WO2016074221A1 - 多杀菌素异源表达菌株及其构建方法和应用 - Google Patents

多杀菌素异源表达菌株及其构建方法和应用 Download PDF

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WO2016074221A1
WO2016074221A1 PCT/CN2014/091118 CN2014091118W WO2016074221A1 WO 2016074221 A1 WO2016074221 A1 WO 2016074221A1 CN 2014091118 W CN2014091118 W CN 2014091118W WO 2016074221 A1 WO2016074221 A1 WO 2016074221A1
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gene cluster
nucleic acid
plasmid
spinosyn
seq
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PCT/CN2014/091118
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French (fr)
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黄隽
余贞
李美红
郑玲辉
李娜
王海彬
白骅
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浙江海正药业股份有限公司
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Priority to BR112017010073-8A priority Critical patent/BR112017010073A2/zh
Priority to CN201480083229.4A priority patent/CN107075461B/zh
Priority to JP2017525575A priority patent/JP6641366B2/ja
Priority to PCT/CN2014/091118 priority patent/WO2016074221A1/zh
Priority to EP14905979.2A priority patent/EP3219788A4/en
Priority to US15/526,075 priority patent/US10844415B2/en
Publication of WO2016074221A1 publication Critical patent/WO2016074221A1/zh

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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
    • 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
    • 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

Definitions

  • the invention belongs to the field of biotechnology and relates to heterologous expression of spinosyn; more specifically, to a spinosad heterologous expression strain, a construction method and application thereof.
  • spinosad is a macrolide compound that combines the safety of biopesticides with the quick-acting properties of chemical pesticides. In June 1999, it received the US President's Green Chemical Challenge Award.
  • spinosyn is fermented by Saccharopolyspora spinosa. Its biosynthesis mainly includes two aspects: one is glycoside ligand, which is synthesized by polyketide synthase (PKS) pathway; the other is buckthorn The addition and modification of sugar and mannose are responsible for the relevant synthetase and modification enzyme, respectively (see Su Jianya et al., Biosynthesis of Spinosad, Chinese Journal of Biotechnology, Vol. 23, No. 5).
  • PKS polyketide synthase
  • other related spinosyn biosynthetic genes are clustered in a DNA fragment of about 80 kb (GenBank AY007564).
  • S. spinosa is a naturally occurring bacterium of spinosad, the yield of this bacterium is not high. Moreover, the genetic manipulation of S. spinosa is difficult, and the transformation space is limited, and it is difficult to obtain a strain with good production performance.
  • a kind of spinosad and Saccharopolyspora erythraea are interspecifically mixed by a method of parental inactivation to select a spinosyn high-yield strain (Wu Ping et al., Protoplast fusion and high spinosyn production) Strain, Grain and Oil Food Technology, 2012, 20 (3): 46-49).
  • a kind of spinosad and Saccharopolyspora erythraea are interspecifically mixed by a method of parental inactivation to select a spinosyn high-yield strain (Wu Ping et al., Protoplast fusion and high spinosyn production) Strain, Grain and
  • the object of the present invention is to provide a method for constructing a spinosyn expression bacterium and to obtain a spinosad heterologous expression bacterium to solve the problem that the genetic background of the recombinant expression bacterium is unclear and the genetic trait is unstable.
  • the present invention provides a method of constructing a spinosyn heterologous expression bacterium, which replaces an erythromycin synthetic gene cluster in S. erythropolis with a spinosyn of S. spinosa Synthesizing a gene cluster and a rhamnose synthesis gene cluster; preferably, the method uses a plurality of homologous recombination methods to replace the erythromycin synthesis gene cluster in S. erythropolis with a polysaccharide A bacteriocin synthesis gene cluster and a rhamnose synthesis gene cluster.
  • the method process comprises:
  • step (1) obtaining a spinosyn synthetic gene cluster sequence covering the S. spinosa and a plurality of nucleic acid fragments thereof in the upstream and downstream sequences, and overlapping sequences between adjacent nucleic acid fragments; (2) using homologous recombination,
  • the plurality of nucleic acid fragments obtained in the step (1) are sequentially ligated into the genome of the S. erythraea, and the erythromycin synthesis gene cluster in the S. erythraea is subjected to the spinosyn synthetic gene cluster sequence of S.
  • spinosa And the upstream and downstream sequences are replaced to obtain a recombinant strain; (3) obtaining a nucleic acid fragment of a rhamnosylose gene cluster of S. spinosa, and replacing the nucleic acid fragment of the rhamnose synthesizing gene cluster by homologous recombination (2) A downstream sequence of the spinosyn synthetic gene cluster of the obtained recombinant strain to obtain a spinosyn heterologous expression bacterium.
