGGCAGCGGCTTCGGCGGCTTCCAGACGGCGATGCTGCTCAGCCGGCCCGGGAGGGCCCGGTGACCCCCGCGGCC
GGGCCGGCCACGCGCACCGCCACCGCCGTCATCACGGGTCTGGGCATCGTCGCGCCGGGCGGCATCGGCGTCGATGA
GTACTGGTCGGCGATGCTGCGCGGACACAGTGCCATCGGCCCCATCACCCGCTTCGACGCCTCCGGCTATCCGGTGC
GGCTGGCCGGCGAGGTGTCCGGCTTCCGCACCGAAGAGCGCATCCCCAGCAGGCTGGCCGTCCAGACCGACCGCTGG
ACCCACCTTGGCCTGGCCGCCGCGGAGGCCGCGCTCGCCGACGCCGGGGCGGGCCCGGGCACCCTGACGGAAAGACC
CCAGCAGTTCGGACTGTCCGAATACGACATGGCGGTCGTGACCGCCTCCTCCTCCGGCGGCACCGAGTTCGGGCAGC
GGGAGATCGAACGGCTGTGGTCGCGCGGGCCCCGGCACGTCGGCGCGTACCAGTCGATCGCCTGGTTCTACGCCGCG
ACCACCGGCCAGATTTCCATCCGCCACGGCATGCGCGGGCCGTGCGGGGTCATCGCGGCCGAGCAGGCCGGCGCCCT
GGACGCGCTCGCGCACGCCCGCCGGGTGGTGCGCGACGGCGCCCGGCTCGTGGTCAGCGGCGGCACCGACGCCTCGC
TGTGCCCGTACGGTCTCGTCGCCCAGCTCTCCAGCGGCCGGCTGTCCACCCGCGAGGACCCGGCACGGGCCTATGTG
CCCTTCGACGCCGGGGCCTGCGGCCACCTCCCGGGCGAGGGCGGGGCGATGCTGATCGTCGAGGACGCCCGCTCGGC
CCGGCGGCGCGGCGCCGCCCACGCGTACGGCGAGATCGCCGGGCACGCGGCCACCTTCGACCCGCCACCCGGCTCCG
GCCGTGAGCCCGGGTTGCGCCGCGCCGTCATGAACGCCCTGATCGACGCCCGGATGGCGCCGGAGGAGATCGACGTC
GTCTTCGCCGACGCCGCCGCCGTACCCGCCCTCGACCGCGCCGAGGCGGACGCCCTCGCCTGGGTCTTCGAGCCGTA
CGGCGTGCCCGTCACCGCGCCCAAGACGCTGACCGGCCGGCTCTACGCGGGCGCCGCGCTGGACGTGGCCACCGCGC
TGCTGGCCATGCGCGACGACGTCATCCCGCCCACCGCGGCCGGGGTGACCCCGCACCCCGACCACCGGCTCGACCTG
GTGGTGGGCGAGCCGCGGCCCGGCCCCGTACGGGCGGTCCTGGTGGCCGCCCGGGGCTTCGGCGGCATGAACGCCGC
GCTGGTCCTGCGCCGGACGGACCCGCGCGGATGAACGAGCAAGGAGACGCCATGACCGAATTCACCATCGACGACCT
GAGGCGGATCATGCGCGGCAGCGCGGGGGCCCAGGACGGGGAGCGCATCGACGGGGAGAACCTCGACGCCGACTTCG
CCGACCTCGGCTACGACTCGCTGGCCGTACTGGAGATCCAGTCGCAGATCGAGAACACCTTCG
ACCTGACGCTGCCCGACGACGCCCTCAGTCGGATGCGCACCCCACGGCAGACCGTCTCCGTGGTCAACGCCTGTATC
GGAAAAGAGGAGTGAGCCGGCTCATGACCGAAGCGACCGAAGCGACCACAGCGACTGAACCGACTGAGCATGCCCGC
ACCGACAACTCCATCGTCATCGACGCGCCCCTGGAGCTGGTCTGGGACATCACGAACGACGTCGAGAACTGGCCGCG
GCTGTTCAGCGAGTACGAGACGGCCGAGATCCTCCACCGGGACGGCGACACCATACGGTTCCGCCTCACCATGCACC
CGGACGAGAACGGCACCAGCTGGAGCTGGGTGTCCGAGCGCACCGCGGACCCGGCCACCCATACCGTCCGCTCCCGC
CGCGTCGAGACGGGCCCCTTCGCATTCATGGACATCTTCTGGGAGTTCACCCCGGAGCCCGAAGGGGTGCGCATGCG
CTGGGTCCAGGAGTTCCACATGTCGCCCCAGGCCCCCGCCGACGACGCCGCCATGACCGCGCACATCAACCGCAACA
