CN104164399A - High-producing strain for cyclic lipopeptide antibiotic--himastatin and construction method thereof - Google Patents
High-producing strain for cyclic lipopeptide antibiotic--himastatin and construction method thereof Download PDFInfo
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- CN104164399A CN104164399A CN201410372728.7A CN201410372728A CN104164399A CN 104164399 A CN104164399 A CN 104164399A CN 201410372728 A CN201410372728 A CN 201410372728A CN 104164399 A CN104164399 A CN 104164399A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a high-producing strain for the cyclic lipopeptide antibiotic--himastatin and a construction method thereof. The high-producing strain for himastatin is constructed by inactivating the hmtA gene or hmtB gene of S. himastatinicus sp.Nov, so the high-producing mutant strain S. himastatinicus sp.Nov for himastatin is obtained, wherein the nucleotide sequence of the hmtA gene is as shown in SEQ ID No. 1 and the nucleotide sequence of the hmtB gene is as shown in SEQ ID No. 3. According to the invention, the hmtA-inactivated mutant strain S. himastatinicus sp.Nov Ju2014 and the hmtB-inactivated mutant strain S. himastatinicus sp.Nov Ju2015 are respectively obtained by inactivating the Mer-family transcriptional regulation gene hmtA and the acetyl glutamic acid kinase gene hmtB at the upper stream of the biosynthesis gene cluster of the cyclic lipopeptide antibiotic--himastatin of S. himastatinicus sp.Nov. The himastatin output of the mutant strain S. himastatinicus sp.Nov Ju2014 and the mutant strain S. himastatinicus sp.Nov Ju2015 is more than 10 times that of a wild strain S. himastatinicus sp.Nov. Thus, successful construction of the two high-producing mutant strains for the cyclic lipopeptide antibiotic--himastatin enables acceleration of the industrialization process of himastatin to be possible and hopeful.
Description
Technical field:
The invention belongs to microbiological genetic engineering and metabolic engineering field, be specifically related to superior strain and the construction process thereof of cyclic ester peptide antibiotics Hei Motading (himastatin).
Background technology:
Cyclic peptide compound is the ring compound that a class forms with amido linkage, is mainly the complicated molecule being combined after a series of modifications by amino acid, glycopeptide, lipopeptid and nucleosides peptide.This compounds is extensively present in invertebrates (sponge, Ascidian, moss animalcules etc.), bacterium, cyanobacteria, fungi and actinomycetes, majority has complicated novel constitutional features, and its most outstanding feature is ring texture and various amino acid derivative structural unit.Ring texture is given the certain physics rigidity of this compounds and to proteolysed tolerance, and different derived structures more makes this compounds possess various Physiology and biochemistry speciality.Because its majority has outstanding biology performance (antibacterial, anti-inflammatory, anticancer, antiviral, immunosuppressive activity, pest-resistant, antimalarial and Physiology and biochemistry regulating and controlling effect etc.), make this compounds become the object of paying close attention to of synthetic chemistry, drug screening and exploitation.Some compounds are wherein successfully for the treatment of clinical disease.Such as antifungic action Tysocidine A (tyrocidine A), for the cyclosporin A (cyclosporin A) of the anti-repulsive interaction of organ transplantation, the golden Portugal bacterium of methicillin resistance is had the vancomycin (Vancomycin) of excellent activity, Mrsa In Rabbits and Vancomycin-resistant Enterococcus is all had to daptomycin (daptomycin) of good anti-microbial activity etc., are all this compounds successful models for clinical treatment.