  • a plurality of nucleic acid fragments are separately constructed into a plasmid, and the plasmid completes homologous recombination with S. erythropolis, wherein the sequence of 5'-3' sequences according to a plurality of nucleic acid fragments is
  • the other plasmids include a sequentially ligated 5' homology arm, a nucleic acid fragment obtained in the step (1), and a resistance gene necessary for conjugative transfer. a cassette in which the 5' homology arm of each plasmid is homologous to the upstream sequence of the erythromycin synthesis gene cluster of S.
  • the plasmid containing the last nucleic acid fragment includes a sequentially linked resistance gene cassette, 5' The source arm, the last nucleic acid fragment, and the 3' homology arm, wherein the 3' homology arm is homologous to the downstream sequence of the erythromycin synthesis gene cluster of S. erythraea.
  • the plasmid containing the last nucleic acid fragment is first homologously recombined with the original S. erythropolis, and the plasmid containing the other nucleic acid fragment is sequentially sequentially homologously recombined with the previous step. Homology recombination was performed.
  • the starting S. erythraea is ATCC 11635.
  • the cosmid supercos-1 is used as a starting plasmid; preferably, the resistance gene cassette comprises an aac(3)IV+oriT sequence.
  • the plasmid construction process of the above step (2) is: first, inserting the upstream and downstream nucleic acid fragments of the erythromycin erythromycin synthetic gene cluster (about 3 kb each) into the cosmid supercos-1, respectively, as 5' homology arm and 3' homology arm, the modified cosmid eryUD-cos2 was obtained; and the nucleic acid fragment obtained in step (1) was further ligated into two homology arms of cosmid eryUD-cos2; Inserting a resistance gene cassette upstream of the 5' homology arm of the plasmid containing the last nucleic acid fragment; in other plasmids, replacing the 3' homology arm of the other plasmid with a resistance gene cassette; further preferably, by homology The method of recombination replaces the 3' homology arms of other plasmids with a resistance gene cassette.
  • sequence of the 5' homology arm is set forth in SEQ ID NO. 46
  • sequence of the 3' homology arm is set forth in SEQ ID NO.
  • the rhamnose synthesis gene cluster is constructed into a plasmid for homologous recombination, and the plasmid is homologously recombined with the recombinant strain obtained in the step (2), and the plasmid includes two The source arm and the rhamnose synthesis gene cluster located between the homologous arms, each of which is homologous to the downstream sequence of the spinosyn synthetic gene cluster, respectively.
  • the sequences of the two homology arms are SEQ ID NO. 49 and SEQ ID NO. 48, respectively.
  • the plurality of nucleic acid fragments are at least 3 nucleic acid fragments, preferably 3, 4, 5, 6, 7 nucleic acid fragments, each of which has a size of 25-40 kb; Further preferably, the plurality of nucleic acid fragments are 4 nucleic acid fragments, the sequences of which are shown in SEQ ID NO. 17-20, respectively.
  • a genomic library is constructed by digesting genomic DNA of S. spinosa with Sau3AI, and a plurality of nucleic acids covering the spinosyn synthetic gene cluster sequence and its upstream and downstream sequences are screened by PCR. Fragment.
  • the present invention provides a spinosyn heterologous expression bacterium constructed by the method of the present invention, wherein the expression bacterium is a erythromycin bacterium, wherein the erythromycin synthesis gene cluster is thorny polysaccharide The spinosyn synthetic gene cluster and the rhamnose synthetic gene cluster are substituted.
  • the expression bacterium is genetically engineered bacteria ES05.
  • the present invention provides the use of the spinosyn heterologous expression bacterium of the present invention for the preparation of spinosyn.
  • the present invention provides a method of preparing spinosyn, which uses the spinosyn heterologous expression construct constructed by the present invention.
  • the present invention replaces the erythromycin biosynthesis gene cluster of erythromycin-producing bacteria Saccharopolyspora erythraea (32 kb, GenBank AY661566.1) with a spinosyn biosynthesis gene cluster (80 kb, GenBank AY007564).
  • the obtained recombinant bacteria also has the potential for transformation, which is superior to the original spinosyn-producing bacteria Saccharomyces cerevisiae; and the fermentation time of the recombinant bacteria is short, it is not easy to be infected, and more profitable. In the production of spinosad, reduce costs, ensure quality, easy to industrialize large-scale production.
