CN101812103B - 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic - Google Patents
6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic Download PDFInfo
- Publication number
- CN101812103B CN101812103B CN200910247567A CN200910247567A CN101812103B CN 101812103 B CN101812103 B CN 101812103B CN 200910247567 A CN200910247567 A CN 200910247567A CN 200910247567 A CN200910247567 A CN 200910247567A CN 101812103 B CN101812103 B CN 101812103B
- Authority
- CN
- China
- Prior art keywords
- ala
- leu
- gly
- arg
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a novel method for synthesizing spirocyclic acetoacetic acid lactone antibiotic with higher activity by using combinatorial biosynthesis based on a genetic engineering method. The method is characterized by carrying out specific locus mutation on the keto-reduction (KR) conservation activity locus of a 6-methy salicylic acid (6-MSA) synthetase and genetically transforming the producing strain Streptomyces antibioticus DSM40725 of chlorothrichin (CHL) and fermenting to generate new chlorothrichin analogues 7 and 8 with improved biological activity. The favorable biological activity of the new chlorothrichin analogues 7 and 8 has values to be developed into medicaments.
Description
Technical field
The invention belongs to biotechnology engineering field, be specifically related to genetic engineering modified 6-MSA synthetic enzyme function, the fermentation at recombinant bacterial strain combination biosynthesizing new compound separates, and structure is identified, determination of activity.
Background technology
Natural compounds is the main source of drug discovery and development; Like anti-infectious penicillium mould and Oxacyclotetradecane,erythromycin deriv, antineoplastic bleomycin and dust Tobramycin, antiparasitic Avrmectin and immunosuppressor S-Neoral etc., be the main medicine of human treatment's disease in the past in decades always.So with the natural compounds is that foundation development and developing new drug are the major fields that chemical boundary and the world of medicine pay close attention to always.Wherein microbe-derived natural product accounts for part and parcel, and the mikrobe natural product of having reported at present has thousands of kinds more than, and wherein polyketone class and non-ribosomal gather the peptide class and accounted for sizable ratio.
Volution etheric acid lactone microbiotic family is in the polyketone class natural product very distinctive one type, and the overwhelming majority in them is produced by actinomycetes, has good antibacterial activity.Wherein representative molecule Chlorothricin (CHL; Fig. 1) be a novel macrolide antibiotics; Separate first from bacterial strain Streptomyces antibioticusT ü 99; Constructional feature is that the big ring skeleton by aglycone partly contains [6,6] and ring and [6,5] volution (Fig. 1): the decahydro naphthalene nucleus that (1) is trans; (2) 4-hydroxyl etheric acid lactone and the volution (volution etheric acid lactone family natural product is gained the name thus) that cyclohexene ring forms, side chain glycosyl and hexamethyl Whitfield's ointment are formed (Helv.Chim.Acta 52 (1969) 127-142).Chlorothricin has good anti-infection activity, and its anti-microbial activity ascribes to the catalytic anaplerotic CO of pyruvate carboxylase
2Fixed suppresses.Further research shows, there are interaction in CHL and Bacillus subtilis cell membrane phospholipid, have the activity of anti-SUV.Current research also show its have the potential anti-tumor activity (J.Antibiot.34,1101-1106, J.Antibiot.36,668-670J.Antibiot.44,207-212).
Chlorothricin unique chemical structure has caused the concern of chemistry, biology and the world of medicine with abundant biological activity, as how become the direction that each side makes great efforts as the more valuable clinical medicine of lead compound development.But; Its complicated chemical structure is that chemosynthesis and structure are derived and brought great challenge; Because too much chiral centre and the functional group that concentrates; Chemosynthesis is very limited for the prospect of production of CHL and analogue thereof, and as the important supplement of organic synthesis, the acquisition that develops into complicated natural product and analogue thereof of combination biosynthesis technology provides novel method.Through clone to its biosynthesis gene; (Chemistry&Biology 13 to have disclosed it biosynthesizing that comprises structural units such as big ring skeleton, side chain glycosyl and hexamethyl Whitfield's ointment mechanism; 575-585); Biosynthetic principle is made up in utilization on this basis, and its biosynthetic pathways metabolism of rational modification is explored and obtained the analog that needed novel biological activity improves.
Summary of the invention
The method that the purpose of this invention is to provide the active chlorothricin analogue that improves of a kind of novel generation; Be specifically related to a kind of amino-acid residue sudden change of the KR structural domain to six-cresotinic acid acid enzyme, finally in chlorothricin produces the recombinant bacterial strain of bacterium, produce the active volution etheric acid lactone microbiotic that improves.
Specific as follows:
At first with the method for PCR in Streptomyces antibioticus DSM40725, obtain the encoding 1.9KbDNA fragment of six-cresotinic acid acid enzyme KR structural domain; Be connected into then in the carrier of pSP72 and obtain containing the segmental plasmid of KR; Next step heavily cuts out the KR structural domain fragment that contains the mutational site from the plasmid that PCR obtains; The KR structural domain that substitutes the 6-MSAS of wild-type is connected among the expression vector pTGV2 together, imports to this pTGV2 deutero-plasmid among the heterogenous expression type strain Streptomyces albus then and detects product.1540 Y is mutated in the recombinant bacterial strain of F and produces OSA as a result.Aforesaid method is simple, only needs conventional molecular biology method, just can obtain the albumen of changing function very soon, and in heterologous host, detects the product of sudden change.The proteic method of point mutation provided by the invention does not rely on any business-like sudden change test kit fully, but a kind of simple, cheapness, the proteic method of point mutation efficiently.
The present invention also provides a kind of strategy and application that in the mutant strain of Streptomyces antibioticus DSM40725 chlB1 gene knockout, produces two new chlorothricin analogue.Concrete grammar be us the above-mentioned expression plasmid that heterogenous expression produces OSA in Streptomyces albus import to Streptomyces antibioticusDSM40725chlB1 gene knockout mutant strain in; Fermentation detects; Separation and purification; Structure identifies that preliminary bioassay has obtained two plain analogues 7 and 8 of Chloronema Dubinina and Gorlenko that activity doubles.
Description of drawings
Fig. 1 representes Chlorothricin (CHL), deschloro-CHL, and new compound 7 that the present invention produced and 8 structure.
Fig. 2 representes that several kinds contain the unitary natural product of type repetition PKS synthetic fragrance, Cholorothricin, Maduroprptin, polyketomycin, Avilamycin, Calicheamicin.
Fig. 3 represent the structural domain of fungi and bacterium 6-MSAS form with and the supposition of the product that produces of structural domain selectivity inactivation, (A) composition of the functional domain of 6-MSAS.(B) ChlB1 of the synthetic route of fungi and bacterium 6-MSAS and DH sudden change (H947F) produces 6-MSA; The ChlB1 of KR sudden change (Y1450F) produces OSA; (6-MSAS G1392A) produces TAL for G1387A, G1389P in fungi KR sudden change.
Fig. 4 representes the product of the ChlB1 heterogenous expression in Streptomyces albus after HPLC analysis DH and the KR sudden change.(I) Streptomyces albus contains the pTGV2 empty plasmid; (II) 6-MSA standard substance; (III) Streptomyces albus contains wild-type 6-MSAS (ChlB1) expression plasmid generation 6-MSA, and output is about 2mg/ml; (IV) the Streptomyces albus ChlB1 expression plasmid that contains DH sudden change (H947A) does not produce 6-MSA; (V) Streptomyces albus contains the ChlB1 expression plasmid continuation generation 6-MSA of DH sudden change (H947F), and output is about 0.3mg/ml; (VI) TAL standard substance; (VII) OSA standard substance; (VIII) the Streptomycesalbus ChlB1 expression plasmid that contains KR sudden change (G1389A) does not produce 6-MSA, TAL, OSA; (IV) Streptomyces albus contains KR sudden change (ChlB1 expression plasmid G1392A) does not produce 6-MSA, TAL, OSA for G1387A, G1389P; (X) Streptomyces albus contains the ChlB1 expression plasmid generation OSA of KR sudden change (Y1540F), and output is about 0.3mg/ml.
Fig. 5 representes that the acyl group of 6-MSA and OSA is modified and transferred on the DMCHL glycosyl after unitary and forms deschloro-CHL; CHL; 7 and 8 synoptic diagram.
Fig. 6 representes that HPLC analyzes generation (I) the wild strain Streptomyces antibioticus DSM40725 generation deschloro-CHL of volution etheric acid lactone new among the Streptomyces antibioticus DSM40725 (Δ chlB1), CHL; (II) mutant strain Streptomyces antibioticus DSM40725 (Δ chlB1) produces DMCHL; (III) complementary Streptomyces antibioticusDSM40725 (Δ chlB1) bacterial strain generation 7,8 of the ChlB1 expression plasmid of KR sudden change (Y1540F) and DMCHL.a，deschloro-CHL；b，CHL；C，DM-CHL；d，7；and e，8。
Fig. 7 representes new compound 7
1H-
1H COSY and 7 and 8 the unitary HMBC synoptic diagram of OSA.
Fig. 8 representes compound 7
1The H-NMR collection of illustrative plates
Fig. 9 representes compound 8
1The H-NMR collection of illustrative plates
Figure 10 representes the H-H COSY of new compound 7
Figure 11 representes the HSQC of new compound 7
Figure 12 representes the HMBC of new compound 7
Figure 13 representes new compound 7ROESY
Figure 14 representes the unitary HMBC collection of illustrative plates of the OSA of new compound 8.The structure that shows compound 8 be the structure of CHL and in the 6-MSA unit contraposition of phenyl ring has increased Sauerstoffatom.
