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WO2012119484A1 - 质粒及利用质粒提高链霉菌抗生素产量的方法 - Google Patents

质粒及利用质粒提高链霉菌抗生素产量的方法 Download PDF

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WO2012119484A1
WO2012119484A1 PCT/CN2012/000276 CN2012000276W WO2012119484A1 WO 2012119484 A1 WO2012119484 A1 WO 2012119484A1 CN 2012000276 W CN2012000276 W CN 2012000276W WO 2012119484 A1 WO2012119484 A1 WO 2012119484A1
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plasmid
gene
streptomyces
vector
expression plasmid
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PCT/CN2012/000276
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English (en)
French (fr)
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由德林
邓子新
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C12P19/626—Natamycin; Pimaricin; Tennecetin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to a method and plasmid in the field of biomedical technology, and in particular to a plasmid and a method for increasing the production of a Streptomyces antibiotic using a plasmid.
  • Bioengineering breeding technology overcomes the randomness and blindness of traditional breeding techniques within a certain scope. It can use the knowledge of microbial secondary metabolic biosynthesis pathways to construct antibiotic high-yield strains by genetic engineering technology, which is helpful for secondary metabolites. Industrial production.
  • the target gene is linked to the vector in vitro by genetic engineering technology and then transferred to the recipient cell, so that the foreign gene is stably expressed in the receptor.
  • the main pathways include: 1 increasing the production of antibiotics by regulating the doubling or inactivation of genes, increasing the active efflux efficiency of antibiotics; 2 introducing foreign genes that increase the supply of precursors and cofactors in host cells, The biological substance of the active substance; 3 by activating or inhibiting the branch metabolism to change the active ingredient or ratio of the biologically active molecule, thereby increasing the accumulation of the optimal active ingredient, reducing or eliminating the accumulation of the low activity secondary component.
  • AdpA is a global positive regulator widely present in Streptomyces and is the central transcriptional regulator of the A-factor regulatory network, which activates the expression of genes required for morphological differentiation and secondary metabolites.
  • AdpA positively regulates melanin biosynthesis in Streptomyces coelicolor, St. Streptomyces lividans, Streptomyces antibioticus, and Streptomyces a verwi til is. There is no literature on the effect of the expression of the ao ⁇ I gene in antibiotic-producing strains on its antibiotic production.
  • Zy ireosci a is a obligate aerobic filamentous Gram-negative bacterium.
  • the gene-encoded hemoglobin of Vitreoscillae has the property of binding oxygen to promote the transport of oxygen in microbial cells and increase oxygen. Utilization. For many cases of insufficient dissolved oxygen, Vgb expression promotes host growth, protein secretion, metabolite production, and enhanced compression resistance.
  • Vitreoscilla hemoglobin gene Integration of the Vitreoscilla hemoglobin gene into a heterologous host strain can increase recombinant protein production and fermentation yield the amount.
  • Wb gene can significantly promote the growth of Streptomyces cinnamensis and the synthesis of antibiotic monensin in antibiotic production (Wen Ying, Song Yuan. Vitreoscilla hemoglobin in Streptomyces cinnafolia)
  • SAM S-adenosylmethionine
  • the present invention designs a novel method and plasmid for improving the production of Streptomyces antibiotics, and constructs a plurality of genes with extensive positive regulation related to antibiotic synthesis and constructs them simultaneously on one vector, and the constructed plasmid can be constructed. Expression in different Streptomyces strains effectively increases the yield of various antibiotics. Summary of the invention
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a novel method for increasing the production of Streptomyces antibiotics and the plasmids used.
  • the present invention provides a plasmid and a method for increasing the production of a Streptomyces antibiotic by using a plasmid, wherein one or several of the three genes having a broad promotion effect on the biosynthesis of antibiotics are simultaneously expressed on one vector, and the junction is transferred. Transfer into Streptomyces for efficient expression to increase the production of antibiotics.
  • the present invention provides a method for increasing the production of antibiotics of Streptomyces i Streptomyces, comprising the steps of: transferring an integrated expression plasmid into Streptomyces, thereby increasing the production of Streptomyces antibiotics; said integrated expression plasmid is ⁇ ZAS "Genes and conventional vectors are constructed, either constructed of ⁇ / ⁇ and b5" genes and conventional vectors, or constructed with the ac ⁇ -c gene and conventional vectors, or metK, and ad P Ac The gene is constructed with a conventional vector.
  • the method for increasing the production of antibiotics of Streptomyces treptomyces comprises the steps of:
  • step (c) Fermentation of the Streptomyces obtained in step (b), obtaining antibiotics.
  • the Streptomyces is Streptomyces i Streptomyces sp.) ZYJ-6 CGMCC NO. 2394.
  • the conventional vector is a PIB139 vector.
  • the base sequence of the zz/ei gene is as shown in SEQ ID NO.
  • the base sequence of the gene is shown in SEQ ID NO.
  • the base sequence of the ac ⁇ 1-c gene is as shown in SEQ ID NO.
  • the present invention also relates to an integrated expression plasmid pFVgb o constructed from a ⁇ / ⁇ gene and a conventional vector.
  • the conventional vector is a PIB139 vector.
  • the present invention relates to a method for constructing the aforementioned integrated expression plasmid pFVgb, comprising the following steps: (a) Plasmid pJTU4405 contains whole gene synthesis vgbS, and Ndel and EcoRI restriction sites are introduced at both ends of gene wb5";
  • the invention also relates to an integrated expression constructed by fflei and "genes and conventional vectors" Plasmid pFMV.
  • the conventional vector is a PIB139 vector.
  • the present invention relates to a method for constructing the integrated expression plasmid pFMV according to claim 11, comprising the steps of: preparing a plasmid pFMetK plasmid; and then cleaving vgbS ⁇ pJTU4405 with Ndel and EcoRI and inserting the corresponding enzyme into pJTU968 At the site, the b5 gene fragment carrying the strong erythromycin promoter was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI to obtain plasmid pFMV.
  • the preparing the plasmid pFMetK plasmid comprises the following steps:
  • the sequence of the primer metKF is: 5, - CAGGGAGCCATATGTCCCGT-3 ' ,
  • the sequence of the primer metKR is: 5 ' -TCGCAAAGGCCACTGACAACA-3 ';
  • the present invention also relates to an integrated expression plasmid pFMA constructed from the ⁇ / ⁇ and SC / 4- C genes and a conventional vector.
  • the conventional vector is a PIB139 vector.
