CN102010871A - Biosynthesis gene cluster of xantholipin - Google Patents
Biosynthesis gene cluster of xantholipin Download PDFInfo
- Publication number
- CN102010871A CN102010871A CN2010102512627A CN201010251262A CN102010871A CN 102010871 A CN102010871 A CN 102010871A CN 2010102512627 A CN2010102512627 A CN 2010102512627A CN 201010251262 A CN201010251262 A CN 201010251262A CN 102010871 A CN102010871 A CN 102010871A
- Authority
- CN
- China
- Prior art keywords
- sequence
- nucleotide
- gene
- called
- base place
- 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.)
- Pending
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a biosynthesis gene cluster of xantholipin in the technical field of biomedicine, which comprises nucleotide sequences of 49 genes or complementary sequences formed by the sequences 1, wherein 29 genes are in charge of synthesizing a carbon framework and translationally modifying the carbon framework; 8 genes are in charge of providing a carbon framework extending unit and a methyl donor during the translational modification; 3 genes are in charge of providing resistance for host bacteria; and 6 genes exist in the biosynthesis process. The xantholipin generated in the invention has the functions of cloning, sequencing, analyzing and applying the biosynthesis gene cluster.
Description
Technical field
What the present invention relates to is a kind of gene cluster of field of biomedicine technology, and what be specifically related to is a kind of biological synthesis gene cluster with microbiotic one yellow fat rhzomorph (xantholipin) of resisting gram-positive bacteria and anti-tumor activity.
Background technology
Xantholipin is a kind of Oxoxanthone (xanthone) class microbiotic that grey yellow streptomycete (Streptomyces flavogriseus) fermentation produces.The xanthones microbiotic is the microorganism natural product that a class has ad hoc structure, found kind more than 20 at present, this type of microbiotic biological activity comprises the inhibition bacterium, fungi and cytotoxicity, its avtive spot mainly acts on cell walls, can with competing property of the precursor substance inhibition of synthetic cell wall, thereby the interference cell wall is synthetic.
Studies confirm that xantholipin has restraining effect more widely to gram-positive microorganism, experiment shows that it can suppress hepatic fibrosis recently simultaneously, glomerulonephritis, the great expression of heat shock protein(HSP) HSP47 in the illness such as matter ephritis between kidney, thus can be used as the potentiality medicine of this type of illness.
Biosynthetic pathway about xantholipin is not illustrated so far as yet, infer that according to structure it extends to form carbon skeleton by 13 malonyl coenzyme As, after through a series of oxydo-reductase, methyltransgerase, the halo enzyme, enzymes such as N synthase carry out complicated back modification to it, have formed highly oxidized Oxoxanthone structure.The biological activity of xantholipin and its structure are closely related, clone and functional analysis along with the whole biological synthesis gene cluster of xantholipin, can illustrate xantholipin unique biological synthesis mechanism from molecular level, use the principle of metabolic engineering on this basis, the biosynthetic pathway of rational modification xantholipin is explored good water solubility, the bioavailability height, active better, and can pass through the mass-produced newtype drug of microbial fermentation.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, disclose a kind of biological synthesis gene cluster of yellow fat rhzomorph, the present invention is by clone, order-checking, analysis and the application thereof of the biological synthesis gene cluster of the microbiotic xantholipin with resisting gram-positive bacteria and anti-tumor activity of streptomycete Streptomyces flavogriseus generation.
The present invention is achieved by the following technical solutions:
The present invention includes the nucleotide sequence of 49 genes or the complementary sequence that forms by sequence 1, wherein: be responsible for synthetic carbon skeleton and it is carried out back modification totally two nineteen gene: xanO1, xanO2, xanO3, xanO4, xanO5, xanO6, xanO7, xanO8, xanO9, xanO10, xanM1, xanM2, xanM3, xanZ1, xanZ2, xanZ3, xanZ4, xanS1, xanS2, xanG, xanA, xanH, xanK, xanD, xanE, xanF, xanC1, xanC2, xanC3; Be responsible for providing the methyl donor in carbon skeleton extender unit and the back modification totally eight gene: xanB1, xanB2, xanB3, xanY1, xanY2, xanY3, xanY4, xanY5; Be responsible for totally three gene: xanR1, xanR2, xanR3 are regulated and control in the biosynthesizing of xantholipin; Be responsible for the host bacterium resistance totally three gene: xanU, xanN, xanQ are provided; In this biosynthetic process, also have six gene: xanJ, xanP, xanL, xanW, xanT, xanV.
The present invention also provides the nucleotide sequence of a coding adenosine homocysteine hydrolase, is made up of the aminoacid sequence in the sequence 2, and called after xanY1, its gene nucleotide series are arranged in sequence 1 1419-1 base place.
The present invention also provides a coding 5, and the nucleotide sequence of 10-Methylene tetrahydrofolate reductase is made up of the aminoacid sequence in the sequence 3, and called after xanY2, its gene nucleotide series are arranged in sequence 1 2299-1397 base place.
The present invention also provides the nucleotide sequence of a coding methionine synthetase, is made up of the aminoacid sequence in the sequence 4, and called after xanY3, its gene nucleotide series are arranged in sequence 1 5802-2305 base place.
The present invention also provides a sugared kinase whose nucleotide sequence of coding, is made up of the aminoacid sequence in the sequence 5, and called after xanY4, its gene nucleotide series are arranged in sequence 1 6791-5799 base place.
The present invention also provides the nucleotide sequence of a coding S-methylthioadenosine synthase, is made up of the aminoacid sequence in the sequence 6, and called after xanY5, its gene nucleotide series are arranged in sequence 1 8037-6817 base place.
The present invention also provides the nucleotide sequence of a coding monooxygenase, is made up of the aminoacid sequence in the sequence 7, and called after xanO1, its gene nucleotide series are arranged in sequence 1 8583-8146 base place.
The present invention also provides the nucleotide sequence of a coding desaturase, is made up of the aminoacid sequence in the sequence 8, and called after xanS1, its gene nucleotide series are arranged in sequence 1 9803-8580 base place.
The present invention also provides the nucleotide sequence of an encoding function agnoprotein, is made up of the aminoacid sequence in the sequence 9, and called after xanJ, its gene nucleotide series are arranged in sequence 1 9867-10595 base place.