Figure 15 representes that the HPLC that MSS4.3 is imported to tunning among the Streptomyces antibioticus DSM40725 (Δ chlB1) analyzes: (I) complementary Streptomyces antibioticus DSM40725 (Δ chlB1) bacterial strain of MSS4.3 plasmid produces 7 and 8 of OSA and trace; (II) OSA standard substance.
The LC-MS of Figure 16 compound 7
The LC-MS of Figure 17 compound 8
Figure 18 representes the anti-Bacillus subtilis determination of activity of new compound: 1, and deschloro-CHL; 2, CHL; 3,7; 4,8.(I) each compound 5 μ g adds the Cowhells cup after being dissolved in 200 μ l methyl alcohol; (II) each compound 10 μ g adds the Cowhells cup after being dissolved in 200 μ l methyl alcohol, 37 degree, 12 hours.7 and 8 anti-microbial activity has improved about 1 times than deschloro-CHL and CHL respectively.
Nomenclature
In the accompanying drawing 1, Chlorothricin (CHL): chlorothricin; Deschloro-CHL: dechlorination chlorothricin; 7 and 8: the analogue of chlorothricin.
In the accompanying drawing 2, PKS: polyketone.
In the accompanying drawing 3,6-MSAS, six-cresotinic acid; AT: acyltransferase; KS: ketone group synthetic enzyme; DH: desaturase; KR: keto reductase; ACP: acyl carrier protein.
In the accompanying drawing 4, Streptomyces albus: the type strain of heterogenous expression belongs to streptomyces; ChlB1: six-cresotinic acid acid enzyme in the chlorothricin biological synthesis gene cluster; HPLC: performance liquid chromatography; OSA: orsellinic acid; TAL:2 hydroxyl 4-methyl-2-pyrone.
In the accompanying drawing 5, CHL: chlorothricin; Deschloro-CHL: dechlorination chlorothricin; DM-CHL: take off six-cresotinic acid chlorothricin.
In the accompanying drawing 6, Streptomyces antibioticus DSM40725 chlorothricin produces bacterium, belongs to streptomyces.7 and 8: new compound.
In the accompanying drawing 7,
1H-
1H COSY: hydrogen-hydrogen two-dimensional correlation spectrum; HMBC: the hydrocarbon relation of multikey.
Accompanying drawing 8
1H-NMR: proton nmr spectra.
In the accompanying drawing 11, HSQC: the single quantum coherent spectrum of heteronuclear
In the accompanying drawing 13, NOESY: two-dimensional nucleus Ao Fuhaoze strengthens spectrum.
In the accompanying drawing 15, MSS4.3: the gene of orsellinic acid synthetic enzyme (aviM) is cloned among the expression vector pWHM3.
In the accompanying drawing 17, LC-MS: performance liquid chromatography and mass spectrometry.
In the accompanying drawing 18, Bacillus subtilis: subtilis.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should know, these instances only be used to the present invention is described and be not used in the restriction scope of the present invention.Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art, and the experimental technique of unreceipted actual conditions in the following example is usually according to the condition described in the document of publishing.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration, but can not limit the content of this aspect.
The DH of embodiment 1 ChlB1 and point mutation of KR structural domain and heterogenous expression
1.DH and the acquisition of the ChlB1 of KR structural domain point mutation
1) sudden change of DH structural domain
The DH structural domain of inactivation ChlB1 is selected its histidine residues of 947 of sudden change, and it is mutated into glycocoll or phenylalanine(Phe).At first get two pairs of primers that design, primer sequence is following:
947 H are mutated into A
The upper reaches: 5 '-C TAC CCC GGC AGC GCC ACC ATC AAC GGC ACG-3 '
Downstream: 5 '-CGT GCC GTT GAT GGT GGC GCT GCC GGG GTA G-3 '
947 H are mutated into F
The upper reaches: 5 '-C TAC CCC GGC AGC TTC ACC ATC AAC GGC ACG-3 '
Downstream: 5 '-CGT GCC GTT GAT GGT GAA GCT GCC GGG GTA G-3 '
With pAL1084 (fragment cloning of wild-type DH structural domain is in pSP72) is the template template, dNTP, and DMSO, no enzyme water, the primestar enzyme of high-fidelity and damping fluid thereof are formed the PCR reaction system and are carried out PCR according to the program that designs.The PCR product is the segmental plasmid of DH structural domain that contains sudden change, it is carried out gel electrophoresis separate, and cuts glue and reclaims purifying, adds the plasmid pAL1084 of restriction enzyme Dpn I chopping as template.Draw 5 μ l enzymes then and cut system Transformed E .coli DH5, picking mono-clonal bacterium colony is overnight cultures in LB nutrient solution (containing the Amp microbiotic), and is denseer to bacterium liquid.Extract plasmid and send order-checking, detect the corresponding amino acid whether 947 H is mutated into.The fragment that from the plasmid of order-checking sudden change, cuts out 1.0kb with BglII/AvrII then is connected in the identical restriction enzyme site of pAL1087 (chlB1 is cloned among the pTGV2), the pTGV2 that obtains the sudden change of the DH plasmid pAL1088 that derives, pAL1089.
2) sudden change of KR
The KR structural domain of inactivation ChlB1, select three kinds of strategies 1. 1389 G be mutated into A, 2. 1387 G is mutated into A, 1389 G is mutated into P, 1392 G is mutated into A, 3. 1540 Y is mutated into F.
Get three pairs of primers that design, primer sequence is following:
1. 1389 G is mutated into A
The upper reaches: 5 '-C ACC GGC GGA CTG GCC ACC CTC GGC CTG G-3 '
Downstream: 5 '-C CAG GCC GAG GGT GGC CAG TCC GCC GGT G-3 '
2. 1387 G is mutated into A, and 1389 G is mutated into P, and 1392 G is mutated into A
The upper reaches: 5 '-C ACC GGC GCA CTG CCC ACC CTC GCC CTG G-3 '
Downstream: 5 '-C CAG GGC GAG GGT GGG CAG TGC GCC GGT G-3 '
3. 1540 Y is mutated into F
The upper reaches: 5 '-GGC CAG GCC GCC TTC GGC TCC GCC AAC G-3 '
Downstream: 5 '-C GTT GGC GGA GCC GAA GGC GGC CTG GCC-3 '
With pAL1085 (fragment cloning of wild-type KR structural domain is in pSP72) is the template template, dNTP, and DMSO, no enzyme water, the primestar enzyme of high-fidelity and damping fluid thereof are formed the PCR reaction system and are carried out PCR according to the program that designs.The PCR product is the plasmid that contains the KR structural domain of sudden change, it is carried out gel electrophoresis separate, and cuts glue and reclaims purifying, adds restriction enzyme Dpn I chopping as template plasmid pAL1085.Draw the 5ul enzyme then and cut system Transformed E .coli DH5, picking mono-clonal bacterium colony is gone into overnight cultures in the LB nutrient solution (containing the Amp microbiotic), and is denseer to bacterium liquid.Extract plasmid and send order-checking, detect 1387G, the corresponding amino acid whether 1389G, 1392G are mutated into.The fragment that from the plasmid of order-checking sudden change, cuts out 1.9kb with AvrII/HindIII then is connected in the identical restriction enzyme site of pAL1087, the pTGV2 that obtains the sudden change of the KR respectively plasmid pAL1090 that derives, pAL1091, pAL1092.
2.DH with the ChlB1 of KR structural domain sudden change at heterogenous expression
1) engages transfer between the genus of bacterial strain ET12567 and bacterial strain Streptomyces albus J1074.
With above-mentioned plasmid pAL1088, pAL1089, pAL1090, pAL1091, pAL1092 Transformed E T12567 cultivates the ET12567 to OD that contains suitable plasmid
600=0.4-0.6, the centrifugal collection of the bacterium in the 25ml nutrient solution washes twice with isopyknic LB, is resuspended among the 1ml LB, as the intestinal bacteria donorcells.Get 20% glycerine spore suspension, the 500 μ L of an amount of frozen Streptomycesalbus J1074 in-80 ℃, wash twice with the TES damping fluid of 500 μ l, be resuspended in isopyknic TES damping fluid, 50 ℃ of heat shocks made spore germination in 10 minutes.Add isopyknic TSB again, 37 ℃ incubation 2-5 hour, centrifugal being resuspended in the 1.5ml LB substratum as the streptomycete recipient cell.The recipient cell 100 μ L of different concns are mixed with isopyknic donorcells directly be coated on MS (the 2.0g mannitol that contains the 10mM magnesium chloride; 2.0g soybean cake powder; 2.0g agar, tap water is settled to 100ml) on the flat board, cultivate after 20 hours for 30 ℃; Wash planar surface gently with the most of intestinal bacteria of flush away with sterilized water, contain nalidixic acid (final concentration is 50 μ g/ml) and corresponding microbiotic (Tsr 50 μ g/ml) at each dull and stereotyped surface coverage 1ml) sterilized water.Cultivate picking zygote more than 5 days for 30 ℃.
2) zygosporic screening.
Choose single bacterium colony and cultivate, import the exactness of plasmid with bacterium liquid PCR checking in TSB (Tsr 50 μ g/ml).
3) fermentation and processing:
With the bacterium liquid of the bacterium that is cultured to logarithmic phase (about 48hr) switching 0.5% in R5A (sucrose 100g/l; Sal epsom 0.25g/l; Magnesium chloride hexahydrate 10.12g/l; Glucose 10g/l; Hy-case amino 0.1g/l; Yeast extract 5g/l; Mops 21g/l; 2ml R5 trace element; Transfer pH=6.85, autoclaving) liquid nutrient medium in continue to cultivate 120hr, transfer to 2-3 to the PH of all fermented products (comprising thalline and bacterium liquid), ultrasonic 15min (10s/50s); With the most of thalline of filter paper elimination, with equal volume of ethyl acetate twice, revolve driedly, heavily be threaded onto in the methyl alcohol.HPLC analyzes tunning.