CCAGGATCCAGATGGCGCTGATCCGCGACCGGATCGAGGCCCGCGCCCGCGCGGCGGCCTCCTGAGCCGGCCCTGTC
CCGACCACCGCACCACCACACCGAGGAGATTCCCATGGCCACGACCTTGATCGTCGCCCGGCTGAAGCCCGGCGACC
ACCACGCCGCGATCAGCAGCCTGTTCGCGGAGTCGGACGCCACCGAACTGCCCGACCTGGTGGGCGTCCAGGAGCGG
CGGCTGCTGACCTTCCAGGACCTGTACTTCCACCTCGTCCGCACGGACGAGGCCCTCTCCAAGACCCTCACCCCGCA
ACACGACAATCCGCTGTTCCGGTCGATCAGCCAGGCGATGGACGAGTACGTCACCCCGTACGAGGGCGCCTGGGGCA
GCGTCCAGGAGGCCTCGGCCCGGCAGTTCTACCACTGGAAGCGCGGCATCGGCCAGGTCCAGTCCTGACGGTTCGGA
GGGAACACCGTGACACACTCAATGGACACGACAGACACAGGCGACCCCGGTACCTCCTACGACGTCGTTGTGATCGG
CGGCGGCCCCGCCGGCTCGAGCACCGCGGCCCAGCTGGCCCGGGCCGGCCACCGGGTGCTGGTGCTGGAGCGGGAGA
AGTTCCCCCGCTACCACATCGGCGAGTCCCTCATCCCCGGCTGCCTCGACCTGGTCAGGGACCTGGGCCTGTGGGAC
CGGCTGGAAGGCCTCGGCTTCACCAAGAAGTACGGCGGCACCCTGCTGTGGGGCGCCACCGAGGGCACCTGGGACTT
CCGGTTCGCCGACGGCAGCGACTACGAGTACTCCTTCCAGGTGCGCCGCGCCGACTTCGACGCCCTGCTGCTCACCC
GCGCCCGGGAGCTCGGCGCCCGGGTGGTCGAGGAGGCCACGGTCAAGGACGTGGAGTTCGACGGCGACCGCGCCACC
GGCCTGGTCTACACCGTCAAGGGCTCGGACGAGCCCGTACGGGTCTCCTCCCGCTTCCTCGTCGACGCCTCCGGACA
GCAGCGGCTCCTCGGCCGCCGGCTCGGGCTCGTCGACTGGCACGAGGACCTGCGCAATGTCGCGGTGTGGAGCTACT
TCCAGGGCTGCGACTTCATCGAGGGCGAGAACCGGGCCGGCGACGTGGTCGTGGAGAACATGTGGCAGGAGGACCAG
CCTCCCGGCTGGTTCTGGTTCATCCCGCTGCACGACGGCACGGTCAGCATCGGCTATGTCACCCGCACCGCTCTGCT
GCAGCGCTCCGGTCTGACCCCGGACCAGCTGTTCGAACAGCAGCTGGCCGCCTCGGACGAGGTCAAGCGGCTCACCC
GGAACGCCACCAGGACCAGCGCCTACCGCACCCTGCGCGACTGGAGCTATGAGTGCACCCGCTTCCATGGCCCCGGC
TGGGCCGTGGTCGGCGACGCCGCGGCCTTTGTCGACCCGCTGTTCTCCACCGGCGTCACCCTGGCCATGCGCGGTGC
GCGGGACCTGGCCCCCGCGGTCGACCAGGCGCTGCGCGACCCGGGCCAGGAGGCCGATCTGCTCAAGGCGTACGAGG
ACGGGTACCGCGAATTCCTCGGCCACGTCCTGACCTTCGTGCGCTTCTTCTACAACATCAAGATGCACAAGGAACAG
TACTGGGAGGGCGCCCAGACCATCGTCGACCCGCAGAAGCTCCAGGCGCCGAAGATCGACTTCGCGGTCCTGCTGGC
CGGCATGACCGGCATCCGGCCCACGCCCGAGATGCAGGCGGACGCCAGGGCCAAGACCAACGACATCCACTCCCGCT
TCGCGACATGAAGACCGCCATGCCGGTGGAGGAGCTGCTGCCGGCCCTCGCGGCGATCGTCCTCGTCGCACGGGGCG
TCCACGCCCTGGCCCGGCGCTGCCGCCAGCCGGCGGTCGTGGCCGAGCTGGCCGCGGGCATCCTCCTCGGCCTGGCG
CTCACCGACGAGGCACAGGCCGAGATCCTCACCCGCCCGGTGCGCGAGGGGCTCACCGCGCTCGGCGGCCTCGGCCT
GCTGCTGTTCCTCTTCGAGACGGGGTGCGGGGCCGATGGCGGCCCGGCGCCCCGGCACGGCCGGCGGGCACCGGCGA