But, some cyclic peptide compounds are wherein because its water-soluble poor, side effect is large or the yielding poorly etc. former thereby exited clinical trial of compound, but this also side light necessity and urgency that these compounds are carried out structure of modification and optimization or improve its output.And the selection that has complex construction, numerous chiral centre and a closed loop position due to these compound majorities is seriously restricting this compounds is being carried out to the efficiency of structure of modification or the process of industrialization.For traditional natural product screening and the limitation of research and development means, develop a kind of taking genetically engineered and enzymology as basic biosynthesis technology in nineteen nineties---Combinatorial biosynthesis (combinatorial biosynthesis), not only make up the deficiency of traditional research method, and provide new Research Thinking and research direction for the further expansion of biomedicine field.And along with the develop rapidly of gene order-checking technology and gene recombination technology etc., Combinatorial biosynthesis has brought revolutionary variation to field of biology, also creates new chemical structure to chemistry subject simultaneously and builds compound library and brought new method and useful supplementing is provided.In addition, antibiotic biosynthesis gene is conventionally in one section of feature that successive zone cluster is arranged of karyomit(e) or huge plasmid, and (comprising structure gene, ego resistance gene, transporter gene and regulatory gene) is for further exploitation and transformation microbiotic provide new opportunity.By 2006, the biological synthesis gene cluster of existing nearly 300 kinds of microbial secondary meta-bolitess is cloned and Function Identification, for utilizing Combinatorial biosynthesis technology, metabolic engineering technology and vitro enzyme to learn a skill to create or transforming to obtain new compound and lay a good foundation.There is many research to be successfully completed the orientation accumulation of structural modification, transformation or some target component of more baroque natural product by biosynthesizing and Combinatorial biosynthesis technology, a series of activity are built quite or active stronger derivative, or high productive mutant, for streptomycete genetic engineering modified provides useful methodological reference sample, bring new hope for solving the day by day thorny resistance problem of pathogenic micro-organism and the treatment of tumor disease.
Cyclic lipopeptide microbiotic Hei Motading (himastatin), its structure as shown in Figure 1, external activity analysis shows that it has good bacteriostatic activity to gram-positive microorganism, and P388 clone and melatonin knurl B16 clone are had to significant cytotoxic activity.
Summary of the invention:
The object of this invention is to provide Hei Motading (himastatin) high productive mutant Streptomyces himastatinicus sp.Nov.
Himastatin high productive mutant Streptomyces himastatinicus sp.Nov of the present invention, it is the Streptomyces himastatinicus sp.Nov of hmtA gene or hmtB gene inactivation, the nucleotide sequence of described hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of described hmtB gene is as shown in SEQ ID NO.3.
The present invention also provides the construction process of himastatin high productive mutant Streptomyces himastatinicus sp.Nov, it is characterized in that, by the hmtA gene of Streptomyces himastatinicus sp.Nov or hmtB gene inactivation, obtain thus himastatin high productive mutant Streptomyces himastatinicus sp.Nov, the nucleotide sequence of described hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of described hmtB gene is as shown in SEQ ID NO.3.
The present invention also provides hmtA gene or the application of hmtB gene in regulation and control himastatin output, and the nucleotide sequence of described hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of described hmtB gene is as shown in SEQ ID NO.3.
Original strain Streptomyces himastatinicus sp.Nov of the present invention is a strain Lu Yuan streptomycete, respectively by the genetic modification of 1 MerR family transcriptional modulatory gene in the biological synthesis gene cluster of its secondary metabolite, 1 acetylglutamate kinase gene having been obtained to mutant strain Streptomyces himastatinicus sp.Nov Ju2014 and the Ju2015 of 2 strain energy high yield himastatin.With the substratum fermentation strain of himastatin, be extracted with ethyl acetate fermented liquid, through its secondary metabolite of analysis of HPLC-DAD-UV.Carry out programanalysis with HPLC program, HPLC analyzes collection of illustrative plates and shows, elution time is at 17.45min place and have a feature ultraviolet absorption value (λ
max210,285) flow point, this flow point should be himastatin.In order to ensure 17.45 min places and there is feature ultraviolet absorption value (λ
max210,285) flow point is himastatin, therefore adopt the fermentative processing of 6L scale, analyzes the sterling that obtains compound 1 subsequently by HPLC-UV; Through nuclear magnetic resonance spectroscopy, its 1D (
1h,
13c) collection of illustrative plates of the himastatin of NMR Spectral data and bibliographical information is in full accord, determines that the compound 1 at retention time 17.45 min places is microbiotic himastatin (its structural formula as shown in Figure 1).
The invention still further relates to the full genome scanning of Streptomyces himastatinicus sp.Nov, and the confirmation of the biological synthesis gene cluster of microbiotic himastatin.In order to find the biosynthetic gene cluster of microbiotic himastatin, we have adopted the method for full genome scanning, utilize Illumina ' s Solexa sequencing technologies, complete genome DNA to Streptomyces himastatinicus sp.Nov bacterial strain carries out sequencing, the result of gene annotation shows that the continuous fragment that a size is 60 Kb may contain the necessary gene information of himastatin biosynthesizing, further the DNA sequence dna of 20 open reading frame approximately 45 Kb sizes of bioinformatic analysis announcement has comprised the regulation and control in microbiotic himastatin biosynthesizing, structure, ego resistance, transhipment and the isogenic information of rear modification (seeing Fig. 2).The sequence of gene cluster is included by EMBL database, sequence number FR823394.