HPLC analyzing and testing condition:
Instrument: Agilent 1100HPLC system
Pillar: Phenomenex C18 column (4.63250nm, part number 00F-3300-E0, S/N115575-1
Detect wavelength: UV=220nm
Moving phase condition: V=1mL/min; A=H
2O (1 ‰ TFA); B=CH
3CN (1 ‰ TFA)
Min A％/B％
0， 90％A/10％B
5， 80％A/20％B
25，30％A/70％B
26，5％A/95％B
29，5％A/95％B
30，90％A/10％B
Analysis through LC-MS detects 947 H respectively and is mutated in the recombinant bacterial strain of A, does not produce 6-MSA; 947 H are mutated in the recombinant bacterial strain of F, produce 6-MSA; 1389 G is mutated in the recombinant bacterial strain of A, does not produce OSA, TAL (2 hydroxyl 4-methyl-2-pyrone), 6-MSA; 1387 G is mutated into A, and 1389 G is mutated into P, and 1392 G is mutated in the recombinant bacterial strain of A, does not produce OSA, TAL, 6-MSA; 1540 Y is mutated in the recombinant bacterial strain of F, produces OSA.
1, E.coli ET12567 and Streptomyces antibioticus DSM40725 (Δ chlB1) and between genus between engage to shift
From receive overnight cultures in the middle of the test tube through picking list bacterium colony on the E.coli ET12567 culture plate that transforms, draw the bacterium liquid of 0.5ml and receive 25ml LB, place 37 ℃ of shaking tables to be cultured to OD
600Be 0.3-0.4, perhaps 0.4-0.6.Centrifugal collection thalline, with isopyknic LB substratum washed twice, centrifugal collection thalline also is suspended in the 1ml LB substratum.As the DNA donor.
Take out-80 ℃, the spore suspension (3 * 10 of the Streptomyces antibioticus DSM40725 (Δ chlB1) that 20% glycerine is preserved
9Individual/mL), and 8,000rpm removed supernatant in centrifugal 3 minutes; Use 0.5ml TES buffer (0.05M, pH 8.0) washed twice then, 500 μ l TES buffer (0.05M; PH 8.0) resuspended, 50 ℃ of heat-shockeds added 500 μ l TSB to excite spore germination in 10 minutes then; Mixing, 37 ℃ of incubation 4-5hr, centrifugal collection spore also is suspended among the 1.5ml LB broth.As recipient bacterium.
Get 100 μ l recipient bacteriums and 100 μ l donor bacterium and mix, be coated in (the MgCl that contains 10mM on two AS-1 or the MS flat board then
2), the spore that will handle equally in addition is coated in (the MgCl that contains 10mM on two AS-1 or the MS flat board
2), respectively as positive control and negative control.The flat board of conjugal transfer is cultivated at 30 ℃ and was adopted sterilized water to wash planar surface gently to remove most intestinal bacteria in 16-20 hour later on, adds a cover 1ml ddH on each dull and stereotyped surface
2O (containing the Tsr final concentration is 50 μ g/ml, Nalidixic acid final concentration prestige 50 μ g/ml) cultivates picking zygote after 3-5 days for 30 ℃.
The zygote that obtains is inoculated among the liquid nutrient medium TSB (Tsr=50 μ g/ml) 30 ℃ of about 28hr of vibration.Take out 200 μ l and be coated on R
2YE (not containing sucrose) (Tsr=50 μ g/ml) cultivated 5 days for 30 ℃, received spore, was stored in-80 ℃.
2, the fermentation of the recombinant bacterial strain of the complementary Streptomyces antibioticus DSM40725 of ChlB1 (Δ chlB1) of KR sudden change
On MS (20ml, Tsr=50 μ g/ml) flat board, be coated with streptomycete spore 100-200 μ l (～109/ml), cultivated 10 days for 30 ℃; Drain cultured solid medium is freezing, add the 50ml anhydrous methanol after smashing to pieces, carrying out ultrasonic bacteria breaking 10 minutes (ultrasonic 10 seconds, 50 seconds at interval), whisking appliance stirred 1 hour; Filter (or centrifugal) and collect organic phase, substratum extracts once with the 50ml anhydrous methanol again, stirs 1 hour, collects and merges twice organic phase; Revolve to steam and remove methyl alcohol, obtain the Vandyke brown resistates, with the dissolving of 1ml anhydrous methanol, centrifugal back HPLC analyzes.
Analyze mutant strain and produced two brand-new peaks, LC-MS detects the analogue that two chemical combination are likely CHL, called after 7 and 8.
In a small amount during the fermentation mutant strain, HPLC and LC-MS detect possible two new compounds 7 and 8, in order to identify the structure of these two compounds; We have carried out bulk fermentation to mutant strain, and fermentation process is seen instance 2, the substratum that fermented is freezing drain after; Smash to pieces and add the equal-volume anhydrous methanol, carrying out ultrasonic bacteria breaking 10 minutes (ultrasonic 10 seconds, 50 seconds at interval); Whisking appliance stirred 1 hour, and filter (or centrifugal) and collect methyl alcohol; Revolve to steam and remove methyl alcohol, disperse with 1/5 water by volume, accent pH is to 2.0-3.0, and with equal-volume ethyl acetate extraction three times, decompressing and extracting obtains the Vandyke brown paste.The paste the first step is carried out rough segmentation earlier, and with the thick silica gel mixed sample paste of 100-200 order, oil pump is drained, the normal phase column of last 300-400 order silica gel prepackage, and condition of gradient elution is:
The eluent proportioning
ETHYLE ACETATE: sherwood oil
80％/20％
60％/40％
40％/60％
20％/80％
Pure ethyl acetate
100％
Methylene dichloride: methyl alcohol
95％/5％
90％/10％
Pure acetone
100％
Find that two new compounds appear at 40% ETHYLE ACETATE/60% sherwood oil; In the wash-out part of 20% ETHYLE ACETATE/80% sherwood oil and 100% ETHYLE ACETATE, be dissolved in 3ml methyl alcohol to the elutriant decompressing and extracting of above-mentioned three parts, the high pressure reversed-phase column of last C18 material, elution order is:
Water: methyl alcohol proportioning
95％/5％
90％/10％
85％/15％
80％/20％
60％/40％
50％/50％
40％/60％
100％
Two new compounds appear at 80% water/20% methyl alcohol, and 60% water/these two portions of 40% methyl alcohol is collected this two portions; Decompressing and extracting, easily in 3ml methyl alcohol, HPLC half preparation then; By the elution requirement of above-mentioned HPLC, collect two new peaks, the sample that has prepared HPLC at last is dissolved in 3ml methyl alcohol; Last appearance superdex gel column, the methanol-eluted fractions with 100% finally obtains pure two new compounds 7 and 8.The ESI-MS/MS collection of illustrative plates of new compound 7,8 and CHL shows that 7 the sodium molecule quasi-molecular ions that adds is 959.17; 8 the sodium molecule quasi-molecular ions that adds is 993.05; The sodium molecule quasi-molecular ions that adds of CHL is 977.36.7,8 with CHL all contain the characteristic fragment peak: 535,665,777; 7 have fragment peak 447; 8 have fragment peak 481; CHL has fragment peak 465; The structure that new compound 7 and 8 are described is to have increased individual Sauerstoffatom at 6-cresotinic acid acid unit.The new compound oil pump is drained and is dissolved in 500 μ l deuterated methanols or the deuterochloroform, carries out
1H-NMR,
13C-NMR, HMBC, HSQC, CONSY, NOESY data gathering, analysis new compound 7,8 and CHL's
1The H-NMR collection of illustrative plates shows that two unimodal chemical shifts are respectively 6.24and 6.29 in 7 structures, explains to contain two fragrant hydrogen; A unimodal chemical shift is 6.53 in 8 structures, explains to contain a fragrant hydrogen; 6.76, one Bimodalization displacement studies 7.37 of a unimodal chemical shift (coupling constant is 8.9 hertz) in the CHL structure are explained and are contained three fragrant hydrogen.Three results compare and draw 7 and 8 structure and the difference of CHL is to have increased individual Sauerstoffatom on the 6-MSA unit phenyl ring.
13The C-NMR collection of illustrative plates.Collection of illustrative plates shows that 7 have identical carbon skeleton with CHL.7 H-H COSY (Figure 10), HSQC (Figure 11), HMBC (Figure 12), and ROESY (Figure 13), show 7 structure be the structure of deschloro-CHL and in the 6-MSA unit contraposition of phenyl ring has increased Sauerstoffatom.The unitary HMBC of 8 OSA (Figure 14) collection of illustrative plates, the structure that shows compound 8 be the structure of CHL and in the 6-MSA unit contraposition of phenyl ring has increased Sauerstoffatom, in sum, confirmed the structure of compound.7 and 8 compounds have been carried out the mensuration of anti-microbial activity, about the activity of finding new compound 7 and 8 resisting gram-positive bacteria Bacillussubtilis doubles than the activity of deschloro-CHL and CHL respectively.7 and 8 hydroxyls in the unitary contraposition introducing of 6-MSA are described, have been increased the water-soluble of compound, improved its biological activity.
