CN109609423A - The autolytic streptin of one plant height production autolytic mycin - Google Patents
The autolytic streptin of one plant height production autolytic mycin Download PDFInfo
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- 230000002358 autolytic effect Effects 0.000 title claims abstract description 48
- 108010043542 streptin Proteins 0.000 title claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 title description 4
- 230000001580 bacterial effect Effects 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 6
- 241001276372 Streptomyces autolyticus Species 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 abstract description 9
- 230000004151 fermentation Effects 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 9
- 150000001875 compounds Chemical class 0.000 abstract description 8
- QTQAWLPCGQOSGP-GBTDJJJQSA-N geldanamycin Chemical compound N1C(=O)\C(C)=C/C=C\[C@@H](OC)[C@H](OC(N)=O)\C(C)=C/[C@@H](C)[C@@H](O)[C@H](OC)C[C@@H](C)CC2=C(OC)C(=O)C=C1C2=O QTQAWLPCGQOSGP-GBTDJJJQSA-N 0.000 abstract description 8
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- 230000000259 anti-tumor effect Effects 0.000 abstract description 7
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- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
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- 241001655322 Streptomycetales Species 0.000 description 1
- HUXWGTSMSXMDBH-UQMARRQJSA-N [(3r,5s,6r,7s,8e,10s,11s,14e)-6,20-dihydroxy-5,11-dimethoxy-3,7,9,15-tetramethyl-16-oxo-17-azabicyclo[16.3.1]docosa-1(22),8,14,18,20-pentaen-10-yl] carbamate Chemical compound C1[C@@H](C)C[C@H](OC)[C@H](O)[C@@H](C)\C=C(C)\[C@H](OC(N)=O)[C@@H](OC)CC\C=C(C)\C(=O)NC2=CC(O)=CC1=C2 HUXWGTSMSXMDBH-UQMARRQJSA-N 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
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- HUXWGTSMSXMDBH-UHFFFAOYSA-N progeldanamycin Natural products C1C(C)CC(OC)C(O)C(C)C=C(C)C(OC(N)=O)C(OC)CCC=C(C)C(=O)NC2=CC(O)=CC1=C2 HUXWGTSMSXMDBH-UHFFFAOYSA-N 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- ATEBXHFBFRCZMA-VXTBVIBXSA-N rifabutin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC(=C2N3)C(=O)C=4C(O)=C5C)C)OC)C5=C1C=4C2=NC13CCN(CC(C)C)CC1 ATEBXHFBFRCZMA-VXTBVIBXSA-N 0.000 description 1
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- 229930000044 secondary metabolite Natural products 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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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
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Abstract
The invention discloses the engineering strain of one plant of autolytic streptin, which has knocked out participation geldanamycin biosynthesis in autolytic streptin using molecular biology methodalmMConstructed by gene.The bacterial strain can largely generate the compound autolytic mycin with very powerful antitumor activity by fermentation.The yield that fermentation is greatly improved autolytic mycin is carried out using bacterial strain provided by the present invention, it is more more economical than other methods and environmentally friendly for producing autolytic mycin.
Description
Technical field
The invention belongs to microorganisms technical fields, it particularly relates to construct streptomycete work using molecular biology method
Journey bacterial strain, which, which can ferment, generates a large amount of autolytic mycins.
Background technique
It take geldanamycin (geldanamycin) as the Geldanamycin class compound (benzoquinone of representative
It ansamycin) is one of current most study, the good antitumoral compounds of effect.The antitumor mechanism of such compound is
With heat shock protein (heat shock protein) Hsp90 specific bond in tumour cell, cause many in tumour cell
The key protein for needing Hsp90 to maintain function is degraded rapidly, to inhibit the growth of tumour cell or initiation tumour cell dead
It dies.Since this kind of compound on tumor cell has very high specificity and extremely strong anti-tumor activity, while having than tradition
Anti-tumor drug more wide spectrum anti-tumor activity and be not easy to develop drug resistance, therefore cause people height close
Note, becomes the research hotspot in tumor therapeutic agent in recent years.Currently, 17-AAG, 17-DMAG and IPI- in this kind of compound
The 504 equal research institution's progress clinical researches just dominated by U.S. NCI, are expected to become new anti-tumor drug.
Autolytic mycin (autolytimycin) be us from actinomyces novel species autolytic streptin (Streptomyces autolyticus) secondary metabolite in find the one geldanamycin structure with very powerful antitumor activity it is similar
Object.Autolytic mycin and geldanamycin and the currently U.S. carry out other knots such as the 17-AAG and 17-DMAG of clinical research
The main distinction of structure analog is that its chromophoric group is phenol rather than benzoquinones, this architectural difference make autolytic mycin have than
Geldanamycin and the stronger target of 17-AAG combine activity and lower toxicity.Therefore, autolytic mycin has good
Development and application prospect.However, autolytic mycin there are the low-down disadvantage of yield, seriously hampers the further research and development to it, then
In addition the chemical synthesis of autolytic mycin is extremely difficult, it is therefore necessary to be improved to its producing strains to improve autolytic mycin production
Rate.
