CN110157724A - Herbicidin biological synthesis gene cluster and its application - Google Patents
Herbicidin biological synthesis gene cluster and its application Download PDFInfo
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- CN110157724A CN110157724A CN201910471680.8A CN201910471680A CN110157724A CN 110157724 A CN110157724 A CN 110157724A CN 201910471680 A CN201910471680 A CN 201910471680A CN 110157724 A CN110157724 A CN 110157724A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
Abstract
The invention belongs to technical field of pharmaceutical biotechnology, and in particular to a method of for improving fermentation herbicidin F and the like yield, the method are as follows: be overexpressed HcdR2 gene in the host strain of fermenting and producing herbicidin F and the like.The host strain is streptomycete, preferably S.mobaraensis US-43 bacterial strain.The HcdR2 is the transcript regutation protein of LuxR protein family, and amino acid sequence is shown in SEQ ID NO.1.The invention further relates to the structurally similar compounds of one group of new herbicidin F.
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, in particular to a kind of Herbicidin biological synthesis gene cluster
And its application.
Background technique
Streptomyces mobaraensis US-43 (is named as S.verticillus before
Var.pingyangensis n.var) it is an isolated streptomycete in Chinese Pingyang. Zhejiang soil, it can be produced
A series of raw glycopeptide antibiotics identical with bleomycin property[1-3], wherein pingyangmycin and boanmycin are
Through being used as anti-tumor drug by SFDA approval.As laboratory preservation strain, we study the bacterial strain under the conditions of different fermentations
Secondary metabolite, piericidin A1 and a series of isocoumarins chemical combination are had discovered that in its fermentation liquid
Object.In addition, detecting the microcomponent of one of fermentation liquid by LC-MS method, this group of lease making UV, MS/MS data authentication is pushed away
Surveying is herbicidin F.
Herbicidins (Fig. 1) is the nucleoside antibiotic being derived by adenosine, and such compound has unique
Furano-pyrano-pyran tricyclic core structure, tricyclic structure postmenstruation processing modification, can be added different side chain radicals
Group, thus structure-rich.According to reports, in S.saganonensis[4-7], S.sp.L-9-10[8], S.scopuliridis
RB72[9]And S.sp.CB01388[10]It being capable of isolated herbicidins compound in these bacterial strains.Herbicidinsization
Closing object has multiple biological activities, and herbicidins compound is in addition to having selection activity of weeding to dicotyledon[4], also have
There are significant algae-resistant and antifungal activity[6], nearest document report herbicidin skeleton structure also has anti-
Cryptosporidium activity[10].The chemical structure and multiple biological activities of Herbicidins complexity cause us to it
Analogue and biosynthesis mechanism are probed into.
The structure of Herbicidin F has determined[6], but before this work starts, biological synthesis gene cluster is also not
It appears in the newspapers.Aureonuclemycin (Figure 1) is the analogue of herbicidin F, their tricyclics having the same
Nuclear structure, but more tiglyl groups and two methyl in herbicidin F ratio aureonuclemycin chemical structure
Group.Tang Gongli seminar[11]One patent report biological synthesis gene cluster of aureonuclemycin,
Aureonuclemycin is catalyzed and synthesized by 4 key genes (anmB, anmC, anmD and anmE), nearest one about
The function of 4 key genes is verified in the article of herbicidins biosynthesis pathway[12], this 4 key genes are to seek
Identification herbicidins biological synthesis gene cluster is looked for provide important clue.Pass through bioinformatic analysis, Wo Men
The gene order homologous with above-mentioned 4 key genes is had found in S.mobaraensis US-43 genome, and then is successfully positioned
With the biological synthesis gene cluster hcd for identifying herbicidin F, the gene cluster with it has been reported that responsible herbicidins it is raw
Her (the S.sp.L-9-10 of object synthesis[13]) and hbc (S.sp.KIB-027[12]) two gene cluster very high homologies.In addition, passing through
It is overexpressed analytical proof, approach specific regulatory control factor hcdR2 has played positive tune in the biosynthesis of herbicidin F
Control effect is overexpressed in bacterial strain in hcdR2, and the yield of herbicidin F significantly improves, and carries out NMR for herbicidin F
Structural Identification is provided convenience.
At the same time, we are overexpressed in bacterial strain fermentation liquor in hcdR2 using GNPS tool and find novel herbicidins
Analog.Based on similar structures can be generated in LC-MS similar fragment it is assumed that being generated according to MS/MS data related
Molecular network figure[14], and the otherness between molecule is visualized[15], thus the similar chemical combination of more efficient discovery
Object[16-19].We analyze the secondary metabolite that hcdR2 is overexpressed in bacterial strain fermentation liquor by LC-MS/MS, flat using GNPS
Platform further explores herbicidins analog.This project has parsed the MS/MS of 6 kinds of novel herbicidins compounds for the first time
Data, nuclear magnetic resoance spectrum (1H NMR) data further demonstrate the structure of herbicidin O.
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Summary of the invention
Present invention firstly relates to a kind of method for improving fermentation herbicidin F and the like yield, the sides
Method are as follows: HcdR2 gene is overexpressed in the host strain of fermenting and producing herbicidin F and the like.
The host strain is streptomycete, preferably S.mobaraensis US-43 bacterial strain.
The HcdR2 is the transcript regutation protein of LuxR protein family, amino acid sequence such as SEQ ID NO.1 institute
Show, its coding nucleotide sequence is as shown in SEQ ID NO.2;
SEQ ID NO.1:
MLVGRECELRRLAEVTAGVRQGRSHALVLRGPAGVGKSALLQHLADHAGPGVRVLSTVGVESEAELPF
AALHQLLRPLLGAADALPPPQSAALRSAFGMDTAPADRFLVALAALTLLSEASRAEPLILLVDDAHWLDSPSADAL
LFVARRLGAEGVAVVFAVRDGSQPFPAPGVPDLRLSPLEADAARSLLTHLLPEAAPAVRERLLREAGGNPLALVEL
SAALSPEQLGGAAALPDELPLTERLQRLFRCRATSLLAEPGNALLLTAAEGDGDLAVIVRAAGDTERALHELSAAA
TAGLVSLDPERVRFRHPLVRSAVYQGAPLSERRAAHLALADALGEGDDRHVWHLAAAAVGPDDRVAHLLAEMAGRS
RRSGGVATATKALRRAAALVSTPRDRARLLVDAAECAWKAAETAQAEALLNEAEPLSDVPALRARLVQVRGAIAHA
SGDPAVACLILLEGARLVQEDDPRLACETLVMAARSAWVADDPARLAEIAGLLARLPAVTPDVRDRFVAHFRYLAG
LSGHAAPDARHDGTALEAHGGGPEGKTGADGKDVPVIWLSGTDPRPWVWPPTFLPYLLHATEPLRDAHQHAVDTLR
RNGAIGSLPMSLAPLVALQLVTGPWPTATANGTEALSLALETGQLGAASHLRAMLAWLAAAQGDGDRCHELARESL
EISTPRRIASAIALAHWAQGLNALAEGQPLNAVKLLAEVCSPEGGAGHFMLRWIVLPDFVEACVRAGEPERAHHVL
TDRDPLHPVPANHPQLRSGWHRSRALLATGEEAEDLFLAALADTGLSPFETGRTHLLYGEWLRRHRRIKPAREQLH
LAEAQLDSVGALPWAELARAELRAAGGRSTQGPSTAAVPMGGEQRLTARELQVMRLAAQGLSNGEIAARLFLSPRT
VGYHLYKIFPKLGVTSRSQLYGRSSG。
The invention further relates to one group of compounds similar with herbicidin F, shown in chemical structure such as following formula (1):
R1 are as follows:-CHO-R4, R4 are linear chain or branched chain alkane or-C (CH containing 1~4 C3)=CHCH3;
R2, R3 are H or CH3;
Preferably, R1 are as follows:
Most preferably, the analog is following compound:
(1) R1 isR2 is CH3, R3 H, molecular weight 522;
(2) R1 isR2 is CH3, R3 CH3, molecular weight 510;
(3) R1 isMolecular weight 510 or 524;
(4) R1 isMolecular weight 524;
The invention further relates to the sides of herbicidin F and the compound similar with herbicidin F described in fermenting and producing
Method, the method are as follows:
(1) the HcdR2 gene is overexpressed in host strain;
(2) host strain described in fermenting and producing;
(3) described herbicidin F and the like is separated.