CGGCCCGGCCGCGGCCCCCCAACCCTCACGAGAGGCGGAAACACTGTGACCGACACCGACTACACCCCCGAGCTGGC
GACCGCGGCCCGGGTGAAGGAACTGGCGATGGGCCTGTGGGTCTCCGCCATGATGATGACGGCCGTTCAGCTGCGGA
TCCCCGACGAGATCGATGACAAGCCGGTCGAGGCGGGCGAGCTCGCCGCCCGGCTGGACCTGCACCCCGAGGCCCTC
GCCCGGTTGCTGCGGGCGCTGGCCGCGCACGGCGTCTTCACGGAGGTGGCCGAGGACCGTTTCGGACACACCGAGCT
CTCCCGGGTGCTCCGCGAGGACCACCCGAACAGCGTGCGCTACCAGGTGCTGTGGACCGTGGCCCCCTGGACCTGGC
AGGCCTGGCCGCACCTGGGGGAGGCGGTGCGCACCGGCGAGCGGGTCTTCCCGCGCCTGTACGGCAAGGAGTTCTTC
GCCTATCTCTCCCAGGACGCCCCGCAGTCCGCCGAGGTGTTCAACCGGGCCATGACCCAGTCCTCCGCCTTCACCTC
GGCCATGGTCGCCGAGGCCCTGGACATGACCGGCGTACGCGTCGTGGTCGACGTCGGCGGCGG
CCGCGGCCACCTGCTGCGCACCGTCCTGGAGCGGTACCCCTGGCTGAGCGGGGTGCTCTACGACCTGGCGCCCGTGG
TGGCCTCCGCGGACCCCGCCCTGCGGCCCGGCGGCGAACTCGCCGACCGCGCCAAGGTGGTCGCGGGCAGCTGCCTG
GACGGCGTCCCGCCCGGCGACCTCCACATCCTGAAGAACCTGCTGGAATGGGACGACGAACGGACCGTCACCACGCT
GCGGCACGTGGCCGGGGCGCTGCGCTCGGGCGGCCGGGCGGTGCTGGTGCAGAACCTGGTCGACGACAGCCCCGAGC
CCAAGGTCACCACCGGGATGGATCTGCTGCTGCTCCTCAACGTCGGCGGGAGGAAGCACACCAGGCGCCAGCTGACC
CGGCTGCTGTCCGAGGCGGGCCTTGAGATCACCGAGATCCGTCCCACGGCCAGCAGCCTCTTCGTCATCGAAATCGC
CCTGCGCGGCTAGCCCGTCCCGCCCCGGGTCCCGTCCGGGCGGTCGCCCTGCCGGTCCGGACGGTCGAGGGCGACCG
GGGGCAGGAACTCGCGGATGAGCGTGCGGTGCCACCACGCGCCGGTGGCCCGCAACTCACTCCAGGTCGTGAAGCGG
TACAGGAACAGGCGGGCGCGGATGTAAGCAGGCGGCGCGTCGGGGAAGGGGCACACCCGCAACAGCTTCAGCGTCGC
CGGGTCGCCCTCCAGCAGCTTGGTGACCAGCGGGCCGAACCAGGACCGGGCGTAGGCCGGCGAGATCCCGGCGAACC
ACATCATCCAGTCGAGGCGCAGGTGGTACGGGGCGAACTGGCGCGGCATCCGGCGCACATCGCCGGGCTTGCCCCGG
AACTCGTACTCCTTCCACACCGTGTCCGACGTGGGCAGCGGCTCGTCGGTGCCCTGGATCACCACCTCGAACCGGGC
GCGGTTGACGCTGCCGAAGGCGCCGTAGGTGTTGACCAGGTGCAGCGGATTGAAGGAGTAGTTCATCAGCTGACTGC
CCGACACCAGGTTCCGGGCGGGCCAGTAGCTGAGCACCACCACCAGAGCGGTGGCAGCCAGCACCAGGGCCTCGAAC
CACACCGGGGGACCGGCGAGCGGCGGCGGCTCGGGCAGTCCCAGCGCCTGGGCCGCGAGCCGCCCGTCGACCGCGGC
GAGGGCCAGCACGATGGTCACCCAGTTCAGCCACGAGAAGTTGCCGGACGCGACCAGCCACAGCTGGGTGACCACGA
TCACGGCCCCGGCCACGCTTGCCCCCGGCTGCGGCGCGAACAGCGCGAGGGGCACGACCAATTGGGCGACGTGGTTG
GCCGCCACCTCGACCCGGTGCAAGGGCCCCGGCAGCCGGTGGAAGAACCAGCTCAGCGGGCCCGGCATCGGCTGTGT
CTCGTGGTGGTAGTACAGACAGGTCAGCTGCCGCCAGCAGCGGTCGCCCCGGATCTTGATCAGCCCGGCGCCGAACT
CGACCCGGAACAGCAGCCAGCGCAGCAGCCACAGCACCAGGACCGGCGGGGCGGTACGGGCGTTGCCGAGGAAGACG