  • the present invention relates to a method for constructing the aforementioned integrated expression plasmid pFMA, comprising the steps of: inserting ad P Ac from pJTU2520 into a corresponding site of plasmid pJTU968 carrying an erythromycin promoter using Ndel and EcoRI, The apA-c gene fragment carrying the strong erythromycin promoter was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI to construct plasmid pFMA.
  • the present invention also relates to an integrated expression plasmid pFMVA constructed from the ei r, ⁇ and atp/i-c genes and a conventional vector.
  • the conventional vector is a PIB139 vector.
  • the present invention relates to a method for constructing the aforementioned integrated expression plasmid pFMVA, comprising the steps of: inserting apA-c carrying an erythromycin promoter from pJTU2522 into an EcoRI site of pFMV using Muni and EcoRI, Plasmid pFMVA.
  • the present invention has the following beneficial effects:
  • the present invention provides a plasmid and a method for increasing the production of a Streptomyces antibiotic by using a plasmid, and one or several of the three genes having a broad promotion effect on the biosynthesis of antibiotics are taken.
  • the manner in which expression is simultaneously carried out on a vector is transferred to Streptomyces by conjugation transfer for efficient expression to increase the production of antibiotics.
  • the Streptomyces sp. CGMCC NO. 2394 ZYJ-6 of the present invention has been submitted to the General Microbiology Center of the China Microbial Culture Collection Management Committee, and its deposit registration number is CGMCC N0. 2394; the deposit address is No. 1 Courtyard of Beichen West Road, Chaoyang District, Beijing. No. 3, Institute of Microbiology, Chinese Academy of Sciences.
  • the Streptomyces CGMCC NO. 2394 ZYJ-6 has been disclosed ((Zhou YJ, Li JL, Zhu J, Chen S, Bai LQ, Zhou XF, Wu HM, Deng ZX.
  • the PIB 139 is described in "11: ⁇ 50:1 CJ, Hughes- Thoraas ZA, Martin CJ, Bohm L Mironenko T, Deacon M, Wheatcroft M, Wirtz G, Staunton J, Leadlay PF. Increasing the efficiency of heterologous promoters in actinomycetes J Mol Microb Biotech, 2002, 4 (4): 417-426.
  • the strain PJTU4405 is described in Chinese Patent Application No. 201010148837. 2, Publication No. CN 101805742 A, Japanese Patent Publication No. 2010- 8-18, the disclosure of the patent document of the disclosure of the disclosure of the disclosure of the patents of the patent of The strain PJTU4405 was prepared by the following method: changing the codon used frequently in Streptomyces to the codon with the highest frequency in Streptomyces, keeping the amino acid sequence unchanged, and generating a new gene Wb5 Ndel and EcoRI cleavage sites were introduced at the wb5M end of the gene, respectively, in which 7 nucleotides were added at the 5' end of the ⁇ b5t-heidate sequence to form a restriction endonuclease Ndel site and a protective base.
  • the E. coli ET12567/pUZ8002 is disclosed in "Paget, MS, Chamberl in, L., Atrih, A., Foster, SJ, and Buttner, MJ. Evidence that the extracytoplasmic function sigma factor ⁇ E is required for normal cell wall structure In Streptomyces coelicolor A3 (2) . J. Bacteriol, 1999 , 181 : 204 - 211 .
  • FIG. 1 is a construction diagram of an expression plasmid.
  • Figure 2 shows the PCR validation of the »ei gene in strains ZYJ-6/pFMetK, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA.
  • Figure 3 is a PCR verification of the wb5" gene in strains ZYJ-6/pFVgb, ZYJ-6/pFMV, ZYJ-6/pF VA.
  • Figure 4 shows strains ZYJ-6/pFAdpA, ZYJ-6/pFMA, ZYJ-6 PCR verification of ap I-c gene in /pFMVA.
  • Figure 5 shows spectrophotometric analysis of strains ZYJ-6 and ZYJ-6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA,
  • ZYJ-6/pFMV, ZYJ-6/pF A, ZYJ-6/pFMVA yield changes of FR-008-III in different culture times.
  • Figure 6 is a quantitative analysis of the yield change of the oxytocin FR-008- ⁇ in the starting strain ZYJ-6 and the high-yield strain ZYJ-6/pFMVA.
  • Example 1 Construction of an integrated expression plasmid for increasing the production of Streptomyces antibiotics
  • the gene expression vector PIB139 is a Streptomyces integrative vector containing a strong promoter of erythromycin resistance gene PermE*, which can be transferred into Streptomyces by the two parents between Escherichia coli and Streptomyces. After point-specific recombination, it is integrated on the chromosome of Streptomyces and replicates and inherited along with the chromosomes.
  • Figure 1 is a diagram showing the construction of all expression plasmids involved in this example.
  • the specific steps include: designing the primer metKF2/metKR in the ffei gene of Streptomyces coelicolor, and adding a Ndel restriction enzyme at the ATG codon
  • the 4334 bp/ ⁇ i/r gene amplified without the original promoter was inserted into the EcoRV site of P BlueS C riptn SK(+) to construct a 4292 bp clone pJTU2524; no promoter was used with Ndel and EcoRI
  • the 7?ei r gene fragment was excised from PJTU2524 and inserted into the pIB139 vector containing the erythromycin resistance gene strong promoter PermE* to obtain plasmid pFMetK.
  • the sequence of the primer metKF2/metKR is:
  • Plasmid PJTU4405 contains a full-length synthetic wb5" with a length of 456 bp, and Ndel and EcoRI restriction sites were introduced at both ends of the gene.
  • the pJTU4405 carrying the Vitreoscilla gene was digested with Ndel and EcoRI.
  • PCR amplification of the aDPA-c gene in Streptomyces coelicolor the specific steps include: designing the primer aDPAc2F/adpAclR, adding a Ndel restriction site at the ATG codon; inserting the 1556 bp ao ⁇ !-c amplified fragment
  • the plasmid pJTU2520 was obtained from the EcoRV site of pBlueScriptl SK (+); the apA-c was excised from P JTU2520 by Ndel and EcoRI and inserted into the corresponding site of plasmid pIB139 carrying the erythromycin promoter to obtain a plasmid.
  • pFAdpA The plasmid pJTU2520 was obtained from the EcoRV site of pBlueScriptl SK (+); the apA-c was excised from P JTU2520 by Ndel and EcoRI and inserted into the corresponding site of plasmid pIB139 carrying the ery
  • the sequence of the primer adpAc2F/adpAc lR ' is:
  • adpAc2F ( 5 ' -GGCTTAGCCATATGAGCCAC- 3 ' );
  • adpAclR (5 ' - CGTTCATGCGGGCCACTTTA- 3, ).