The biosynthesis of Geldanamycin class compound is with 3- amino -5- hydroxybenzoic acid (3-amino-5-
Hydroxybenzoic acid, AHBA) it is starting material, gradually added by polyketide synthase (polyketide synthase, PKS)
Enter some extending elements such as acetic acid, propionic acid and hydroxyacetic acid etc., the nuclear structure of a polyketone is formed, finally using rear modification
Enzyme modify.The current biosynthesis to such compound both at home and abroad is studies have shown that its PKS for synthesizing nuclear structure
Enzyme is much like, and the main distinction is modified after being, i.e. C-17 O- methylation, C-21 oxidations, C-7 carbamoylations and C-4, and 5
Position oxidation etc., these modifications have critically important influence to the anti-tumor activity and cytotoxicity of such compound.
We have cloned the full genome cluster of autolytic streptin geldanamycin biosynthesis and have carried out sequence, pass through
Bioinformatic analysis has selected the gene for participating in its biosynthesisalmM, autolytic streptin wild-type strain is lost
Pass transformation.After the gene is knocked, bacterial strain can generate a large amount of autolytic mycin.
Autolytic mycin of the present invention can pass through buildingalmMGene knockout engineering bacteria bulk fermentation generates, with chemistry
Synthetic method is compared, more economical, environmental protection, and sustainable use.
Summary of the invention
The purpose of the present invention is to provide the autolytic streptin genetic engineering bacteriums that one kind can largely generate autolytic mycin.
To achieve the goals above, present invention employs following technical solutions:
1) use Red/ET One_step PCR method, using plasmid pHY773 as template, by PCR amplification obtain both ends have withalmMBase
Because aac (3) the IV gene of homologous sequence is inserted into box.
2) by electrotransformation institute's amplified fragments are imported containing pIJ790 andalmMThe Escherichia coli of gene cosmid plasmid
(Escherichia coli) in, the transformant acquisition of screening apramycin (Apramycin) resistance contains insertion mutationalmMGene.
3) preparation contains insertion mutation from above-mentioned transformantalmMThe clay plasmid of gene is imported big by electrotransformation
In enterobacteria ET12567/pUZ8002.
4) using engagement transfer containing insertion mutationalmMIn channel genes wild type autolytic streptin, A Bo is screened
The engagement of chloramphenicol resistance and thiostrepton sensitivity is drawn to shift son.
5) this mutant strain is through Southern hybridization verificationalmMThe autolytic streptin mutant strain of gene knockout.
6) autolytic streptin is obtained using the culture of MS culture mediumalmMThe Fresh spores of gene mutation strain are inoculated with Fresh spores
Into TSB culture medium, culture prepares fermentation seed liquid, is then inoculated with seed liquor fermented and cultured into fermentation medium.
7) fermented liquid supernatant is collected, twice with ethyl acetate extracting.Tunning is through 45 DEG C of evaporated under reduced pressure of Rotary Evaporators
After be dissolved in proper amount of methanol.Tunning is analyzed using HPLC.Then pure through normal-phase silica gel column chromatography and recrystallization separation
Change tunning.
8) compound structure of separated purifying through mass spectrum and1H、13The confirmation of C nuclear magnetic resonance spectroscopy.
Genetic engineering bacterium autolytic streptin of the present inventionalmMGene knockout mutant strain is preserved in China typical culture collection
Center (CCTCC), address: Wuhan University, deposit number: CCTCC NO:M 2013156, preservation date: on April 24th, 2013.
Detailed description of the invention
Fig. 1 is autolytic streptinalmMThe construction strategy of gene knockout mutant strain.
Fig. 2 is autolytic streptinalmMThe Southern hybridization verification of gene knockout mutant strain.Wherein 1 is DNA
Molecular Weight Marker VII, DIG labeled；2 areBssThe wild-type strain genomic DNA of H II digestion；
3 areBssThe mutant strain genomic DNA of H II digestion.
Fig. 3 is autolytic streptinalmMThe tunning of gene knockout mutant strain detects.
Specific embodiment
Below by way of specific embodiment and embodiment, the present invention is described in detail, but protection scope of the present invention is not
It is limited to this.The processes such as the preparation, conversion, engagement transfer, electrophoresis and the Southern hybridization that are previously mentioned in embodiment, such as without special theory
It is bright, then it can be achieved using the conventional method of this field, repeated no more as the paralleling method recorded with the present invention.