  • Figure 1 Construction process of plasmid pBS-eryUD
  • FIG. 2 Plasmid map of plasmids Supercos-1 (A) and eryUD-cos2 (B);
  • Figure 3 Digestion results of total DNA of spinosyn producing bacteria; each lane shows: 1, total DNA without digestion; 2, enzyme digestion for 5 min; 3, enzyme digestion for 8 min; 4, enzyme digestion for 11 min; Digestion for 14 min; 6, digestion for 17 min; 7, enzymatic digestion for 20 min; M, ⁇ / Hind III;
  • Figure 4 Enzyme digestion results of total DNA of spinosyn producing bacteria; each lane shows: 1, digestion for 5 min; 2, enzyme digestion for 6 min; 3, enzyme digestion for 7 min; M, ⁇ /Hind III;
  • Figure 5 Schematic diagram of the screening of a genomic plasmid library using a 96-well plate
  • Figure 6 Schematic diagram of the position of each gene and the location of the genomic plasmid library in the spinosyn synthetic gene cluster
  • Figures 8A-8F PCR electrophoresis results for further screening of the positive results obtained in Figures 7A-7F;
  • Figures 9A-9D PCR electrophoresis results of screening a plasmid library comprising spnD and spnE;
  • Figures 10A-10D PCR electrophoresis results for further screening of the positive results obtained in Figures 9A-9D;
  • Figure 11 Schematic diagram of fragment modification and homologous recombination of the spinosyn synthetic gene cluster
  • FIG. 13 Map of plasmid pUAmT14
  • Figure 14 Map of plasmid pAT-DgegU
  • Figure 16 Schematic diagram of PCR detection of overlapping portions of each fragment.
  • the apramycin resistance gene cassette aac(3)IV+ oriT replaces the extra homologous arm fragment on the spinosyn synthetic gene cluster fragment so that its homology arm is equivalent to the length of the homology arm designed in eryUD-cos2, and has the elements necessary for the junction transfer. Since the resistance gene cassette after PCR targeting replacement is outside the two homology arms, when the homologous double exchange occurs and the target fragment is transferred to the chromosome of S. erythropolis, it is lost, so the next round of pruning And can be reused in DNA fragment transfer.
  • the four plasmid plasmids which were trimmed were sequentially transformed into S. erythraea by the method of conjugative transfer in the order of the contained DNA fragments, and the transfer of the gene fragments was carried out by screening the double exchanger. After 4 steps of transfer, the 80 kb spinosyn synthetic gene cluster was successfully transferred to the original location of the erythromycin synthesis gene cluster on the chromosome of S. erythraea. Also transferred are fragments of about 10 kb upstream and downstream of the spinosyn synthetic gene cluster, which can serve as target sites for subsequent genetic manipulation, referred to as "operable zone 1" and "operable zone 2", respectively.
  • the exogenous fragment was formulated in a molar ratio of 3:1 to 9:1 with a linearized vector in a total volume of 3 ⁇ l.
  • 3 ⁇ l of Solution I (TaKaRa, Cat. No. D6020A) was added, and the water bath was kept at 16 ° C for 30 min or more.
  • the final concentration of each antibiotic is as follows: the final concentration of ampicillin (Ap) is 100 ⁇ g/mL, kanamycin ( The final concentration of Km) and apramycin (Am) were both 50 ⁇ g/mL, the final concentration of chloramphenicol (Cm) was 25 ⁇ g/mL, 37 ° C (BW25113 was 30 ° C), and the culture was carried out at 220 rpm until the OD600 was 0.4. Between -0.6;
  • the cells were collected by centrifugation, suspended in 1/10 volume of 100 mM CaCl 2 /15% (W/V) glycerol, and packed in a 1.5 ml centrifuge tube at 100 ⁇ l/tube to obtain competent cells;
  • Bacterial solution (if the plasmid is transformed, take 50 ⁇ l; if the ligation product is transformed, centrifuge and remove most of the supernatant, and leave the remaining liquid suspension cells) on the LB plate containing the corresponding screening antibiotic, 37 ° C ( BW25113 was cultured at 30 ° C for 14-18 h, and transformants were grown.
  • reaction solution was prepared in the following proportions: 2 ⁇ PrimeSTAR GC buffer (Mg 2+ Plus), 25 ⁇ l; dNTP mixture (2.5 mM each), 4 ⁇ l; upstream primer (25 ⁇ M), 1 ⁇ l; downstream primer (25 ⁇ M), 1 ⁇ l; ddH 2 O, 18 ⁇ l; template DNA, 0.5 ⁇ l; HS DNA polymerase, 0.5 ⁇ l;
  • Reaction procedure 95 ° C ⁇ 5 min; (98 ° C ⁇ 10 sec, 68 ° C ⁇ 1 min / 1 kb target fragment length) ⁇ 25 cycles; 72 ° C ⁇ 2 min, 16 ° C ⁇ 1 min.
  • the PCR product recovery kit (No. W5202) and the glue recovery kit (No. W5203) of Shanghai Huasheng Biotechnology Co., Ltd. were used for recycling.