GCGAGGAAGCCGCTCTCCAGCAGCAGGGACTCCCAGCCGAACCCGTACCACACCTGCCCCACGTTGACGATCGACAG
ATACAGCAGCCACAGCACCAGCCACATCAGCATCGCGGCCCACAGCGGCACCCGGTCCGCGGCCCCCGCCACCACCG
CGGCGGCGAGCGCGGCCCCCAGCCAGGCGCAGCCGGTGAAGAAGCGGTCCGAGAAGTGGAGGTGGAAGAGGCTCGGG
GAGCGCCGGAACGGCATCCGCGCGGTGAACCGCGGCACCGGCGTCAGGCCCCGCTCGCCGATCAGGGCCCGCCCCTG
GTTCGCCGCGACCACAAAGGCGATCAGATAGAGGGCCGCCAGGCCGCGTTGGAAGACCAGTCGGCTCAGCCAGTAGT
CGGATGAGGTGAACCACTCCATGCGGAACCGCTCCCTGGGCCTGTTTCCCGGCTCGGTTCGCCCTTGCCGCCGGGCA
CTCGGCTATCCGTACCACCTACCGTTAAACGCGTGCGGCCCGGCGGGCCCCGGGGCCGGCCGAGTTCATCCGGTCGG
CGCGCCCGGCCGCCGGTCCTCGCTTAACCGGTTCAGGGCCGCCGTGGCGGGGCGGTGCTGCCGCGCGGGGTGAGCGC
CGGGGTGGGCGCCTGGTGGGTGCTGGGCGCCGCCTGGCCGAGCACATCGAACAGGCGGCGGGCGACCTCGGCCCCGA
AGCCGAAGACGTCATGGCTCATCGCCGACAGCGTGGGGCGGGTGAGCTGGCACAGCTGGGAGTCGTCCCAGGCCAGC
AGCGAGACGTCGACCGGAACCGCAAGACCCATCTCGGCGGTCACCGACAGGCCCGCCACCGCCATGATGTCGTTGTC
GTAGATGACCGCCGTCGGCCGGTCGGCCGAGGCCAGCAGCGCCCGCGTGGCGCGCGCCCCCTCCTCACCCGAGAAGT
CGGTGGCGACCGTGCGCCCGTCGGCCAGCCCCAGTTCCCGTACGGCCGCCTCGAAGGCGGCGGTACGGATCACGCTG
TGCCCCAGCGTGGCGGGGCCGCTCACCCGGGCGATCCGCCGGTGGCCCAGCGCCGCCAGATAGCGCACCGCCTCCGT
GACAGCGGTGCCGTCGTCGGTCCATACACAGGTGAAGCCCCCGGCCAGGGACGGGTGCCCGGCGGCCACGGCGGGCA
CCCCGAGCCGGCGCAGCTCGGGGACGCGCGGATCCCCGTCGTGGAGGTCGACGAGAATCGACCCGCCGACCTGCCGC
CCCCGCCACCACGCCTCCTGGACCGCGATCTCCTCCCGCACGTCCCGCACCAGCCGCAGCAGCAGCGAGCAGGACCG
CTCGACGAGCACGCTCTCGATGCCCGAGACGAACTCCATGAACCACGGCTCCAGCCCCAGCATCCGGGCCGGCCGGG
CGATGGCCAGGCCCACGGCGTCCACCCGCGCGTTGGACAGCGACCGGGCGGAGAGATGGGGCGCCCAGCCCAGCTCC
CGGGCGGCCTCGAAGATCCGGGCCCGGGTGGCCTGGGAGACGCCCGGGCGGTCGTTGAAGGCGAGCGAGACCGCGCC
CTTGGAGACCCCGGCCCGCGCGGCGACGTCCTTGATGGTGACGCGCCGCGGTGGCCCCGCCGTGACCTCGCTGTCCA
TGCACCGACTCCCGCTGCTCGGCTGCCGCTCGCGACGAACGCCACACCCTACCGCGTCGGTAA
ACCGGTCAAGGTCGCTTTTCGCGTGCGCAGCGCCGCGAACAGCCGCGGGCCGAACCGTTCGCGGGCCCCTCACCCGC
TCCGCCGGTACGCCGACGGAGCCGGGGAGGGGCCGCGGCCGAGGACCTGGCCGAG
Table 1：Zunyimycins biological synthesis gene cluster information：
Wherein, after 3 skeleton synthetic genes are abxP, abxK, abxS, 5 modifying gene be abxO, abxC, abxD,
AbxH, abxM, controlling gene is abxA, and resistant gene is abxR.