The present invention also provides a proteic nucleotide sequence of coding spore coat, is made up of the aminoacid sequence in the sequence 10, and called after xanP, its gene nucleotide series are arranged in sequence 1 10592-12211 base place.
The present invention also provides the nucleotide sequence of an encoding glycosyl transferring enzyme, is made up of the aminoacid sequence in the sequence 11, and called after xanG, its gene nucleotide series are arranged in sequence 1 12379-13554 base place.
The present invention also provides the nucleotide sequence of a coding N synthase, is made up of the aminoacid sequence in the sequence 12, and called after xanA, its gene nucleotide series are arranged in sequence 1 13542-15386 base place.
The present invention also provides the nucleotide sequence of an encoding transcription regulatory factor, is made up of the aminoacid sequence in the sequence 13, and called after xanR1, its gene nucleotide series are arranged in sequence 1 15392-16075 base place.
The present invention also provides the nucleotide sequence of a Codocyte cytochrome p 450 hydroxylase, is made up of the aminoacid sequence in the sequence 14, and called after xanO2, its gene nucleotide series are arranged in sequence 1 16114-17316 base place.
The present invention also provides a coding 3Fe-S to reduce proteic nucleotide sequence, is made up of the aminoacid sequence in the sequence 15, and called after xanK, its gene nucleotide series are arranged in sequence 1 17333-17602 base place.
The present invention also provides the nucleotide sequence of a coding resistance translocator, is made up of the aminoacid sequence in the sequence 16, and called after xanU, its gene nucleotide series are arranged in sequence 1 17599-19728 base place.
The present invention also provides the nucleotide sequence of a coding flavine monooxygenase, is made up of the aminoacid sequence in the sequence 17, and called after xanO3, its gene nucleotide series are arranged in sequence 1 19908-21086 base place.
The present invention also provides the nucleotide sequence of a coding lactoylglutathione lyase, is made up of the aminoacid sequence in the sequence 18, and called after xanL, its gene nucleotide series are arranged in sequence 1 21707-21147 base place.
The present invention also provides the nucleotide sequence of a coding non-heme chlB4, is made up of the aminoacid sequence in the sequence 19, and called after xanH, its gene nucleotide series are arranged in sequence 1 22057-23472 base place.
The present invention also provides a proteic nucleotide sequence of encoded peptide chain transfer, is made up of the aminoacid sequence in the sequence 20, and called after xanQ, its gene nucleotide series are arranged in sequence 1 23469-25070 base place.
The present invention also provides the nucleotide sequence of a coding oxygen methyltransgerase, is made up of the aminoacid sequence in the sequence 21, and called after xanM1, its gene nucleotide series are arranged in sequence 1 25067-26086 base place.
The present invention also provides the nucleotide sequence of a coding reductase enzyme, is made up of the aminoacid sequence in the sequence 22, and called after xanZ1, its gene nucleotide series are arranged in sequence 1 26651-27508 base place.
The present invention also provides the nucleotide sequence of a reverse transport protein of coding film ion, is made up of the aminoacid sequence in the sequence 23, and called after xanN, its gene nucleotide series are arranged in sequence 1 28888-27593 base place.
The present invention also provides the nucleotide sequence of an encoding function agnoprotein, is made up of the aminoacid sequence in the sequence 24, and called after xanW, its gene nucleotide series are arranged in sequence 1 29473-28985 base place.
The present invention also provides an encoding transcription to regulate proteic nucleotide sequence, is made up of the aminoacid sequence in the sequence 25, and called after xanR2, its gene nucleotide series are arranged in sequence 1 29600-30061 base place.
The present invention also provides the nucleotide sequence of a coding oxygen methyltransgerase, is made up of the aminoacid sequence in the sequence 26, and called after xanM2, its gene nucleotide series are arranged in sequence 1 31105-30083 base place.
The present invention also provides the nucleotide sequence of a coding flavine monooxygenase, is made up of the aminoacid sequence in the sequence 27, and called after xanO4, its gene nucleotide series are arranged in sequence 1 32742-31120 base place.
The present invention also provides the nucleotide sequence of a coding methyltransgerase, is made up of the aminoacid sequence in the sequence 28, and called after xanM3, its gene nucleotide series are arranged in sequence 1 33084-34118 base place.
The present invention also provides the nucleotide sequence of a coding flavine monooxygenase, is made up of the aminoacid sequence in the sequence 29, and called after xanO5, its gene nucleotide series are arranged in sequence 1 34203-35798 base place.
The present invention also provides the nucleotide sequence of a coding reductase enzyme, is made up of the aminoacid sequence in the sequence 30, and called after xanS2, its gene nucleotide series are arranged in sequence 1 35868-36554 base place.
The present invention also provides the nucleotide sequence of a coding oxydo-reductase, is made up of the aminoacid sequence in the sequence 31, and called after xanZ2, its gene nucleotide series are arranged in sequence 1 37667-36759 base place.
The present invention also provides an encoding transcription to regulate proteic nucleotide sequence, is made up of the aminoacid sequence in the sequence 32, and called after xanR3, its gene nucleotide series are arranged in sequence 1 37715-38722 base place.
The present invention also provides the nucleotide sequence of a plain carboxylase of encoding human, is made up of the aminoacid sequence in the sequence 33, and called after xanB1, its gene nucleotide series are arranged in sequence 1 40187-38844 base place.
The present invention also provides the nucleotide sequence of a plain carboxyl carrier protein of encoding human, is made up of the aminoacid sequence in the sequence 34, and called after xanB2, its gene nucleotide series are arranged in sequence 1 40714-40190 base place.
The present invention also provides the nucleotide sequence of a coding carboxyltransferase, is made up of the aminoacid sequence in the sequence 35, and called after xanB3, its gene nucleotide series are arranged in sequence 1 42468-40774 base place.
The present invention also provides the nucleotide sequence of a coding monooxygenase, is made up of the aminoacid sequence in the sequence 36, and called after xanO6, its gene nucleotide series are arranged in sequence 1 42857-42546 base place.
The present invention also provides the nucleotide sequence of a coding monooxygenase, is made up of the aminoacid sequence in the sequence 37, and called after xanO7, its gene nucleotide series are arranged in sequence 1 43194-42847 base place.
The present invention also provides the nucleotide sequence of a coding 3-oxygen acyl-acyl carrier protein reductase enzyme, is made up of the aminoacid sequence in the sequence 38, and called after xanZ3, its gene nucleotide series are arranged in sequence 1 43946-43194 base place.