Following gene that content provides according to the present invention and protein sequence:
Amino acid/nucleotides sequence tabulation:
SEQUENCE LISTING
< 110>Shanghai organic chemistry institute
< 120>genetic engineering modified 6-cresotinic acid acid enzyme and combinatorial biosynthesis of spirocyclic etheric acid lactone microbiotic
< 130>specification sheets, claims
<160>2
<170>PatentIn version 3.3
<210>1
<211>5271
<212>DNA
<213>Streptomyces antibioticus DSM40725
<220>
<221>CDS
<222>(1)..(5271)
<400>1
gtg cag agt cac gac gtt gcc cgt gcg ggc ggc agg gaa gtc gtc gag 48
Val Gln Ser His Asp Val Ala Arg Ala Gly Gly Arg Glu Val Val Glu
1 5 10 15
gag ccg atc gcc gtg ctc ggg atg gcg tgc cgg ttc gca ggt ggt gcc 96
Glu Pro Ile Ala Val Leu Gly Met Ala Cys Arg Phe Ala Gly Gly Ala
20 25 30
gac acc ctg gag gcg ttc tgg gag ttg ctg ctg gag ggc cgg gac ggc 144
Asp Thr Leu Glu Ala Phe Trp Glu Leu Leu Leu Glu Gly Arg Asp Gly
35 40 45
atc ggt gag gtg cct gag aag cgg tgg cgc gcc tac gag gag gcc ggc 192
Ile Gly Glu Val Pro Glu Lys Arg Trp Arg Ala Tyr Glu Glu Ala Gly
50 55 60
ccc gat cat gcg gcg gcg gtg cgg agg gcg acg cgg tgg ggt ggg ttc 240
Pro Asp His Ala Ala Ala Val Arg Arg Ala Thr Arg Trp Gly Gly Phe
65 70 75 80
ctc gat gac atc gag ggg ttc gac gcg gag ttc ttc ggg ttg tcg ccg 288
Leu Asp Asp Ile Glu Gly Phe Asp Ala Glu Phe Phe Gly Leu Ser Pro
85 90 95
cgt gag gcg gag ttg atg gat ccg cag cag cgg ttg ctg ctg gag gtg 336
Arg Glu Ala Glu Leu Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Val
100 105 110
gcg tgg gag gcg ttg gag cac gcg ggt att gcg ccg cgg gag ttg gcg 384
Ala Trp Glu Ala Leu Glu His Ala Gly Ile Ala Pro Arg Glu Leu Ala
115 120 125
ggg acg gac gcg ggt gtg ttc gtg ggg atc ggt tcg gat gat tac ggc 432
Gly Thr Asp Ala Gly Val Phe Val Gly Ile Gly Ser Asp Asp Tyr Gly
130 135 140
cgg cgg ttg ttg gag gat ctg ccg ggg atc gag gcg tgg acg ggg atc 480
Arg Arg Leu Leu Glu Asp Leu Pro Gly Ile Glu Ala Trp Thr Gly Ile
145 150 155 160
ggc agt gcg atg tgt gcg gcg gcg aac cgg atc tcg tat gcg ctg gat 528
Gly Ser Ala Met Cys Ala Ala Ala Asn Arg Ile Ser Tyr Ala Leu Asp
165 170 175
ctg aag ggg ccg agt ctg gcg gtg gac acg gcg tgt tcg gcg tcg ttg 576
Leu Lys Gly Pro Ser Leu Ala Val Asp Thr Ala Cys Ser Ala Ser Leu
180 185 190
gtg gcg gtg cat ctg gcg tgt cag agt ctg cgg gcg ggt gag agt gag 624
Val Ala Val His Leu Ala Cys Gln Ser Leu Arg Ala Gly Glu Ser Glu
195 200 205
gtg tcg ctc gcg gcg ggt gtg aat ctg atg atc tca ccg ggg ttg acg 672
Val Ser Leu Ala Ala Gly Val Asn Leu Met Ile Ser Pro Gly Leu Thr
210 215 220
ctg acg ctg gat gcg gcg ggt gcg acg gcg ccg gac ggg cgg tcg aag 720
Leu Thr Leu Asp Ala Ala Gly Ala Thr Ala Pro Asp Gly Arg Ser Lys
225 230 235 240
tcc ttc gat gcc tcc gcg gac ggt tat ggc cgg ggc gag ggg tgt ggg 768
Ser Phe Asp Ala Ser Ala Asp Gly Tyr Gly Arg Gly Glu Gly Cys Gly
245 250 255
ctg ctc gtg ctg aag cgg ttg tcg gac gcg gtg cgg gac ggg gat ccg 816
Leu Leu Val Leu Lys Arg Leu Ser Asp Ala Val Arg Asp Gly Asp Pro
260 265 270
gtg ctg gcg gtg atc cgg ggc agt tcg gtg aac cag gac ggg aag acg 864
Val Leu Ala Val Ile Arg Gly Ser Ser Val Asn Gln Asp Gly Lys Thr
275 280 285
aac ggg atc atg gcg ccg agt ggt tcg gcg cag gag cat gtg ctg gat 912
Asn Gly Ile Met Ala Pro Ser Gly Ser Ala Gln Glu His Val Leu Asp
290 295 300
ctg gcg tgc cgg cgg gcg ggg gtg gat ccg gcg tcg gtg gat tac gtc 960
Leu Ala Cys Arg Arg Ala Gly Val Asp Pro Ala Ser Val Asp Tyr Val
305 310 315 320
gag gcg cat ggc acg ggg acg cgg ctt gga gac ccg ttg gaa gcg ggt 1008
Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro Leu Glu Ala Gly
325 330 335
gcg ctg agc gcg gtg ttc ggg cgg ggg cgg ccc aag gat gag ccg tgt 1056
Ala Leu Ser Ala Val Phe Gly Arg Gly Arg Pro Lys Asp Glu Pro Cys
340 345 350
ctg atc ggt tcg gtg aag tcg aac atc ggg cat ctg gag gcg gcg gcg 1104
Leu Ile Gly Ser Val Lys Ser Asn Ile Gly His Leu Glu Ala Ala Ala
355 360 365
ggg att gcg agc ctg atc aag gcg acg ctg gcg ttg agc aag gga gag 1152
Gly Ile Ala Ser Leu Ile Lys Ala Thr Leu Ala Leu Ser Lys Gly Glu
370 375 380
atc ccg ccg agt ctg aac ttc tcg cag ggc aat ccg gcg atc gac tgg 1200
Ile Pro Pro Ser Leu Asn Phe Ser Gln Gly Asn Pro Ala Ile Asp Trp
385 390 395 400
gcg gag tcc ggg ctg cgg gtg gtg acc gag cgg acg gcc tgg ccc gag 1248
Ala Glu Ser Gly Leu Arg Val Val Thr Glu Arg Thr Ala Trp Pro Glu
405 410 415
cgg gag gac cga ccg gtc cgt gcg ggc gtt tcc ggc ttc ggc tat ggc 1296
Arg Glu Asp Arg Pro Val Arg Ala Gly Val Ser Gly Phe Gly Tyr Gly
420 425 430
ggc acc atc gcg cat gtg gtc atg gag cag gcg cct gag gtg agt cgg 1344
Gly Thr Ile Ala His Val Val Met Glu Gln Ala Pro Glu Val Ser Arg
435 440 445
ccc gat gac gcg gcg ggt gat gag ggg tct gcc gag gtc gtg acg gag 1392
Pro Asp Asp Ala Ala Gly Asp Glu Gly Ser Ala Glu Val Val Thr Glu
450 455 460
cgg ctg ttc ccg ctc tcg ggt gga acg cag gcc gga ctc cgg gcg tat 1440
Arg Leu Phe Pro Leu Ser Gly Gly Thr Gln Ala Gly Leu Arg Ala Tyr
465 470 475 480
gcg gga cgc ctc gcg gac cgg ctg tcg gac gac gac gcc gag gaa ctg 1488
Ala Gly Arg Leu Ala Asp Arg Leu Ser Asp Asp Asp Ala Glu Glu Leu
485 490 495
ccc ctg gag tcg gtc ggg cac acc ctg gcc ttg cgc agg tcg gcg ctg 1536
Pro Leu Glu Ser Val Gly His Thr Leu Ala Leu Arg Arg Ser Ala Leu
500 505 510
gcg cac cgg gcc gcc gtc gtg gcc tcg gac cgc aag gac ctg gtg gcc 1584
Ala His Arg Ala Ala Val Val Ala Ser Asp Arg Lys Asp Leu Val Ala
515 520 525
aag ctg cgg ttg atc acg ctg ggg gag cag acc cgg gaa gcc gtg atc 1632
Lys Leu Arg Leu Ile Thr Leu Gly Glu Gln Thr Arg Glu Ala Val Ile
530 535 540
ggg tcg gta ccc tcc gat gcc ggt gcg ggg ccg gtg tgg gtg ttc tcc 1680
Gly Ser Val Pro Ser Asp Ala Gly Ala Gly Pro Val Trp Val Phe Ser
545 550 555 560
ggg cat ggt tcg cag tgg tcg ggg atg ggg cgt gaa ctg ctg gcg tcc 1728
Gly His Gly Ser Gln Trp Ser Gly Met Gly Arg Glu Leu Leu Ala Ser
565 570 575
gag ccc gcg ttc gca gcg gtg atc gac gag atc gat ccc gtt ttc cgt 1776
Glu Pro Ala Phe Ala Ala Val Ile Asp Glu Ile Asp Pro Val Phe Arg
580 585 590
gcg gag atc ggg ttc tcg gcc cgg cag gct ctg ctc gac ggt gac ttc 1824
Ala Glu Ile Gly Phe Ser Ala Arg Gln Ala Leu Leu Asp Gly Asp Phe
595 600 605
gac acc gtc gac cgt gtt cag acg atg att ttc gcg gtg cag gtc gcg 1872
Asp Thr Val Asp Arg Val Gln Thr Met Ile Phe Ala Val Gln Val Ala
610 615 620
ctg gcg gcg gtc tgg cac tct tat ggt gcc gcc ccg tcg gcg gtg atc 1920
Leu Ala Ala Val Trp His Ser Tyr Gly Ala Ala Pro Ser Ala Val Ile
625 630 635 640
ggg cac tcc gtg ggg gag atc gcg gcg gct gtg gcg gcg ggt gcg ctg 1968
Gly His Ser Val Gly Glu Ile Ala Ala Ala Val Ala Ala Gly Ala Leu
645 650 655