  • step 4) Repeat step 3) and pour off the liquid in the recovery tube;
  • reaction solutions were prepared: purified DNA fragment, 4.2 ⁇ l; BKL buffer, 0.5 ⁇ l; BKL enzyme mixture, 0.25 ⁇ l; 37 ° C water bath for 30 min or more, and then transferred to a 70 ° C water bath for 5 min to inactivate the enzyme mixture.
  • Escherichia coli ET12567 (pUZ8002) (Gust B, Kieser T, Chater KF. REDIRECT Technology: PCR-targeting system in Streptomyces coelicolor. Norwich: John Innes Centre. 2002: 13-35) Competent cells were prepared according to Method 2, cultured When adding Km, Cm to the culture medium;
  • step 6 the cells containing 10 6 -10 8 cells suspended in 500 ⁇ l of 2 ⁇ YT medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%) The spore solution was heat-shocked at 50 ° C for 10 min and cooled at room temperature;
  • step 7) Take 500 ⁇ l of the bacterial solution of step 5) and heat-sprayed spore solution of step 6), centrifuge at 9000rcf for 1 min, discard about 800 ⁇ l of the supernatant, and suspend the cells in residual liquid, and apply the MS (soybean cake). Powder 2%, mannitol 2%, agar powder 1.5%) plate (the plate was previously blown in a sterile environment for 1 h to partially dehydrate), cultured at 34 ° C for 14-18 h;
  • Method 8 Preparation of plasmid DNA by alkaline lysis
  • reaction solutions were prepared: 2 ⁇ GC I buffer (TaKaRa, Cat. No. DRR20GCI), 25 ⁇ l; 2.5 mM dNTP, 4 ⁇ l; Primer 1 (25 ⁇ M), 1 ⁇ l; Primer 2 (25 ⁇ M), 1 ⁇ l; Template DNA, 1 ⁇ l (10-100 ng) ); H 2 O, 32.5 ⁇ l.
  • the PCR product is less than 3 kb, and rTaq DNA polymerase (TaKaRa, product number R001) is added, 0.5 ⁇ l;
  • the PCR product was larger than 3 kb, and LATaq DNA polymerase (TaKaRa, product number DRR002B) was added, 0.5 ⁇ l.
  • PCR test uses ordinary Taq DNA polymerase, see Method 9 for details;
  • the "ligation-derived plasmid” is a continuous operation process, including the following aspects of operation: (1) ligation reaction of DNA fragments (see method 1); (2) transformation of ligation reaction products To E. coli DH5 ⁇ (see method 2); (3) pick the transformant to extract the plasmid; (4) the plasmid was digested with an endonuclease, and the fragment size of the digested product was checked to be consistent with the expected;
  • PCR amplification refers to the use HS DNA polymerase and GC buffer for amplification, see Method 3;
  • reagents used in the following examples are commercially available from chemical or biological reagent stores or suppliers; the instruments used are also conventional in the art.
  • steps (1) and (2) are to combine the upstream and downstream fragments of the erythromycin biosynthesis gene cluster (ie, two homology arms for the double exchange, the 5' homology arm and the 3' homology arm)
  • the vector was cloned into the vector pBluscript KS(+) (GenBank: X52331.1), and a BamH I site was introduced between the two homologous arms, and a Not I position was introduced at the front end of the upstream fragment and the end of the downstream fragment. Point to cut a fragment containing two homology arms with Not I.
  • the build process is shown in Figure 1:
  • PCR product After the PCR product was recovered, it was digested with Not I (TaKaRa) for 1 h, and the digested product was recovered, and then digested with BamH I (TaKaRa) for 1 h to recover the double-cut product; the vector pBluscript KS(+) was also followed by Not I and BamH I were digested and recovered, and ligated with the above double-digested PCR product, and screened and tested to obtain recombinant plasmid 1: pBS-eryU;
  • primer edF SEQ ID NO. 3, introduced Pst I site at the 5' end
  • edR SEQ ID NO. 4, introduced at the 3' end
  • the Not I and Xho I sites were PCR amplified a 2642 bp fragment 2 of the erythromycin biosynthesis gene cluster: eryD (SEQ ID NO. 47, comprising the restriction site and the protected base sequence, and thus the size was 2667 bp).
  • telomere sequence After recovery of the PCR product, double digestion with Xho I and Pst I, respectively, with recombinant plasmid 1: pBS-eryU: 10 ⁇ H buffer, 5 ⁇ l; PCR product (or plasmid 1: pBS-eryU), 25 ⁇ l; ddH 2 O , 18 ⁇ l; Pst I (TaKaRa, Cat. No. 1073A), 1 ⁇ l; Xho I (TaKaRa, Cat. No. 1094A), 1 ⁇ l.