The present invention also provides the nucleotide sequence of a coding monooxygenase, is made up of the aminoacid sequence in the sequence 39, and called after xanO8, its gene nucleotide series are arranged in sequence 1 44431-43970 base place.
The present invention also provides the nucleotide sequence of a coding cyclase, is made up of the aminoacid sequence in the sequence 40, and called after xanC1, its gene nucleotide series are arranged in sequence 1 44909-44442 base place.
The present invention also provides the nucleotide sequence of a coded beta-keto acyl synthase β, is made up of the aminoacid sequence in the sequence 41, and called after xanE, its gene nucleotide series are arranged in sequence 1 46303-45017 base place.
The present invention also provides the nucleotide sequence of a coded beta-keto acyl synthase α, is made up of the aminoacid sequence in the sequence 42, and called after xanF, its gene nucleotide series are arranged in sequence 1 47583-46306 base place.
The present invention also provides the nucleotide sequence of a coding cyclase, is made up of the aminoacid sequence in the sequence 43, and called after xanC2, its gene nucleotide series are arranged in sequence 1 48014-47580 base place.
The present invention also provides the nucleotide sequence of a coding CurD homologous protein, is made up of the aminoacid sequence in the sequence 44, and called after xanT, its gene nucleotide series are arranged in sequence 1 48198-48584 base place.
The present invention also provides a glairy nucleotide sequence of coding oxidases, is made up of the aminoacid sequence in the sequence 45, and called after xanO9, its gene nucleotide series are arranged in sequence 1 48596-50014 base place.
The present invention also provides the nucleotide sequence of a coding monooxygenase, is made up of the aminoacid sequence in the sequence 46, and called after xanO10, its gene nucleotide series are arranged in sequence 1 50513-50067 base place.
The present invention also provides the nucleotide sequence of an encoding function agnoprotein, is made up of the aminoacid sequence in the sequence 47, and called after xanV, its gene nucleotide series are arranged in sequence 1 50908-50528 base place.
The present invention also provides the nucleotide sequence of a coding 3-oxygen acyl-acyl carrier protein reductase enzyme, is made up of the aminoacid sequence in the sequence 48, and called after xanZ4, its gene nucleotide series are arranged in sequence 1 51663-50908 base place.
The present invention also provides the nucleotide sequence of a coding acyl carrier protein, is made up of the aminoacid sequence in the sequence 49, and called after xanD, its gene nucleotide series are arranged in sequence 1 51920-51660 base place.
The present invention also provides the nucleotide sequence of a coding cyclase, is made up of the aminoacid sequence in the sequence 50, and called after xanC3, its gene nucleotide series are arranged in sequence 1 52255-51917 base place.
The complementary sequence of described sequence 1 can obtain at any time according to DNA base complementrity principle, and the nucleotide sequence of sequence 1 or partial nucleotide sequence can obtain by polymerase chain reaction (PCR) or with digestion with restriction enzyme DNA.
Described nucleotide sequence or partial nucleotide sequence, the method that the DNA that can utilize the method for polymerase chain reaction (PCR) or comprise sequence of the present invention carries out Southern hybridization as probe obtains the homologous gene of xantholipin biosynthesis gene from other microorganisms.
Described from carry partial sequence 1 recombinant vectors at least, or from the microorganism library, or from microbe genome DNA, separate the approach of xantholipin biosynthesis gene.
The described approach that is comprised the recombinant DNA carrier of dna sequence dna in the partial sequence 1 at least.
The described approach that the microbe that the xantholipin biosynthesis gene transformed or lack is arranged in the genome that is created in.
Nucleotide sequence provided by the invention or a plurality of sequence can obtain recombination sequence and corresponding D NA molecule with the fusion of carrier sequence.
Comprise nucleotide sequence provided by the present invention or the clone gene or the dna fragmentation of partial sequence can obtain new xantholipin derivative by interrupting biosynthetic one or several synthesis step of xantholipin at least.
Comprise nucleotide sequence provided by the present invention or the clone gene or the dna fragmentation of partial sequence can obtain new xantholipin derivative by interrupting biosynthetic one or several modification step of xantholipin at least.
Comprise the output that dna fragmentation or gene can be used for improving the xantholipin or derivatives thereof.For example, change the positive regulator gene of multiple copied more or interrupt negative regulator gene over to.
Comprise nucleotide sequence provided by the present invention or at least the cloned DNA of partial sequence can be used to from streptomycete xantholipin genomic library more library, location clay.These library plasmids include the partial sequence among the present invention at least, also include the DNA that former adjacent domain is not cloned in the streptomycete xantholipin genome.
Described nucleotide sequence can be modified or be suddenlyd change.These approach comprise and inserting or displacement, the polymerase chain reaction, and mistake mediation polymerase chain reaction, the locus specificity sudden change, or directly evolve with the homologous sequence in other source (DNA shuffling) etc.
Comprise sequence of the present invention or at least the clone gene of partial sequence can in foreign host, express with the enzyme that obtains modifying or higher biological activity or higher output by appropriate expression system.These foreign host comprise streptomycete, intestinal bacteria, genus bacillus, yeast, plant and animal cell etc.
Xantholipin biosynthesizing modifying factor, regulatory gene, the nucleotide sequence of resistance related gene provides by disappearance or has transformed these modifying factors, regulatory gene, transporter gene and obtain the approach that xantholipin structural derivative or xantholipin and derivative output thereof improve.
Comprise aminoacid sequence of the present invention or at least the polypeptide of partial sequence (sequence 2-50) may after remove or substitute certain or some amino acid, still have biological activity even new biologic activity is arranged, perhaps improved output or optimization albumen dynamic characteristic or other character of being devoted to obtain.
Aminoacid sequence that the present invention comprises or gene order can be transferred to other and nucleoside antibiotics by modes such as conversion, transduction, conjugal transfers and produce in the bacterium, thereby produce this antibiotic derivative.
All genes and albumen information that xantholipin biosynthesizing provided by the invention is relevant, help to illustrate and understand the biosynthetic molecule mechanism of xantholipin and relevant xanthones microbiotic family, thereby for further utilizing the genetic engineering means transformation to provide fundamental basis and material.Gene provided by the invention and coded protein thereof also can be used for searching and development can be used for medicine, industry, the compound or the albumen of agricultural.