The method of host strain described in fermenting and producing described in step (2) are as follows: use secondary liquid fermentation process, fermentation training
Support base are as follows: 0.3% high nitrogen Dried Corn Steep Liquor Powder, 2% soybean cake powder, 2.5% glucose, 2% starch, 2% maltose, 0.2%
K2HPO4, 0.3%NaCl.
The secondary liquid fermentation parameter are as follows:
The spore inoculating of host strain is in seed fermentation liquid, and 28 DEG C, 220rpm is cultivated 48 hours;
Seed fermentation liquid is inoculated in liquid fermentation medium, and for liquid fermentation medium in 28 DEG C, 220rpm cultivates 7
It.
Separation described in step (3) are as follows: the supernatant of fermentation liquid is by C18Chromatography column enrichment, respectively with 50% He
The elution of 100% methanol solution.
The invention further relates to the application of the analog of the herbicidin F in medicine preparation, the drug is
Drug that is antimycotic or inhibiting tumour.
Detailed description of the invention
The chemical structure and biological synthesis gene cluster of Fig. 1, Aureonuclemycin and herbicidin F.
(A) chemical structure of Aureonuclemycin and herbicidin F (compound 1).
(B) the biological synthesis gene cluster anm and hcd of Aureonuclemycin and herbicidin F;Hbc and her is
Herbicidins biological synthesis gene cluster through reporting.The gene cluster top gene that the place that lines mark is that FIMO is analyzed
Spacer region.Black lines indicate that the spacer region contains consistency binding sequence;Grey lines indicate the spacer region without matched one
Cause property sequence.
Fig. 2, it is overexpressed influence of the transcription regulaton factor to herbicidin F biosynthesis and expression conditions.
(A) HPLC analyzes S.mobaraensis US-43 and is overexpressed the yield of herbicidin F in bacterial strain.i,
S.mobaraensis US-43;ii,US43/pSET152;iii,US43/pL-hcdR2;iv,US43/pL-hcdR1;v,
US43/pL-hcdR3。
(B-D) the transcriptional expression situation US43/pL-hcdR2 (B), US43/pL-hcdR1 of different genes in bacterial strain are overexpressed
(C)US43/pL-hcdR3(D).Obtained data are all from three biology and repeat, and there are two multiple holes for each sample.
The transcriptional expression level of gene in control strain US43/pSET152 is defined as 1.
Fig. 3, the biological synthesis gene cluster for finding other 15 similar hcd in GenBank using genome mining.
The gene cluster top intergenic region that the place that lines mark is that FIMO is analyzed.Black lines indicate that the spacer region contains unanimously
Property binding sequence;Grey lines indicate the spacer region without matched consensus sequence.
DNA binding domain compares the consistency binding sequence of analysis result and prediction in Fig. 4, LuxR regulatory protein.(A)
The DNA binding domain structure alignment of homologous protein in DNA binding domain and different strains in HcdR2.The sequence of red collimation mark note is one
Cause sequence;The sequence of yellow collimation mark note is the similar sequence of physicochemical properties.The secondary structure sequence of top is from TraR
The three-dimensional structure of (PDB 1L3L).The GenBank accession numbers of these LuxR albumen is as follows: S.sp.L-9-10
(Her12,RYJ25228.1),S.scopuliridis RB72(PVE09951.1),S.sp.NRRL F-5135(WP_
078854789.1),S.acidiscabies strain a10(GAQ51936.1),S.sp.V2(PWG13438.1),
S.ipomoeae 91-03(EKX64212.1),S.sp.NRRL S-31(WP_079165723.1),S.mobaraensis
NBRC 13819(EMF01239.1),S.sp.CB02959(PJN35945.1),S.albulus CCRC 11814
(EPY92754.1),S.caeruleatus NRRL B-24802(KUN91385.1),S.corchorusii DSM 40340
(KUN18075.1) the HcdR2 consensus-binding site of (B) MEME Suite platform prediction.Reverse arrow indicates binary symmetric.
The guidance of Fig. 5, GNPS tool finds novel herbicidin analog.(A) hcdR2 is detected by LC-MS method
The molecular compound being overexpressed in bacterial strain constitutes molecular network.(B) it is found using herbicidin F as target potential
Herbicidin analog, and form molecular network figure.The molecular network figure contains quasi-molecular ion [M+H]+M/z value from
508 to 536 10 molecules.(C) it is analyzed according to GNPS as a result, having found 10 peak herbicidin (1- in LC-MS map
10)。
The structure and (+)-ESI-MS/MS data of Fig. 6,10 herbicidin analogs.(A) MS/ of 10 compounds
MS analyzes result.Parent ion is represented by dashed line, and it is identical to correspond to molecular weight color in color and GNPS.It is shown as diagnosing in figure
Quasi-molecular ions.(B) the tentative structure of compound 1-9, red font herbicidin analog are novel herbicidin analog.
Fig. 7,7 compounds 1,2,31H NMR compares (solvent DMSO-d6)。
Fig. 8, S.mobaraensis US-43 draft genome and bioinformatic analysis prediction result.
The MS/MS fragment of Fig. 9, compound herbicidin F
The HPLC of Figure 10, compound 1,2,3, UV and HRMS map
Specific embodiment
MATERIALS METHODS
Bacterial strain, plasmid and condition of culture
Wild type S.mobaraensis US-43 herein derives bacterium there are also it to be listed in table 2.
Table 2, bacterial strain and plasmid
Amr, apramycin (apramycin) resistance;Kmr, kanamycins (kanamycin) resistance;Cmr, chloramphenicol
(chloramphenicol) resistance
Wild type S.mobaraensis US-43 is an isolated streptomycete from Chinese Pingyang. Zhejiang soil,
S.mobaraensis US-43 wild mushroom and its derivative bacterium in 28 DEG C constant temperature incubation 7 days, the breeding culture of bacterial strain uses solid
S5 culture medium[32], engagement, which is shifted, uses MS culture medium[33], bacterial strain extract genomic DNA when use liquid φ culture medium[34]。
Compound herbicidin F is synthesized using secondary liquid fermentation process, (0.3% is high for liquid seeds fermentation medium
Nitrogen Dried Corn Steep Liquor Powder, 2% soybean cake powder, 2.5% glucose, 2% starch, 2% maltose, 0.2%K2HPO4, 0.3%NaCl)
Firsts and seconds fermentation is respectively used to fermentation medium (consistent with liquid seeds fermentation medium).
