CN102533896B - Method for obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene - Google Patents
Method for obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene Download PDFInfo
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Abstract
The invention provides a method for obtaining geldanamycin derivatives by knocking out an oxygen-methyltransferase gene, relating to a method for obtaining a chemical compound by using a molecular biology method for knocking out genes participating in the biosynthesis of a streptomyces active metabolite. In the method, the o-methyl-transferase gene in the biosynthesis gene cluster of a benzoquinone ansamycin compound which is not reported is found and proved by experiments by cloning and sequencing a biosynthesis gene cluster of the geldanamycin in streptomyces autolyticus. A Red/ET single-step PCR (Polymerase Chain Reaction) method is used for building the insertion mutation of the o-methyl-transferase gene, and the knockout mutant of the o-methyl-transferase gene is obtained through homologous recombination. The 17-o-demethyl-geldanamycin and the 17-amino-17 -demethoxy geldanamycin of geldanamycin derivatives can be obtained by knocking out the strain fermentation of the built o-methyl-transferase gene and can be used as anti-tumor medicines or precursors for synthesizing similar anti-tumor medicines. The method provided by the invention is used for building engineering bacteria to synthesize two compounds and is more economical and environment-friendly in comparison with other methods.
Description
Technical field
The present invention relates to utilize molecular biology method, by knocking out the method that the biosynthetic gene of streptomycete active metabolite obtains new compound that participates in.
Background technology
The benzoquinones AMSA compound (benzoquinone ansamycin) that the geldanamycin (geldanamycin) of take is representative is one of current most study, the good antineoplastic compound of effect.The antitumor mechanism of this compounds be with tumour cell in heat shock protein(HSP) (heat shock protein) Hsp90 specific combination, the key protein that causes the interior many Hsp90 of needs of tumour cell to maintain function is degraded rapidly, thereby the growth of inhibition tumor cell or triggering tumor cell are dead.Although geldanamycin has very strong anti-tumor activity, because its toxic side effect is large, poorly water-soluble and poor stability, be not suitable for clinical use.By some chemical groups of geldanamycin are modified, can improve its activity and reduce toxicity.The multiple geldanamycin derivant that has potentiality has entered clinical experiment at present, and the common feature of these compounds is to have carried out modification in various degree in carbon-17 of geldanamycin.
The biosynthesizing of benzoquinones peace Sha compound is to take 3-amino-hydroxy-benzoic acid as initiator, by polyketide synthase (polyketide synthase, PKS) progressively add some extending elements as acetic acid, propionic acid and oxyacetic acid etc., form the core texture of a polyketone, finally through modifying enzyme later, modify and form again.The domestic and international research of the biosynthesizing for this compounds at present shows, the PKS enzyme of its synthetic kernel core structure is very similar, the key distinction is rear modification, the wherein most importantly modification of carbon-17, the result of modification has very important impact to the anti-tumor activity of this compounds and cytotoxicity.Although the biosynthetic gene cluster of a plurality of benzoquinones peace Sha's compound is cloned, as streptomyces hygroscopicus (
streptomyces hygroscopicus) herbimycin (herbimycin) gene cluster in NRRL 3602 in geldanamycin biological synthesis gene cluster and streptomyces hygroscopicus AM 3672 etc., but participating in the gene of the methylated enzyme of 17-oxygen, coding fails to find always.