  • the vgbS PJTU4405 was excised into the corresponding restriction sites of pJTU968 by Ndel and EcoRI, and the w ⁇ S" gene fragment carrying the strong promoter of erythromycin was inserted into the EcoRI site of the pFMetK plasmid by Muni and EcoRI. Plasmid pFMV.
  • the apA_c was cleaved from pJTU2520 with Ndel and Eco PI and inserted into the corresponding site of the plasmid PJTU968 carrying the erythromycin promoter, and the atp-c gene fragment carrying the strong promoter of erythromycin was amplified by Muni and Eco ⁇ .
  • the plasmid pFMA was constructed by inserting into the EcoRI site of the pFMetK plasmid.
  • the ac/ ⁇ -c carrying the erythromycin promoter was excised from P JTU2522 into the EcoRI site of pFMV using Muni and EcoRI to obtain plasmid pFMVA.
  • the constructed plasmid was transformed into E. coli ET12567/pUZ8002, and the transformant was selected to culture E. coli ET12567/pUZ8002 carrying the transfer plasmid in liquid LB medium.
  • the final concentration of the liquid LB medium was 25 ⁇ g/ ⁇ .
  • the cells were collected after incubation at 37 ° C for 8 hours, and the cells were washed 3 times with fresh LB medium for use. 0.
  • the pH of the pH is 8. 0. 0. 05. Then, it was heat-shocked in a water bath at 55 ° C for 10 min. After cooling to room temperature, it was mixed with E. coli cells in an equal amount of 10 8 : 10 8 and then applied to the SFM medium plate.
  • the composition of the medium plate was: 2 %. Agarose (m/v), 2% mannitol (m/v), 2% soy cake powder (m/v), the pH of the culture plate was 7. 2 ⁇ 7. 5, dried and placed at 30 ° C The culture is carried out in an incubator.
  • the plate was covered with 1 ml of sterile water containing nalidixic acid and apramycin, the final concentration of nalidixic acid on the plate was 50 ng/mL, and the apramycin was 30 ng/mL.
  • the transfer zygote can be seen after 30 days of culture.
  • 2, 1 is a marker
  • 2, 3, 4, and 5 are strains ZYJ-6/pFMetK, ZYJ-6/pFMV, ZYJ-6/pFMA, and ZYJ-6/pFMVA chromosomes can be amplified by a template of 986 bp. Amplification band.
  • the pZVgb, pFMV, pFMVA three plasmids all contain the gZ ⁇ gene, and the ZYJ_6/pFVgb, ZYJ-6/pFMV, ZYJ-6/pFMVA strains obtained by the transformation of the plasmid were verified by PCR to be vgbTF/vgbTR, and the PCR product was 436 bp (see Figure 3).
  • 1 is marker
  • 2, 3, and 4 are strains ZYJ-6/pFVgb, ZYJ-6/pFMV, and ZYJ-6/pFMVA chromosomes can be used to amplify a 436 bp amplification band.
  • the ppdpA, pFMA, pFMVA three plasmids all contain the adpA gene, and the ZYJ-6/pFAdpA, ZYJ-6/pF A, ZYJ6-/pFMVA strains obtained by the transformation of the plasmid were verified by PCR as adpATF/adpATR, PCR products. It is 620 bp (see Figure 4).
  • 1 is marker
  • 2, 3, and 4 are strains ZYJ-6/pFAdpA, ZYJ-6/pFMA, and ZYJ-6/pFMVA chromosomes are used as templates to amplify a 620 bp amplification band.
  • metKTF 5' GAACAGACCCACGGGCTCGG 3'
  • metKTR 5' TGTCCCGTCGCCTGTTCACC 3'
  • vgbTF 5' GTGGACCAGCAGACCATCAA 3'
  • vgbTR 5' ACTCGACCGCCTGGGCGTAC 3'
  • adpATF 5' CGCAGGGACTGGAGGCGATC 3'
  • adpATR 5' CACCCGCTGGGTGATCAGCC 3' PCR reaction system: 0. 1 ⁇ ⁇ template DNA, primers 50 pmol each, 4 ⁇ DMSO, 4 ⁇ dNTP, 5 ⁇ PCR buffer and 1 unit Tag DNA polymerase.
  • the polymerase is a product of Japan T0Y0B0, plus pure Water to
  • the cycling conditions of the PCR reaction were: 94 ° C, 5 min; 94 ° C, 30 s; 57 ° C, 30 s; 72 ° C, 80 s (30 cycles); 72 ° C extension for 5 min, 4 ° C End the reaction.
  • the starting strain ZYJ-6 and the obtained ZYJ-6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA were sequentially liquid. Fermentation and antibiotic testing.
  • the strain ZYJ-6 was used as the host, and the yield of the FR-008 III component was used as the detection standard.
  • the strain was first activated on SFM plates and cultured for 30 days for 30 days. Then inoculated in 25 ml liquid medium TSBY ( 10. 3% sucrose), wherein the liquid medium TSBY is prepared by: tryptone (TSB) 30 g, Difco yeast powder 5 g, sucrose 340 g (34%) or 103 g ( 10. 3 %), to a volume of 1000 ml, sterilized.
  • TSBY tryptone
  • the cells were incubated at 30 ° C for 24 hours, sampled, centrifuged, collected, dried, and weighed to determine the difference in bacterial density between the strains. It was inoculated into 50 ml of liquid medium YEME at a volume ratio of about 1/100, and cultured at 30 ° C for 84 hours on a shaker.
  • liquid medium YEME The preparation method of liquid medium YEME is as follows: Take Digco yeast powder 3 g, Difco peptone 5 g, Oxoid malt powder 3 g, sucrose 103 g, glucose 10 g, then dilute to 1000 ml, sterilize, add 2 before use a sterile 2. 5 M MgCl 2 ⁇ 60 solution in ml.
  • the fermentation broth was taken at different culture times for extraction and detection of antibiotics.
  • the fermentation broth was taken at 36 h, 48 h, 60 h, 72 h and 84 h, respectively, then 2 volumes of n-butanol were added, shaken and extracted, and the supernatant was centrifuged, and the antibiotics in the broth were extracted three times.
  • the extracts were combined at a wavelength of 380 nm with a spectrophotometer (PerkinElmer®) and a non-antibiotic-producing mutant HJ-5 of Streptomyces FR-008 (Yirong Zhang, Linquan Bai and Zixin Deng.
  • Figure 5 shows the strain ZYJ-6 and the six strains constructed ZYJ-6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA Yield analysis of oxytocin.