Escherichia coli DH5α[35]As general escherichia coli cloning host, E.coli ET12567/
pUZ8002[36]It is for carrying out engagement transfer between Escherichia coli and streptomycete[33]Host, the two cultivated using LB
Base[35]37 DEG C of constant temperature incubations.When needing antibiotic, working concentration is as follows: A Pu mycin (apramycin, Am, 50 μ g/ml),
Ammonia benzyl mycin (ampicillin, Amp, 100 μ g/ml), kanamycins (kanamycin, Km, 50 μ g/ml), chloramphenicol
(chloramphenicol, Cm, 25 μ g/ml).
The building of hcdR1, hcdR2 and hcdR3 gene overexpression plasmid
In order to use primer pL-hcdR1-F/pL- for hcdR1 gene overexpression in S.mobaraensis US-43
HcdR1-R expands complete hcdR1 gene order.
The PCR product of hcdR1 gene is cloned into the site NdeI-BamHI of pL646.Carrier pL646 is from integrated
Plasmid pSET152 can be integrated into streptomyces gene group without streptomycete replicon containing the site φ C31attP
The SD sequence of strong promoter ermE*p and tuf1 gene is contained in multiple cloning sites upstream in the site attB.
Using same strategy, hcdR2 is cloned into the NdeI-BamHI and NdeI-XbaI of pL646 with hcdR3 gene respectively
Site.Thus recombinant expression pL-hcdR1, pL-hcdR2 and pL-hcdR3, the inverted importing of recombinant plasmid is obtained
E.coli ET12567/pUZ8002, and S.mobaraensis US-43 is directed respectively by engaging transfer.By plasmid
PSET152 is imported in S.mobaraensis US-43 simultaneously, as control.
The analysis of product herbicidin F
S.mobaraensis US-43 wild-type strain and its derivative strain are in 28 DEG C of constant temperature incubations of solid S5 culture medium
7 days.By the spore inoculating of S.mobaraensis US-43 and its derivative in 100mL seed fermentation liquid, 28 DEG C, 220rpm
Culture 48 hours.5mL seed fermentation liquid is taken to be inoculated in 100mL liquid fermentation medium.Liquid fermentation medium in 28 DEG C,
220rpm is cultivated 7 days.Obtained supernatant is analyzed using LC-MS, in order to analyze the analog of herbicidin F, hair
The supernatant of zymotic fluid is by Sep-Pak C18Classic Cartridge (Waters Associates) enrichment, respectively with 50% He
The elution of 100% methanol solution.Sample is through Agilent 1100instrument (Agilent Technologies, Santa
Clara, CA, USA) HPLC detection is carried out, using C18column (Agilent, 150mm × 4.6mm, 5 μm), Detection wavelength is
210nm and 254nm.Testing conditions are as follows, mobile phase: CH3OH-H2O;Flow velocity: 1mL/min;Using gradient elution, 0-5min,
5%CH3OH;5-45min, 5%-100%CH3OH;45-55min, 100%;56-60min, 5%CH3OH。
RT-PCR transcription analysis
The thallus for having cultivated the S.mobaraensis US-43 of 48h in fermentation medium is collected, freezes and protects in liquid nitrogen
It deposits.According to extracting method (Promega), bacterial strain RNA is extracted with TRIzol reagent, is handled with DNaseI and removes remaining chromosome
DNA.With the quality and concentration of the RNA that 8000 spectrophotometer of NanoDrop (Thermo Scientific) measurement is extracted.With 2
μ g total serum IgE is that template is used for reverse transcription, is utilizedOne-Step gDNA Removal and cDNA Synthesis
SuperMix (Transgen) amplifies genetic fragment from target gene, utilizes Real-Time PCR Detection
System (Bio-Rad) is detected, and each reaction system includes 12.5 μ L FastStart UniversalGreen
Master (ROX) (Roche), 2.5 μ L templates, 5 μ L primers, 5 μ L RNase-free H2O。
Bioinformatic analysis
Using hcdB/C/D/E as target spot, potential herbicidin/aureonuclemycin biology is excavated using BLASTP
Synthetic gene cluster.Each gene in each gene cluster is labeled and annotates, and analyzes its three-dimensional using HHpred and BLASTP
Structure and conservative structural domain.Intergenic region in each gene cluster is selected out and is submitted to MEME Suite sever
Carry out MEME-ChIP analysis.Collect give a mark in each gene cluster highest sequence and submit for MEME analyze to obtain one
Consensus sequence.In order to further verify the consensus sequence of discovery, FIMO analysis is carried out to all intergenic regions, is found
With the matched individual of the consensus sequence.
Global Natural product Social Molecular Networking(GNPS)
To obtain the LC-MS/MS data for GNPS analysis, using 4006 column of macroporous absorbent resin to US-43/pL-
HcdR2 fermentation liquid is enriched with, and is eluted respectively with 30% and 80% aqueous acetone solution.It is dense to the eluent pressurization of 80% acetone
Contracting, is fractionated using ODS column.Component containing herbicidins is synthesized into crude extract.Crude extract is analyzed through LC-MS,
Through Agilent 1200 (Agilent Technologies, Santa Clara, CA, USA) and ion trap mass spectrometer
(Thermo Fisher Scientific, Waltham, MA, USA), using VP-ODS column (150mm × 4.6mm, 5 μm,
SHIMADZU), flow velocity 1mL/min, 60 minutes gradient elutions (with upper consistent).LC-ESI (+) MS/MS data are collided with 35eV
Energy is obtained as .raw file format, is converted into .mzxml file using the MS conversion program of ProteoWizard 3.0
Format simultaneously uploads to MassIVE server (massive.ucsd.edu).
According tohttps://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash2.jspWebsite provides
Guidance, data are analyzed using GNPS molecular network tool.