Description of drawings
The chemical structure of Fig. 1 Xantholipin.
The structure of Fig. 2 Xantholipin biological synthesis gene cluster is formed and restriction mapping.
Wherein: (A) 3 eclipsed clays have been represented the DNA zone of sallow streptomyces gene group 92kb, and probe 1-3 represents probe portion;
(B) structure of Xantholipin biological synthesis gene cluster is formed.
The structure and the detection of Fig. 3 40kb fragment deletion mutant strain.
Wherein: (A) the process synoptic diagram of structure 40kb fragment deletion mutant strain;
(B) the growth phenotype at substratum ISP-3 of large fragment deletion mutant strain, (1) dull and stereotyped positive mycelia and spore phenotype a: wild-type, b-c: large fragment deletion mutant strain; (2) dull and stereotyped reverse side produces pigment phenotype a: wild-type, b-c: large fragment deletion mutant strain;
(C) HPLC of large fragment deletion mutant strain (high performance liquid chromatography) detects, Wild type:Streptomyces flavogriseus wild-type, ZWK1:40kb fragment deletion mutant strain.
The MS (mass spectrum) of Fig. 4 sallow streptomycete fermentation product detects.
The biosynthetic pathway synoptic diagram of Fig. 5 xantholipin of the present invention.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Culture presevation information related in following examples is as follows:
Ash yellow streptomycete (Streptomyces flavogriseus): be located away from tideland, Chang Mount island, south, Shandong plant rhizosphere soil, now be preserved in Chinese typical culture collection center (CCTCC, (the wuchang, wuhan Luo Jiashan of Wuhan University, postcode: 430072, phone: (027)-68752319), China Wuhan), preservation date on July 20th, 2010, deposit number CCTCC No.M2010183.
Intestinal bacteria DH10B: buy from the Maryland, USA lid look Regensburg biotechnological formulation Gibco-BRL of company.
1.xantholipin the clone of biological synthesis gene cluster, analysis:
As shown in Figure 1, xantholipin belongs to II type polyketide synthase synthetic xanthones microbiotic, with the lysolipin structural similitude.Because both all have the chlorine atom in similar position, inferring in its synthetic gene bunch has the higher halo enzyme of homology.Utilize Nucleotide conserved sequence design one couple of PCR (polymerase chain reaction) primer (5 '-CCGACGGCTGGTTCTGGTCCATCCC-3 ') of lysolipin halo enzyme and (5 '-ACAGCAGCGGGTCGA GGAAGCAGGC-3 ') successfully to produce the bacterium ash yellow streptomycete from xantholipin and amplify a specificity product that expection is big or small, sequential analysis shows that the homology of both aminoacid sequences reaches 70%.Set out according to this primer, utilize round pcr from grey yellow streptomycete, to clone positive colony.Further the gene interrupt experiments shows that the deletion mutantion strain of 40kb completely loses the generation ability of xantholipin (Fig. 3 C).(chromosome walking) obtained 3 overlapped clays by chromosome walking, covered the zone of about 92kb on the karyomit(e) (Fig. 2 A), dna sequencing 92445bp successive zone on the karyomit(e), GC content is 70.8%.Bioinformatic analysis has comprised 86 opening code-reading frames, wherein 58 opening code-reading frames is carried out further experiment research, shown in Fig. 2 B.
2.40kb fragment deletion
28B8 comprises xanU to the segmental carrier of the about 40kb of orf10, by with the KpnI fragment cloning at its external source fragment two ends to shuttle vectors, import in the grey yellow streptomycete xanU is lacked to the about 40kb fragment of orf10, studies confirm that this 40kb fragment deletion mutant strain lost the ability that produces xantholipin, illustrate that this 40kb fragment is relevant with the biosynthesizing of xantholipin.Xantholipin itself is a yellow compound, wild-type ash yellow streptomycete can produce xantholipin makes substratum show yellow, and ZWK1 mutant strain substratum still is the primary oyster white, has confirmed that xanU is directly related with the biosynthesizing of xantholipin to the about 40kb fragment of orf10.HPLC detects explanation, the characteristic peak of xantholipin can appear in retention time in xantholipin standard substance and wild-type about 28.6 minutes, the ZWK1 mutant strain does not then have (Fig. 3 C), thereby confirms that further xanU is directly related with the biosynthesizing of xantholipin to orf10.
3.Xantholipin determining of biological synthesis gene cluster border
According to the functional analysis of gene coded protein, the biological synthesis gene cluster of xantholipin is confirmed as from gene xanY1 having contained the zone of 52kb on the karyomit(e) to xanC3, comprises 49 opening code-reading frames.Further confirmed the border of xantholipin biological synthesis gene cluster by the gene interrupt experiments.Interrupt orf1, orf2, orf3, orf4, orf5 finds can not influence the biosynthesizing of xantholipin.Biosynthesizing according to coded albumen of bioinformatic analysis orf1-orf5 and xantholipin is irrelevant, and modify relevantly behind the coded albumen of xanY1-xanY5 and the xantholipin, so xanY1 is confirmed as the left margin of xantholipin biological synthesis gene cluster.Interrupt orf6-orf11 simultaneously to the biosynthesizing of xantholipin also not influence, and be to be responsible for the biosynthetic structure gene of xantholipin, so xanC3 is confirmed as the biosynthetic right margin of xantholipin according to bioinformatic analysis xanC3.
4.Xantholipin biosynthetic pathway
(A) carbon skeleton is synthetic
13 malonyl coenzyme As are at xanD, the acyl carrier protein that xanE, xanF encode respectively; beta-keto acyl synthase α; formed the polyketone long-chain of 26 carbon atoms under the acting in conjunction of beta-keto acyl synthase β, then at xanZ1, xanZ3; under the catalysis of the keto reductase of xanZ4 coding; 11 ketone group is reduced to hydroxyl, thereby causes the folding of carbochain space structure, then xanC1; xanC2; the cyclase catalysis of xanC3 coding the further folding and C9 of carbochain, C14 cyclisation, C7; the C16 cyclisation; C5, C18 cyclisation, C4; the C21 cyclisation; C2, the C23 cyclisation, asparagine synthase will be from transamination to a carbon atom of l-asparagine hydrolysis and the hydroxyl generation replacement(metathesis)reaction the carboxyl then; spontaneous cyclization takes place in newly-generated amino and 26 ketone groups, forms the carbon skeleton structure of core.