  • the recombinant plasmid 2: pBS-eryUD was obtained by a 37 ° C water bath for 1 h, separately recovered, ligated, and screened and identified.
  • the spore solution of spinosad-producing bacteria (NRRL 18538) was stored in 30 ml of TSB medium, and total DNA was extracted according to method 10.
  • reaction solutions were prepared: total spinosyn producing bacteria DNA prepared in Example 2, 100 ⁇ l; 10 ⁇ H buffer, 15 ⁇ l; ddH 2 O, 35 ⁇ l.
  • reaction solutions were prepared: spinosyn producing bacteria total DNA, 1000 ⁇ l; 10 ⁇ H buffer, 150 ⁇ l; ddH 2 O, 350 ⁇ l.
  • reaction solution was prepared: a partially digested product of the total DNA of the spinosyn producing strain, 100 ⁇ l; FastAP buffer, 11.5 ⁇ l; FastAP, 3 ⁇ l.
  • reaction solutions were prepared: Cosmid EryUD-Cos2, 50 ⁇ l; 10 ⁇ H buffer, 10 ⁇ l; ddH 2 O, 37 ⁇ l; Xho I, 3 ⁇ l.
  • reaction solutions were prepared: 5' dephosphorylated linearized cosmid EryUD-Cos2, 50 ⁇ l; 10 x BamH I buffer, 10 ⁇ l; ddH 2 O, 37 ⁇ l; BamH I, 3 ⁇ l.
  • reaction solution was prepared: a partially digested product of dephosphorylated spinosyn producing total DNA, 3 ⁇ l; cosmid EryUD-Cos2 double-digested with Xho I and BamH I, 1 ⁇ l; 10 ⁇ T4 ligase buffer 3 ⁇ l; ddH 2 O, 21 ⁇ l; T4 ligase (TaKaRa, Cat. No. D2011A), 2 ⁇ l.
  • Packaging kit is III XL Packaging System (Stratagene Inc., Cat. No.: 200101), follow these steps:
  • the LB medium containing 100 ⁇ g/ml Cb was dispensed into 15 96-well plates at 150 ⁇ l/well, and the numbers of the 15 plates were 1# to 15#, respectively, and each well was inoculated with a toothpick to pick up one of the above transformants. Incubate at 37 ° C ⁇ 220 rpm for about 16 h.
  • the longitudinal direction of the 16#96 well plate the A1-A12 hole in the 1#96 well plate is mixed into the A1 hole of the 16#96 plate, the B1-B12 hole is mixed into the B1 hole of the 16#96 plate, and so on, H1 The -H12 well was mixed into the H1 well of the 16#96 well plate.
  • A1-A12 hole in 1#96 plate is mixed into A1 hole of 16#96 plate
  • A1-A12 hole in 2#96 plate is mixed to A2 of 16#96 plate Holes, and so on, the A1-A12 wells in the 12#96 well plates were mixed into the A12 wells of the 16#96 well plates. See Figure 5 for details.
  • the longitudinal direction of the 17#96 well plate the A1-A12 hole in the 13#96 well plate is mixed into the A1 hole of the 17#96 plate, and the B1-B12 hole is mixed into the B1 hole of the 17#96 plate, H1- The H12 well was mixed into the H1 well of a 17#96 well plate.
  • the transverse direction of the 17#96 well plate the A1-A12 hole in the 13#96 well plate is mixed into the A1 hole of the 17#96 plate (renumbered as A13), and the A1-A12 hole in the 14#96 plate is mixed to 17
  • the A2 well of the #96 well plate (renumbered A14)
  • the A1-A12 well of the 15#96 well plate was mixed into the A3 well of the 17#96 well plate (renumbered A15).
  • spnR the relative positions on the spinosyn biosynthetic gene cluster are 4168-5330, 20151-21020, 34049-34639, respectively.
  • the relative positions of these three fragments in the spinosyn biosynthetic gene cluster are shown in Figure 6 (the arrow on the spinosyn biosynthetic gene cluster indicates the location of the fragment).
  • the PCR product sizes of partial fragments of these three genes were 1163 bp (spnR), 870 bp (spnF) and 591 bp (spnB), respectively.
  • the primer sequences used for PCR amplification of these three genes are:
  • spnR primer spnRF (SEQ ID NO. 5) and spnRR (SEQ ID NO. 6);
  • spnF primer spnFF (SEQ ID NO. 7) and spnFR (SEQ ID NO. 8);
  • spnB primers spnBF (SEQ ID NO. 9) and spnBR (SEQ ID NO. 10).