Embodiment 1:
Autolytic streptinalmMThe building of gene knockout mutant strain: by ORF finder of the website NCBI, glimmer and
The tools such as BLAST analyze the geldanamycin gene cluster cloned and be sequenced, thus it is speculated that each open reading frame (open
Reading frame, ORF) function.Then, it is constructed using Red/ET One_step PCR methodalmMGene (SEQ ID NO:1) is inserted
Enter mutation (Fig. 1).In this method, first using pHY773 as template, using PCR amplification both ends have withalmMDNA homolog sequence
Aac (3) the IV gene of column is inserted into box.The primer of PCR amplification are as follows: 5 '-GTTCCAGGACACGGTCGATCTGATCCTGGCGGGC
GAATGATTCCGGGGATCCGTCGACC-3 ' (SEQ ID NO:2) and 5 '-GAAGGGGACCAGTTCGTGTTCGGGTGGGGG
AGTGCCTCATGTAGGCTGGAGCTGCTTC-3 ' (SEQ ID NO:3), PCR amplification condition are as follows: 95 DEG C preheat 5 minutes, 1
Circulation；94 DEG C be denaturalized 1 minute, 50 DEG C renaturation 45 seconds, 72 DEG C expand 2.5 minutes, 10 circulation；94 DEG C are denaturalized 1 minute, and 55 DEG C multiple
Property 45 seconds, 72 DEG C expand 1 minute, 20 circulation；72 DEG C expand 5 minutes；4 DEG C 1 hour.Then PCR is expanded by electrotransformation
Increase segment import containing pIJ790 andalmMIn the Escherichia coli of gene cosmid plasmid, the transformant of apramycin resistance is screened,
Acquisition contains insertion mutationalmMGene.Preparation contains insertion mutation from above-mentioned transformantalmMThe clay matter of gene
Grain is imported in Escherichia coli ET12567/pUZ8002 by electrotransformation.Using engagement transfer containing insertion mutationalmMBase
Because importing in wild type autolytic streptin, son is shifted in the engagement for screening apramycin resistance and thiostrepton sensitivity
The autolytic streptin of energyalmMGene knockout mutant strain.Using Southern hybridization verification autolytic streptinalmMKnockout mutant strain
(Fig. 2).Southern hybridization probe, PCR primer are as follows: 5 '-are prepared by PCR amplification first
GACCTGGTGTTCGAGCATGTGG-3 ' (SEQ ID NO:4) and 5 '-GCCGATCAGCACGATCTTTCC-3 ' (SEQ ID
NO:5), PCR amplification condition are as follows: 95 DEG C preheat 5 minutes, 1 circulation；94 DEG C be denaturalized 1 minute, 50 DEG C renaturation 45 seconds, 72 DEG C amplification
2.5 minutes, 10 circulations；94 DEG C be denaturalized 1 minute, 55 DEG C renaturation 45 seconds, 72 DEG C expand 1 minute, 20 circulation；72 DEG C of amplifications 5
Minute；4 DEG C 1 hour.Then, autolytic streptin is preparedalmMThe genomic DNA of mutant strain and wild-type strain, DNA is through limiting
Property restriction endonucleaseBssH II complete degestion is transferred to nitrocellulose filter after electrophoresis, carries out Southern hybridization with above-mentioned probe and tests
Card.
Embodiment 2:
Autolytic streptin wild-type strain andalmMThe detection of gene knockout mutant strain tunning: autolytic streptin wild-type bacteria
Strain andalmMThe Fresh spores of gene knockout mutant strain are seeded to respectively in 50 mL TSB culture mediums, in 28oC, 200 rpm are trained
It supports 72 hours and prepares fermentation seed liquid, 0.5 mL seed liquor (2 % beans into fermentation medium are then inoculated with the inoculum concentration of 1 %
Powder, 0.2 % peptone, 2 % glucose, 0.5 % soluble starch, 0.2 % yeast extract, 0.4 % NaCl, 0.05 %
K2HPO4, 0.05 % MgSO4, 0.2 % CaCO3), in 28oC, 250 rpm fermented and cultured 7 days.Fermentation liquid centrifugation (3500 g, 30
Minute) after take supernatant, with ethyl acetate extracting twice, then through 45 DEG C of evaporated under reduced pressure of Rotary Evaporators.Tunning difference is molten
Analyzed (Fig. 3) in proper amount of methanol and using HPLC, autolytic mycin in autolytic streptin wild-type strain tunning not
It detects, andalmMThe yield of autolytic mycin is every milliliter of 33.8 microgram of fermentation liquid in gene knockout mutant strain.Autolytic mycin
Through chloroform: after the normal-phase silica gel column chromatography and recrystallization of methanol (20:1) isolate and purify, structure through mass spectrum and1H、13C nuclear-magnetism
Resonance analyzing confirmation.