(B) back modification approach
C3 has taken place in the new carbon skeleton that forms under the catalysis of a series of monooxygenases and oxydo-reductase, C4, C6, C8, C15, C17, C19, the redox of eight positions of C22, then at xanO4, the Bayer-Villiger oxidizing reaction takes place at C14 down in the effect of the flavine monooxygenase of xanO5 coding, insert Sauerstoffatom in the C15 carbon-carbon bond and form lactone structure, the molecular transposition that 7 yuan of rings take place subsequently becomes Sauerstoffatom with C15, next xanM1, xanM2, the Methyl transporters enzyme catalysis of xanM3 coding C13, the oxygen of C19 position hydroxyl methylates, then the structure of the hydroxyl of C17 position and the oxygen methyl generation cyclisation formation methylene-bis oxo bridge of C19 position under the Cytochrome P450 oxydase effect of xanO2 coding, under the catalysis of the halo enzyme that xanH encodes, the chlorine atom adding is downloaded to the chloro of C12 position generation phenyl ring at last, thereby has finally formed compound xantholipin.
Xantholipin produces the extraction of bacterium sallow streptomyces gene group DNA:
Inoculate seed culture medium (glucose 10g, Zulkovsky starch 25g, the yeast extract 2g of the grey yellow streptomycete spore of 20 μ l, 20% glycerine preservation to 5ml, fish meal 5g, cottonseed meal 3g, casein acidolysis thing 3g, lime carbonate 2g, distilled water 1000ml, pH 7.2) in, place 30 ℃ of shaking tables to cultivate 36 hours, centrifugal collection thalline is stand-by, with the 500 μ l lysozyme solns 50mg mycelium that suspends again,, or be incubated to cell and become translucent at 37 ℃ of about 30min of incubation.Add 500 μ l 2%SDS, mix the about 1min of vibration and significantly descend up to the viscosity of solution, 37 ℃ of incubation 20min add the neutral phenol/chloroform of 25 μ l then, and after the mixing vibration evenly, the centrifugal 5min of 12000r/min pipettes supernatant liquor, discards white middle layer.Repeat the secondary extracting till cannot see (or considerably less) middle layer with neutral phenol/chloroform, the 3M sodium-acetate (pH nature) that adds 0.1 times of volume at last, mix the Virahol (or dehydrated alcohol of 2.2 times of volumes) that the back adds 1 times of volume, at room temperature place 5min (perhaps placing 30min) after mixing once more at-20 ℃, 12000r/min is centrifugal, abandoning supernatant, precipitate twice with 70% washing with alcohol DNA, discard all supernatant liquors at last, after treating the ethanol volatilization, be dissolved in a certain amount of TE damping fluid.
Xantholipin produces the foundation of bacterium ash yellow streptomycete conjugal transfer system:
It is standby that collection grows in the fresh spore of ISP-3 substratum ash yellow streptomycete.The target plasmid that contains oriT must could import in the receptor chain fungal cell by conjugal transfer under the assistance of helper plasmid pUZ8002.To be transferred into the plasmid transformation escherichia coli ET12567/pUZ8002 in the grey yellow streptomycete earlier, and cultivate the intestinal bacteria ET12567/pUZ8002 contain target plasmid then, and collect thalline behind the 12h, it is standby to wash thalline 2 times with fresh LB substratum.Streptomycete spore as acceptor needs to handle through heat shock and pre-the sprouting.The streptomycete spore is suspended in again in the TES damping fluid of 5ml 0.05M pH8.0, heat shock 10min in 50 ℃ of water-baths is cooled to and adds the pre-germination medium of equal-volume 2 * spore (Oxide yeast extract paste 10%, Oxide casamino acids 1%, CaCl after the room temperature
20.01M) (need the stoste of preparation 5M, separately be added in the yeast extract paste casamino acids solution after the sterilization) sprout 2-3h in advance, centrifugal collection spore also is suspended among an amount of LB again, and spore is broken up in vibration on mixing tank, by 10
8: 10
8With the Bacillus coli cells balanced mix, contain an amount of microbiotic with the 1ml sterilized water behind the 26-30h and nalidixic acid (being used for suppressing colibacillary growth) covers, put 30 ℃ and can see zygote after cultivating a couple of days.
Xantholipin produces the structure and the screening in bacterium sallow streptomyces gene group library:
The construction process reference reagent box CopyControl of genomic library
TMThe experimental technique that Fosmid Library Production Kit provides.
Utilize Nucleotide conserved sequence design one couple of PCR (polymerase chain reaction) primer (5 '-CCGACGGCTGGTTCTGGTCCATCCC-3 ') of lysolipin halo enzyme and (5 '-ACAGCAGCGGGTCGA GGAAGCAGGC-3 ') successfully to produce the bacterium ash yellow streptomycete from xantholipin and amplify a specificity product that expection is big or small, sequential analysis shows that the homology of both aminoacid sequences reaches 70%.Set out according to this primer, utilize PCR (polymerase chain reaction) to come screening-gene group library, at first with clone's mixed culture of each 96 orifice plate, carry the mixing clay and carry out the PCR reaction then as template, earlier positive signal is positioned each 96 orifice plate, same method is the row of being positioned then, is positioned a little at last.
Embodiment 4
The fermentation of Xantholipin, separation and purification and Analysis and Identification:
The fermentation flow process:
Bacterial classification is applied to the ISP3 substratum after passing a generation with seed culture medium in the freeze-drying pipe, receives spore after 7 days and preserves in the glycerine pipe.Get an amount of spore (about 50ul) in the glycerine pipe and be seeded to 80ml seed culture medium (500ml triangular flask) (glucose 10g, Zulkovsky starch 25g, yeast extract 2g, fish meal 5g, cottonseed meal 3g, casein acidolysis thing 3g, lime carbonate 2g, distilled water 1000ml, pH 7.2), 30 ℃, 220rpm cultivated 2 days.Inoculum size with 5% is seeded to (500ml triangular flask) (glucose 10g in the 80ml fermention medium with seed, dextrin 25g, oatmeal 20g, cottonseed meal 10g, fish meal 5g, yeast extract 2g, lime carbonate 3g, distilled water is settled to 1000ml, before the sterilization PH is transferred to 7.2), 30 ℃, 220rpm cultivated 6 days.