  • 2 ⁇ GC I buffer 750 ⁇ l; 2.5 mM dNTP, 120 ⁇ l; spnRF (25 ⁇ M), 15 ⁇ l; spnRR (25 ⁇ M), 15 ⁇ l; spnFF (25 ⁇ M), 15 ⁇ l; spnFR (25 ⁇ M), 15 ⁇ l; spnBF (25 ⁇ M), 15 ⁇ l; spnBR (25 ⁇ M), 15 ⁇ l; ddH 2 O, 535 ⁇ l; rTaq (TaKaRa, Cat. No. DR001A), 7.5 ⁇ l.
  • E1-E12 (denoted as 7E1-7E12), H1-H12 (denoted as 9H1-9H12) of 9#96 plate, A1-A12 (denoted as 10A1-10A12), D1-D12 of 10#96 plate ( The 10S1-10D12), G1-G12 (denoted as 10G1-10G12) and H1-H12 (denoted as 10H1-10H12) bacteria have corresponding genes of interest, and the above 7 are recorded as the second group.
  • the first set of results showed that 1F9, 4E11, 4F11, 9B5 and 9D10 respectively had corresponding target fragments; the second set of results showed that 7E9, 9H9, 10A5, 10D5, 10D6 and 10G3 respectively had corresponding target fragments.
  • the sequencing primers were: cosF: SEQ ID NO. 11; cosR: SEQ ID NO.
  • spnD primer spnDF (SEQ ID NO. 13), spnDR (SEQ ID NO. 14);
  • spnE primer spnEF (SEQ ID NO. 15), spnER (SEQ ID NO. 16).
  • 2 ⁇ GC I buffer 750 ⁇ l; 2.5 mM dNTP, 120 ⁇ l; spnDF (25 ⁇ M), 15 ⁇ l; spnDR (25 ⁇ M), 15 ⁇ l; spnEF (25 ⁇ M), 15 ⁇ l; spnER (25 ⁇ M), 15 ⁇ l; ddH 2 O, 580 ⁇ l; rTaq , 7.5 ⁇ l.
  • B8, C2, D4, E4, E12, F4, A15 and D15 had corresponding target bands, namely B1-B12 of 8#96 well plate (denoted as 8B1-8B12) and C1 of 2#96 plate.
  • -C12 denoted as 2C1-2C12
  • D1-D12 denoted as 4D1-4D12
  • E1-E12 denoted as 4E1-4E12
  • F1-F12 denoted as 4F1-4F12
  • the samples were sequenced by the above-mentioned numbering.
  • the sequencing primers are also cosF and cosR.
  • the relative positions of the spinosyn biosynthetic gene cluster fragments contained in each library plasmid are shown in the following table.
  • the sequence position disclosed in GenBank AY007564.1 is set to 1-80161, and 9D10, 10G3, 15A11 and 15D1 have partial sequences outside this range.
  • 10G3 SEQ ID NO. 17
  • 15D1 SEQ ID NO. 20
  • the fragments carried by the four plasmids 10G3, 9B5, 8B8 and 15D1 can cover the complete spinosyn biosynthesis
  • the gene cluster and its upstream and downstream sequences, the sequences of the fragments carried by the four plasmids are as follows:
  • FIG. 11 is a schematic illustration of the entire process in which "US" is the upstream fragment eryU of the erythromycin synthesis gene cluster, "DS", the downstream fragment eryD of the erythromycin synthesis gene cluster.
  • the purpose of this example is to insert a resistance fragment containing the aac(3)IV gene (apramycin resistance gene) and oriT (the junction transfer initiation site, an essential element for junctional transfer) into the library plasmid 15D1.
  • the engineered plasmid can be used for conjugative transfer.
  • This example was carried out in two steps: first, the resistance gene cassette was excised from the plasmid pIJ773 with Cla I and EcoR I, and after blunt-endification (SEQ ID NO: 65), Hinc was ligated to the vector pUC118 (TaKaRa, Cat. No. D3322). II site.
  • the transformant in the correct insertion direction can be screened, and the resistant fragment can be excised with Hind III;
  • the second step is to insert the Hind III excision fragment into the Hind III site of 15D1. Since the resistant fragment only functions as a screening and junction transfer, its insertion direction does not affect the results of subsequent experiments, so it is not necessary to determine its insertion direction.
  • the specific operation is as follows (shown in Figure 12):
  • reaction solutions were prepared: pIJ773, 20 ⁇ l; 10 ⁇ H buffer, 5 ⁇ l; ddH 2 O, 23 ⁇ l; Cla I (TaKaRa, Cat. No. D1034A), 1 ⁇ l; EcoR I (TaKaRa, Cat. No. D1040A), 1 ⁇ l.