The purifying and characterization of compound 1-3
In order to isolate and purify to obtain novel herbicidin analog, bacterial strain US43/pL-hcdR2 is overexpressed to hcdR2 and is expanded
Big fermentation.Fermentation liquid is centrifuged off mycelium, supernatant (about 4L) is passed through into 4006 column of macroporous absorbent resin (about 400mL),
After being washed with water, active material is afforded using gradient elution (30%, 80%, 100% aqueous acetone solution), three groups is obtained and evaporates
Point, Fr 1 arrives Fr 3.According to HPLC analysis as a result, we have found herbicidin F and its similar in Fr 2 (1.197g)
Object.We utilize reversed flash chromatography column (RediSep column:40g C18), with 14.8% acetonitrile containing 0.01%TFA
Aqueous solution separates Fr 2, obtains 5 fraction Fr 2-1 to Fr 2-5.Pass through semi-preparative HPLC (ReproSil-Pur Basic-
C18column, 5 μm, 250 × 10mm) Fr 2-3 (262mg) is isolated and purified, it obtains compound 1 (11mg), 2 (1mg), 3
(0.5mg).Using SHIMADZU LC-20A instrument analyze sample, chromatographic column be ReproSil-Pur Basic-C18 (5 μm,
150 × 4.6mm), using eluent identical with half preparation HPLC, Detection wavelength 254nm.HRESIMS data are by being equipped withG2-S QTof'sUPLC detects to obtain.Bruker spectrometer detects to obtain NMR data, and the solvent of compound is
DMSO-d6Or CD3OD。
Compound 11H and 13C NMR:1H NMR(600MHz,CD3OD)δ8.36(s,1H,H-2),8.09(s,1H,
), H-8 6.71 (q, J=7.1Hz, 1H, H-3 "), 6.09 (d, J=1.6Hz, 1H, H-1 '), 5.00 (d, J=3.2Hz, 1H, H-
8 '), 4.52 (dd, J=10.3,5.7,1H, H-6 ') and and 4.50 (d, J=1.8Hz, 1H, H-3 '), 4.45 (s, 1H, H-
10 '), 4.41 (q, J=2.4Hz, 1H, H-4 '), 4.30 (dd, J=3.2,1.0Hz, 1H, H-9 '), 4.08 (d, J=1.1Hz,
1H,H-2’),3.61(s,3H,H-11’-OCH3),3.41(s,3H,H-2’-OCH3),2.30–2.22(m,2H,H-5’),1.90
(d, J=7.0Hz, 3H, H-4 "), 1.85 (s, 3H, H-5 ")13C NMR(150MHz,CD3OD)δ171.4(C-11’),167.2
(C-1”),154.4(C-6),150.3(C-2),149.5(C-4),142.1(C-3”),141.8(C-8),128.6(C-2”),
119.9(C-5),93.5(C-7’),91.8(C-2’),89.1(C-1’),79.4(C-4’),78.4(C-10’),74.7(C-
3’),72.0(C-8’),70.6(C-9’),66.7(C-6’),58.5(C-2’-OCH3),52.8(C-11’-OCH3),26.7(C-
5’),15.2(C-4”),12.5(C-5”)。
Compound 11H NMR (there are tautomerisms for hemiacetal and free carbonyl form):1H NMR(500MHz,DMSO)δ
8.37(s,1H,H-2)/,8.35(s,1H,H-2),8.19(s,2H,H-NH2) 7.88 (s, 1H, H-8), 6.63 (qd, J=6.9,
1.2Hz, 1H, H-3 "), 5.93 (d, J=1.9Hz, 1H, H-1 ')/6.00 (d, J=1.6Hz, 1H, H-1 '), 4.91 (d, J=
3.1Hz, 1H, H-8 '), 4.46 (s, 1H, H-10 '), 4.40 (d, J=2.2Hz, 1H, H-3 '), 4.33 (dd, J=11.6,
5.4Hz, 1H, H-6 '), 4.21 (d, J=1.8Hz, 1H, H-4 '), 4.17 (s, 1H, H-2 ') 4.16 (dd, J=3.1,0.9Hz,
1H,H-9’),3.50(s,3H,H-11’-OCH3)/3.67(s,3H,H-11’-OCH3),3.32(s,3H,H-2’-OCH3)/3.34
(s,3H,H-2’-OCH3), 2.16-2.05 (m, 2H, H-5 '), 1.89 (dd, J=7.1,1.0Hz, 3H, H-4 "), 1.81 (s,
3H,H-5”).
Compound 21H NMR (there are tautomerisms for hemiacetal and free carbonyl form):1H NMR(600MHz,DMSO-
d6)δ8.33(s,1H,H-2)/8.30(s,1H,H-2),8.04(s,2H,H-NH2)7.90(s,1H,H-8)/7.88(s,1H,H-
8), 6.61 (dd, J=13.7,6.8Hz, 1H, H-3 "), 5.91 (s, 1H, H-1 ')/5.88 (s, 1H, H-1 '), 4.97 (d, J=
4.1Hz, 1H, H-8 ')/4.90 (d, J=3.1Hz, 1H, H-8 '), 4.64-4.18 (m, 6H, H-4 ', H-6 ', H-10 ', H-2 ',
H-3’,H-9’,signals from hemiacetal form and free carbonyl form overlapped with
each other),3.50(s,3H,H-11’-OCH3)/3.48(s,3H,H-11’-OCH3),2.24–1.93(m,2H,H-5’),
1.88 (d, J=7.1Hz, 3H, H-4 ")/1.85 (d, J=7.1Hz, 3H, H-4 "), 1.82 (s, 3H, H-5 ")/1.81 (s, 3H,
H-5”H).
Compound 31H NMR (there are tautomerisms for hemiacetal and free carbonyl form):1H NMR(500MHz,DMSO-
d6)δ13.00(s,1H,H-COOH),8.26(s,1H,H-2),7.81(s,1H,H-8),7.72(s,2H,H-NH2),6.72(m,
1H, H-3 "), 5.95 (d, J=1.9Hz, 1H, H-1 ')/5.92 (d, J=1.9Hz, 1H, H-1 '), 4.97 (d, J=4.1Hz,
1H, H-8 ')/4.92 (d, J=3.1Hz, 1H, H-8 '), and 4.57-4.14 (m, 6H, H-4 ', H-6 ', H-10 ', H-2 ', H-3 ' and, H-
9’,signals from hemiacetal form and free carbonyl form overlapped with each
other),3.34(s,3H,H-2’-OCH3)/3.32(s,1H,H-2’-OCH3),2.20–1.93(m,2H,H-5’),1.89(dd,
J=7.0,0.9Hz, 2H, H-4 ")/1.87 (dd, J=7.1,0.8Hz, 2H, H-4 "), 1.81 (d, J=1.0Hz, 3H, H-5 ")
Herbicidin F biosynthesis base in embodiment 1, bioinformatic analysis identification S.mobaraensis US-43
Because of cluster (hcd)
S.mobaraensis US-43 is the more mature bacterial strain of this laboratory research (CPCC 204095), right for many years
The Metabolite the study found that it can also generate a large amount of other kinds of compounds in addition to glycopeptide antibiotics.In order to
System in depth excavates other types metabolite, we determine the whole genome sequence of S.mobaraensis US-43, benefit
With in its genome of antiSMASH 4.0.0 software prediction at least contain 47 secondary metabolite biological synthesis gene clusters[20]。
By carrying out LC-MS analysis to the bacterial strain fermentation liquor, detects microcomponent compound 1, analyzed through UV, MS/MS data, chemical combination
Object 1 is speculated as herbicidin F.
Though the structure of Herbicidin F it has been determined that but its biosynthesis pathway have not been reported.Compound
Aureonuclemycin (Figure 1A) and herbicidin F tricyclic core skeleton having the same, to study herbicidin F
Biological synthesis gene cluster, with the key gene (anmB, anmC, anmD and anmE) of aureonuclemycin biosynthesis
(Figure 1B) is target, and herbicidin biology that may be present is searched in S.mobaraensis US-43 genome and is closed
At gene cluster.Pass through BLASTP analysis and antiSMASH prediction, our gene in S.mobaraensis US-43
Four corresponding homologous sequences of key gene (anmBCDE) are had found in cluster (Figure 1B).Contain two in the downstream of the homologous sequence
A transmethylase, it is presumed that they are responsible for the modification of the methylation in herbicidin F structure;The Sequences upstream has one
β-ketoacyl synzyme, it is presumed that it is related to the biosynthesis of side chain tiglyl group in herbicidin F structure;
In addition to this nearby there are one transport protein, three transcription regulatory factors and some other albumen.We are by this gene cluster
It is named as hcd, thus it is speculated that hcd may be responsible for the biosynthesis of herbicidin F.The functional annotation of gene cluster hcd is shown in Table 2.