(nucleotide sequence is as shown in SEQ ID NO.1 to the present invention relates to the gene hmtA that has been responsible for transcribing regulating and controlling effect in himastatin biological synthesis gene cluster, the aminoacid sequence of the albumen HmtA of its coding is as shown in SEQ ID NO.2), (nucleotide sequence is as shown in SEQ ID NO.3 for acetylglutamate kinase gene hmtB, the aminoacid sequence of the albumen HmtB of its coding is as shown in SEQ ID NO.4), it is carried out separately can obtaining after replacement mutation the engineering strain of high yield microbiotic himastatin.In the upstream of himastatin biological synthesis gene cluster, there is the gene of individual called after hmtA, its one of coding has 247 amino acid whose polypeptide, identifies that by bioinformatic analysis this peptide chain belongs to MerR family transcriptional control.In order to verify protein HmtA and the concrete effect of HmtB in the biosynthesizing of microbiotic himastatin of hmtA and hmtB genes encoding, carry out respectively replacing in reading frame with a DNA fragmentation with Apramycin sulfate resistance, make hmtA and hmtB inactivation.Sudden change clay is transferred in the E.coli ET12567/pUZ8002 bacterial strain of the defect that methylates, and wild-type S.himastatinicus sp.Nov bacterial strain plant between conjugal transfer, obtain some zygotes, respectively choose respectively (the A Pula tolerance of 3 resistant phenotypes, kantlex sensitivity) and all correct mutant strain S.himastatinicus sp.Nov Ju2014 of genotype, mutant strain S.himastatinicus sp.Nov Ju2015, simultaneously with condition fermentation, wild type strain is as positive control with wild-type S.himastatinicus sp.Nov bacterial strain.Analyze the fermented liquid ethyl acetate extraction of mutant strain Δ hmtA by HPLC, result shows two high yield himastatin that mutant strain clone is consistent, and the output of this target product has exceeded more than 10 times compared with wild type strain.These digital proofs HmtA and HmtB albumen in the biosynthesizing of himastatin, there is the effect of negative regulatory factor.
Nucleotide sequence provided by the present invention or partial nucleotide sequence, the DNA of 1st~744 that can utilize the method for polymerase chain reaction (PCR) or comprise sequence shown in SEQ ID NO.1 of the present invention obtains the gene similar to hmtA as probe with methods such as Southern hybridization from other biological body.
Comprise nucleotide sequence provided by the present invention or at least partly nucleotide sequence can modify outward or suddenly change by body endosome, comprise insertion, displacement or disappearance, polymerase chain reaction, mistake mediation polymerase chain reaction, mutation site-specific, not homotactic reconnecting, the different piece of sequence or carry out orthogenesis with the homologous sequence in other sources, or by ultraviolet ray or chemical reagent mutagenesis etc.
The clone gene that comprises nucleotide sequence provided by the present invention or at least part of nucleotide sequence can express by suitable expression system to obtain corresponding enzyme or other higher biologically active substance or output in foreign host.These foreign host comprise intestinal bacteria, streptomycete, micromonospora, pseudomonas, genus bacillus, yeast, plant and animal etc.
Aminoacid sequence provided by the present invention can be used for separating needed albumen and can be used for the preparation of antibody.
The polypeptide that comprises aminoacid sequence provided by the present invention or at least part of sequence may still have biological activity even to have new biologic activity after removing or substituting some amino acid, or has improved output or optimized albumen dynamic characteristic or other character of being devoted to obtain.
Comprise the mutant strain that DNA fragmentation or gene can be used for building the output that improves himastatin or derivatives thereof, the invention provides the approach that improves output in genetically engineered microorganism.
The Mer family transcriptional modulatory gene hmtA of the present invention by the biological synthesis gene cluster upstream of the cyclic lipopeptide microbiotic himastatin of inactivation S.himastatinicus sp.Nov and acetylglutamate kinase gene hmtB and obtain respectively the mutant strain S.himastatinicus sp.Nov Ju2014 of hmtA inactivation and the mutant strain S.himastatinicus sp.Nov Ju2015 of hmtB inactivation.The output of the himastatin of mutant strain S.himastatinicus sp.Nov Ju2014 and Ju2015 is the more than 10 times of wild-type S.himastatinicus sp.Nov.Therefore, the success of two high productive mutants of this cyclic lipopeptide microbiotic himastatin be configured to accelerate himastatin industrialization process brought may with hope.