Preparation method includes：
1st, the extraction experimental procedure of pET-32a plasmid
(1) the escherichia coli pET-32a bacterium solution of 1～5mL incubated overnight, 12000rpm, centrifugation 2min, supernatant discarded are taken；
(2) 1mL deionized water is added, piping and druming is mixed, 12000rpm is centrifuged 2min, supernatant discarded；
(3) bacterial sediment adds appropriate solution P1 (plus RNase), places 1-2min；
(4) equal-volume solution P2 is added, leniently spins upside down 6-8 time, 5min is placed, so that thalline is fully cracked；
(5) a certain amount of solution P3 (P1 is added:P2:P3=5:5:7=V:V:V), leniently spin upside down 6-8 immediately
Secondary, fully mix, now will appear from white flock precipitate, 12000rpm, 4 DEG C, 10min is centrifuged, obtains supernatant；
(6) supernatant for collecting step (5) is transferred in adsorption column, tries not to suction out precipitation, and 12000rpm is centrifuged
30s, outwells the waste liquid in collecting pipe, adsorption column is put in collecting pipe；
(7) 300 μ L protein liquid removal PE, 12000rpm centrifugation 30s is added in adsorption column, outwells the waste liquid in collecting pipe,
Adsorption column is put in collecting pipe；
(8) 500 μ L rinsing liquid WB, 12000rpm centrifugation 30s is added in adsorption column, outwells the waste liquid in collecting pipe, weight
Multiple operation is once；
(9) adsorption column is put in collecting pipe, 12000rpm, is centrifuged 3min, it is therefore an objective to by remaining rinsing in adsorption column
Liquid is removed；
(10) adsorption column is placed in the centrifuge tube of 1 sterilizing, most ethanol waved by 50 DEG C of baking ovens, drips to adsorbed film middle part
Plus the elution buffer EB of 50-60 DEG C of preheating, room temperature is placed 2min, 12000rpm and is centrifuged 2min, obtains pET-32a plasmid
DNA, plasmid DNA is collected in centrifuge tube, and ultraviolet spectrophotometer measures concentration and quality.
Solution P1：Glucose makes cell suspension, and EDTA can be with Ca2+And Mg2+Sting and suppress the activity of DNase, RNase
A degradation of rna, it is to avoid RNA pollutes.
Solution P2：NaOH and SDS cracking antibacterial, denatured protein and a small amount of plasmid.
Solution P3：Neutralization NaOH, is that protein is co-precipitated together with bacterial genomes DNA, plasmid DNA renaturation.
Protein liquid removal PE：Nuclease is removed, is easy to plasmid to preserve for a long time.
Rinsing liquid WB：Remove organic faciess and inorganic salt residual.
2nd, pET-32a plasmid enzyme restriction
(1) following 10 μ L enzyme action system is prepared in PCR pipe：
Table 2：10 μ L plasmid enzyme restriction systems
(2) low-speed centrifugal, mixes reaction system, reaction tube is placed in 37 DEG C of constant incubators, endonuclease reaction 3h, obtains
Enzyme action linear expression vector pET-32a；(plasmid DNA is ring-type, is changed into linear after restriction enzyme enzyme action, could connect and insert
The genes of interest for entering)
(3) detection of endonuclease reaction product：5 μ L digestion products are taken, is mixed with DNA sample-loading buffer, point sample, while take 5 μ
The appropriate DNA molecular amount standard of L point sample in the sample adjacent holes, agarose gel electrophoresiies are analyzed, and gel imaging instrument is taken a picture, according to
Molecular weight standard judges amplified production size and substantially concentration.
3rd, the plasmid extraction experimental procedure of synthetic gene
(1) the streptomycete Streptomyces sp.FJS31-2zunyimycins for taking 1～5mL incubated overnight is series of halogenating
The bacterium solution of two type polyketone biological synthesis gene clusters, 12000rpm, it is centrifuged 2min, supernatant discarded；
(2) 1mL deionized water is added, piping and druming is mixed, 12000rpm is centrifuged 2min, supernatant discarded；
(3) bacterial sediment adds appropriate solution P1 (plus RNase), places 1-2min；
(4) equal-volume solution P2 is added, leniently spins upside down 6-8 time, 5min is placed, so that thalline is fully cracked；
(5) a certain amount of solution P3 (P1 is added:P2:P3=5:5:7=V:V:V), leniently spin upside down 6-8 immediately
Secondary, fully mix, now will appear from white flock precipitate, 12000rpm, 4 DEG C, 10min is centrifuged, obtains supernatant；
(6) supernatant for collecting step (5) is transferred in adsorption column, tries not to suction out precipitation, and 12000rpm is centrifuged
30s, outwells the waste liquid in collecting pipe, adsorption column is put in collecting pipe；
(7) 300 μ L protein liquid removal PE, 12000rpm centrifugation 30s is added in adsorption column, outwells the waste liquid in collecting pipe,
Adsorption column is put in collecting pipe；
(8) 500 μ L rinsing liquid WB, 12000rpm centrifugation 30s is added in adsorption column, outwells the waste liquid in collecting pipe, weight
Multiple operation is once；
(9) adsorption column is put in collecting pipe, 12000rpm, is centrifuged 3min, it is therefore an objective to by remaining rinsing in adsorption column
Liquid is removed；
(10) adsorption column is placed in the centrifuge tube of 1 sterilizing, most ethanol waved by 50 DEG C of baking ovens, drips to adsorbed film middle part
Plus the elution buffer EB of 50-60 DEG C of preheating, room temperature is placed 2min, 12000rpm and is centrifuged 2min, obtains the matter of synthetic gene
Grain DNA, plasmid DNA is collected in centrifuge tube, and ultraviolet spectrophotometer measures concentration and quality.