We produce the actinomycetes novel species of geldanamycin from a strain--autolytic streptin (
streptomyces autolyticuscGMCC 0516) clone and the biosynthetic full gene cluster of the geldanamycin that checked order in (ZL 00134063.8), found that first coding participates in the chlB5 gene of carbon-17 modification, and by proved the function of this gene in body with experiment in vitro, the research of relevant this gene there is not yet report.On this basis, we have carried out genetic modification to autolytic streptin wild type strain, a carbon-17 methylated chlB5 gene in responsible geldanamycin biosynthetic pathway (
gdmMT) be knocked after, bacterial strain has completely lost the ability that produces geldanamycin, but can produce 17-oxygen-demethyl geldanamycin (17-O-Demethyl-geldanamycin) and 17-amino-17-de-methoxy geldanamycin (17-Amino-17-demethoxy-geldanamycin).Wherein 17-oxygen-demethyl geldanamycin can be used as the initial substance of geldanamycin carbon-17 transformation, by structure of modification, may obtain the antitumor drug with better pharmacological characteristics.And 17-amino-17-de-methoxy geldanamycin is the most promising geldanamycin series antineoplastic medicament at present, just by American I nfinity Pharmaceuticals, is being developed at present, and is entering first phase clinical trial.But the biosynthesizing of 17-amino-17-de-methoxy geldanamycin so far there are no report.
17-oxygen-demethyl the geldanamycin the present invention relates to and 17-amino-17-de-methoxy geldanamycin can produce by constructed chlB5 gene knockout engineering bacterium fermentation, compare with chemical synthesis process, more economically, environmental protection, and sustainable use.
Summary of the invention
The present invention has built the full gene library of autolytic streptin, with
gdmNwith
gdmAIgene fragment is that probe obtains geldanamycin biological synthesis gene cluster by Colony Hybridization In Situ For Screening and Southern screening by hybridization, adopt shotgun to measure institute's clone gene bunch DNA sequence dna, and therefrom found all to have no in benzoquinones AMSA compound biological synthesis gene cluster the chlB5 gene of report, the function of this genes encoding chlB5 in body and experiment in vitro confirm.Adopt Red/ET mono-step PCR method to build the knockout mutant strain of this chlB5 gene, tunning analysis shows, constructed chlB5 gene knockout engineering bacteria can produce 17-oxygen-demethyl geldanamycin and 17-amino-17-de-methoxy geldanamycin.The invention provides and build the method that this oxygen methyl transferase gene knocks out engineering bacteria, and the method that obtains 17-oxygen-demethyl geldanamycin and 17-amino-17-de-methoxy geldanamycin by fermentation.678 Nucleotide of 17-chlB5 full length gene involved in the present invention, 225 amino acid of protein total length.Genetic engineering bacterium autolytic streptin of the present invention (
streptomyces autolyticus, CGMCC 0516) and chlB5 (GdmMT) mutant strain is preserved in Chinese Typical Representative culture collection center (CCTCC), address: Wuhan University, deposit number: M 2011200, preservation date: on June 16th, 2011.
accompanying drawing explanation:
Fig. 1. geldanamycin biological synthesis gene cluster in autolytic streptin CGMCC 0516.(A) with probe 1 and probe 2 screenings, obtain covering the full gene cluster clay of geldanamycin biosynthesizing plasmid pBS17001, pBS17002 and pBS17003; (B) the supposition function of the genetic composition of geldanamycin biological synthesis gene cluster and relevant ORF; (C) the possible route of synthesis of geldanamycin.
Fig. 2. the chlB5 of expression and purification.1 is low molecular weight protein (LMWP) standard, the 2 chlB5 albumen that are purifying.
Fig. 3. the impact of different pH on chlB5 activity.
Fig. 4. the impact of different metal ion pair chlB5 activity.
Fig. 5. the structure of chlB5 knockout mutant strain and Southern hybridization analysis.(A) construction strategy of mutant strain; (B) Southern hybridization analysis, wherein 1 is
bamthe wild type strain genomic dna that H I enzyme is cut, 2 are
bamthe mutant strain genomic dna that H I enzyme is cut.