  • the present invention provides a plasmid and a method for increasing the production of a Streptomyces antibiotic by using a plasmid, and one or several of the three genes having a broad promotion effect on the biosynthesis of antibiotics are simultaneously expressed on one vector. In a manner, the transfer is transferred to Streptomyces for efficient expression to increase the production of antibiotics.

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Description

说 明 书 质粒及利用质粒提高链霉菌抗生素产量的方法 技术领域
本发明涉及的是一种生物医药技术领域的方法及质粒， 具体涉及质粒及利用质粒提 高链霉菌抗生素产量的方法。
背景技术
随着细胞生物学以及生物工程技术在微生物尤其是抗生素育种领域的应用， 基因工 程育种技术已成为主要的菌种改良手段， 并且在改良工业生产菌种方面已获得巨大的成 功。 生物工程育种技术在一定范围内克服了传统育种技术的随机性和盲目性， 可以借助 对微生物次级代谢生物合成途径的认识， 利用基因工程技术构建抗生素高产菌株， 有助 于次级代谢产物的工业生产。
在微生物菌种选育领域中， 通过基因工程技术将目的基因与载体在体外连接然后转 入受体细胞， 使外源基因在受体中稳定表达遗传。 主要途径包括有： ①通过调节基因的 加倍或失活、 提高抗生素的主动外排效率等来提高抗生素的产量； ②在宿主细胞中引入 增加前体物和辅因子的供给的外源基因， 提高活性物质的生物^ ~成量； ③通过激活或抑 制支路代谢以改变生物活性分子的活性成分或比例， 提高最佳活性组分的积累、 降低或 消除低活性次级组分的积累。
AdpA是广泛存在于链霉菌中的一个全局性正调节因子， 是 A-因子调控网络的中心 转录调控因子， 可激活形态分化和次级代谢产物所需基因的表达。 在天蓝色链霉菌 ( Streptomyces coelicolor)、 变铅青链霉菌 i Streptomyces lividans) ^ 抗生链霉菌 ( Streptomyces antibioticus)和阿维链霉菌 ( Streptomyces a verwi til is) 中， AdpA 正调节黑色素的生物合成。 目前还没有文献报道 ao^I基因在抗生素产生菌株里的表达 对其抗生素产量的影响。
透明颤菌（ z' ireosci a)是一种专性好氧的丝状革兰氏阴性细菌， ^ 基因编码的 透明颤菌的血红蛋白具有结合氧的特性可以促进微生物细胞内氧的传输， 提高氧的利用 率。 对许多在溶氧不足的情况下， Vgb表达会促进宿主的生长， 蛋白的分泌， 代谢物的 产生以及增强抗压性。
将透明颤菌血红蛋白基因整合到异源宿主菌中可以增加重组蛋白产量和发酵物产 量。 经过对现有技术的检索发现， 在抗生素生产中， Wb基因可明显促进肉桂地链霉菌 的菌体生长和抗生素莫能菌素合成 （文莹， 宋渊. 透明颤菌血红蛋白在肉桂地链霉菌
{ Streptomyces cinnamonensis^ 中的表达对其细胞生长及抗生素合成的影响. 生物 工程学报， 2001， 17 (1 ) : 24-28 )。
Figure imgf000004_0001
基因的表达 使红霉素的产量提高 60% (Brunker, P. Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stabl e expression of the Vi treoscill hemoglobin gene (vhb ) . Microbiology 1998， 144 (9)： 2441-2448 )。
S-腺苷甲硫氨酸 （S- adenosylmethionine, 简称 SAM) ， 由三磷酸腺苷和 L-甲硫氨 酸经 SAM合成酶催化反应合成。 作为生物合成过程中的重要中间代谢物质， SAM合成酶 基因 ^i/r在很多链霉菌中的高效表达都能够有效提高其代谢物的产生。 在天蓝色链霉 菌 Streptomyces coelicolor) 里， SAM合成酶基因/ ffe 的高效表达能够大大提高放 线紫红素的产量 ( Okamoto S ， Lezhava A. Enhanced expressi on of S- Adenosylmethionine synthetase causes overproduct ion of act inorhodin in Streptomyces coelicolor k?> (2 ) . J Bacteriol , 2003， 185 (2)： 601 - 609 ) ； 在 Saccharopolyspora erythraea中异源表达 S-腺苷甲硫氨酸合成酶基因可以提高红霉素 A的产量 (Wang Y, Wang YG. Improved producti on of erythromycin A by expression of a heterologous gene encoding S- adenosylmethionine synthetase. Appl Microbiol Biotechnol , 2007, 75 (4) : 837-842 )； Streptomyces actuosus Φ SAM 成酶基因 ffiei r的高效表达促进了那西肽的合成 （Zhang XC, Fen MQ. Overexpression of yeast S-adenosylmethionine synthetase metK in Streptomyces ac tuosus leads to increased product ion of nosiheptide. Appl Microbiol Biotechnol , 2008， 78 (6)： 991 - 995 )。 '
但是上述现有技术仅是单一基因在某些微生物菌株中的表达， 其对代谢产物的影响 作用还是有限的， 对于整个代谢工程仍是非常不充分的。 微生物菌株自身复杂的代谢途 径以及细胞循环中复杂的调节方式及其特异性， 决定了对其代谢流中的某个反应进行定 向改变依然是不够的， 对微生物代谢途径的生化改造可以多步进行， 最大限度的增加代 谢产物的积累。 近年来， 由于 DNA重组技术的发展， 使得用基因技术改造代谢途径成为 可能。 本发明根据上述技术的不足， 设计了一个全新的提高链霉菌抗生素产量的方法及 质粒，将多个与抗生素合成相关的具有广泛正调控作用的基因构建在一个载体上同时表 达， 构建的质粒可以在不同链霉菌菌株中进行表达， 有效提高各种抗生素的产量。 发明内容