tcg ctg acg gac gga gcg cgg ctg atc tgc cgc cgc tcc cga ctc ttg 2016
Ser Leu Thr Asp Gly Ala Arg Leu Ile Cys Arg Arg Ser Arg Leu Leu
660 665 670
cgg cgg gtg gcc ggc cag gga gcg atg gct atg gcg agc atc tcc ttc 2064
Arg Arg Val Ala Gly Gln Gly Ala Met Ala Met Ala SerIle Ser Phe
675 680 685
gag gag gcg gcc gag cgg ctg gcg ggc cgt acg gat gtg gtg ccg gcg 2112
Glu Glu Ala Ala Glu Arg Leu Ala Gly Arg Thr Asp Val Val Pro Ala
690 695 700
att gcc gcg tcc ccg ctc tcc gcg gtc gtg gca ggt gac cct gca gcg 2160
Ile Ala Ala Ser Pro Leu Ser Ala Val Val Ala Gly Asp Pro Ala Ala
705 710 715 720
atc aac gcg ctg atc gac gag tgg cag gca cag gac atc cag atg cgc 2208
Ile Asn Ala Leu Ile Asp Glu Trp Gln Ala Gln Asp Ile Gln Met Arg
725 730 735
cgg gtc gcc tcg gac gtg gcc ttc cac agc ccg cac atg gac ccg ctg 2256
Arg Val Ala Ser Asp Val Ala Phe His Ser Pro His Met Asp Pro Leu
740 745 750
ctc acc gaa atc gcg gcc gct gcc gag gac ttg acg ccg cgc cag ccc 2304
Leu Thr Glu Ile Ala Ala Ala Ala Glu Asp Leu Thr Pro Arg Gln Pro
755 760 765
gaa ctc ccg gtg tac tcc acg gcc atg gag gac ccc cgc tcc cag gcg 2352
Glu Leu Pro Val Tyr Ser Thr Ala Met Glu Asp Pro Arg Ser Gln Ala
770 775 780
acc ctc gac ggc tcc tac tgg gcc gcc aac ctg cgt aac ccg gtg cgg 2400
Thr Leu Asp Gly Ser Tyr Trp Ala Ala Asn Leu Arg Asn Pro Val Arg
785 790 795 800
ttg cag ccg gcg gtg acg gcg gcg gtc gag gac ggc cac cgc gcg ttc 2448
Leu Gln Pro Ala Val Thr Ala Ala Val Glu Asp Gly His Arg Ala Phe
805 810 815
atc gaa gtg tcc gcg cat ccc gtg gtc acg cac tcc atc ggc gag acg 2496
Ile Glu Val Ser Ala His Pro Val Val Thr His Ser Ile Gly Glu Thr
820 825 830
ctc tcc gag ctc ggc cag gag gac gcc ttc acc ggc tcc tcc ctg cgc 2544
Leu Ser Glu Leu Gly Gln Glu Asp Ala Phe Thr Gly Ser Ser Leu Arg
835 840 845
cgc aac cag ccc gaa cgc gcc acc ctc ctg tcc gcc gtc ggc gcg gcg 2592
Arg Asn Gln Pro Glu Arg Ala Thr Leu Leu Ser Ala Val Gly Ala Ala
850 855 860
cac tgc cat ggc atc gcg gtg gac tgg gcg cgt ctg cac ccg acc ggt 2640
His Cys His Gly Ile Ala Val Asp Trp Ala Arg Leu His Pro Thr Gly
865 870 875 880
gac ctg gtc gcc ctg ccg ctg gtg gcc tgg cag cgc agc ccg cac tgg 2688
Asp Leu Val Ala Leu Pro Leu Val Ala Trp Gln Arg Ser Pro His Trp
885 890 895
cac gag cgg gcc tcc gcc gcc acc ggc cag ggc ttg cag cac gac ctt 2736
His Glu Arg Ala Ser Ala Ala Thr Gly Gln Gly Leu Gln His Asp Leu
900 905 910
gac tcc cac gcg ctg ctc ggg ccg cgc gtc ccg gtc gcg gga cgg ccg 2784
Asp Ser His Ala Leu Leu Gly Pro Arg Val Pro Val Ala Gly Arg Pro
915 920 925
ctg gaa ctg tgg cgc aca ctg ctc gac gac gag acg cgc ccc tac ccc 2832
Leu Glu Leu Trp Arg Thr Leu Leu Asp Asp Glu Thr Arg Pro Tyr Pro
930 935 940
ggc agc gcc acc atc aac ggc acg gag atc gtg ccc gcc gcc gtc ctg 2880
Gly Ser Ala ThrIle Asn Gly Thr Glu Ile Val Pro Ala Ala Val Leu
945 950 955 960
atc aac acg ttc ctc gac gcg gca cgc gcc gcc gac ggg gcc cgc ccg 2928
Ile Asn Thr Phe Leu Asp Ala Ala Arg Ala Ala Asp Gly Ala Arg Pro
965 970 975
gtc ctg cgg gac atg gcg ctg cgg ctg ccg ctg atc acc acc gag cgg 2976
Val Leu Arg Asp Met Ala Leu Arg Leu Pro Leu Ile Thr Thr Glu Arg
980 985 990
cgc gaa ctc cag gtc gtc agg gac gac aac tcc ttg cgt ctg gcc tcg 3024
Arg Glu Leu Gln Val Val Arg Asp Asp Asn Ser Leu Arg Leu Ala Ser
995 1000 1005
cgt tca ctg gag gac ggt gcc gcg tgg ctg acc cac acc acc gcc 3069
Arg Ser Leu Glu Asp Gly Ala Ala Trp Leu Thr His Thr Thr Ala
1010 1015 1020
acc gcc gca ccg gcg ggc agc ggc gaa gcg ctc cag gac ctg gcc 3114
Thr Ala Ala Pro Ala Gly Ser Gly Glu Ala Leu Gln Asp Leu Ala
1025 1030 1035
gcc ggt gcc gtg ttg cgc ccg gcg gac ccg ggt gat gtg cag cgc 3159
Ala Gly Ala Val Leu Arg Pro Ala Asp Pro Gly Asp Val Gln Arg
1040 1045 1050
cac ctg acc tcg gtg ggc gtg ccg acc atg gga ttt gag tgg acc 3204
His Leu Thr Ser Val Gly Val Pro Thr Met Gly Phe Glu Trp Thr
1055 1060 1065
atc gag gaa ctc gcc cgg agc gag ggc atg ttg gcc gca cgt gtg 3249
Ile Glu Glu Leu Ala Arg Ser Glu Gly Met Leu Ala Ala Arg Val
1070 1075 1080
agt gtc gag cgg ccg cag cgg gcc cag gag acg tgg gcg ccc ttg 3294
Ser Val Glu Arg Pro Gln Arg Ala Gln Glu Thr Trp Ala Pro Leu
1085 1090 1095
ctg gac gcc gcg ctg tcc atc gcg ccg acg gcc atc ccc ggc ccg 3339
Leu Asp Ala Ala Leu Ser Ile Ala Pro Thr Ala Ile Pro Gly Pro
1100 1105 1110
ccg gcc ctg cgc atg gtg gcc tcc ttc gag gag atc gtc acc gaa 3384
Pro Ala Leu Arg Met Val Ala Ser Phe Glu Glu Ile Val Thr Glu
1115 1120 1125
ggc gcc ccg ccg gcc ggt ccg gcg acc atc cag gtc gcg gcc gac 3429
Gly Ala Pro Pro Ala Gly Pro Ala Thr Ile Gln Val Ala Ala Asp
1130 1135 1140
ccg gtc cac gag aac acc gtc gac gtc cgg atc gcc gac acc gac 3474
Pro Val His Glu Asn Thr Val Asp Val Arg Ile Ala Asp Thr Asp
1145 1150 1155
ggg cag gcc gtg gcg tgg gtg cgc ggc ctg cgc tac gac ggc atg 3519
Gly Gln Ala Val Ala Trp Val Arg Gly Leu Arg Tyr Asp Gly Met
1160 1165 1170
gac cag ggc ggc atg acg gcg gcg cac ccc cgc gac ctg gtc ttc 3564
Asp Gln Gly Gly Met Thr Ala Ala His Pro Arg Asp Leu Val Phe
1175 1180 1185
gag atg gcc tgg cgg ccc ttc gag gcc ccc gcg ccg cag gac gtg 3609
Glu Met Ala Trp Arg Pro Phe Glu Ala Pro Ala Pro Gln Asp Val
1190 1195 1200
tcc gcc cgc cgg atc gtc ctg atc gcc gca cac gac gtg aag ccc 3654
Ser Ala Arg Arg Ile Val Leu Ile Ala Ala His Asp Val Lys Pro
1205 1210 1215
ctg cgc acg gcc ctc acc cgt gcc ggc gct cac gtc gac gtc ggg 3699
Leu Arg Thr Ala Leu Thr Arg Ala Gly Ala His Val Asp Val Gly
1220 1225 1230
ctg gac ggc acg cte gac gag aac acc gac gtc gtc gtg gtg ccc 3744
Leu Asp Gly Thr Leu Asp Glu Asn Thr Asp Val Val Val Val Pro
1235 1240 1245
gac ctc acc gcg gac atc ccc gtc ccc gag gcc gca gcc cgt tcc 3789
Asp Leu Thr Ala Asp Ile Pro Val Pro Glu Ala Ala Ala Arg Ser
1250 1255 1260
gca tgg ctg ctg ctg agc acc gcg cag cgc atc gcc gcc ctg gac 3834
Ala Trp Leu Leu Leu Ser Thr Ala Gln Arg Ile Ala Ala Leu Asp
1265 1270 1275
acc ctg cgc ttc ccc cgc ctg tgg tgc ctg acc acc gca gtc cgt 3879
Thr Leu Arg Phe Pro Arg Leu Trp Cys Leu Thr Thr Ala Val Arg
1280 1285 1290
gaa agc cag gcc gaa acc cac ctc gcg cag tcc acc ctg tgg ggc 3924
Glu Ser Gln Ala Glu Thr His Leu Ala Gln Ser Thr Leu Trp Gly
1295 1300 1305
ctg ggc cgg gtg atc gcg ggc gag cac agc gaa ctg tgg ggc ggc 3969
Leu Gly Arg Val Ile Ala Gly Glu His Ser Glu Leu Trp Gly Gly
1310 1315 1320
gtc atc gac ctg gcc ccc ggc acc ccg gac gcc acc acc ctg ctc 4014
Val Ile Asp Leu Ala Pro Gly Thr Pro Asp Ala Thr Thr Leu Leu
1325 1330 1335
agc gtc ctg cac acc ggc ggc ggc gag gac gtc atc gcc ctc cgc 4059
Ser Val Leu His Thr Gly Gly Gly Glu Asp Val Ile Ala Leu Arg
1340 1345 1350
gac ggc acc gcc acc acg gcc cgc ctc acc acg acg caa cgc gag 4104