Fig. 6. the product of chlB5 knockout mutant strain detects.I: geldanamycin standard specimen; II:17-oxygen-demethyl geldanamycin standard specimen; III: △
gdmAImutant strain product; IV: wild type strain product: V: △
gdmMTproduct; VI: complementary bacterial strain product; VII: chlB5 catalysis 17-oxygen-demethyl geldanamycin reaction product of inactivation; VIII: chlB5 catalysis 17-oxygen-demethyl geldanamycin reaction product; ◇: 17-amino-17-de-methoxy geldanamycin.
embodiment:
1. the Cloning and sequencing of geldanamycin biological synthesis gene cluster in autolytic streptin:
The full gene library that the clay plasmid pHAQ34 of take is vector construction autolytic streptin, first uses
sauthe partially digested autolytic streptin chromosomal DNA of 3AI, reclaims 40 kb left and right fragments, dephosphorylation process after with
bampHAQ34 that H I enzyme is cut connects, connect product after packing transfection Escherichia coli (
escherichia coli) XL1 Blue MR sets up gene library.With pcr amplification
gdmN(probe 1, amplimer is 5 '-AAGGTGATGGGCCTGGCGCC-3 ' and 5 '-CGCGCGGTGCCGTCCACATG-3 ') and
gdmAI(probe 2, amplimer is 5 '-TGCTGAGGCTGGATTGGG-3 ' and 5 '-ACAGAACCAGGATCAGGAGACC-3 ') gene fragment is probe, by Colony Hybridization In Situ For Screening and Southern screening by hybridization positive colony, utilize DNA restriction analysis and end sequence to measure geldanamycin biological synthesis gene cluster is positioned to clay plasmid pBS17001, pBS17002 and pBS17003(Figure 1A).Take pSmart as carrier has built respectively 3 sequencing plasmid libraries, then the Insert Fragment in constructed sequencing library is carried out to determined dna sequence, sequence obtains the complete sequence of geldanamycin biological synthesis gene cluster after SeqScape splicing.
2. the checking of chlB5 gene:
ORF finder by NCBI website, the instrument such as glimmer and BLAST is analyzed geldanamycin gene cluster, infer the route of synthesis (Fig. 1 C) that each open reading frame (open reading frame, ORF) function (Figure 1B) and geldanamycin are possible.According to amino acid sequence homology determine the chlB5 of may encoding gene (
gdmMT), the methyltransgerase structural domain that it contains three distinctive SAM of depending on and the DXDXD structural domain of being combined with divalent cation.Take pBS17003 as template employing pcr amplification
gdmMTgene (amplimer 5 '-GGAATTCCATATGCCGTCCACACTGCACAC-3 ' and 5 '-CCCAAGCTTCAGCCGAGCCGCACACCCG-3 '), is cloned into construction expression plasmid pBS17009 in carrier pET-28a (+).PBS17009 is converted in e. coli bl21, by this chlB5 of IPTG abduction delivering (GdmMT), and adopts affinitive layer purification GdmMT.The GdmMT of purifying is single band in SDS-PAGE electrophoresis, and apparent molecular weight is 26 kDa, conforms to (Fig. 2) with 26.2 kDa that calculate by sequence.This GdmMT under SAM exists in vitro catalysis 17-demethyl geldanamycin generate geldanamycin, the optimal ph of catalyzed reaction is 7.0(Fig. 3), best metal ion is Mg
2+(Fig. 4).
3. the structure of chlB5 gene knockout mutant strain:
Adopt Red/ET mono-step PCR method to build the insertion mutation (Fig. 5 A) of chlB5 gene.In this method, first the pHY773 of take inserts box (primer is 5 '-CTATCCGGTGACCGTGTCGACCCTGAAGGAGATCGCATGATTCCGGGGATCCGTCG ACC-3 ' and 5 '-GGCCGCCCCCGGGAACAGCGCCGTCCAGCGGAGCGCTCATGTAGGCTGGAGCTGCT TC-3 ') as template adopts pcr amplification two ends with the aac with chlB5 DNA homolog sequence (3) IV gene, then by electricity, transforming pcr amplified fragment importing is in the intestinal bacteria of chlB5 gene containing pIJ790 and goal gene, the combination of screening apramycin (Apramycin) resistance is shifted son and is obtained insertion mutation gene.Utilize in conjunction with shifting mutator gene is imported in wild-type autolytic streptin, by the knockout mutant strain SB17002 of homologous recombination construction chlB5 gene.This mutant strain is through PCR and Southern hybridization checking (Fig. 5 B), wherein PCR verifies that all primers are 5 '-TGCACCCCGACTACCAGG-3 ' and 5 '-CGCCGGAAGAAGAATCTATCC-3 ', and the PCR primer of preparation Southern hybridization probe is 5 '-GCGACTGGTCGAAGTGGTGTTC-3 ' and 5 '-CGGTGGAGCGGCAGATTGA-3 '.Build the complementary bacterial strain of this mutant strain simultaneously, take pBS17003 as template pcr amplification wild-type
gdmMTgene (primer is 5 '-GGAATTCCATATGGGGCCTGAGATCGAGATTGC-3 ' and 5 '-CGGGATCCTCAGCCGAGCCGCACACCCGCCAGGATG-3 '), amplified fragments be cloned into
ermE*in the plasmid pBS9010 of promotor, then utilize in conjunction with shifting complementary plasmid is imported in SB17002 and obtains complementary bacterial strain SB17003.