Solution P1：Glucose makes cell suspension, and EDTA can be with Ca2+And Mg2+Sting and suppress the activity of DNase, RNase
A degradation of rna, it is to avoid RNA pollutes.
Solution P2：NaOH and SDS cracking antibacterial, denatured protein and a small amount of plasmid.
Solution P3：Neutralization NaOH, is that protein is co-precipitated together with bacterial genomes DNA, plasmid DNA renaturation.
Protein liquid removal PE：Nuclease is removed, is easy to plasmid to preserve for a long time.
Rinsing liquid WB：Remove organic faciess and inorganic salt residual.
4th, the plasmid enzyme restriction of synthetic gene
(1) following 10 μ L enzyme action system is prepared in PCR pipe：
Table 3：10 μ L plasmid enzyme restriction systems
(2) low-speed centrifugal, mixes reaction system, reaction tube is placed in 37 DEG C of constant incubators, endonuclease reaction 3h, obtains
Synthetic gene digestion products；
(3) detection of endonuclease reaction product：5 μ L synthetic gene digestion products are taken, are mixed with DNA sample-loading buffer, point sample,
While taking the appropriate DNA molecular amount standard of 5 μ L point sample in the sample adjacent holes, agarose gel electrophoresiies are analyzed, gel imaging instrument
Photograph, judges amplified production size and substantially concentration according to molecular weight standard.
5th, enzyme action synthetic gene product agarose gel is reclaimed
(1) with clean blade, the agarose gel containing target DNA is cut after electrophoresis terminates under uviol lamp, as far as possible quickly
Glue is cut, the liquid of the gel surface that gently exhausted with napkin, shreds gel on free of contamination clean Thin film glove；
(2) gel fragment is transferred in a weighed clean 1.5mL centrifuge tube, calculates gel after weighing again
Weight, add 3 times of gel volumes solution bufferDE-A (redness), mix homogeneously after 75 DEG C heating, in heating process
The mixing of turning upside down that can be interrupted, until gel piece is completely melt (about 6-8min)；
(3) bufferDE-B (solution is changed into yellow) of 1/2bufferDE-A volume is added, is fully mixed；
(4) with pipettor, mixed solution addition DNA is prepared in pipe, 12000rpm is centrifuged 30s, abandons liquid；
(5) DNA is prepared pipe to put back in 2mL centrifuge tube, 500 μ LbufferW1,12000rpm centrifugation 30s is added, abandons liquid
Body；
(6) DNA is prepared pipe to put back in 2mL centrifuge tube, 700 μ LbufferW2,12000rpm centrifugation 30s is added, abandons liquid
Body, the step is repeated once；
(7) pipe will be prepared put back in 2mL centrifuge tube, 12000rpm is centrifuged 3min；
(8) pipe will be prepared to be put in clean 1.5mL centrifuge tube, 50 DEG C of 25-30 μ for preheating are added film central authorities are prepared
LEB elution buffer, room temperature standing 2min, 12000rpm centrifugation 3min, eluted dna；
(9) the enzyme action synthetic gene needed for ttom of pipe solution is, ultraviolet spectrophotometer surveys DNA concentration and quality, by DNA
- 20 DEG C are stored in for a long time can.
6th, purpose fragment connection
(1) 10 μ L coupled reaction system
Table 4：Linked system
(2) the of short duration centrifugation of above-mentioned mixed liquor, then connects 4～6h in 16 DEG C of room temperatures and obtains connection product；
(3) connection product can be useable immediately for transformed competence colibacillus cell or to put 4 DEG C of refrigerators standby.