The functional annotation of table 2, gene cluster hcd
The confirmation of approach specific regulatory control albumen in embodiment 2, hcd
Transcriptional modulatory gene hcdR1, hcdR2 and hcdR3 there are three containing in the hcd gene cluster of prediction, we construct
The overexpression bacterial strain of each transcription regulator gene is detected by HPLC and is overexpressed herbicidin F in bacterial strain fermentation liquor
Change of production to judge whether the gene cluster hcd of prediction is responsible for the biosynthesis of herbicidin F, and further determines that
Whether hcdR1, hcdR2 and hcdR3 are approach specific regulatory control genes in herbicidin F biosynthesis.We by each
Transcriptional modulatory gene is cloned into pSET152[21]The expression plasmid carrier pL646 being derived[22]On, contain φ C31attP
Point can be integrated into the site attB in streptomyces gene group, multiple cloning sites upstream contain strong promoter ermE*p and
The SD sequence of tuf1 gene.Building is overexpressed plasmid pL-hcdR1, pL-hcdR2 and passes through the side for engaging and shifting with pL-hcdR3
Formula is directed respectively into S.mobaraensis US-43 wild type, obtains being overexpressed bacterial strain US43/pL-hcdR1, US43/
PL-hcdR2 and US43/pL-hcdR3.Plasmid pSET152 is imported into S.mobaraensis US-43 wild-type strain simultaneously
In obtain US43/pSET152 bacterial strain as experiment contrast bacterial strain.By wild-type strain S.mobaraensis US-43, bacterium is compareed
Strain US43/pSET152, is overexpressed bacterial strain US43/pL-hcdR1, US43/pL-hcdR2 and US43/pL-hcdR3 are in the same terms
Lower fermentation handles fermentation liquid with identical method, fermented liquid supernatant is tested and analyzed through HPLC.
HPLC is analyzed as the result is shown relative to wild-type strain S.mobaraensis US-43 and control strain US43/
PSET152, only in being overexpressed bacterial strain US43/pL-hcdR2, the yield of compound 1 significantly improves (Fig. 2A), is compound 1
Isolate and purify and provide convenience.Compound 1 is analyzed through MS/MS data, and compared with reported H NMR spectroscopy, determines chemical combination
Object 1 is herbicidin F.
Microcomponent confirmation in bacterial strain fermentation liquor is herbicidin F, and is only overexpressed bacterial strain in hcdR2
In US43/pL-hcdR2 fermentation liquid, the yield of herbicidin F is significantly improved, and shows that hcdR2 may be herbicidin F
The approach specificity positive regulation factor in biosynthesis.
Next, we are speculated in the middle using quantitative RT-qPCR technology detection overexpression bacterial strain is responsible for herbicidin F
The transcriptional expression situation of biosynthesis related genes.When we detect fermentation 48h in gene cluster hcd each gene it is opposite
Expression, analysis are overexpressed bacterial strain US43/pL- in hcdR2 the results show that compared with control strain US43/pSET152
In hcdR2, adjusts gene hcdR2 and hcdB~T-phase significantly improves expression;The relative expression of hcd1, hcd2 and hcd3
Horizontal substantially no variation (Fig. 2 B).
In US43/pL-hcdR1 and US43/pL-hcdR3, adjusts gene hcdR1 and hcdR3 relative expression levels raise
Obviously, other genes relative expression levels are without significant change (Fig. 2 C, 2D).Gene relative expression's feelings of quantitative RT-qPCR detection
The synthesis for being overexpressed herbicidin F in bacterial strain of condition and HPLC detection is horizontal consistent, this show hcdR1, hcdR2 with
In hcdR3, only hcdR2 is approach specificity positive regulation gene in herbicidin F biosynthesis.
At the same time, be overexpressed bacterial strain US43/pL-hcdR2 in hcd gene relative expression levels show hcdB~H and
HcdT is responsible for compound herbicidin F biosynthesis;The relative expression levels of hcd1~3 without significant change, show hcd1~
3 may be not engaged in the biosynthesis of herbicidin F.
Embodiment 3, HcdR2 are approach specific regulatory control albumen relatively conservative in herbicidin biosynthesis
Approach specific regulatory control albumen HcdR2 belongs to LuxR family, is analyzed by BLASTp, in the C-terminal of the regulatory protein
There is typical helix-turn-helix (HTH) DNA binding domain (DBD).We using hcdB/C/D/E as target,
Similar sequences are searched in GenBank, discovery contains hcd/hbc/her/anm similar genes cluster (figure at least in 15 plants of actinomyces
3), and wherein contain the transcript regutation protein for belonging to LuxR protein family in 11 gene clusters.It is adjusted in these LuxR families
In albumen, in addition in 13819 gene cluster of bacterial strain S.mobaraensis NBRC LuxR albumen (compare analysis with HcdR2,
99.78% amino acid similarity), remaining LuxR regulatory protein and HcdR2 compare analysis, and comparison result is shown in length range
Their interior amino acid similarities with 30-43%.HHpred and BLASTp analysis, analysis are carried out to these LuxR regulatory proteins
Their three-dimensional structure has very high similitude as the result is shown, is N-terminal AAA respectively including two relatively conservative regions
ATPase structural domain and C-terminal helix-turn-helix (HTH) DNA binding domain (DBD).
In addition, the DBD structural domain of the DBD structural domain of the homologous protein of these HcdR2 and LuxR family regulatory factor TraR
It compares analysis shows that going out whole homology, and use online ESPript[23]Platform shows four helical structures of HTH motif
Domain.HTH structural domain adjusts the expression of gene by the specific site DNA near combining target promoter.We are using online
MEME Suite 5.0.4[24-26]Program analyzes the intergenic region in each gene cluster, finds possible HcdR2 consistency knot
Close sequence.
48 intergenic regions picked out are analyzed first with the MEME-ChIP of MEME-Suite, from analysis
As a result the highest HcdR2 consistency binding sequence of selection marking in.This consistency binding sequence has potential palindromic sequence,
With LuxR[27], TraR[28], LasR[29], QscR[30]Etc. regulatory proteins known binding site it is consistent.Then we utilize FIMO
48 intergenic regions are scanned, searching can be with the matched sequence of motif.Search obtains 30 promoter regions can be with motif
Match and they are sorted into (Fig. 1 and 3) by p value.It include hcd in search, 14 gene clusters discovery including her, anm,
At least one size is the consistency binding sequence of 21-bp in these gene clusters, and is predominantly located in the promoter of hcdB
Region (Fig. 4 B).These results imply that the homologous protein of HcdR2 may be by adjusting in conjunction with the consensus sequence in different strains
Save the synthesis of herbicidin analog.It is interesting that we have found consistency binding sequence in 4 special gene clusters,
But it is not found the homologous protein of HcdR2 near the sequence, but is had found in the genome other positions of wherein 3 plants of bacterium
The homologous protein of HcdR2.We have also discovered binding site by the anmB promoter region in gene cluster anm.These the results show that
In the different strains containing similar hcd/hbc/her/anm gene cluster, herbicidins biosynthesis pathway specificity is adjusted
Mechanism may be conservative.This prompts us, can activate new similar hcd/ by being overexpressed the homologous protein of HcdR2
Hbc/her/anm gene cluster.