4. the detection of chlB5 gene knockout mutant strain tunning:
In fresh spore inoculating to the 50 mL TSB substratum of autolytic streptin bacterial strain, in 28
oc, 250 rpm cultivate and within 40 hours, prepare fermentation seed liquid, then with the inoculum size of 1 %, inoculate 0.5 mL seed liquor (2 % bean powderes, 0.2 % peptone to fermention medium, 2 % glucose, 0.5 % Zulkovsky starch, 0.2 % yeast extract, 0.4 % NaCl, 0.05 % K
2hPO
4, 0.05 % MgSO
4, 0.2 % CaCO
3), in 28
oc, 250 rpm fermentation culture 5 days.Fermented liquid centrifugal (3500 g, 30 minutes) is got supernatant, with ethyl acetate extracting twice, then through silica gel column chromatography and half preparative HPLC, carries out purifying, is dissolved in acetone and analyzes after elutriant evaporate to dryness.Constructed chlB5 gene knockout mutant strain loses the ability that produces geldanamycin, and produces new compound 17-oxygen-demethyl geldanamycin and 17-amino-17-de-methoxy geldanamycin (Fig. 6).Compound structure through mass spectrum and
1h,
13c nuclear magnetic resonance spectroscopy is confirmed.
sequence table
<110> Yunnan University
The method of <120> obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene
<130> oxygen methyltransgerase
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 678
<212> DNA
<213> autolytic streptin (
streptomyces autolyticus)
<400> 1
tcagccgagc cgcacacccg ccaggatccc gtgtgcgacg ggcaggcgca gtgcgagata 60
gccgctggag gggtcggcga ggtagtcccg catctcacgg tgcatatcga agccgaagtc 120
catgtccatg tcgtccacga gcaccagcgc gccccggcgc agccggggtt ccaccacctc 180
gagcaccgcg cggttgaggt tgggccagcc gtccatcagc aggaggtcca ccgactcggg 240
cagctcccgc agggtctccc gtgcgtcgcc gacccggacg ctcgcgatgt cggagaggcc 300
ggcggcggcg atggcctccg tggccctggc caccttgtcc gcctggagct ccgtgccgat 360
cacctgcccg ccgccgttgt cccgtaccgc gctggccagg tagagggtgg acacgccgaa 420
agaggtcccg tactcgacga cggtcctggc gccggtcgcg cgggtgagca ggtagagcag 480
ctcaccgcct tccttggaca ccgacatgga ggcgtccttg aacgtctcgg ccatctggcc 540
ggcatccagc tcggcccagg ccgccttcgg gtcggtcaca ccggtctcgg cgaacgcgcg 600
ctcgtcgcct tcgcgctcgg cctccagcag tccgtccagg acggtcgcca cgggctcggt 660
gtgcagtgtg gacggcat 678
<210> 2
<211> 225
<212> PRT
<213> autolytic streptin (
streptomyces autolyticus)
<400> 2
Met Pro Ser Thr Leu His Thr Glu Pro Val Ala Thr Val Leu Asp Gly
1 5 10 15
Leu Leu Glu Ala Glu Arg Glu Gly Asp Glu Arg Ala Phe Ala Glu Thr
20 25 30
Gly Val Thr Asp Pro Lys Ala Ala Trp Ala Glu Leu Asp Ala Gly Gln
35 40 45
Met Ala Glu Thr Phe Lys Asp Ala Ser Met Ser Val Ser Lys Glu Gly
50 55 60
Gly Glu Leu Leu Tyr Leu Leu Thr Arg Ala Thr Gly Ala Arg Thr Val
65 70 75 80