本发明的目的在于克服现有技术的不足， 提供一种全新的提高链霉菌抗生素产量的 方法以及使用到的质粒。 本发明提供了质粒及利用质粒提高链霉菌抗生素产量的方法， 通过对抗生素的生物合成有广泛促进作用的 3个基因中的一个或者几个采取在一个载体 上同时进行表达的方式， 利用接合转移转入链霉菌中进行高效表达以提高抗生素的产 量。
第一方面， 本发明提供了一种提高链霉菌 i Streptomyces 抗生素产量的方法， 包 括如下步骤： 将整合表达质粒转入链霉菌中， 进而实现链霉菌抗生素产量提高； 所述整 合表达质粒为 ^ZAS"基因与常规载体构建而得， 或者为 ζτ^ί/Γ和 b5 "基因与常规载体构 建而得，或者为 和 ac^-c基因与常规载体构建而得，或者为 metK、 和 adPA-c 基因与常规载体构建而得。
优选地， 所述提高链霉菌 treptomyces 抗生素产量的方法包括如下步骤：
(a)构建整合表达质粒；
(b)将整合表达质粒转入链霉菌中；
(c)发酵 (b)步获得的链霉菌， 获得抗生素。
优选地， 所述链霉菌为链霉菌 i Streptomyces sp. ) ZYJ- 6 CGMCC NO. 2394。
优选地， 所述常规载体为 PIB139载体。
优选地， 所述 zz/ei 基因的碱基序列如 SEQ ID NO. 1所示。
优选地， 所述 基因的碱基序列如 SEQ ID NO. 2所示。
优选地， 所述 ac^l- c基因的碱基序列如 SEQ ID NO. 3所示。
第二方面， 本发明还涉及一种由 ^/^基因与常规载体构建而得的整合表达质粒 pFVgb o
优选地， 所述常规载体为 PIB139载体。
进一步， 本发明还涉及前述整合表达质粒 pFVgb的构建方法， 包括如下步骤： (a)质粒 pJTU4405含有全基因合成 vgbS, 在基因 wb5"两端分别引入了 Ndel和 EcoRI酶切位点；
(b)用 Ndel和 EcoRI双酶切带有透明颤菌（ Vitreoscilla) 基因的 pJTU4405 ;
(c)将 wb5"基因插入含有红霉素抗性基因强启动子 PermE*的链霉菌整合型载体 PIB139的对应位点， 得到质粒 pFVgb。
第三方面， 本发明还涉及一种由 fflei 和 "基因与常规载体构建而得的整合表达 质粒 pFMV。
优选地， 所述常规载体为 PIB139载体。
进一步， 本发明还涉及一种如权利要求 11所述的整合表达质粒 pFMV的构建方法， 包括如下步骤： 制备质粒 pFMetK质粒； 之后用 Ndel和 EcoRI将 vgbS } pJTU4405上切 下插入 pJTU968的相应酶切位点， 再用 Muni和 EcoRI将带有红霉素强启动子的 b5基 因片段插入到 pFMetK质粒的 EcoRI位点， 得到质粒 pFMV。
优选地， 所述制备质粒 pFMetK质粒包括如下步骤：
(a)在天蓝色链霉菌 Streptomyces coelicolor) 的 metK 基因内部设计引物 metKF2/metKR, 在 ATG密码子处加入了一个 Ndel酶切位点；
引物 metKF的序列为： 5， - CAGGGAGCCATATGTCCCGT-3 ' ，
引物 metKR的序列为： 5 ' -TCGCAAAGGCCACTGACAACA-3 ' ；
(b)将扩增到的不带原始启动子的 1334bp的 metK基因插入 pBlueScriptll SK (+) 的 EcoRV位点构建 4292bp的克隆 pJTU2524;
(c)用 Ndel和 EcoRI将无启动子的 metK基因片段从 pJTU2524上切下插入到含有红 霉素抗性基因强启动子 PermE*的 pIB139载体中， 得到质粒 pFMetK。
第四方面， 本发明还涉及一种由 ζ^ί/Γ和 SC/ 4-C基因与常规载体构建而得的整合表 达质粒 pFMA。
优选地， 所述常规载体为 PIB139载体。
进一步地， 本发明还涉及前述整合表达质粒 pFMA的构建方法， 包括如下步骤： 用 Ndel和 EcoRI将 adPA-c从 pJTU2520上切下插入到带有红霉素启动子的质粒 pJTU968 的相应位点， 再用 Muni 和 EcoRI 将带有红霉素强启动子的 adpA-c基因片段插入到 pFMetK质粒的 EcoRI位点， 构建质粒 pFMA。
第五方面， 本发明还涉及一种由 ei r、 ^和 atp/i-c基因与常规载体构建而的整 合表达质粒 pFMVA。
优选地， 所述常规载体为 PIB139载体。
进一步地， 本发明还涉及前述整合表达质粒 pFMVA的构建方法， 包括如下步骤： 用 Muni和 EcoRI将带有红霉素启动子的 adpA- c从 pJTU2522上切下插入到 pFMV的 EcoRI 位点， 得到质粒 pFMVA。
本发明具有如下的有益效果： 本发明提供了质粒及利用质粒提高链霉菌抗生素产量 的方法，通过对抗生素的生物合成有广泛促进作用的 3个基因中的一个或者几个采取在 . 一个载体上同时进行表达的方式，利用接合转移转入链霉菌中进行高效表达以提高抗生 素的产量。
本发明涉及的链霉菌 CGMCC NO. 2394 ZYJ-6已提交中国微生物菌种保藏管理委员会 普通微生物中心， 其保藏登记编号为. CGMCC N0. 2394； 保藏地址为北京市朝阳区北辰西 路 1号院 3号， 中国科学院微生物研究所。 所述链霉菌 CGMCC NO. 2394 ZYJ-6已经公开 于 ((Zhou YJ， Li JL, Zhu J, Chen S, Bai LQ， Zhou XF， Wu HM, Deng ZX. Incomplete β -Ketone Processing as a Mechanism for Polyene Structural Variation in the FR-008/Candicidin Complex. Chemi stry & Biology, 2008, 15 : 629-638》 。 所述链 霉菌 CGMCC NO. 2394 ZYJ-6定向生产杀念菌素单一组份 FR-008- III， 其产量是野生型菌 株该组份产量的 120-130 %，而 FR- 008-ΠΙ是杀念菌素 3个主要组分中最具药用价值的 成份， 通过该工程菌可以大规模生产高纯度的杀念菌素有效组份， 具有显著的工业应用 价值。