Embodiment 4 finds novel herbicidin class in hcdR2 overexpression bacterial strain fermentation liquor using molecular network tool
Like object
It being overexpressed in bacterial strain US43/pL-hcdR2 in hcdR2, the yield of herbicidin F is significantly improved, meanwhile,
We have found some in S.mobaraensis US-43 wild strain hair in being overexpressed bacterial strain US43/pL-hcdR2 fermentation liquid
The micro analog of the herbicidin that can't detect in zymotic fluid.A kind of effective tool of the GNPS as discovery noval chemical compound, we
It is utilized to analysis and finds the herbicidin analog that hcdR2 is overexpressed in bacterial strain fermentation liquor.US43/pL-hcdR2 bacterial strain hair
The LTQ XL that the crude extract of zymotic fluid is coupled through Agilent 1200 (Agilent Technology, Santa Clara, CA, USA)
Ion trap mass spectrometer tests and analyzes.Obtained LC-MS/MS data are uploaded into MassIVE platform (massive.ucsd.edu),
It is analyzed using the molecular network tool based on GNPS, generates molecular network figure.Use Cytoscape V3.5.1[31]Tool
Make molecular network relationship visualization.By the analysis to US43/pL-hcdR2 bacterial strain fermentation liquor crude extract, depict with compound
Molecular network figure centered on herbicidin F, this network contain 10 different moleculars of the m/z value from 508 to 536
(Fig. 5).We analyze LC-ESI (+) MS (Fig. 5) and ESI (+)-MS/MS (Fig. 6 A) data of these compounds, identification
5) and 6 kinds of novel herbicidins structure (compounds 3/ (the compound 1,2/3,4 and of herbicidins compound known to 4 kinds out
2,6-10) (Fig. 6 B).
Compound 1, molecular weight 536 are herbicidin F according to its MS/MS fragmentation patterns (Fig. 6,9) confirmation.?
In ESI (+)-MS/MS map of compound 1, m/z value is that two peaks of 418 (F3) and 283 (F6) respectively correspond [M-tigly-
2H2O+H]+With [M-tigly-adenyl-2H2O+H]+, the two peaks can be used as diagnosis daughter ion peak.M/z value is 518,
The peak of 500,454 (F1), 436 (F2), 319 (F4) and 301 (F5) respectively correspond [M-H2O+H]+, [M-2H2O+H]+, [M-
tigly+H]+, [M-tigly-H2O+H]+, [M-tigly-adenyl+H]+, and [M-tigly-adenyl-H2O+H]+, they with
F3, F6 constitute the characteristic peak in herbicidins compound MS/MS map together.
Compound 2 and 3 and herbicidin F (1) have similar MS/MS map, the standard having the same of compound 2 and 3
Molecular ion peak and fragment, and than compound 1 14Da small (Fig. 6), this shows in R2Or R3Methyl may be not present in place.In order to
The structure for further parsing them isolates and purifies to obtain compound 2 (1mg) and 3 (0.5mg).Compound 2 and 3 has at 260nm
There is maximum absorption wavelength, meets nucleoside compound characteristic absorption spectrum (Figure 10).According to high-resolution electrospray ionization mass spectrometry
(HR-ESIMS)[M+H]+M/z value is 522.1857, determines the molecular formula C having the same of compound 2 and 322H27O10N5, than
Herbicidin F lacks a CH2, this also further demonstrates the structure in R2Or R3Lack the supposition of a methyl in position.It utilizes1H NMR spectra further determines that the position of methyl, by collecting compound 2 and 31H H NMR spectroscopy obtains to assist determining first
The hydroxyl proton signal of base location.In DMSO-d6, compound 2 and 3 has two groups1H NMR signal, this phenomenon may be
Since there are hemiketal structure and free carbonyl forms in herbicidins compound there are caused by tautomerism, according to
Report[13], in D2There is mutually variation in the hemiketal structure and its free carbonyl form for forming B ring in O in herbicidin structure
Structure may cause map and two sets of nuclear magnetic signals be presented.For the ease of comparative analysis, the solvent of compound 1 is changed to DMSO-d6。
By comparing compound 2 and 11H H NMR spectroscopy (Fig. 7) finds to lack H-2 '-OCH in compound 23Signal, it was demonstrated that compound 2 exists
R2Place lacks a methyl, with herbicidin K structure having the same.By comparing compound 3 and 1,21H H NMR spectroscopy
(Fig. 7) has found to lack H-11 '-OCH in compound 33Signal contains H-11 '-COOH signal, it was demonstrated that compound 3 is in R3Place lacks
One methyl, and contain free carboxy at C-11 ', being analyzed and identified compound 3 is a kind of novel herbicidin F class
Like object, it is named as herbicidin O.
It is preliminary to infer compound 4-10's by the MS/MS fragment comparative analysis (Fig. 6) of compound 4-10 and compound 1-3
Structure.According to the quasi-molecular ion peak (respectively than the 28Da small and 82Da of compound 1) and diagnostic fragment of compound 4 and 5, determinization
Conjunction object 4 is herbicidin G, and compound 5 is herbicidin B.Compound 6,7 and compound 2,3 F3 having the same
([M-tigly-2H2O+H]+,m/z 404)、F6([M-tigly-adenyl-2H2O+H]+, m/z 269) and fragment segment, show
Compound 6,7 and compound 2,3 are in R1The substituent group at place is different.The molecular weight of compound 6 is smaller than the molecular weight of compound 7
14Da illustrates the R of compound 61Place lacks a methyl, and contains an isobutyl group, the substitution base phase with herbicidin E
Together.Since the amount for purifying obtained compound 6 and 7 is fewer, the position of methyl can't be determined in R2Or R3.8 He of compound
9 and compound 1 (herbicidin F) F3 ([M-tigly-2H having the same2O+H]+,m/z 418)、F6([M-tigly-
adenyl-2H2O+H]+, m/z 283) and cleaved fragment, only R1The substituent group at place is different.The molecular weight ratio of compound 8
The small 26Da of the molecular weight of herbicidin F shows compound 8 in R1The substituent group of position is propiono.The standard of compound 9 point
The daughter ion ratio herbicidin small 12Da of F, shows the R of compound 91May contain an isobutyl group, with herbicidin E and
The substituent group of compound 6 is identical.The quasi-molecular ion ratio herbicidin small 18Da of F of compound 10, and and herbicidin
F diagnosis daughter ion fragments F3 ([M-tigly-2H having the same2O+H]+,m/z 418)、F6([M-tigly-adenyl-2H2O
+H]+, m/z 283), illustrate there is a H in compound 102O molecule is lost.Since the amount of the compound isolated and purified compares
Few, the structure of these herbicidins analogs cannot determine completely, but GNPS is as discovery herbicidins analog
Effective tool plays huge advantage really.
Finally, it should be noted that above embodiments are used only as helping skilled in the art to understand essence of the invention,
Limiting the scope of the present invention that it goes without doing.