7th, connection product transformed clone bacterial strain JM109 experimental procedure：
(1) clone strain JM109 being taken out in -80 DEG C of refrigerators, is positioned over immediately on ice；
(2) 1 μ L connection product being added in 100 μ L clone strain JM109, is statically placed in ice bath 30min on ice, this process
In should not shake centrifuge tube；
(3) centrifuge tube is placed in 42 DEG C of metal baths accurately and places 90s；
(4) quick centrifuge tube is transferred in ice bath cools down 2～3min, should not shake centrifuge tube during this；
(5) 600 μ LLB culture medium being added in centrifuge tube, is positioned over 37 DEG C, cultivates 45-60min on 200rpm shaking table；
(6) LB solid medium heating and melting, be cooled to 50 DEG C or so (non-scald on hand), add final concentration of 100ng/mL ammonia
Benzylpcnicillin gently shakes up, and is down flat plate standby in super-clean bench；
(7) by the cell that has recovered take out, 4000rpm, be centrifuged 3min, abandon supernatant in super-clean bench, by precipitation rifle blow even,
Spreading rod paves plate, each reaction two flat board of paving, is statically placed in 37 DEG C of incubator culture 14-16h, and enzyme action identification PCR identification is surveyed
Plasmid expression vector pET-abx is obtained after sequence.
8th, plasmid expression vector pET-abx conversion expression competent cell experimental procedure：
(1) expression strain BL21 (DE3) Rosetta being taken out in -80 DEG C of refrigerators, is positioned over immediately on ice；
(2) 1 μ L plasmid expression vector pET-abx being added in 100 μ L expression strain BL21 (DE3) Rosetta, is statically placed in
Ice bath 30min, should not shake centrifuge tube during this on ice；
(3) centrifuge tube is placed in 42 DEG C of metal baths accurately and places 90s；
(4) quick centrifuge tube is transferred in ice bath cools down 2～3min, should not shake centrifuge tube during this；
(5) 600 μ LLB culture medium being added in centrifuge tube, is positioned over 37 DEG C, cultivates 45-60min on 200rpm shaking table；
(6) LB solid medium heating and melting, be cooled to 50 DEG C or so (non-scald on hand), add final concentration of 100ng/mL ammonia
Benzylpcnicillin gently shakes up, and is down flat plate standby in super-clean bench；
(7) by the cell that has recovered take out, 4000rpm, be centrifuged 3min, abandon supernatant in super-clean bench, by precipitation rifle blow even,
Spreading rod paves plate, each reaction two flat board of paving, is statically placed in 37 DEG C of incubator culture 14-16h, and enzyme action identification PCR identification is surveyed
Positive colony bacterium is obtained after sequence.
9th, positive colony bacterium induction synthesis
(1) 50 μ L of positive colony bacterium, is inoculated in LB liquid of 4 5mL containing final concentration of 100ng/ μ L ampicillin and trains
In foster base；
(2) 37 DEG C of constant-temperature table cultures, 220rpm, cultivates to OD600=0.3-0.5；
(3) IPTG of final concentration of 1.0mM/L is added, 18 DEG C, 28 DEG C and 37 DEG C inducing culture 12h, 6h is respectively placed in,
3h, while 1 pipe is not added with IPTG as blank.
10th, induction synthetic product HPLC detection
The bacterial strain of a large amount of induction synthetic proteinses, and other negative control bacterial strains and blank bacterial strain, HPLC is tested and analyzed
Extraction product.
10.1st, tunning pretreatment
Equal-volume ethyl acetate extracts 5h, collects organic phase solvent, 37 DEG C of Rotary Evaporators concentrating samples, the extractum for obtaining
Being dissolved using methanol, 12000rpm, centrifugation 10min, 0.2 μm of filtering with microporous membrane, 200 μ L of supernatant is taken to sample bottle.
10.2nd, sample HPLC analysis
Shimadzu LC-20AHPLC analysis condition is as shown in table 5：
Table 5：Shimadzu LC-20AHPLC analysis condition
Column system：
Chromatographic column：wondaSilC18-WR(5um,1.6×160nm)
Sampling volume：50μL
Column oven：30℃
Type of elution：Gradient elution
Analysis liquid phase result, by positive and negative control, determines target product and carries out MS analysis.
11st, product purification, structure elucidation
Target product is enriched with, purification prepares monomeric compound, using multiple chromatograph means, crystallization and efficient liquid
Equal method isolates and purifies compound, determines the spatial configuration of compound using analytic methods such as one-dimensional two dimension spectroscopy information, most
Identification obtains the structure of monomeric compound eventually.
11.1, product purification
The extraction of induced product ethyl acetate is concentrated to give extractum, prepares Agilent Agilent liquid phase purification using half and prepares mesh
Mark compound.
HPLC analysis condition is as follows：
Table 6：Agilent HPLC analysis condition
Column system：
Chromatographic column：Agilent-C18(5um,9.4×250mm)
Sampling volume：100μL
Column oven：30℃
Type of elution：Gradient elution
11.2 product structure is parsed
Target product purity reaches more than 95%, true using one-dimensional nuclear magnetic resonance (1D-NMR) and high resolution mass spectrum technology etc.