Fermenting process was surveyed the pH value, a few days ago all at 5-6, and pH 8-8.5 when putting bottle.
Separation and purification:
The 80ml fermented liquid is soaked ambient temperature overnight with isopyknic acetone.Centrifugal then, abandon thalline, get supernatant, 45 ℃ of rotations steam acetone, and the residue water rotates evaporate to dryness for 45 ℃ with ethyl acetate then with about 80ml ethyl acetate extraction, with about 1ml dissolve with methanol ,-70 ℃ of preservations.
The HPLC separation condition:
After crude product was dissolved in methyl alcohol, the centrifuging and taking supernatant was crossed the organic phase filter membrane of 0.22 μ m, was used for that HPLC analyzes or LC/MS analyzes.The separation condition of HPLC is mobile phase A: 0.2% formic acid, and 0.1% trifluoroacetic acid ultrapure water, Mobile phase B: HPLC level acetonitrile, detect wavelength 384nm, in the 0-3min, Mobile phase B is corresponding to rise to 20% from 10%; 3-50min, Mobile phase B rises to 55% from 20%; 50-52min, Mobile phase B rises to 90% from 55%; 52-60min, moving phase maintains 90%.Chromatographic column is Waters 150 * 46mm C
18The SB chromatographic column, flow velocity 0.5ml/min.Program is provided with as follows:
| Time (min) | Mobile phase B (%) | Flow velocity (ml/min) |
| ?0? | ?10? | 0.5? |
| ?3? | ?20? | 0.5? |
| ?50? | ?55? | 0.5? |
| ?52? | ?90? | 0.5? |
| ?60? | ?90? | 0.5? |
Embodiment 5
Gene interrupts the structure of mutant strain:
The interruption carrier that the makes up mode by conjugal transfer is gone in the grey yellow streptomycete, with the zygote resistance checking on the ISP-3 flat board that adds apramycin (apramycin) that grows out, after checking is correct, enlarging on the ISP-3 flat board of apramycin relaxes cultivates not adding, treat the spore plentiful back of growth (about 7 days), collect spore, adding the about 50-100 of the every flat board of gradient dilution single bacterium colony on the corresponding antibiotic ISP-3 flat board then, choosing colony draws antibiotic ISP-3 dull and stereotyped and contain enlarged culturing on the ISP-3 flat board of thiostrepton containing A Bo respectively, be chosen on the thiostrepton flat board long but even up well-grown bacterium colony on the plate at A Bo, be transferred to and extract total DNA in the seed culture medium, after carrying out PCR checking correctly, mutant strain carries out fermentation culture.
Claims (6)
1. the biological synthesis gene cluster of a yellow fat rhzomorph, it is characterized in that, comprise the nucleotide sequence of 49 genes or the complementary sequence that forms by sequence 1, wherein: be responsible for synthetic carbon skeleton and it is carried out back modification totally two nineteen gene: xanO1, xanO2, xanO3, xanO4, xanO5, xanO6, xanO7, xanO8, xanO9, xanO10, xanM1, xanM2, xanM3, xanZ1, xanZ2, xanZ3, xanZ4, xanS1, xanS2, xanG, xanA, xanH, xanK, xanD, xanE, xanF, xanC1, xanC2, xanC3; Be responsible for providing the methyl donor in carbon skeleton extender unit and the back modification totally eight gene: xanB1, xanB2, xanB3, xanY1, xanY2, xanY3, xanY4, xanY5; Be responsible for totally three gene: xanR1, xanR2, xanR3 are regulated and control in the biosynthesizing of xantholipin; Be responsible for the host bacterium resistance totally three gene: xanU, xanN, xanQ are provided; In this biosynthetic process, also have six gene: xanJ, xanP, xanL, xanW, xanT, xanV.
2. the biological synthesis gene cluster of yellow fat rhzomorph according to claim 1, it is characterized in that, described sequence 1, the nucleotide sequence of a coding adenosine homocysteine hydrolase is made up of the aminoacid sequence in the sequence 2, called after xanY1, its gene nucleotide series are arranged in sequence 1 1419-1 base place;
A coding 5, the nucleotide sequence of 10-Methylene tetrahydrofolate reductase is made up of the aminoacid sequence in the sequence 3, and called after xanY2, its gene nucleotide series are arranged in sequence 1 2299-1397 base place;
The nucleotide sequence of a coding methionine synthetase is made up of the aminoacid sequence in the sequence 4, and called after xanY3, its gene nucleotide series are arranged in sequence 1 5802-2305 base place;
The sugared kinase whose nucleotide sequence of coding is made up of the aminoacid sequence in the sequence 5, and called after xanY4, its gene nucleotide series are arranged in sequence 1 6791-5799 base place.