SEQUENCE LISTING
<110>Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
<120>Herbicidin biological synthesis gene cluster and its application
<130> CP11902318C
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 930
<212> PRT
<213>HcdR2 amino acid sequence
<400> 1
Met Leu Val Gly Arg Glu Cys Glu Leu Arg Arg Leu Ala Glu Val Thr
1 5 10 15
Ala Gly Val Arg Gln Gly Arg Ser His Ala Leu Val Leu Arg Gly Pro
20 25 30
Ala Gly Val Gly Lys Ser Ala Leu Leu Gln His Leu Ala Asp His Ala
35 40 45
Gly Pro Gly Val Arg Val Leu Ser Thr Val Gly Val Glu Ser Glu Ala
50 55 60
Glu Leu Pro Phe Ala Ala Leu His Gln Leu Leu Arg Pro Leu Leu Gly
65 70 75 80
Ala Ala Asp Ala Leu Pro Pro Pro Gln Ser Ala Ala Leu Arg Ser Ala
85 90 95
Phe Gly Met Asp Thr Ala Pro Ala Asp Arg Phe Leu Val Ala Leu Ala
100 105 110
Ala Leu Thr Leu Leu Ser Glu Ala Ser Arg Ala Glu Pro Leu Ile Leu
115 120 125
Leu Val Asp Asp Ala His Trp Leu Asp Ser Pro Ser Ala Asp Ala Leu
130 135 140
Leu Phe Val Ala Arg Arg Leu Gly Ala Glu Gly Val Ala Val Val Phe
145 150 155 160
Ala Val Arg Asp Gly Ser Gln Pro Phe Pro Ala Pro Gly Val Pro Asp
165 170 175
Leu Arg Leu Ser Pro Leu Glu Ala Asp Ala Ala Arg Ser Leu Leu Thr
180 185 190
His Leu Leu Pro Glu Ala Ala Pro Ala Val Arg Glu Arg Leu Leu Arg
195 200 205
Glu Ala Gly Gly Asn Pro Leu Ala Leu Val Glu Leu Ser Ala Ala Leu
210 215 220
Ser Pro Glu Gln Leu Gly Gly Ala Ala Ala Leu Pro Asp Glu Leu Pro
225 230 235 240
Leu Thr Glu Arg Leu Gln Arg Leu Phe Arg Cys Arg Ala Thr Ser Leu
245 250 255
Leu Ala Glu Pro Gly Asn Ala Leu Leu Leu Thr Ala Ala Glu Gly Asp
260 265 270
Gly Asp Leu Ala Val Ile Val Arg Ala Ala Gly Asp Thr Glu Arg Ala
275 280 285
Leu His Glu Leu Ser Ala Ala Ala Thr Ala Gly Leu Val Ser Leu Asp
290 295 300
Pro Glu Arg Val Arg Phe Arg His Pro Leu Val Arg Ser Ala Val Tyr
305 310 315 320
Gln Gly Ala Pro Leu Ser Glu Arg Arg Ala Ala His Leu Ala Leu Ala
325 330 335
Asp Ala Leu Gly Glu Gly Asp Asp Arg His Val Trp His Leu Ala Ala
340 345 350
Ala Ala Val Gly Pro Asp Asp Arg Val Ala His Leu Leu Ala Glu Met
355 360 365
Ala Gly Arg Ser Arg Arg Ser Gly Gly Val Ala Thr Ala Thr Lys Ala
370 375 380
Leu Arg Arg Ala Ala Ala Leu Val Ser Thr Pro Arg Asp Arg Ala Arg
385 390 395 400
Leu Leu Val Asp Ala Ala Glu Cys Ala Trp Lys Ala Ala Glu Thr Ala
405 410 415
Gln Ala Glu Ala Leu Leu Asn Glu Ala Glu Pro Leu Ser Asp Val Pro
420 425 430
Ala Leu Arg Ala Arg Leu Val Gln Val Arg Gly Ala Ile Ala His Ala
435 440 445
Ser Gly Asp Pro Ala Val Ala Cys Leu Ile Leu Leu Glu Gly Ala Arg
450 455 460
Leu Val Gln Glu Asp Asp Pro Arg Leu Ala Cys Glu Thr Leu Val Met
465 470 475 480
Ala Ala Arg Ser Ala Trp Val Ala Asp Asp Pro Ala Arg Leu Ala Glu
485 490 495
Ile Ala Gly Leu Leu Ala Arg Leu Pro Ala Val Thr Pro Asp Val Arg
500 505 510
Asp Arg Phe Val Ala His Phe Arg Tyr Leu Ala Gly Leu Ser Gly His
515 520 525
Ala Ala Pro Asp Ala Arg His Asp Gly Thr Ala Leu Glu Ala His Gly
530 535 540
Gly Gly Pro Glu Gly Lys Thr Gly Ala Asp Gly Lys Asp Val Pro Val
545 550 555 560
Ile Trp Leu Ser Gly Thr Asp Pro Arg Pro Trp Val Trp Pro Pro Thr
565 570 575
Phe Leu Pro Tyr Leu Leu His Ala Thr Glu Pro Leu Arg Asp Ala His
580 585 590
Gln His Ala Val Asp Thr Leu Arg Arg Asn Gly Ala Ile Gly Ser Leu
595 600 605
Pro Met Ser Leu Ala Pro Leu Val Ala Leu Gln Leu Val Thr Gly Pro
610 615 620
Trp Pro Thr Ala Thr Ala Asn Gly Thr Glu Ala Leu Ser Leu Ala Leu
625 630 635 640
Glu Thr Gly Gln Leu Gly Ala Ala Ser His Leu Arg Ala Met Leu Ala
645 650 655
Trp Leu Ala Ala Ala Gln Gly Asp Gly Asp Arg Cys His Glu Leu Ala
660 665 670
Arg Glu Ser Leu Glu Ile Ser Thr Pro Arg Arg Ile Ala Ser Ala Ile
675 680 685
Ala Leu Ala His Trp Ala Gln Gly Leu Asn Ala Leu Ala Glu Gly Gln
690 695 700
Pro Leu Asn Ala Val Lys Leu Leu Ala Glu Val Cys Ser Pro Glu Gly
705 710 715 720
Gly Ala Gly His Phe Met Leu Arg Trp Ile Val Leu Pro Asp Phe Val
725 730 735
Glu Ala Cys Val Arg Ala Gly Glu Pro Glu Arg Ala His His Val Leu
740 745 750
Thr Asp Arg Asp Pro Leu His Pro Val Pro Ala Asn His Pro Gln Leu
755 760 765
Arg Ser Gly Trp His Arg Ser Arg Ala Leu Leu Ala Thr Gly Glu Glu
770 775 780
Ala Glu Asp Leu Phe Leu Ala Ala Leu Ala Asp Thr Gly Leu Ser Pro
785 790 795 800
Phe Glu Thr Gly Arg Thr His Leu Leu Tyr Gly Glu Trp Leu Arg Arg
805 810 815
His Arg Arg Ile Lys Pro Ala Arg Glu Gln Leu His Leu Ala Glu Ala
820 825 830
Gln Leu Asp Ser Val Gly Ala Leu Pro Trp Ala Glu Leu Ala Arg Ala
835 840 845
Glu Leu Arg Ala Ala Gly Gly Arg Ser Thr Gln Gly Pro Ser Thr Ala
850 855 860
Ala Val Pro Met Gly Gly Glu Gln Arg Leu Thr Ala Arg Glu Leu Gln
865 870 875 880
Val Met Arg Leu Ala Ala Gln Gly Leu Ser Asn Gly Glu Ile Ala Ala
885 890 895
Arg Leu Phe Leu Ser Pro Arg Thr Val Gly Tyr His Leu Tyr Lys Ile
900 905 910
Phe Pro Lys Leu Gly Val Thr Ser Arg Ser Gln Leu Tyr Gly Arg Ser
915 920 925
Ser Gly
930
<210> 2
<211> 2793
<212> DNA
<213>HcdR2 coding nucleotide sequence
<400> 2
atgctggtcg gacgggagtg tgagttacgg cggttggcgg aggtcacggc cggcgtacgc 60
caggggcgca gccacgccct cgtactgcgc ggcccggccg gggtgggcaa gagcgcgttg 120
ctccagcacc tcgcggacca cgccggaccc ggggtgcggg tgctgtccac cgtcggcgtg 180
gagagcgagg ccgagctgcc cttcgccgcc ctgcaccagt tgctgcgccc gttgctcggc 240
gcggccgacg ccctgccgcc cccgcagagc gcggcgctgc gctcggcctt cggcatggac 300
accgcacccg ccgaccggtt cctggtggcg ctcgccgcgc tgacgctcct gtcggaggcg 360
tcccgcgcgg aaccgctgat cctgctggtg gacgacgccc actggctgga ctccccctcc 420
gccgacgccc tcctcttcgt ggcccggcgg ctcggcgccg aaggcgtcgc ggtggtcttc 480