Black not its fourth streptomycete Streptomyces himastatinicus sp.Nov of the present invention, purchased from US mode culture collection warehousing (American type culture collection), be numbered: ATCC53653, this bacterial strain is for sale, and anyone can buy from this preservation storehouse.
Brief description of the drawings:
Fig. 1 is the chemical structural formula of black not its fourth (himastatin).
Fig. 2 is himastatin biological synthesis gene cluster organization chart in streptomycete Streptomyces himastatinicus sp.Nov.
Fig. 3 is the structure schematic diagram of hmtA double exchange mutant strain Streptomyces himastatinicus sp.Nov Ju2014, and wherein Mutant Ju2014 represents mutant strain Streptomyces himastatinicus sp.Nov Ju2014;
Fig. 4 is the PCR proof diagram of hmtA double exchange mutant strain Streptomyces himastatinicus sp.Nov Ju2014, wt represents wild-type Streptomyces himastatinicus sp.Nov, and Δ hmtA1, Δ hmtA2, Δ hmtA3 represent three clones of mutant strain Streptomyces himastatinicus sp.Nov Ju2014;
Fig. 5 is the structure schematic diagram of hmtB double exchange mutant strain Streptomyces himastatinicus sp.Nov Ju2015, and wherein Mutant Ju2015 represents mutant strain Streptomyces himastatinicus sp.Nov Ju2015;
Fig. 6 is the PCR proof diagram of hmtB double exchange mutant strain Streptomyces himastatinicus sp.Nov Ju2015, wherein wt represents wild-type Streptomyces himastatinicus sp.Nov, and Δ hmtB1, Δ hmtB2, Δ hmtB3 represent three clones of mutant strain Streptomyces himastatinicus sp.Nov Ju2015;
Fig. 7 is the HPLC analytical results after fermenting respectively under hmtA double exchange mutant strain Streptomyces himastatinicus sp.Nov Ju2014 and wild-type equal conditions, wherein wt represents wild-type Streptomyces himastatinicus sp.Nov, Δ hmtA1, Δ hmtA2, Δ hmtA3 represent three clones of mutant strain Streptomyces himastatinicus sp.Nov Ju2014, and Δ hmtA1, Δ hmtA2, Δ hmtA3 are the HPLC results after sample introduction after diluting 10 times.
Fig. 8 is the HPLC analytical results after fermenting respectively under hmtB double exchange mutant strain Streptomyces himastatinicus sp.Nov Ju2015 and wild-type equal conditions, wherein wt represents wild-type Streptomyces himastatinicus sp.Nov, Δ hmtB1, Δ hmtB2, Δ hmtB3 represent three clones of mutant strain Streptomyces himastatinicus sp.Nov Ju2015, and Δ hmtB1, Δ hmtB2, Δ hmtB3 are the HPLC results after sample introduction after diluting 10 times.
Fig. 9 is the H spectrum of active compound 1.
Embodiment:
Following examples are to further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
The cultivation of 1.S.himastatinicus sp.Nov
At S.himastatinicus sp.Nov wild type strain of the present invention and the mutant strain substratum using of growing, every liter is to prepare like this: fish meal 10g, glycerine 20g, calcium carbonate 5g and yeast extract 5g are added in 1L water, pH is between 7.2-7.4, and conventional sterilizing is for subsequent use.The temperature of its most suitable growth is at 28 DEG C-30 DEG C.