  • the 1389 bp fragment containing the aac(3)IV gene and oriT was recovered by electrophoresis at 37 ° C for 1 h. After blunt-endization with the BKL kit, it was ligated with pUC118/Hinc II, BAP (TaKaRa, Cat. No. D3322). The transformant was picked and the plasmid was digested with Hind III, and the recombinant plasmid with the result of digestion was 3502 bp + 1402 bp.
  • a 1402 bp fragment containing the aac(3)IV gene and oriT was recovered by electrophoresis, and ligated with HindIII-digested and dephosphorylated plasmid 15D1 to obtain a recombinant plasmid 15D1-AmT.
  • Example 5 Spinosyn biosynthesis gene cluster The first fragment migrated to Saccharopolyspora
  • Recombinant plasmid 15D1-AmT was transformed into S. erythraea (ATCC 11635) by conjugative transfer (see Method 7). After the transformants were passaged in two relaxation cultures, the apramycin-sensitive colonies were screened. Genomic DNA was extracted (according to Method 10, but the volume of TSB was changed to 3 ml, and the volume was still 500 ⁇ l except for 70% alcohol washing, and the volume of other reagents was also reduced accordingly. The same as the primer spnEF (SEQ ID NO. 15) /spnER (SEQ ID NO. 16) and primer ery1F (SEQ ID NO. 21) / ery1R (SEQ ID NO. 22) were subjected to PCR assay.
  • the primer spnEF/spnER amplifies the internal sequence of the DNA fragment carried by the library plasmid 15D1, and the primer ery1F/ery1R amplifies the sequence inside the erythromycin synthesis gene cluster. Therefore, spnEF/spnER can amplify the target band and ery1F/ery1R can not amplify the target band, indicating that the erythromycin synthesis gene cluster has been replaced by the DNA fragment carried by the library plasmid 15D1, which is the target strain.
  • the genetically engineered strain ES01 was screened.
  • Example 6 Example 8 and Example 10
  • the purpose of the following Example 6, Example 8 and Example 10 is to utilize the resistance gene cassette aac(3)IV+oriT (the resistance
  • the sex gene cassette was PCR amplified from plasmid pIJ773, and positions 14-1382 of SEQ ID NO. 65) replaced the DS on the corresponding library plasmid and the partial sequence at the 3' end of the carried genomic fragment. It is mainly divided into two steps: firstly, positive and negative primers with lengths of 59 nt and 58 nt, respectively, 39 nt at the 5' end of the primer as a homology arm, and 20 nt at 3' (forward primer, SEQ ID NO. 63) and 19 nt (reverse primer, SEQ ID NO.
  • the resistance gene cassette was amplified from pIJ773 by PCR, and the PCR product introduced a 39 bp homologous arm at both ends of the resistance gene cassette. The two homologous arms were respectively located on the library plasmid to be replaced by the fragment. Both sides; the PCR product was then transformed into E. coli BW25113 (pIJ790) containing the library plasmid, and the target sequence was replaced with a resistance gene cassette using the recombinant system within the strain. See Table 2 for information on the primers used and the fragments to be replaced in the examples.
  • the library plasmid 8B8 was transformed according to Method 2 to BW25113 (pIJ790) (see Document 2) competent cells;
  • the cells were collected by centrifugation at 4 ° C, washed twice with 10% glycerol, and then suspended in 100 ⁇ l of 10% glycerol to obtain electroporation competent cells;
  • Reaction system 5 ⁇ PrimeSTAR buffer (Mg 2+ Plus), 25 ⁇ l; dNTP mixture (2.5 mM each), 4 ⁇ l; primer 8BA-L (SEQ ID NO. 23, 25 ⁇ M), 1 ⁇ l; primer 8BA-R (SEQ ID NO. 24, 25 ⁇ M), 1 ⁇ l; ddH 2 O, 18 ⁇ l; plasmid pIJ773, 0.5 ⁇ l; HS DNA polymerase, 0.5 ⁇ l.
  • Reaction procedure 95 ° C ⁇ 5 min; (98 ° C ⁇ 10 sec, 50 ° C ⁇ 10 sec, 72 ° C ⁇ 90 sec) ⁇ 10 cycles; 72 ° C ⁇ 2 min, 16 ° C ⁇ 1 min. (98 ° C ⁇ 10 sec, 68 ° C ⁇ 90 sec) ⁇ 15 cycles; 72 ° C ⁇ 1 min, 16 ° C ⁇ 1 min.
  • the gel was recovered to recover a target fragment of about 1.4 kb.
  • step (6) Take 3 ⁇ l of the resistant fragment obtained in step (5) and add 50 ⁇ l of BW25113 (pIJ790/8B8) obtained in step (4).
  • the competent cells were electrotransformed and transferred to a 2 mm electrotransformation cup (BioRad).