gccgtacggg acgggtccca gccctttccg gcgccgggcg tgcccgacct gcgcctctcc 540
cccctggagg cggacgcggc ccggtcgctg ctcacccacc tgctgcccga agccgcgccc 600
gcggtgcgcg agcgactgct gcgggaggcg ggcggcaacc ccctcgccct ggtcgaactg 660
tccgccgccc tcagccccga acagctcggc ggcgcggcgg ccctaccgga cgaactgccg 720
ctgaccgagc ggctgcagcg gctcttccgc tgccgggcca ccagcctgct ggccgagccc 780
ggcaacgccc tgctgctgac cgccgccgag ggcgacggag acctggcggt catcgtccgc 840
gccgccgggg acaccgaacg cgccctgcac gagctgtccg ccgcggccac cgcggggctg 900
gtctccctgg acccggaacg ggtccggttc cgccaccccc tggtccgctc ggccgtctac 960
cagggcgcgc cgctcagcga acggcgcgcc gcgcacctcg ccctggccga cgcgctgggc 1020
gagggcgacg accggcacgt ctggcacctg gccgccgccg cggtgggccc cgacgacaga 1080
gtcgcgcacc tgctggccga gatggccggc cgctcccggc gctccggtgg agtggccacc 1140
gccaccaagg cactgcgccg ggccgccgca ctggtctcca ccccgcgcga ccgcgcccgg 1200
ttgctcgtcg acgccgccga gtgcgcctgg aaggccgccg agaccgccca ggccgaggcg 1260
ctgctcaacg aggccgagcc gctgtccgac gtgcccgcgc tgcgcgcccg gctcgtccag 1320
gtgcgcgggg ccatcgccca cgcctccggc gatccggccg tcgcctgcct gatcctgctg 1380
gagggtgcgc gcctcgtaca ggaggacgat ccccggctgg cctgcgagac gctggtgatg 1440
gcggcgcgtt cggcctgggt ggccgacgac ccggcgcggc tggcggagat cgccggtctg 1500
ctggcgcggc tcccggcggt gacaccggac gtccgggacc gcttcgtggc gcatttccgg 1560
tacctcgcgg gcctgtccgg ccacgcggcc ccggacgccc gccacgacgg tacggctctg 1620
gaggcgcacg gcggcgggcc ggaggggaag acgggcgccg acggcaagga cgtccccgtc 1680
atctggctgt ccggcaccga ccccagaccg tgggtgtggc cgcccacctt cctgccgtac 1740
ctcctccatg ccaccgagcc gttgcgcgac gcccatcagc acgcggtcga cacgctgcgc 1800
cgcaacggcg cgatcggctc cctcccgatg tccctggccc ctctcgtggc gctccagctc 1860
gtcaccggcc cctggcccac cgccaccgcc aacggcaccg aggccctctc cctggccctg 1920
gagaccggcc agctgggcgc cgcctcccac ctgcgggcga tgctcgcctg gctggccgcg 1980
gcacagggcg acggcgaccg ctgccacgaa ctggcccgcg agtcgctgga gatctccacc 2040
ccgcgccgga tcgcctccgc catcgccctg gcccactggg cgcagggcct caacgccctg 2100
gccgaggggc agcccctgaa cgcggtcaag ctgctggccg aggtctgttc ccccgagggc 2160
ggtgccggac acttcatgct gcgctggatc gtcctgcccg acttcgtcga ggcgtgcgtc 2220
agagccggcg agcccgagcg cgcccaccac gtgctgaccg accgcgaccc cctccacccg 2280
gtgccggcga accatccgca gctgcgctcc ggctggcacc gcagccgggc cctgctcgcc 2340
accggcgagg aagcggagga cctgttcctg gccgccctgg ccgacaccgg cctctccccc 2400
ttcgagaccg gccgcaccca cctgctctac ggcgagtggc tccgccgcca ccgccgcatc 2460
aaaccggccc gcgagcaact gcacctcgcc gaggcccaac tggacagcgt gggcgcgctg 2520
ccatgggcgg aactggcacg cgccgaacta cgggccgcgg gcggccgctc cacccagggg 2580
ccttcgacgg cggccgttcc catgggcggg gaacagcgtc tgacagcacg cgaactccag 2640
gtcatgcggc tcgccgcaca gggcctgagc aacggcgaga tcgccgcccg gctgttcctg 2700
agcccccgca ccgtcggcta tcacctctac aagatcttcc cgaagctggg ggtcacgtcg 2760
cggtcccagt tgtacggacg ctcctccggc tga 2793
Claims (9)
1. a kind of method for improving fermentation herbicidin F and the like yield, the method are as follows: in fermenting and producing
HcdR2 gene is overexpressed in the host strain of herbicidin F and the like.
2. the method according to claim 1, wherein
The host strain is streptomycete, preferably S.mobaraensis US-43 bacterial strain;
The HcdR2 is the transcript regutation protein of LuxR protein family, and amino acid sequence is shown in SEQ ID NO.1.
3. one group of compound similar with herbicidin F, shown in chemical structure such as following formula (1):
Wherein,
R1 are as follows:-CHO-R4;
R2, R3 are H or CH3;
R4 is linear chain or branched chain alkane or-C (CH containing 1~4 C3)=CHCH3。
4. compound according to claim 3, which is characterized in that the compound is following four groups of compounds
(1) R1 isR2 is CH3, R3 H, molecular weight 522;
(2) R1 isR2 is CH3, R3 CH3, molecular weight 510;
(3) R1 isMolecular weight 510 or 524;
(4) R1 isMolecular weight 524.
5. the invention further relates to similar with herbicidin F described in fermenting and producing herbicidin F or claim 3 or 4
The method of compound, the method are as follows:
(1) the HcdR2 gene is overexpressed in host strain;
(2) host strain described in fermenting and producing;
(3) described herbicidin F and the like is separated.
6. according to the method described in claim 5, it is characterized in that,
The method of host strain described in fermenting and producing described in step (2) are as follows: use secondary liquid fermentation process, fermentation medium
Are as follows: 0.3% high nitrogen Dried Corn Steep Liquor Powder, 2% soybean cake powder, 2.5% glucose, 2% starch, 2% maltose, 0.2%K2HPO4,
0.3%NaCl.
7. according to the method described in claim 6, it is characterized in that, the secondary liquid fermentation parameter are as follows:
The spore inoculating of host strain is in seed fermentation liquid, and 28 DEG C, 220rpm is cultivated 48 hours;
Seed fermentation liquid is inoculated in liquid fermentation medium, and in 28 DEG C, 220rpm is cultivated 7 days liquid fermentation medium.
8. according to any method of claim 5-7, which is characterized in that
Separation described in step (3) are as follows: the supernatant of fermentation liquid is by C18Chromatography column enrichment, respectively with 50% and 100% first
Alcoholic solution elution.
9. the application of any compound of claim 3 or 4 in medicine preparation, the drug are antimycotic or inhibit
The drug of tumour.
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