2. separation and purification and the Structural Identification of the secondary metabolite himastatin of streptomycete S.himastatinicus sp.Nov
First, S.himastatinicus sp.Nov bacterial strain ferments 7 days in the 250 mL triangular flasks of substratum that 50mL step 1 is housed, 28~30 DEG C of temperature, rotating speed 200 rpm; Then, the ethyl acetate extraction of 2 times of volumes for fermented liquid, 50 DEG C of underpressure distillation of upper organic phase, use 1mL dissolve with methanol, obtain crude extract, carry out HPLC analysis, carry out the demarcation (210nm, 285nm) of target compound according to characteristic ultraviolet absorption.Then utilize same fermentation condition, S.himastatinicus sp.Nov is carried out to the fermentation of 4L scale.The runic thing of tunning is carried out carrying out after HPLC analysis to the separation of target compound.HPLC analyzes the composed as follows of moving phase used: A phase, 15% acetonitrile+85% water+0.1% Glacial acetic acid; B phase, 20% water+80% acetonitrile+0.1% Glacial acetic acid.HPLC program is: 0~20 min, and B is 0%~80% gradient elution mutually; 21~23min, B is 80%~100% gradient elution mutually; 24~30min, 100%B phase wash-out.UV detects wavelength, and (flow velocity is 1 min/mL for 210 nm, 285nm.In this program, the about 17.45min of the retention time of target compound 1.Active compound 1 is carried out to HR-ESI-MS, 1D (
1h,
13c) NMR (Fig. 9) test, the comprehensive spectral data of analyzing, with document: JOHN E.LEET, DANIEL R.SCHROEDER, BALA S.KRISHNAN and JAMES A.MATSON, HIMASTATIN, A NEW ANTITUMOR ANTIBIOTIC FROM STREPTOMYCES HYGROSCOPICUS
II .ISOLATION AND CHARACTERIZATION, THE JOURNAL OF ANTIBIOTICS, VOL.XL III NO.8,961~966,1990, the nuclear magnetic data of the himastatin in 8. contrasts, and carries out the confirmation of target compound 1, identify that target compound 1 is for himastatin, its structural formula as shown in Figure 1.
3. contain the screening of the Cosmid 12H8 of gene hmtA and hmtB
The structure of S.himastatinicus sp.Nov genomic library: taking SuperCos I as carrier, detailed process is with reference to the operational manual of SuperCos 1 Cosmid Vector Kit.Operating process can roughly be summarized as follows: from S.himastatinicus sp.Nov mycelium, extract genomic dna, through Sau3AI Partial digestion, with calf intestinal Phosphoric acid esterase dephosphorylation, then be connected to by SuperCos 1 carrier of XbaI and BamHI enzymolysis processing, carry out in vitro package by Packagene Lambda DNA packaging system, infect intestinal bacteria LE392, screening on LB+kanamycin (Kan, final concentration 50 μ g/mL) flat board.Choose approximately 1920 clones and select in 20 96 orifice plates, cultivate after 12h, in every hole, add the glycerine of equal-volume 50%.-80 DEG C of preservations.
The gene order-checking of S.himastatinicus sp.Nov transfers to Hua Da company to complete.The sequence providing according to Hua Da company.In order to find the biosynthetic gene cluster of microbiotic himastatin, we have adopted the method for full genome scanning, utilize Illumina ' s Solexa sequencing technologies, complete genome DNA to Streptomyces himastatinicus sp.Nov bacterial strain carries out sequencing, the result of gene annotation shows that the continuous fragment that a size is 60 Kb may contain the necessary gene information of himastatin biosynthesizing, further the DNA sequence dna of 20 open reading frame approximately 45 Kb sizes of bioinformatic analysis announcement has comprised the regulation and control in microbiotic himastatin biosynthesizing, structure, ego resistance, transhipment and the isogenic information of rear modification, infer the weave construction of himastatin biological synthesis gene cluster as shown in Figure 2.The sequence of himastatin gene cluster is included by EMBL database, sequence number FR823394.The wherein hmtA gene in gene cluster (its nucleotide sequence is as shown in SEQ ID NO.1) coding MerR family transcriptional regulator; HmtB gene (its nucleotide sequence is as shown in SEQ ID NO.3) coding acetylglutamate kinase.In order to study hmtA and the function of hmtB gene in himastatin biosynthetic process, we carry out respectively knocking out of gene to it.According to PCR-targeting operational requirement, for the clay that obtains knocking out hmtA and hmtB, we have designed two pairs of primers near hmtA sequence, SAF (5 '-CGCAGATCCACGCCGCACTC-3 '), SAR (5 '-AGT TCCGCGTGACGGAGGTG-3 ') and SBF (5 '-CGCCACCTGCTCGTCACCG-3 '), SBR (5 '-TCG GCG TAC AGC CAC AGC AG-3 ').Two pairs of primer PCR gained object bands are respectively 622 bp and 1679bp.Utilize respectively this two pairs of primers, from the genomic library building, utilize round pcr screening positive clone.PCR program is as follows: 95 DEG C of 5min; 95 DEG C of 45s, 58 DEG C of 45s, 72 DEG C of 90s, 30 circulations; 72 DEG C of 10min.Wherein screen multiple positive colonies.The positive colony of our selected the 12nd 96 orifice plate correspondences (H, 8) position, called after Cosmid 12H8, it contains complete hmtA and hmtB gene, carries out next step hmtA and knocking out of hmtB gene.