Val Glu Tyr Gly Thr Ser Phe Gly Val Ser Thr Leu Tyr Leu Ala Ser
85 90 95
Ala Val Arg Asp Asn Gly Gly Gly Gln Val Ile Gly Thr Glu Leu Gln
100 105 110
Ala Asp Lys Val Ala Arg Ala Thr Glu Ala Ile Ala Ala Ala Gly Leu
115 120 125
Ser Asp Ile Ala Ser Val Arg Val Gly Asp Ala Arg Glu Thr Leu Arg
130 135 140
Glu Leu Pro Glu Ser Val Asp Leu Leu Leu Met Asp Gly Trp Pro Asn
145 150 155 160
Leu Asn Arg Ala Val Leu Glu Val Val Glu Pro Arg Leu Arg Arg Gly
165 170 175
Ala Leu Val Leu Val Asp Asp Met Asp Met Asp Phe Gly Phe Asp Met
180 185 190
His Arg Glu Met Arg Asp Tyr Leu Ala Asp Pro Ser Ser Gly Tyr Leu
195 200 205
Ala Leu Arg Leu Pro Val Ala His Gly Ile Leu Ala Gly Val Arg Leu
210 215 220
Gly
225
sequence table
<110> Yunnan University
The method of <120> obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene
<130> chlB5
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 678
<212> DNA
<213> autolytic streptin (Streptomyces autolyticus)
<400> 1
tcagccgagc cgcacacccg ccaggatccc gtgtgcgacg ggcaggcgca gtgcgagata 60
gccgctggag gggtcggcga ggtagtcccg catctcacgg tgcatatcga agccgaagtc 120
catgtccatg tcgtccacga gcaccagcgc gccccggcgc agccggggtt ccaccacctc 180
gagcaccgcg cggttgaggt tgggccagcc gtccatcagc aggaggtcca ccgactcggg 240
cagctcccgc agggtctccc gtgcgtcgcc gacccggacg ctcgcgatgt cggagaggcc 300
ggcggcggcg atggcctccg tggccctggc caccttgtcc gcctggagct ccgtgccgat 360
cacctgcccg ccgccgttgt cccgtaccgc gctggccagg tagagggtgg acacgccgaa 420
agaggtcccg tactcgacga cggtcctggc gccggtcgcg cgggtgagca ggtagagcag 480
ctcaccgcct tccttggaca ccgacatgga ggcgtccttg aacgtctcgg ccatctggcc 540
ggcatccagc tcggcccagg ccgccttcgg gtcggtcaca ccggtctcgg cgaacgcgcg 600
ctcgtcgcct tcgcgctcgg cctccagcag tccgtccagg acggtcgcca cgggctcggt 660
gtgcagtgtg gacggcat 678
<210> 2
<211> 225
<212> PRT
<213> autolytic streptin (Streptomyces autolyticus)
<400> 2
Met Pro Ser Thr Leu His Thr Glu Pro Val Ala Thr Val Leu Asp Gly
1 5 10 15
Leu Leu Glu Ala Glu Arg Glu Gly Asp Glu Arg Ala Phe Ala Glu Thr
20 25 30
Gly Val Thr Asp Pro Lys Ala Ala Trp Ala Glu Leu Asp Ala Gly Gln
35 40 45
Met Ala Glu Thr Phe Lys Asp Ala Ser Met Ser Val Ser Lys Glu Gly
50 55 60
Gly Glu Leu Leu Tyr Leu Leu Thr Arg Ala Thr Gly Ala Arg Thr Val
65 70 75 80
Val Glu Tyr Gly Thr Ser Phe Gly Val Ser Thr Leu Tyr Leu Ala Ser
85 90 95
Ala Val Arg Asp Asn Gly Gly Gly Gln Val Ile Gly Thr Glu Leu Gln
100 105 110
Ala Asp Lys Val Ala Arg Ala Thr Glu Ala Ile Ala Ala Ala Gly Leu
115 120 125
Ser Asp Ile Ala Ser Val Arg Val Gly Asp Ala Arg Glu Thr Leu Arg
130 135 140
Glu Leu Pro Glu Ser Val Asp Leu Leu Leu Met Asp Gly Trp Pro Asn
145 150 155 160
Leu Asn Arg Ala Val Leu Glu Val Val Glu Pro Arg Leu Arg Arg Gly