Asp Gly Thr Ala Thr Thr Ala Arg Leu Thr Thr Thr Gln Arg Glu
1355 1360 1365
ccc act ggc acc ccg ctg gaa tgc cgg gcg gac gga acg tac ctg 4149
Pro Thr Gly Thr Pro Leu Glu Cys Arg Ala Asp Gly Thr Tyr Leu
1370 1375 1380
atc acc ggc gga ctg ggc acc ctc ggc ctg gaa gtc gcc ggc cgg 4194
Ile Thr Gly Gly Leu Gly Thr Leu Gly Leu Glu Val Ala Gly Arg
1385 1390 1395
ctc gcc gaa cgc ggc gcc cgc cgt ctc gtc ctc gcc gga cgc acc 4239
Leu Ala Glu Arg Gly Ala Arg Arg Leu Val Leu Ala Gly Arg Thr
1400 1405 1410
gga ctg cca ccc cgc tcc acc tgg ggc gag acc acc gac acg cac 4284
Gly Leu Pro Pro Arg Ser Thr Trp Gly Glu Thr Thr Asp Thr His
1415 1420 1425
acc agg cag cgc atc gag gcc gtc aag gcc ctc gaa gac cag ggc 4329
Thr Arg Gln Arg Ile Glu Ala Val Lys Ala Leu Glu Asp Gln Gly
1430 1435 1440
gtc acc gtc cgt gtc atc ccc ctc gac atc acc gac acg gcc aag 4374
Val Thr Val Arg Val Ile Pro Leu Asp Ile Thr Asp Thr Ala Lys
1445 1450 1455
gcc gcc gaa cag ctc acc ccc gac gcc ctg ggc ctg cca ccc atc 4419
Ala Ala Glu Gln Leu Thr Pro Asp Ala Leu Gly Leu Pro Pro Ile
1460 1465 1470
cgc ggc atc gtc cac ctc gcc ggc gtc ctc gac aac cgc atg gtg 4464
Arg Gly Ile Val His Leu Ala Gly Val Leu Asp Asn Arg Met Val
1475 1480 1485
acc gcg gtc gac gag aca tcc ctg cgc acc gtg ctg cgg ccc aag 4509
Thr Ala Val Asp Glu Thr Ser Leu Arg Thr Val Leu Arg Pro Lys
1490 1495 1500
gcc gac ggc gcc tgg acc ctg cac acc ctc ttc ccg ccc ggc acc 4554
Ala Asp Gly Ala Trp Thr Leu His Thr Leu Phe Pro Pro Gly Thr
1505 1510 1515
atc gac ttc ctg atc ctg ttc tcc tcc tgc ggc cag ctc ctc ggc 4599
Ile Asp Phe Leu Ile Leu Phe Ser Ser Cys Gly Gln Leu Leu Gly
1520 1525 1530
ctg ccc ggc cag gcc gcc tac ggc tcc gcc aac gcc ttc ctc gac 4644
Leu Pro Gly Gln Ala Ala Tyr Gly Ser Ala Asn Ala Phe Leu Asp
1535 1540 1545
gcc ctc gcc gtc cac cgc aac acc acc acc ccg acc gcc gcc gac 4689
Ala Leu Ala Val His Arg Asn Thr Thr Thr Pro Thr Ala Ala Asp
1550 1555 1560
acc acc agc ttc ggc tgg acc tcc tgg cgc ggc cag ggc atg gcc 4734
Thr Thr Ser Phe Gly Trp Thr Ser Trp Arg Gly Gln Gly Met Ala
1565 1570 1575
gtc aac gac gtc gtc gac gcc gaa ctg cgc gcc cga ggc gtc acc 4779
Val Asn Asp Val Val Asp Ala Glu Leu Arg Ala Arg Gly Val Thr
1580 1585 1590
gac atc acc acc cag gaa gcc ttc gcc gcc tgg gac ttc gcc gca 4824
Asp Ile Thr Thr Gln Glu Ala Phe Ala Ala Trp Asp Phe Ala Ala
1595 1600 1605
caa cac ggc ccc gga aac tac ccc gtc cta cgc cgg ctg ccc cac 4869
Gln His Gly Pro Gly Asn Tyr Pro Val Leu Arg Arg Leu Pro His
1610 1615 1620
gag ccg gac atg gac cag ctc ccc ctc ctc agc gag atc cac cac 4914
Glu Pro Asp Met Asp Gln Leu Pro Leu Leu Ser Glu Ile His His
1625 1630 1635
acc cag ccc acc gcc ccc acc tcc ggc gcc gca acc gac tcc tac 4959
Thr Gln Pro Thr Ala Pro Thr Ser Gly Ala Ala Thr Asp Ser Tyr
1640 1645 1650
gcg ggc ctc gcc ccc gac gaa ctg cgc gcc cgc ctc atc gac gag 5004
Ala Gly Leu Ala Pro Asp Glu Leu Arg Ala Arg Leu Ile Asp Glu
1655 1660 1665
gtc gcc gca cac atc tcg gcc gag atg aaa ctc gcc gcc tcc cag 5049
Val Ala Ala His Ile Ser Ala Glu Met Lys Leu Ala Ala Ser Gln
1670 1675 1680
ctc gac cac cgc aag tcc ctg gtc gag cag ggc ctg gac tcg gtg 5094
Leu Asp His Arg Lys Ser Leu Val Glu Gln Gly Leu Asp Ser Val
1685 1690 1695
atg acg atc gtg atc cgg cgc cgc ctg gag aag tgg ttc ggt cac 5139
Met Thr Ile Val Ile Arg Arg Arg Leu Glu Lys Trp Phe Gly His
1700 1705 1710
aaa ctc ccc gcg acc ctg ctg tgg cac cag ccc acc gtc acc gcc 5184
Lys Leu Pro Ala Thr Leu Leu Trp His Gln Pro Thr Val Thr Ala
1715 1720 1725
atc agc gaa cac ctg gcc gaa ctc ctg gcc ccc acc acg tcc cag 5229
Ile Ser Glu His Leu Ala Glu Leu Leu Ala Pro Thr Thr Ser Gln
1730 1735 1740
ccc gac aac acg gca ccc gcc gaa ccg gcg gca acg gcc tga 5271
Pro Asp Asn Thr Ala Pro Ala Glu Pro Ala Ala Thr Ala
1745 1750 1755
<210>2
<211>1756
<212>PRT
<213>Streptomyces antibioticus DSM40725
<400>2
Val Gln Ser His Asp Val Ala Arg Ala Gly Gly Arg Glu Val Val Glu
1 5 10 15
Glu Pro Ile Ala Val Leu Gly Met Ala Cys Arg Phe Ala Gly Gly Ala
20 25 30
Asp Thr Leu Glu Ala Phe Trp Glu Leu Leu Leu Glu Gly Arg Asp Gly
35 40 45
Ile Gly Glu Val Pro Glu Lys Arg Trp Arg Ala Tyr Glu Glu Ala Gly
50 55 60
Pro Asp His Ala Ala Ala Val Arg Arg Ala Thr Arg Trp Gly Gly Phe
65 70 75 80
Leu Asp Asp Ile Glu Gly Phe Asp Ala Glu Phe Phe Gly Leu Ser Pro
85 90 95
Arg Glu Ala Glu Leu Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Val
100 105 110
Ala Trp Glu Ala Leu Glu His Ala Gly Ile Ala Pro Arg Glu Leu Ala
115 120 125
Gly Thr Asp Ala Gly Val Phe Val Gly Ile Gly Ser Asp Asp Tyr Gly
130 135 140
Arg Arg Leu Leu Glu Asp Leu Pro Gly Ile Glu Ala Trp Thr Gly Ile
145 150 155 160
Gly Ser Ala Met Cys Ala Ala Ala Asn Arg Ile Ser Tyr Ala Leu Asp
165 170 175
Leu Lys Gly Pro Ser Leu Ala Val Asp Thr Ala Cys Ser Ala Ser Leu
180 185 190
Val Ala Val His Leu Ala Cys Gln Ser Leu Arg Ala Gly Glu Ser Glu
195 200 205
Val Ser Leu Ala Ala Gly Val Asn Leu Met Ile Ser Pro Gly Leu Thr
210 215 220
Leu Thr Leu Asp Ala Ala Gly Ala Thr Ala Pro Asp Gly Arg Ser Lys
225 230 235 240
Ser Phe Asp Ala Ser Ala Asp Gly Tyr Gly Arg Gly Glu Gly Cys Gly
245 250 255
Leu Leu Val Leu Lys Arg Leu Ser Asp Ala Val Arg Asp Gly Asp Pro
260 265 270
Val Leu Ala Val Ile Arg Gly Ser Ser Val Asn Gln Asp Gly Lys Thr
275 280 285
Asn Gly Ile Met Ala Pro Ser Gly Ser Ala Gln Glu His Val Leu Asp
290 295 300
Leu Ala Cys Arg Arg Ala Gly Val Asp Pro Ala Ser Val Asp Tyr Val
305 310 315 320
Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro Leu Glu Ala Gly
325 330 335
Ala Leu Ser Ala Val Phe Gly Arg Gly Arg Pro Lys Asp Glu Pro Cys
340 345 350
Leu Ile Gly Ser Val Lys Ser Asn Ile Gly His Leu Glu Ala Ala Ala
355 360 365
Gly Ile Ala Ser Leu Ile Lys Ala Thr Leu Ala Leu Ser Lys Gly Glu
370 375 380
Ile Pro Pro Ser Leu Asn Phe Ser Gln Gly Asn Pro Ala Ile Asp Trp
385 390 395 400
Ala Glu Ser Gly Leu Arg Val Val Thr Glu Arg Thr Ala Trp Pro Glu
405 410 415
Arg Glu Asp Arg Pro Val Arg Ala Gly Val Ser Gly Phe Gly Tyr Gly
420 425 430
Gly Thr Ile Ala His Val Val Met Glu Gln Ala Pro Glu Val Ser Arg
435 440 445
Pro Asp Asp Ala Ala Gly Asp Glu Gly Ser Ala Glu Val Val Thr Glu
450 455 460
Arg Leu Phe Pro Leu Ser Gly Gly Thr Gln Ala Gly Leu Arg Ala Tyr
465 470 475 480
Ala Gly Arg Leu Ala Asp Arg Leu Ser Asp Asp Asp Ala Glu Glu Leu
485 490 495
Pro Leu Glu Ser Val Gly His Thr Leu Ala Leu Arg Arg Ser Ala Leu
500 505 510
Ala His Arg Ala Ala Val Val Ala Ser Asp Arg Lys Asp Leu Val Ala
515 520 525
Lys Leu Arg Leu Ile Thr Leu Gly Glu Gln Thr Arg Glu Ala Val Ile
530 535 540
Gly Ser Val Pro Ser Asp Ala Gly Ala Gly Pro Val Trp Val Phe Ser
545 550 555 560