The structure of 4.hmtA gene deletion mutants Streptomyces himastatinicus sp.Nov Ju2014
In the reading frame of hmtA gene, inactivation is the PCR-Targeting technology that has adopted λ-RED mediation.The Cosmid 12H8 that includes hmtA gene is transformed in E.coli BW25113/pIJ790, called after E.coli BW25113/pIJ790/12H8.Taking the pIJ773 plasmid crossed by EcoRI and HindIII enzymolysis as template, use with hmtA gene have respectively 39bp homology arm knock out primer (hmtAdelF:5 '-
acgctcggcgcgacatacgcccggacccgacgacccgataTTCCGGGGATCCGT CGAC-3 ', hmtAdelR:5 '-
tagccggtacccgacgtcgatggcctcgagcccgaggagtGTAGGCTGGAGCTGCT TC-3 ' underscore part representative and hmtA DNA homolog) there is the pcr amplification reaction of aac (3) the IV-oriT resistance fragment of Apr resistance.The aac having increased (3) IV-oriT resistance fragment electricity is transformed in E.coli BW25113/pIJ790/12H8 competent cell, homologous recombination by λ-RED mediation obtains improved restructuring cosmid, called after restructuring cosmid pJu2014.Carry out amplified reaction and determine the exactness of restructuring cosmid pJu2014 with the PCR checking primer of hmtA gene (IDhmtAF:5 '-GAACCGATTCCGCCTGACC-3 ', IDhmtAR:3 '-ACATCGCCCTCGCCCTCAC-3 ').Subsequently, restructuring cosmid pJu2014 is proceeded in E.coli ET12567/pUZ8002, called after E.coli ET12567/pUZ8002/p Ju2014, and itself and wild type strain S.himastatinicus sp.Nov carry out conjugal transfer.
The process of conjugal transfer is as follows: by the spore of wild-type S.himastatinicus sp.Nov, in TSB nutrient solution, 12-18 hour is cultivated in 28 DEG C of concussions, makes its spore germination; E.coli ET12567/pUZ8002/pJu2014 is being added with kantlex (Kan, final concentration is 50 μ g/mL), penbritin (Amp, final concentration is 100 μ g/mL), paraxin (Cml, final concentration is 25 μ g/mL) and A Baila mycin (Apm, final concentration is 50 μ g/mL) LB nutrient solution in be cultured to optical density value OD=0.6, centrifugal collecting cell, by aseptic LB liquid nutrient medium washed twice, then mix with the wild-type spore of having sprouted, mixed solution is laid in and is added with 10mM MgSO
4m-ISP
4on solid medium; 28 DEG C growth 23 hours after, on each solid medium flat board, be coated with 800 μ L and be added with the sterilized water of the antibiotic medicine of Trimethoprim and Apramycin sulfate, wherein containing trimethoprim (Tmp, 50 mg/mL) 30 μ L, A Baila mycin (Apm, 50 mg/mL) 30 μ L; Be allowed to condition at the 4-5 days that grows under 30 DEG C of incubators, until there is being double exchange mutant strain in zygote, called after Streptomyces himastatinicus sp.Nov Ju2014.This double exchange mutant strain passes through it to antibiotic resistant phenotype (Kan
saMP.AMp.Amp Apm
r) and genotype and screened and qualification are out, wherein the PCR of hmtA genetically deficient checking utilizes the pcr amplification reaction that carries out of primer (IDhmtAF, IDhmtAR) to be confirmed.Obtain thus the mutant strain Streptomyces himastatinicus sp.Nov Ju2014 that hmtA is knocked.
The building process of hmtB deletion mutant bacterial strain is with hmtA gene knockout, but the following hmtBdelF:5 ' of the deletion-primers of hmtB-
cttggcgtccggtacgaccgtggtgccggaccgctcgtcaTTCCGGGGATCCGTCGAC-3 ', hmtBdelR:5 '-
gaggcgtcgttcgccgacgacgtcgtacggatgcaccggthe representative of TGTAGGCTGGAGCTGCTTC-3 ' underscore part and hmtB DNA homolog.The PCR checking primer of hmtB gene is: IDhmtBF:5 '-aggctcgtgtcggaggtagt-3 ', IDhmtBR:5 '-cggcgggacagaatcttg-3 '.Obtain thus the mutant strain Streptomyces himastatinicus sp.Nov Ju2015 that hmtB is knocked.