所述 PIB139记载于《 11 :^50：1 CJ， Hughes- Thoraas ZA, Martin CJ，Bohm L Mironenko T， Deacon M， Wheatcroft M， Wirtz G， Staunton J , Leadlay PF. Increasing the efficiency of heterologous promoters in actinomycetes. J Mol Microb Biotech, 2002, 4 (4)： 417-426》。
所述的菌株 PJTU4405记载于中国专利申请 201010148837. 2， 公开号 CN 101805742 A, 公开日 2010- 8- 18的发明专利文献 《透明颤菌血红蛋白 vgbS核苷酸序列及其质粒和制备 方法》 中。 所述的菌株 PJTU4405通过以下方式制备得到： 将 ^b基因在链霉菌中使用频 率不高的密码子改为在链霉菌中使用频率最高的密码子， 保持氨基酸序列不变， 产生新 的基因 Wb5"。 在基因 wb5M端分别引入 Ndel和 EcoRI的酶切位点， 其中在 ^b5t亥苷酸序 列的 5 ' 端添加 7个核苷酸， 形成限制性内切酶 Ndel位点和保护碱基， 在 ^/λ5核苷酸序列 的 3 ' 端添加 8个核苷酸， 形成限制性内切酶 EcoRI位点和保护碱基。 将;^ 5"全基因合成， 全长 456bp。 合成的 456bp的 b ¾DNA被插在载体 pGH的 Smal位点， 产生质粒 pJTU4405， 测序证明合成无误。
所述大肠杆菌 ET12567/pUZ8002公开于《Paget， M. S. , Chamberl in, L.， Atrih, A. , Foster , S. J. , and Buttner , M. J. . Evidence that the extracytoplasmic function sigma factor σ E is required for normal cell wall structure in Streptomyces coelicolor A3 (2 ) . J. Bacteriol , 1999 , 181 : 204 - 211》 。
附图说明 图 1为表达质粒的构建图。
图 2为菌株 ZYJ- 6/pFMetK， ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ- 6/pFMVA中 »ei 基因 的 PCR验证。
图 3为菌株 ZYJ-6/pFVgb, ZYJ-6/pFMV, ZYJ-6/pF VA菌株中 wb5"基因的 PCR验证。 图 4 为菌株 ZYJ- 6/pFAdpA， ZYJ-6/pFMA, ZYJ-6/pFMVA中 a p I- c基因的 PCR验证。 图 5 为分光光度法分析菌株 ZYJ- 6和 ZYJ- 6/pFMetK， ZYJ-6/pFVgb, ZYJ-6/pFAdpA,
ZYJ-6/pFMV, ZYJ-6/pF A, ZYJ- 6/pFMVA中杀念菌素 FR- 008- III在不同培养时间的产量 变化。
图 6为定量分析出发菌株 ZYJ-6和高产菌株 ZYJ-6/pFMVA中杀念菌素 FR- 008-ΙΠ 的产量变化。
具体实施方式
下面结合具体实施例， 进一步阐述本发明。 这些实施例仅用于说明本发明而不 用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规 条件， 例如 Sambrook 等分子克隆： 实验室手册 （New York : Cold Spring Harbor Laboratory Press, 1989 ) 中所述的条件， 或按照制造厂商所建议的条件。
实施例 1、 提高链霉菌抗生素产量的整合表达质粒的构建
本实施例涉及的基因表达载体 PIB139是一个含有红霉素抗性基因强启动子 PermE* 的链霉菌整合型载体， 可以通过大肠杆菌和链霉菌之间的两亲本接合转移进入链霉菌， 发生位点特异性重组后整合在链霉菌的染色体上， 随着染色体一起复制和遗传。 图 1为 本实施例中涉及的所有表达质粒构建图。
1. 1质粒 pFMetK的构建
PCR扩增天蓝色链霉菌 M145中不带原始启动子的 /^ί 基因， 具体步骤包括： 在天 蓝色链霉菌的 ffei 基因内部设计引物 metKF2/metKR，在 ATG密码子处加入了一个 Ndel 酶切位点；将扩增到的不带原始启动子的 1334bp的/^ i/r基因插入 PBlueSCriptn SK(+) 的 EcoRV位点构建 4292bp的克隆 pJTU2524; 用 Ndel和 EcoRI将无启动子的 7?ei r基因 片段从 PJTU2524上切下插入到含有红霉素抗性基因强启动子 PermE*的 pIB139载体中， 得到质粒 pFMetK。
所述的引物 metKF2/metKR的序列为：
met F (5， -CAGGGAGCCATATGTCCCGT-3 ' )；
raetKR (5' - TCGCAAAGGCCACTGACAACA-3， )<, 1. 2质粒 pFVgb的构建
质粒 PJTU4405含有全基因合成 wb5"，长度 456bp，在基因 两端分别引入了 Ndel 和 EcoRI酶切位点。 用 Ndel和 EcoRI双酶切带有透明颤菌 基因的 pJTU4405。 将
"基因 （441bp ) 插入含有红霉素抗性基因强启动子 PermE*的链霉菌整合型载体 PIB139的对应位点， 得到质粒 pFVgb。
1. 3质粒 pFMV的构建
PCR 扩增天蓝色链霉菌中的 adpA-c基因， 具体步骤包括： 设计引物 adpAc2F/ adpAclR， 在 ATG密码子处加入了一个 Ndel酶切位点； 将 1556bp的 ao^!-c扩增片段插 入到 pBlueScriptl l SK (+ ) 的 EcoRV位点， 得到质粒 pJTU2520 ; 再用 Ndel和 EcoRI将 adpA-c从 PJTU2520上切下插入到带有红霉素启动子的质粒 pIB139的相应位点，得到质 粒 pFAdpA。
所述的引物 adpAc2F/ adpAc lR '的序列为：
adpAc2F ( 5 ' -GGCTTAGCCATATGAGCCAC- 3 ' )；
adpAclR (5 ' - CGTTCATGCGGGCCACTTTA- 3， )。
1. 4含有 metK和 ^bS"两个基因的质粒 pFMV的构建
用 Ndel和 EcoRI将 vgbS PJTU4405上切下插入 pJTU968的相应酶切位点， 再用 Muni和 EcoRI将带有红霉素强启动子的 w^S"基因片段插入到 pFMetK质粒的 EcoRI位点， 得到质粒 pFMV。
1. 5含有膨卿 adpA-c两个基因的质粒 pFMA的构建