The nucleotide sequence of a coding S-methylthioadenosine synthase is made up of the aminoacid sequence in the sequence 6, and called after xanY5, its gene nucleotide series are arranged in sequence 1 8037-6817 base place;
The nucleotide sequence of a coding monooxygenase is made up of the aminoacid sequence in the sequence 7, and called after xanO1, its gene nucleotide series are arranged in sequence 1 8583-8146 base place;
The nucleotide sequence of a coding desaturase is made up of the aminoacid sequence in the sequence 8, and called after xanS1, its gene nucleotide series are arranged in sequence 1 9803-8580 base place;
The nucleotide sequence of an encoding function agnoprotein is made up of the aminoacid sequence in the sequence 9, and called after xanJ, its gene nucleotide series are arranged in sequence 1 9867-10595 base place;
The proteic nucleotide sequence of coding spore coat is made up of the aminoacid sequence in the sequence 10, and called after xanP, its gene nucleotide series are arranged in sequence 1 10592-12211 base place;
The nucleotide sequence of an encoding glycosyl transferring enzyme is made up of the aminoacid sequence in the sequence 11, and called after xanG, its gene nucleotide series are arranged in sequence 1 12379-13554 base place;
The nucleotide sequence of a coding N synthase is made up of the aminoacid sequence in the sequence 12, and called after xanA, its gene nucleotide series are arranged in sequence 1 13542-15386 base place;
The nucleotide sequence of an encoding transcription regulatory factor is made up of the aminoacid sequence in the sequence 13, and called after xanR1, its gene nucleotide series are arranged in sequence 1 15392-16075 base place;
The nucleotide sequence of a Codocyte cytochrome p 450 hydroxylase is made up of the aminoacid sequence in the sequence 14, and called after xanO2, its gene nucleotide series are arranged in sequence 1 16114-17316 base place;
A coding 3Fe-S reduces proteic nucleotide sequence and is made up of the aminoacid sequence in the sequence 15, and called after xanK, its gene nucleotide series are arranged in sequence 1 17333-17602 base place;
The nucleotide sequence of a coding resistance translocator is made up of the aminoacid sequence in the sequence 16, and called after xanU, its gene nucleotide series are arranged in sequence 1 17599-19728 base place;
The nucleotide sequence of a coding flavine monooxygenase is made up of the aminoacid sequence in the sequence 17, and called after xanO3, its gene nucleotide series are arranged in sequence 1 19908-21086 base place;
The nucleotide sequence of a coding lactoylglutathione lyase is made up of the aminoacid sequence in the sequence 18, and called after xanL, its gene nucleotide series are arranged in sequence 1 21707-21147 base place;
The nucleotide sequence of a coding non-heme chlB4 is made up of the aminoacid sequence in the sequence 19, and called after xanH, its gene nucleotide series are arranged in sequence 1 22057-23472 base place;
The proteic nucleotide sequence of encoded peptide chain transfer is made up of the aminoacid sequence in the sequence 20, and called after xanQ, its gene nucleotide series are arranged in sequence 1 23469-25070 base place;
The nucleotide sequence of a coding oxygen methyltransgerase is made up of the aminoacid sequence in the sequence 21, and called after xanM1, its gene nucleotide series are arranged in sequence 1 25067-26086 base place;
The nucleotide sequence of a coding reductase enzyme is made up of the aminoacid sequence in the sequence 22, and called after xanZ1, its gene nucleotide series are arranged in sequence 1 26651-27508 base place;
The nucleotide sequence of a reverse transport protein of coding film ion is made up of the aminoacid sequence in the sequence 23, and called after xanN, its gene nucleotide series are arranged in sequence 1 28888-27593 base place;
The nucleotide sequence of an encoding function agnoprotein is made up of the aminoacid sequence in the sequence 24, and called after xanW, its gene nucleotide series are arranged in sequence 1 29473-28985 base place;
An encoding transcription is regulated proteic nucleotide sequence and is made up of the aminoacid sequence in the sequence 25, and called after xanR2, its gene nucleotide series are arranged in sequence 1 29600-30061 base place;
The nucleotide sequence of a coding oxygen methyltransgerase is made up of the aminoacid sequence in the sequence 26, and called after xanM2, its gene nucleotide series are arranged in sequence 1 31105-30083 base place;
The nucleotide sequence of a coding flavine monooxygenase is made up of the aminoacid sequence in the sequence 27, and called after xanO4, its gene nucleotide series are arranged in sequence 1 32742-31120 base place;
The nucleotide sequence of a coding methyltransgerase is made up of the aminoacid sequence in the sequence 28, and called after xanM3, its gene nucleotide series are arranged in sequence 1 33084-34118 base place;
The nucleotide sequence of a coding flavine monooxygenase is made up of the aminoacid sequence in the sequence 29, and called after xanO5, its gene nucleotide series are arranged in sequence 1 34203-35798 base place;
The nucleotide sequence of a coding reductase enzyme is made up of the aminoacid sequence in the sequence 30, and called after xanS2, its gene nucleotide series are arranged in sequence 1 35868-36554 base place;
The nucleotide sequence of a coding oxydo-reductase is made up of the aminoacid sequence in the sequence 31, and called after xanZ2, its gene nucleotide series are arranged in sequence 1 37667-36759 base place;
An encoding transcription is regulated proteic nucleotide sequence and is made up of the aminoacid sequence in the sequence 32, and called after xanR3, its gene nucleotide series are arranged in sequence 1 37715-38722 base place;
The nucleotide sequence of a plain carboxylase of encoding human is made up of the aminoacid sequence in the sequence 33, and called after xanB1, its gene nucleotide series are arranged in sequence 1 40187-38844 base place;
The nucleotide sequence of a plain carboxyl carrier protein of encoding human is made up of the aminoacid sequence in the sequence 34, and called after xanB2, its gene nucleotide series are arranged in sequence 1 40714-40190 base place;
The nucleotide sequence of a coding carboxyltransferase is made up of the aminoacid sequence in the sequence 35, and called after xanB3, its gene nucleotide series are arranged in sequence 1 42468-40774 base place;
The nucleotide sequence of a coding monooxygenase is made up of the aminoacid sequence in the sequence 36, and called after xanO6, its gene nucleotide series are arranged in sequence 1 42857-42546 base place;
The nucleotide sequence of a coding monooxygenase is made up of the aminoacid sequence in the sequence 37, and called after xanO7, its gene nucleotide series are arranged in sequence 1 43194-42847 base place;
The nucleotide sequence of a coding 3-oxygen acyl-acyl carrier protein reductase enzyme is made up of the aminoacid sequence in the sequence 38, and called after xanZ3, its gene nucleotide series are arranged in sequence 1 43946-43194 base place;
The nucleotide sequence of a coding monooxygenase is made up of the aminoacid sequence in the sequence 39, and called after xanO8, its gene nucleotide series are arranged in sequence 1 44431-43970 base place;
The nucleotide sequence of a coding cyclase is made up of the aminoacid sequence in the sequence 40, and called after xanC1, its gene nucleotide series are arranged in sequence 1 44909-44442 base place;
The nucleotide sequence of a coded beta-keto acyl synthase β is made up of the aminoacid sequence in the sequence 41, and called after xanE, its gene nucleotide series are arranged in sequence 1 46303-45017 base place;
The nucleotide sequence of a coded beta-keto acyl synthase α is made up of the aminoacid sequence in the sequence 42, and called after xanF, its gene nucleotide series are arranged in sequence 1 47583-46306 base place;
The nucleotide sequence of a coding cyclase is made up of the aminoacid sequence in the sequence 43, and called after xanC2, its gene nucleotide series are arranged in sequence 1 48014-47580 base place;
The nucleotide sequence of a coding CurD homologous protein is made up of the aminoacid sequence in the sequence 44, and called after xanT, its gene nucleotide series are arranged in sequence 1 48198-48584 base place;
The glairy nucleotide sequence of coding oxidases is made up of the aminoacid sequence in the sequence 45, and called after xanO9, its gene nucleotide series are arranged in sequence 1 48596-50014 base place;
The nucleotide sequence of a coding monooxygenase is made up of the aminoacid sequence in the sequence 46, and called after xanO10, its gene nucleotide series are arranged in sequence 1 50513-50067 base place;
The nucleotide sequence of an encoding function agnoprotein is made up of the aminoacid sequence in the sequence 47, and called after xanV, its gene nucleotide series are arranged in sequence 1 50908-50528 base place;
The nucleotide sequence of a coding 3-oxygen acyl-acyl carrier protein reductase enzyme is made up of the aminoacid sequence in the sequence 48, and called after xanZ4, its gene nucleotide series are arranged in sequence 1 51663-50908 base place;
The nucleotide sequence of a coding acyl carrier protein is made up of the aminoacid sequence in the sequence 49, and called after xanD, its gene nucleotide series are arranged in sequence 1 51920-51660 base place;
The nucleotide sequence of a coding cyclase is made up of the aminoacid sequence in the sequence 50, and called after xanC3, its gene nucleotide series are arranged in sequence 1 52255-51917 base place.