165 170 175
Ala Leu Val Leu Val Asp Asp Met Asp Met Asp Phe Gly Phe Asp Met
180 185 190
His Arg Glu Met Arg Asp Tyr Leu Ala Asp Pro Ser Ser Gly Tyr Leu
195 200 205
Ala Leu Arg Leu Pro Val Ala His Gly Ile Leu Ala Gly Val Arg Leu
210 215 220
Gly
225
Claims (2)
1. utilize the method for obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene, it is characterized in that using autolytic streptin (
streptomyces autolyticus) oxygen methyl transferase gene sudden change engineering bacteria, its deposit number is CCTCC NO:M2011200, fermentation produces 17-oxygen-demethyl geldanamycin and 17-amino-17-de-methoxy geldanamycin.
2.
according tothe method of utilizing obtaining geldanamycin derivatives by knocking out o-methyl-transferase gene claimed in claim 1, it is characterized in that described fermentation carries out fermentation culture for engineering bacteria that chlB5 is suddenlyd change, to tunning extract, HPLC detection and mass spectrum and
1h,
13c nuclear magnetic resonance spectroscopy.
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| CN104862326B (en) * | 2015-06-02 | 2017-10-27 | 中国农业科学院生物技术研究所 | A kind of green muscardine fungus oxygen transmethylase and its application |
| CN106191156B (en) * | 2016-08-01 | 2019-05-07 | 上海交通大学 | The method for improving Ge Erdeng element fermentation level |
| CN111269867B (en) * | 2019-12-12 | 2022-05-10 | 中国科学院南海海洋研究所 | Gene engineering strain for directionally producing antiviral and antibacterial active compound and application thereof |
| CN111607578B (en) * | 2020-05-27 | 2021-11-12 | 江南大学 | Oxygen methyltransferase mutant and application thereof in prodigiosin production |
Citations (1)
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| CN101054557A (en) * | 2007-04-09 | 2007-10-17 | 中国医学科学院医药生物技术研究所 | Construction of geldanamycin gene engineering high yield strain |
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| CN101054557A (en) * | 2007-04-09 | 2007-10-17 | 中国医学科学院医药生物技术研究所 | Construction of geldanamycin gene engineering high yield strain |
Non-Patent Citations (4)
| Title |
|---|
| Isolation and Structure Elucidation of Autolytimycin, A New Compound Produced by Streptomyces Autolyticus JX-47.;Ming Gang LI et al.;《Chinese Chemical Letters》;20011231;903-906 * |
| Ming Gang LI et al..Isolation and Structure Elucidation of Autolytimycin, A New Compound Produced by Streptomyces Autolyticus JX-47..《Chinese Chemical Letters》.2001,903-906. |
| 朱皖宜 等.格尔德霉素产生菌的诱变育种及其发酵的研究.《2010年中国药学大会暨第十届中国药师周论文集》.2010,1-4. |
| 格尔德霉素产生菌的诱变育种及其发酵的研究;朱皖宜 等;《2010年中国药学大会暨第十届中国药师周论文集》;20101231;1-4 * |
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