用 Ndel和 Eco PI将 adpA_c从 pJTU2520上切下插入到带有红霉素启动子的质粒 PJTU968的相应位点， 再用 Muni和 Eco^将带有红霉素强启动子的 atp - c基因片段插 入到 pFMetK质粒的 EcoRI位点， 构建质粒 pFMA。
1. 6含有 metJ、 和 c的质粒 pFMVA 的构建
用 Muni和 EcoRI将带有红霉素启动子的 ac/^-c从 PJTU2522上切下插入到 pFMV的 EcoRI位点， 得到质粒 pFMVA。
实施例 2、 把质粒转入链霉菌宿主以及接合转移子的筛选和验证
将构建的质粒转入大肠杆菌 ET12567/pUZ8002中， 挑转化子在液体 LB培养基中培 养携带有转移质粒的大肠杆菌 ET12567/pUZ8002， 该液体 LB培养基内存在有终浓度为 25 μ g/ μ L的氯霉素， 50 μ g/ μ L的卡那霉素和 30 μ g/ μ L的阿泊拉霉素。
37°C培养 8小时后收集菌体， 用新鲜 LB培养基洗涤菌体 3次备用。 将链霉菌孢子悬浮于 5ml TES缓冲液中，该缓冲液浓度为 0. 05 mol/L, pH值为 8. 0。 然后在 55 °C水浴中热激. 10 min， 冷却至室温后， 按 108： 108与大肠杆菌细胞等量 混合后涂在 SFM培养基平板上， 该培养基平板组分为： 2 %琼脂糖 （m/v )， 2 % 甘露醇 (m/v ) , 2 % 黄豆饼粉 （m/v)， 培养平板 pH值为 7. 2〜7. 5， 吹干后放在 30°C培养箱中 进行培养。
12小时后用含萘啶酮酸和阿泊拉霉素的 1 ml无菌水覆盖平板， 平板上萘啶酮酸的 终浓度为 50 ng/mL, 阿泊拉霉素为 30 ng/mL, 置 30Ό培养 3天后即可看到转移接合子。
从覆盖板上挑选单个接合转移子接种到阿泊拉霉素抗性平板上进一步确认抗性。 以 备选菌株的总 DNA作为 PCR模板， pFMetK, pFMA, pFMV ， pFMVA四个质粒中都含有 y7?ei r 基因， 质粒经转化获得的 ZYJ-6/pFMetK， ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ- 6/pFMVA菌株 用 PCR验证使用的引物为 metKTF/metKTR, PCR产物为 986 bp (见图 2 ) 。 图 2中 1为 marker , 2、 3、 4、 5分别为菌株 ZYJ- 6/pFMetK， ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA 的染色体为模板可以扩增得到 986 bp扩增带。
pFVgb , pFMV, pFMVA三个质粒中都含有 gZ^基因，质粒经转化获得的 ZYJ_6/pFVgb， ZYJ-6/pFMV, ZYJ- 6/pFMVA菌株用 PCR验证使用的引物为 vgbTF/vgbTR, PCR产物为 436 bp (见图 3 ) 。 图 3中 1为 marker, 2、 3、 4分别为菌株 ZYJ-6/pFVgb , ZYJ-6/pFMV, ZYJ-6/pFMVA的染色体为模板可以扩增得到 436 bp扩增带。
pFAdpA , pFMA , pFMVA 三个质粒中都含有 adpA 基因， 质粒经转化获得的 ZYJ-6/pFAdpA, ZYJ-6/pF A, ZYJ6-/pFMVA菌株用 PCR验证使用的引物为 adpATF/adpATR, PCR产物为 620 bp (见图 4 ) 。 图 4中 1为 marker , 2、 3、 4分别为菌株 ZYJ- 6/pFAdpA， ZYJ-6/pFMA, ZYJ- 6/pFMVA的染色体为模板可以扩增得到 620 bp扩增带。
引物序列 ：
metKTF: 5' GAACAGACCCACGGGCTCGG 3'
metKTR: 5' TGTCCCGTCGCCTGTTCACC 3'
vgbTF: 5' GTGGACCAGCAGACCATCAA 3'
vgbTR: 5' ACTCGACCGCCTGGGCGTAC 3'
adpATF: 5' CGCAGGGACTGGAGGCGATC 3'
adpATR: 5' CACCCGCTGGGTGATCAGCC 3' PCR反应体系： 0. 1 μβ模板 DNA, 引物各 50 pmol , 4 μΐ DMSO, 4 μΐ dNTP， 5 μΐ PCR缓冲液和 1个单位 Tag DNA聚合酶， 该聚合酶为日本 T0Y0B0公司产品， 加纯水到
PCR反应的循环条件为： 94°C， 5 min; 94 °C , 30 s; 57 °C , 30 s; 72°C， 80 s (30 个循环） ； 72°C延伸 5 min, 4°C结束反应。
实施例 3、 菌株发酵以及抗生素的提取和检测
将出发菌株 ZYJ-6和获得的 ZYJ - 6/pFMetK, ZYJ-6/pFVgb, ZYJ-6/pFAdpA, ZYJ-6/ pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA六个菌株依次进行液体发酵及抗生素检测。以菌株 ZYJ - 6 作为宿主， 以 FR-008 III组分的产量作 ¾检测标准。
首先将菌株在 SFM平板上活化， 30 培养 3天。然后接种在 25 ml液体培养基 TSBY ( 10. 3%蔗糖） 中， 其中液体培养基 TSBY的制备方法为： 胰蛋白胨 （TSB) 30 g， Difco 酵母粉 5 g， 蔗糖 340 g (34 %) 或 103 g ( 10. 3 %) ， 定容至 1000 ml , 分装灭菌。
30°C摇床培养 24小时， 取样， 离心， 收菌体， 烘干， 称干重， 确定各菌种之间菌 体密度的差异， 调整到一致。 按大约 1/100体积比接种到 50 ml液体培养基 YEME中， 30°C摇床培养 84小时。
液体培养基 YEME的制备方法为： 取 Difco酵母粉 3 g， Difco蛋白胨 5 g， Oxoid 麦芽粉 3 g， 蔗糖 103 g， 葡萄糖 10 g， 然后定容至 1000 ml， 灭菌， 使用前补加 2 ml 的 无菌 2. 5 M MgCl2 · 6 0溶液。
在不同的培养时间取发酵液进行抗生素的提取和检测。 分别在 36 h， 48 h， 60 h, 72 h和 84 h取发酵液， 然后加入 2倍体积的正丁醇， 振荡萃取， 离心取上清， 重复三 次， 将菌液中的抗生素萃取出来， 合并萃取液， 在波长 380 nm处用分光光度计检测 (PerkinElmer®) ， 同时以链霉菌 FR-008的不产抗生素的突变株 HJ- 5 (Yirong Zhang, Linquan Bai and Zixin Deng. Functional characterization of the first two actinomycete 4'- amino- 4- deoxychorismate lyase genes. Microbiology, 2009, 155 : 2450 - 2459) 的发酵液作为空白对照。 实验重复三次。