Sequence table
<110>Yunnan University
The autolytic streptin of<120>one plant heights production autolytic mycin
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1623
<212> DNA
<213>autolytic streptin (Streptomyces autolyticus)
<400> 1
gtggacgtcg cgcgggacac ggacaacggc gggcccaaga ccgatgtgct gatcgtcggg 60
tacggaccgg tggggcagct gctgtcggtg ctgctggccc agcgcggccg ccgggtgacg 120
gtggtggagc gctggccgga gccgtaccgg cacccccggg cggtcgggtt cgacagtgag 180
gccgcgcgca tcctggcctc ggccgggatc ggcgactcgc tcgagaagtt caccgaaccc 240
gcgcgggacc acgcctggca gaacaccaag ggcgagacgc tgatcgacca cgaggtcgcc 300
gaccgggggc actgcacctg gccggaggcg ctgtcggcct atcagccggc cctggagtcc 360
gcactgatcg aacacggggc gacgctgccg cggctgcggg tgctgcgcgg ctacgaggcg 420
gtgggactcg cggacaacgg tgaccgggtg accttgaccg tggtcggtcc ggacggcgag 480
aggacggacc tcatcgcttc gtgggtggtc ggctgcgacg gcgccaacag cctggtgcgg 540
gcgagtgtcg gcaccacggt gacggacctc gacttctcct acgactggct gatctgcgat 600
gtgcggctgc atgagcaccg cgagttccgg ccgaacaatc tggagatctg cgatccggcc 660
cgcccgcgga ccgcggtgtc cgcggggccc ggacaccggc ggtacgagtt catgcgggtg 720
cccgcggacg accccgcgca cttcggcacc gtggagagcg cgtgggagct gctgcggctg 780
ttcgacgtga cgcccgacaa cggcgtcctg gaccggcatg cggtctacac cttccaggcc 840
cgctgggcgg agcactggcg ggccggacgg ctgctgctgg ccggggactc ggcgcacctc 900
atgccgccgt tcgcggggca gggcatgtgc tccggattcc gtgacgcggc gaatctggcc 960
tggaagctgg acctggtgct gagcgggcac gcggcaccgg cgctgctgga cacctacacc 1020
agcgagcggc gggcgcatgt gcggcacgcg gtggagatgt ccgtgggcct gggccgggtg 1080
gtgtgcatgg cggacccggc cgcggcggcg gaccgggacg cggcgatgct ggccgcgcgg 1140
aagcggaaca tcgggccgag cgccgcgcgc cgctcggtgg tgcgcccgct cgtggacggg 1200
ttgctgcggc gggacgggca gggccggccg gcgccgtacg ccggacaggc ggccccacag 1260
tggcgggtgg gccgcgcggg cggcaccggg ctgttcgacg atgtggtggg caccggtttc 1320
atcctgctgt acggcgagga cgtggtcccg gcgctggacg cgcggcagct gacgttcctc 1380
gacaacatcg gggcccggct ggtgcgtgtg gtgcgcgcgg aaacgcccgc ggccgacctg 1440
gaaccagggg acgcgcggga cgtggagggc cggtatctgc cgtatctgtc ggagatgggc 1500
gcgctggcgg tgctggtacg cccggacttc tacgtgttcg gcatcgcggg cgacaaggcc 1560
gaactgccct cgctggtgga cgacttggcg gaacaactca ccccgaccgc cgccccgtcc 1620
tga 1623
<210> 2
<211> 59
<212> DNA
<213>artificial sequence (Artificial)
<400> 2
gttccaggac acggtcgatc tgatcctggc gggcgaatga ttccggggat ccgtcgacc 59
<210> 3
<211> 58
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
gaaggggacc agttcgtgtt cgggtggggg agtgcctcat gtaggctgga gctgcttc 58
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
gacctggtgt tcgagcatgt gg 22
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
gccgatcagc acgatctttc c 21
Claims (2)
1. a kind of autolytic streptin, it is characterised in that: the bacterium is to knock outalmMGene autolytic streptin (Streptomyces autolyticus), it is preserved in China typical culture collection center (CCTCC), Wuhan, China, deposit number is CCTCC NO:
M 2013156。
2. the purposes of genetic engineering bacterium according to claim 1, it is characterised in that: the bacterial strain is used for autolytic mycin
Fermenting and producing.
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| CN103409358A (en) * | 2013-05-28 | 2013-11-27 | 云南大学 | Gene engineering strain of high-yield autolytic mycin |
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