Determine the planar structure of compound, using two dimensional NMR (2D-NMR), two-dimentional HMBC spectrum, two-dimentional hsqc spectrum, two-dimentional ROESY
Spectrum, two-dimentional COSY spectrum analytic method determines the spatial configuration of compound, obtains Fig. 1～Fig. 7, wherein Fig. 1 for 2-n- heptyl -4- hydroxyl
Base quinoline mass spectral analyses figure, it is 2-n- heptyl -4- hydroxyquinoline that Fig. 2 is 2-n- heptyl -4- hydroxyquinoline hydrogen analysis of spectrum figure, Fig. 3
Carbon analysis of spectrum figure, it is 2-n- heptyl -4- hydroxyquinoline that Fig. 4 is 2-n- heptyl -4- hydroxyquinoline HMBC spectrum analysis figure, Fig. 5
Hsqc spectrum map analysis figure, it is 2-n- heptyl -4- hydroxyl quinoline that Fig. 6 is 2-n- heptyl -4- hydroxyquinoline ROESY spectrum analysis figure, Fig. 7
Quinoline COSY spectrum analysis figure, final identification obtains the structure of 2-n- heptyl -4- hydroxyquinoline structure.
2-n- heptyl -4- hydroxyquinoline structure
White powder, m.p.364～408 DEG C,
ESI-MSm/z:244[M+H]+；1HNMR(400MHz,MeOH)δH:12.32(1H,brs,OH-4),8.35(1H,t,
J=7.6Hz, H-5), 7.58 (1H, t, J=6.8Hz, H-6, H-8), 7.32 (1H, t, J=7.6Hz, H-7), 6.24 (1H,
Brs, H-3), 2.68 (2H, brs, H-1 ') (1.694H, d, J=6.4Hz, H-2 ', H-6 '), 1.23 (6H, m, H-3 ', H-4 ',
H-5 '), 0.80 (3H, t, J=7.2Hz, H-7 ')；13CNMR(100MHz,CDCl3)δC:179.1(C-4),155.5(C-2),
140.9(C-8a),132.0(C-5),125.5(C-6),125.2(C-4a),123.8(C-7),118.8(C-8),108.3(C-
3),34.6(C-1′),31.8(C-5′), 29.4(C-2′),29.3(C-3′),29.2(C-4′),22.8(C-6′),14.2(C-
7′).
Above-described is only embodiments of the invention, and in scheme, the known general knowledge here such as concrete structure and characteristic is not made
Excessive description.It should be pointed out that for a person skilled in the art, on the premise of without departing from present configuration, acceptable
Make some deformation and improve, these should also be considered as protection scope of the present invention, these are all without impact present invention enforcement
Effect and practical applicability.This application claims protection domain should be defined by the content of its claim, in description
Specific embodiment etc. records the content that can be used for explaining claim.
Claims (4)
- The preparation method of 1.2-n- heptyl -4- hydroxyquinoline, it is characterised in that comprise the following steps：First, genetic engineering skill is used Art is cloned to the zunyimycins biological synthesis gene cluster of streptomycete Streptomyces sp.FJS31-2 bacterial strain and is reflected Fixed；Then, the gene cluster is integrated in prokaryotic expression plasmid vector pET32a, the heterogenous expression plasmid for building the gene cluster is carried Body pET-abx；Again, expression plasmid carrier pET-abx is converted competent escherichia coli cell BL21 (DE3) Rosetta, most Afterwards, by IPTG induce synthesis destination protein bacterial strain, obtain tunning after fermentation, tunning is carried out product purification and Structure elucidation, obtains 2-n- heptyl -4- hydroxyquinoline compounds.
- 2. the preparation method of 2-n- heptyl -4- hydroxyquinoline according to claim 1, it is characterised in that：Streptomycete The preserving number of Streptomyces sp.FJS31-2 bacterial strain is：CGMCC 4.7321.
- 3. the preparation method of 2-n- heptyl -4- hydroxyquinoline according to claim 1, it is characterised in that：Described The NCBI of zunyimycins biological synthesis gene cluster submits Serial No. No.KU243130 to.
- 4. the preparation method of 2-n- heptyl -4- hydroxyquinoline according to claim 1, it is characterised in that：The IPTG is lured The temperature for leading synthesis is 18～28 DEG C.
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| CN102925529B (en) * | 2011-10-26 | 2014-07-02 | 苏州同力生物医药有限公司 | Method for producing intermediate of levo-praziquantel and levo-praziquantel |
| JP2015065910A (en) * | 2013-09-30 | 2015-04-13 | 株式会社カネカ | Production method of optically active nitrogen-containing cyclic alcohol compound |
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| JP2015065910A (en) * | 2013-09-30 | 2015-04-13 | 株式会社カネカ | Production method of optically active nitrogen-containing cyclic alcohol compound |
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| YUHONG LÜ, ET AL.: "Molecular Genetic Characterization of an Anthrabenzoxocinones Gene Cluster in Streptomyces Sp. FJS31-2 for the Biosynthesis of BE-24566B and Zunyimycin Ale", 《MOLECULES》 * |
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