图 5为菌株 ZYJ-6和构建的六个菌株 ZYJ- 6/pFMetK， ZYJ-6/pFVgb, ZYJ-6/pFAdpA, ZYJ-6/pFMV, ZYJ-6/pFMA, ZYJ-6/pFMVA产生的杀念菌素的产量分析。
六个菌株与出发菌株同时进行平行发酵检测， 结果表明， 前五个菌株使杀念菌素的 产量依次提高了 90% (pFMetK)、 130% (pFVgb)、 80% (pFAdpA)、 130% (pFMV) 、 150% (pFMA) (见图 5 ) ; 图 6表示三个基因的整合型质粒 pFMVA在 ZYJ-6中的表达最终将 杀念菌素的产量提高了 2. 1倍， 产量从 424ug/ml (ZYJ-6 ) 提高到 1301ug/ml
(ZYJ-6/pFMVA) 。
综上所述， 本发明提供了质粒及利用质粒提高链霉菌抗生素产量的方法， 通过对抗 生素的生物合成有广泛促进作用的 3个基因中的一个或者几个采取在一个载体上同时进 行表达的方式， 利用接合转移转入链霉菌中进行高效表达以提高抗生素的产量。

Claims

权 利 要 求 书
1、 一种提高链霉菌 StrePtomyces 抗生素产量的方法， 其特征在于， 包括如下 步骤： 将整合表达质粒转入链霉菌中， 进而实现链霉菌抗生素产量提高； 所述整合表达 质粒为 wb5"基因与常规载体构建而得， 或者为 y^i 和 wb5"基因与常规载体构建而得， 或者为 Hei/r和 acp - c基因与常规载体构建而得， 或者为 fflei T、 和 3φ Ι- c基因与 常规载体构建而得。
2、 如权利要求 1 所述的提高链霉菌抗生素产量的方法， 其特征在于， 包括如下步 骤：
(a)构建整合表达质粒；
(b)将整合表达质粒转入链霉菌中；
(c)发酵 (b)步获得的链霉菌， 获得抗生素。
3、 如权利要求 1所述的提高链霉菌抗生素产量的方法， 其特征在于， 所述链霉菌 为链霉菌 Streptomyces sp. ) ZYJ-6 CGMCC NO. 2394。
' 4、 如权利要求 1所述的提高链霉菌抗生素产量的方法， 其特征在于， 所述常规载 体为 PIB139载体。
5、 如权利要求 1所述的提高链霉菌抗生素产量的方法， 其特征在于， 所述 ^ίΤΓ基 因的碱基序列如 SEQ ID NO. 1所示。
6、 如权利要求 1所述的提高链霉菌抗生素产量的方法， 其特征在于， 所述 "基 因的碱基序列如 SEQ ID NO. 2所示。
7、 如权利要求 1所述的提高链霉菌抗生素产量的方法， 其特征在于， 所述 ao^I - c 基因的碱基序列如 SEQ ID NO. 3所示。
8、 一种由 基因与常规载体构建而得的整合表达质粒 pFVgb。
9、 如权利要求 8所述整合表达质粒 pFVgb, 其特征在于， 所述常规载体为 PIB139 载体
10、 一种如权利要求 8所述整合表达质粒 pFVgb的构建方法， 其特征在于， 包括如 下步骤：
(a)质粒 pJTU4405含有全基因合成 vgbS, 在基因 两端分别引入了 Ndel和 EcoRI酶切位点；
(b)用 Ndel和 EcoRI双酶切带有透明颤菌（ Vi treoscilla 基因的 pJTU4405; (c)将 b5"基因插入含有红霉素抗性基因强启动子 PermE*的链霉菌整合型载体 PIB139的对应位点， 得到质粒 pFVgb。
11、 一种由 z/ei r和 wb5"基因与常规载体构建而得的整合表达质粒 pFMV。
12、 如权利要求 11所述整合表达质粒 pFMV， 其特征在于， 所述常规载体为 pIB139 载体 <=
13、 一种如权利要求 11所述的整合表达质粒 pFMV的构建方法， 其特征在于， 包括 如下步骤： 制备质粒 pFMetK质粒； 之后用 Ndel和 EcoRI将 vgbS pJTU4405上切下插 入 PJTU968的相应酶切位点， 再用 Muni和 EcoRI将带有红霉素强启动子的 基因片 段插入到 pFMetK质粒的 EcoRI位点， 得到质粒 pFMV。
14、 如权利要求 13所述的整合表达质粒 pFMV的构建方法， 其特征在于， 所述制备 质粒 pFMetK质粒包括如下步骤：
(a)在天蓝色链霉菌 <i Streptomyces coeli color) 的 metK 基因内部设计引物 metKF2/metKR, 在 ATG密码子处加入了一个 Ndel酶切位点；
引物 metKF的序列为： 5， -CAGGGAGCCATATGTCCCGT- 3， ，
弓 I物 metKR的序列为： 5 ' -TCGCAAAGGCCACTGACAACA- 3 ' ；
(b)将扩增到的不带原始启动子的 1334bp的 metK基因插入 pBlueScriptll SK (+ ) 的 EcoRV位点构建 4292bp的克隆 pJTU2524;
(c)用 Ndel和 EcoRI将无启动子的 metK基因片段从 PJTU2524上切下插入到含有红 霉素抗性基因强启动子 PermE*的 pIB139载体中， 得到质粒 PFMetK。
15、 一种由
Figure imgf000014_0001
ao^!-c基因与常规载体构建而得的整合表达质粒 pFMA。
16、 如权利要求 15所述整合表达质粒 pFMA， 其特征在于， 所述常规载体为 PIB139 载体
17、 一种如权利要求 15所述的整合表达质粒 pFMA的构建方法， 其特征在于， 包括 如下步骤: 用 Ndel和 EcoRI将 adpA-c k pJTU2520上切下插入到带有红霉素启动子的 质粒 pJTU968的相应位点， 再用 Muni和 EcoRI将带有红霉素强启动子的 ac^4- c基因片 段插入到 pFMetK质粒的 EcoRI位点， 构建质粒 pFMA。
18、 一种由 ^、 "和 ao^l-c基因与常规载体构建而的整合表达质粒 pFMVA。
19、如权利要求 18所述整合表达质粒 pFMVA,其特征在于，所述常规载体为 pIB139 载体
20、 一种如权利要求 18所述的整合表达质粒 pFMVA的构建方法， 其特征在于， 包 括如下步骤： 用 Muni和 EcoRI将带有红霉素启动子的 adpA_c从 pJTU2522上切下插入 到 pFMV的 EcoRI位点， 得到质粒 pFMVA。
PCT/CN2012/000276 2011-03-08 2012-03-05 质粒及利用质粒提高链霉菌抗生素产量的方法 WO2012119484A1 (zh)

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