Gly His Gly Ser Gln Trp Ser Gly Met Gly Arg Glu Leu Leu Ala Ser
565 570 575
Glu Pro Ala Phe Ala Ala Val Ile Asp Glu Ile Asp Pro Val Phe Arg
580 585 590
Ala Glu Ile Gly Phe Ser Ala Arg Gln Ala Leu Leu Asp Gly Asp Phe
595 600 605
Asp Thr Val Asp Arg Val Gln Thr Met Ile Phe Ala Val Gln Val Ala
610 615 620
Leu Ala Ala Val Trp His Ser Tyr Gly Ala Ala Pro Ser Ala Val Ile
625 630 635 640
Gly His Ser Val Gly Glu Ile Ala Ala Ala Val Ala Ala Gly Ala Leu
645 650 655
Ser Leu Thr Asp Gly Ala Arg Leu Ile Cys Arg Arg Ser Arg Leu Leu
660 665 670
Arg Arg Val Ala Gly Gln Gly Ala Met Ala Met Ala Ser Ile Ser Phe
675 680 685
Glu Glu Ala Ala Glu Arg Leu Ala Gly Arg Thr Asp Val Val Pro Ala
690 695 700
Ile Ala Ala Ser Pro Leu Ser Ala Val Val Ala Gly Asp Pro Ala Ala
705 710 715 720
Ile Asn Ala Leu Ile Asp Glu Trp Gln Ala Gln Asp Ile Gln Met Arg
725 730 735
Arg Val Ala Ser Asp Val Ala Phe His Ser Pro His Met Asp Pro Leu
740 745 750
Leu Thr Glu Ile Ala Ala Ala Ala Glu Asp Leu Thr Pro Arg Gln Pro
755 760 765
Glu Leu Pro Val Tyr Ser Thr Ala Met Glu Asp Pro Arg Ser Gln Ala
770 775 780
Thr Leu Asp Gly Ser Tyr Trp Ala Ala Asn Leu Arg Asn Pro Val Arg
785 790 795 800
Leu Gln Pro Ala Val Thr Ala Ala Val Glu Asp Gly His Arg Ala Phe
805 810 815
Ile Glu Val Ser Ala His Pro Val Val Thr His Ser Ile Gly Glu Thr
820 825 830
Leu Ser Glu Leu Gly Gln Glu Asp Ala Phe Thr Gly Ser Ser Leu Arg
835 840 845
Arg Asn Gln Pro Glu Arg Ala Thr Leu Leu Ser Ala Val Gly Ala Ala
850 855 860
His Cys His Gly Ile Ala Val Asp Trp Ala Arg Leu His Pro Thr Gly
865 870 875 880
Asp Leu Val Ala Leu Pro Leu Val Ala Trp Gln Arg Ser Pro His Trp
885 890 895
His Glu Arg Ala Ser Ala Ala Thr Gly Gln Gly Leu Gln His Asp Leu
900 905 910
Asp Ser His Ala Leu Leu Gly Pro Arg Val Pro Val Ala Gly Arg Pro
915 920 925
Leu Glu Leu Trp Arg Thr Leu Leu Asp Asp Glu Thr Arg Pro Tyr Pro
930 935 940
Gly Ser Ala Thr Ile Asn Gly Thr Glu Ile Val Pro Ala Ala Val Leu
945 950 955 960
Ile Asn Thr Phe Leu Asp Ala Ala Arg Ala Ala Asp Gly Ala Arg Pro
965 970 975
Val Leu Arg Asp Met Ala Leu Arg Leu Pro Leu Ile Thr Thr Glu Arg
980 985 990
Arg Glu Leu Gln Val Val Arg Asp Asp Asn Ser Leu Arg Leu Ala Ser
995 1000 1005
Arg Ser Leu Glu Asp Gly Ala Ala Trp Leu Thr His Thr Thr Ala
1010 1015 1020
Thr Ala Ala Pro Ala Gly Ser Gly Glu Ala Leu Gln Asp Leu Ala
1025 1030 1035
Ala Gly Ala Val Leu Arg Pro Ala Asp Pro Gly Asp Val Gln Arg
1040 1045 1050
His Leu Thr Ser Val Gly Val Pro Thr Met Gly Phe Glu Trp Thr
1055 1060 1065
Ile Glu Glu Leu Ala Arg Ser Glu Gly Met Leu Ala Ala Arg Val
1070 1075 1080
Ser Val Glu Arg Pro Gln Arg Ala Gln Glu Thr Trp Ala Pro Leu
1085 1090 1095
Leu Asp Ala Ala Leu Ser Ile Ala Pro Thr Ala Ile Pro Gly Pro
1100 1105 1110
Pro Ala Leu Arg Met Val Ala Ser Phe Glu Glu Ile Val Thr Glu
1115 1120 1125
Gly Ala Pro Pro Ala Gly Pro Ala Thr Ile Gln Val Ala Ala Asp
1130 1135 1140
Pro Val His Glu Asn Thr Val Asp Val Arg Ile Ala Asp Thr Asp
1145 1150 1155
Gly Gln Ala Val Ala Trp Val Arg Gly Leu Arg Tyr Asp Gly Met
1160 1165 1170
Asp Gln Gly Gly Met Thr Ala Ala His Pro Arg Asp Leu Val Phe
1175 1180 1185
Glu Met Ala Trp Arg Pro Phe Glu Ala Pro Ala Pro Gln Asp Val
1190 1195 1200
Ser Ala Arg Arg Ile Val Leu Ile Ala Ala His Asp Val Lys Pro
1205 1210 1215
Leu Arg Thr Ala Leu Thr Arg Ala Gly Ala His Val Asp Val Gly
1220 1225 1230
Leu Asp Gly Thr Leu Asp Glu Asn Thr Asp Val Val Val Val Pro
1235 1240 1245
Asp Leu Thr Ala Asp Ile Pro Val Pro Glu Ala Ala Ala Arg Ser
1250 1255 1260
Ala Trp Leu Leu Leu Ser Thr Ala Gln Arg Ile Ala Ala Leu Asp
1265 1270 1275
Thr Leu Arg Phe Pro Arg Leu Trp Cys Leu Thr Thr Ala Val Arg
1280 1285 1290
Glu Ser Gln Ala Glu Thr His Leu Ala Gln Ser Thr Leu Trp Gly
1295 1300 1305
Leu Gly Arg Val Ile Ala Gly Glu His Ser Glu Leu Trp Gly Gly
1310 1315 1320
Val Ile Asp Leu Ala Pro Gly Thr Pro Asp Ala Thr Thr Leu Leu
1325 1330 1335
Ser Val Leu His Thr Gly Gly Gly Glu Asp Val Ile Ala Leu Arg
1340 1345 1350
Asp Gly Thr Ala Thr Thr Ala Arg Leu Thr Thr Thr Gln Arg Glu
1355 1360 1365
Pro Thr Gly Thr Pro Leu Glu Cys Arg Ala Asp Gly Thr Tyr Leu
1370 1375 1380
Ile Thr Gly Gly Leu Gly Thr Leu Gly Leu Glu Val Ala Gly Arg
1385 1390 1395
Leu Ala Glu Arg Gly Ala Arg Arg Leu Val Leu Ala Gly Arg Thr
1400 1405 1410
Gly Leu Pro Pro Arg Ser Thr Trp Gly Glu Thr Thr Asp Thr His
1415 1420 1425
Thr Arg Gln Arg Ile Glu Ala Val Lys Ala Leu Glu Asp Gln Gly
1430 1435 1440
Val Thr Val Arg Val Ile Pro Leu Asp Ile Thr Asp Thr Ala Lys
1445 1450 1455
Ala Ala Glu Gln Leu Thr Pro Asp Ala Leu Gly Leu Pro Pro Ile
1460 1465 1470
Arg Gly Ile Val His Leu Ala Gly Val Leu Asp Asn Arg Met Val
1475 1480 1485
Thr Ala Val Asp Glu Thr Ser Leu Arg Thr Val Leu Arg Pro Lys
1490 1495 1500
Ala Asp Gly Ala Trp Thr Leu His Thr Leu Phe Pro Pro Gly Thr
1505 1510 1515
Ile Asp Phe Leu Ile Leu Phe Ser Ser Cys Gly Gln Leu Leu Gly
1520 1525 1530
Leu Pro Gly Gln Ala Ala Tyr Gly Ser Ala Asn Ala Phe Leu Asp
1535 1540 1545
Ala Leu Ala Val His Arg Asn Thr Thr Thr Pro Thr Ala Ala Asp
1550 1555 1560
Thr Thr Ser Phe Gly Trp Thr Ser Trp Arg Gly Gln Gly Met Ala
1565 1570 1575
Val Asn Asp Val Val Asp Ala Glu Leu Arg Ala Arg Gly Val Thr
1580 1585 1590
Asp Ile Thr Thr Gln Glu Ala Phe Ala Ala Trp Asp Phe Ala Ala
1595 1600 1605
Gln His Gly Pro Gly Asn Tyr Pro Val Leu Arg Arg Leu Pro His
1610 1615 1620
Glu Pro Asp Met Asp Gln Leu Pro Leu Leu Ser Glu Ile His His
1625 1630 1635
Thr Gln Pro Thr Ala Pro Thr Ser Gly Ala Ala Thr Asp Ser Tyr
1640 1645 1650
Ala Gly Leu Ala Pro Asp Glu Leu Arg Ala Arg Leu Ile Asp Glu
1655 1660 1665
Val Ala Ala His Ile Ser Ala Glu Met Lys Leu Ala Ala Ser Gln
1670 1675 1680
Leu Asp His Arg Lys Ser Leu Val Glu Gln Gly Leu Asp Ser Val
1685 1690 1695
Met Thr Ile Val Ile Arg Arg Arg Leu Glu Lys Trp Phe Gly His
1700 1705 1710
Lys Leu Pro Ala Thr Leu Leu Trp His Gln Pro Thr Val Thr Ala
1715 1720 1725
Ile Ser Glu His Leu Ala Glu Leu Leu Ala Pro Thr Thr Ser Gln
1730 1735 1740
Pro Asp Asn Thr Ala Pro Ala Glu Pro Ala Ala Thr Ala
1745 1750 1755
Claims (5)
1. volution etheric acid lactone microbiotic is characterized in that structural formula is following:
R in the formula
1=OH, R
2=H;
Perhaps R
1=OH, R
2=Cl.
2. one kind is come the antibiotic method of combinatorial biosynthesis of spirocyclic etheric acid lactone through genetic engineering modified 6-MSAS, it is characterized in that this method comprises:
1) Y in catalytic activity site that in the PCR primer, has introduced the KR structural domain of 6-MSAS is mutated into the mutational site of F; To contain the segmental plasmid of KR is template; PCR obtains corresponding KR fragment; Cut connection through enzyme and substitute original KR structural domain, and finally be cloned among the expression vector pTGV2, be built into the expression plasmid of the 6-MSAS of KR point mutation;