As shown in Figure 3, the PCR proof diagram of mutant strain Streptomyces himastatinicus sp.Nov Ju2014 as shown in Figure 4 for the structure schematic diagram of concrete mutant strain Streptomyces himastatinicus sp.Nov Ju2014.
As shown in Figure 5, the PCR proof diagram of mutant strain Streptomyces himastatinicus sp.Nov Ju2015 as shown in Figure 6 for the structure schematic diagram of concrete mutant strain Streptomyces himastatinicus sp.Nov Ju2015.
5. the fermentation of the mutant strain Streptomyces himastatinicus sp.Nov Ju2015 that the mutant strain Streptomyces himastatinicus sp.Nov Ju2014 that wild type strain and double exchange mutant strain hmtA are knocked and hmtB are knocked and HPLC thereof analyze
Wild type strain and double exchange mutant strain are cultivated 7 days dull and stereotyped upper 30 DEG C of ISP4 (M-ISP4) solid medium of improveing respectively, the appropriate spore inoculating of picking, in the triangular flask of substratum that 50mL himastatin step 1 is housed, is cultivated 7 days on the shaking table of 28 DEG C and 200rpm rotating speed.The ethyl acetate of 2 times of volumes extraction for fermented liquid, the extraction liquid of organic phase is dissolved in 1mL methyl alcohol after revolving steaming instrument evaporate to dryness, after the centrifugal 5min of 12000rpm, gets supernatant liquor and carries out HPLC analysis.HPLC analytical results is shown in Fig. 7 and Fig. 8.Calculate the himastatin multiple that output improves in mutant strain by the HPLC analyzing and testing of mutant strain and wild type strain tunning and the integration radiometer of HPLC collection of illustrative plates.As can be seen from Figures 7 and 8, wild-type Streptomyces himastatinicus sp.Nov, the mutant strain Streptomyces himastatinicus sp.Nov Ju2015 that mutant strain Streptomyces himastatinicus sp.Nov Ju2014 and hmtB are knocked under equal conditions ferments, with wild-type comparison, the output of the target product himastatin of the mutant strain Streptomyces himastatinicus sp.Nov Ju2015 that mutant strain Streptomyces himastatinicus sp.Nov Ju2014 and hmtB are knocked has exceeded more than 10 times compared with wild type strain.Therefore, the success of two kinds of high productive mutants of this cyclic lipopeptide microbiotic himastatin be configured to the industrialization process that accelerates microbiotic himastatin brought may with hope.
M-ISP4 substratum (the ISP4 liquid nutrient medium of improvement): be to add peptone, yeast extract and sea salt on the basis of ISP4 substratum, whole massfraction is respectively: 0.1%, 0.05%, 3%.The formula that is M-ISP4 substratum is: every liter of M-ISP4 substratum contains: Zulkovsky starch 10g, K
2hPO
41g, MgSO
4.7H
2o 1g, NaCl 1g, (NH
4)
2sO
42g, CaCO
32g, FeSO
4.7H
2o 0.001g, MnCl
2.4H
2o 0.001g, ZnSO
4.7H
2o 0.001g, peptone 1g, yeast extract 0.5g, thick sea salt 30g, surplus is water, pH7.0~7.4.Solid medium is on the basis of liquid medium within, to add 1.5% agar.
Claims (3)
1.himastatin high productive mutant Streptomyces himastatinicus sp.Nov, it is characterized in that, it is the Streptomyces himastatinicus sp.Nov of hmtA gene or hmtB gene inactivation, the nucleotide sequence of described hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of described hmtB gene is as shown in SEQ ID NO.3.
2. the construction process of a himastatin high productive mutant Streptomyces himastatinicus sp.Nov claimed in claim 1, it is characterized in that, by the hmtA gene of Streptomyces himastatinicus sp.Nov or hmtB gene inactivation, obtain thus himastatin high productive mutant Streptomyces himastatinicus sp.Nov, the nucleotide sequence of described hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of described hmtB gene is as shown in SEQ ID NO.3.
The application in regulation and control himastatin output of 3.hmtA gene or hmtB gene, the nucleotide sequence of described hmtA gene is as shown in SEQ ID NO.1, and the nucleotide sequence of described hmtB gene is as shown in SEQ ID NO.3.
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