3. according to the biological synthesis gene cluster of claim 1 or 2 described yellow fat rhzomorphs, it is characterized in that the nucleotide sequence of described sequence 1 or partial nucleotide sequence obtain by the polymerase chain reaction or with digestion with restriction enzyme DNA.
4. according to the biological synthesis gene cluster of claim 1 or 2 described yellow fat rhzomorphs, it is characterized in that, described nucleotide sequence or partial nucleotide sequence are utilized the method for polymerase chain reaction or are obtained the homologous gene of xantholipin biosynthesis gene with the method that the DNA of the local sequence of going on the market carries out Southern hybridization as probe.
5. according to the biological synthesis gene cluster of claim 1 or 2 described yellow fat rhzomorphs, it is characterized in that described nucleotide sequence or the clone gene or the dna fragmentation of partial sequence obtain the xantholipin derivative by interrupting biosynthetic one or several synthesis step of xantholipin at least.
6. according to the biological synthesis gene cluster of claim 1 or 2 described yellow fat rhzomorphs, it is characterized in that described nucleotide sequence or the clone gene or the dna fragmentation of partial sequence obtain new xantholipin derivative by interrupting biosynthetic one or several modification step of xantholipin at least.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102512627A CN102010871A (en) | 2010-08-11 | 2010-08-11 | Biosynthesis gene cluster of xantholipin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102512627A CN102010871A (en) | 2010-08-11 | 2010-08-11 | Biosynthesis gene cluster of xantholipin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102010871A true CN102010871A (en) | 2011-04-13 |
Family
ID=43841201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102512627A Pending CN102010871A (en) | 2010-08-11 | 2010-08-11 | Biosynthesis gene cluster of xantholipin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102010871A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820474A (en) * | 2012-11-16 | 2014-05-28 | 中国医药集团总公司四川抗菌素工业研究所 | Biosynthesis gene cluster of polyenoid and polyol macrolide compound |
| CN105237544A (en) * | 2015-10-10 | 2016-01-13 | 上海交通大学 | Xantholipin B and preparation method and use thereof |
-
2010
- 2010-08-11 CN CN2010102512627A patent/CN102010871A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820474A (en) * | 2012-11-16 | 2014-05-28 | 中国医药集团总公司四川抗菌素工业研究所 | Biosynthesis gene cluster of polyenoid and polyol macrolide compound |
| CN105237544A (en) * | 2015-10-10 | 2016-01-13 | 上海交通大学 | Xantholipin B and preparation method and use thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sucipto et al. | Exploring Chemical Diversity of α‐Pyrone Antibiotics: Molecular Basis of Myxopyronin Biosynthesis | |
| CN102144030A (en) | Pyripyropene A biosynthetic gene | |
| CN105200072B (en) | The biological synthesis gene cluster of aromatic polyketones class atypia square ring element fluostatins a kind of and its application | |
| CN102181470B (en) | Method for improving yield of Streptomyces antibiotics and plasmid thereof | |
| Meiser et al. | DKxanthene biosynthesis—Understanding the basis for diversity-oriented synthesis in myxobacterial secondary metabolism | |
| CN103131716A (en) | Biosynthetic gene cluster of salinomycin and application thereof | |
| JP6882330B2 (en) | Carrimycin biosynthetic gene cluster | |
| CN101402929B (en) | A alkali-fast sorangium cellulosum and uses of the same in producing epothilone | |
| CN104017839B (en) | Method for manufacturing a pyripyropene | |
| Suroto et al. | Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis | |
| CN103820474A (en) | Biosynthesis gene cluster of polyenoid and polyol macrolide compound | |
| CN102703495A (en) | Method for improving yield of streptomycete antibiotic and plasmid thereof | |
| CN102660488B (en) | Genetic engineering bacterium capable of promoting biological synthesis of medermycin and application thereof | |
| CN102010871A (en) | Biosynthesis gene cluster of xantholipin | |
| CN102719388A (en) | Method for improving yield of streptomyces antibiotics and plasmids thereof | |
| CN103224905A (en) | Identification and characterization of the spinactin biosysnthesis gene cluster from spinosyn producing saccharopolyspora spinosa | |
| CN103215282A (en) | Biosynthesis gene cluster of kosinostatin and application thereof | |
| CN102911957B (en) | Biosynthesis gene cluster of griseoviridin and viridogrisein and application of biosynthesis gene cluster | |
| CN102373171B (en) | Nucleoside antibiotic A201A superior strain and construction method thereof | |
| CN112795587B (en) | Escherichia coli engineering bacteria producing surfactant, construction method and application thereof | |
| CN104911196A (en) | Trioxacarcin biosynthetic gene cluster and application thereof | |
| CN102732534B (en) | Biosynthetic gene cluster of xiamycin A and oxiamycin, and application thereof | |
| CN102174531B (en) | Biosynthetic gene cluster of Yatakemycin | |
| CN102884187A (en) | Nucleic acid structure containing a pyripyropene biosynthesis gene cluster and a marker gene | |
| CN104427870B (en) | UK 2 biosynthesis gene and the method improving UK 2 productivity ratio using it |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110413 |