2) expression plasmid with the 6-MSAS of KR structural domain rite-directed mutagenesis imports to heterogenous expression among the Streptomyces albus, in the recombinant bacterial strain that obtains, produces OSA;
3) expression plasmid that produces OSA after the point mutation of 6-MSA KR structural domain is imported among the chlorothricin generation bacterium Streptomyces antibioticus DSM40725 of six-cresotinic acid acid synthase gene interruption; The recombinant bacterial strain fermentation that obtains, tunning obtains the described volution etheric acid of claim 1 lactone microbiotic through separation and purification;
Wherein 6-MSAS represents six-cresotinic acid acid enzyme, and OSA represents orsellinic acid.
3. method as claimed in claim 2 is characterized in that it is the sequence that the Y in 1540 in KR structural domain catalytic activity site through 6-MSAS is mutated into the corresponding mutational site of F that described reorganization produces OSAS; Wherein OSAS represents the orsellinic acid synthetic enzyme.
4. method as claimed in claim 2 is characterized in that it is that the Y in 1540 in KR structural domain catalytic activity site through 6-MSAS is mutated into the mutant strain that Streptomyces antibioticusDSM40725 that expression plasmid that the sequence in the corresponding mutational site of F is built into for the PCR primer imports to Streptomyces albus and chlB1 gene knockout obtains that described reorganization produces OSAS.
5. be used to prepare antibiotic or antitumor drug like claim 1 volution etheric acid lactone microbiotic.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910247567A CN101812103B (en) | 2009-12-30 | 2009-12-30 | 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910247567A CN101812103B (en) | 2009-12-30 | 2009-12-30 | 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101812103A CN101812103A (en) | 2010-08-25 |
| CN101812103B true CN101812103B (en) | 2012-08-29 |
Family
ID=42619482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910247567A Active CN101812103B (en) | 2009-12-30 | 2009-12-30 | 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101812103B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118063531A (en) * | 2024-04-17 | 2024-05-24 | 中国科学院南海海洋研究所 | Preparation and application of macrolide compound PA-46101s C-E |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100465277C (en) * | 2005-07-01 | 2009-03-04 | 中国科学院上海有机化学研究所 | Chlorothricin biological synthesis gene cluster and its uses |
-
2009
- 2009-12-30 CN CN200910247567A patent/CN101812103B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100465277C (en) * | 2005-07-01 | 2009-03-04 | 中国科学院上海有机化学研究所 | Chlorothricin biological synthesis gene cluster and its uses |
Non-Patent Citations (1)
| Title |
|---|
| Xin-Ying Jia et. al..Genetic Characterization of the Chlorothricin Gene Cluster as a Model for Spirotetronate Antibiotic Biosynthesis.《Chemistry & Biology》.2006,第13卷(第6期),第575-585页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101812103A (en) | 2010-08-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhou et al. | Utilization of canola oil and lactose to produce biosurfactant with Candida bombicola | |
| Kunze et al. | Aurafuron A and B, new bioactive polyketides from Stigmatella aurantiaca and Archangium gephyra (myxobacteria) | |
| CN1316001A (en) | Polyketides and their synthesis | |
| IE904072A1 (en) | New polypeptide compound and a process for preparation¹thereof | |
| US7745198B2 (en) | Method of producing macrolide compound | |
| TANAKA et al. | Brasilinolide A, a new macrolide antibiotic produced by Nocardia brasiliensis: producing strain, isolation and biological activity | |
| CN101812103B (en) | 6-methy salicylic acid synthetase transformed by genetic engineering and combinatorial biosynthesis of spirocyclic acetoacetic acid lactone antiboitic | |
| CN109810919B (en) | Ansha all-carbon cyclic polyketone antibiotics and application thereof in preparation of antibacterial drugs or antitumor drugs | |
| JP5826406B2 (en) | Streptomyces, antitumor compound Spiro-Indymycin AD, production method and use thereof, and antitumor agent and drug containing spiroindimycin | |
| CN108660093B (en) | Marine streptomycete and tunicamycin compound and preparation method thereof | |
| CA2107110C (en) | Novel immunosuppressant agent from streptomyces braegensis | |
| CA2007680A1 (en) | Microbial transformation product of l-683,590 | |
| CN113337432B (en) | Methylophilus for producing pyrroloquinoline quinone and application thereof | |
| Sasamura et al. | Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887 | |
| Takaichi et al. | Carotenoids in a Corynebacterineae, Gordonia terrae AIST-1: carotenoid glucosyl mycoloyl esters | |
| CN104263738A (en) | Biosynthesis gene cluster of FAS II inhibitor ABX | |
| Bundale et al. | Isolation of aromatic polyketide producing soil Streptomyces using combinatorial screening strategies | |
| WO2010122669A1 (en) | Novel compound amycolamicin, method for production thereof, and use thereof | |
| Zhi-hua et al. | Improved production of spiramycin by mutant Streptomyces ambofaciens | |
| WO2005063963A1 (en) | Microorganism producing tacrolimus and mass-productive method tacrolimus using the same | |
| UMEZAWA et al. | Isolation from Streptomyces of a novel naphthoquinone compound, naphthablin, that inhibits Abl oncogene functions | |
| JP5823733B2 (en) | Antibiotic-producing microorganism and antibiotic produced by the same | |
| Pistorius et al. | Genetic engineering of Chromobacterium vaccinii DSM 25150 for improved production of FR900359 | |
| JP2006314248A (en) | Method for producing triterpene derivative | |
| Lee et al. | Production of tylosin by Streptomyces fradiae in palm oil medium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230523 Address after: 202152 room 196-8, building 3, No. 1999, Beixing highway, Sanxing Town, Chongming District, Shanghai (Shanghai yuhaitang science and Technology Park) Patentee after: Guoke Xinyan international technology transfer Co.,Ltd. Address before: 200032 Shanghai city Xuhui District Fenglin Road No. 354 Patentee before: SHANGHAI INSTITUTE OF ORGANIC CHEMISTRY, CHINESE ACADEMY OF SCIENCES |
|
| TR01 | Transfer of patent right |