  • the electric shock parameters are: 2500V, 25 ⁇ F, 200 ⁇ .
  • 1 ml of SOC medium (2 ml of 1 mol/L glucose per 100 ml of SOB medium) was added, and the whole amount was transferred to a 1.5 ml centrifuge tube.
  • the larger transformant was picked up in 3 ml of LB liquid medium containing Am, cultured at 37 ° C ⁇ 200 rpm for 6 h, and the plasmid was extracted using primer 8BD-L (SEQ ID NO. 25) / 8BD-R (SEQ ID NO) .26) Perform PCR test (extension time in PCR reaction program is 4 min). Primers 8BD-L and 8BD-R are located on both sides of the substituted sequence on library plasmid 8B8, respectively. If the replacement is successful, the PCR product should be a 1963 bp target band plasmid; if the substitution is unsuccessful, the PCR product is 3957 bp. After screening, recombinant plasmid 8B8-AmT was obtained.
  • Example 7 Spinosyn biosynthesis gene cluster The second fragment migrated to Saccharopolyspora
  • the recombinant plasmid 8B8-AmT was transformed into the genetically engineered strain ES01 obtained in Example 5 by conjugative transfer. After the transformants were passaged in two relaxation cultures, the apramycin-sensitive colonies were screened. Total DNA was extracted and PCR assay was performed with primer 8BD-L (SEQ ID NO. 25) / 8BD-R (SEQ ID NO. 26). The principle is the same as that of the step (8) of the embodiment 6. The PCR product is only a 1963 bp band which is the target strain. The genetically engineered strain ES02 was screened.
  • the primer for the amplification resistance gene cassette was 9B5-L (SEQ ID NO. 27) / 9B5-R (SEQ ID NO. 28).
  • Plasmid PCR Assay A plasmid capable of amplifying a 1881 bp band of interest was screened using primers 95A-L (SEQ ID NO. 29)/95A-R (SEQ ID NO. 30) to obtain a recombinant plasmid 9B5-AmT.
  • Example 9 The third fragment of the spinosyn biosynthesis gene cluster migrated to Saccharopolyspora
  • the recombinant plasmid 9B5-AmT was transformed into the genetically engineered strain ES02 obtained in Example 7 by conjugative transfer. After the transformants were passaged in two relaxation cultures, the apramycin-sensitive colonies were screened. Total DNA was extracted and PCR assay was performed with primers 95A-L (SEQ ID NO. 29) / 95A-R (SEQ ID NO. 30). The PCR product was only a 1881 bp band which was the target strain. The genetically engineered strain ES03 was screened.
  • the primer for the amplification resistance gene cassette was 10G3-L (SEQ ID NO. 31) / 9B5-R (SEQ ID NO. 28).
  • the PCR assay was carried out by using primer 10G-L (SEQ ID NO. 32)/10G-R (SEQ ID NO. 33), and a plasmid capable of amplifying a 1676 bp target band was selected to obtain a recombinant plasmid 10G3-AmT.
  • Example 11 The fourth fragment of the spinosyn biosynthetic gene cluster migrated to Saccharopolyspora
  • the recombinant plasmid 10G3-AmT was transformed into the genetically engineered strain ES03 obtained in Example 9 by conjugative transfer. After the transformants were passaged in two relaxation cultures, the apramycin-sensitive colonies were screened. Total DNA was extracted and PCR was performed with primer 10G-L/10g-R. The PCR product was only a 1676 bp band and was the target strain. The genetically engineered strain ES04 was screened.
  • Example 12 Construction of a recombinant plasmid containing a polysaccharide synthase gene cluster of S. spinosa
  • the purpose of this example is to collect four rhamnose synthesis genes and clone them together between the upper and lower homology arms for the genetic engineering obtained by inserting these four genes into the same example using homologous double exchange.
  • the 80 kb spinosyn biosynthetic gene cluster was inserted into the erythromycin synthesis gene cluster by the previous examples, and the erythromycin synthesis gene cluster was deleted, but also introduced.
  • Two sequences derived from spinosad spp., which are not related to spinosyn biosynthesis, are called "operable regions" (as shown in Figure 11, KCZ1). KCZ2). While this embodiment selects two homology arms in one of the "operable regions", the four genes inserted will not destroy the spinosyn biosynthetic gene cluster. Therefore, this embodiment includes the following steps:
  • Endonuclease Xba I is an enzyme affected by methylation.
  • the plasmid extracted from a host strain having methylation function such as DH5 ⁇ cannot. It is cleaved by Xba I, but the PCR product is not methylated. Therefore, when the PCR product is directly digested, regardless of the sequence of the base behind the recognition site, the digestion is not affected.
  • the downstream homology arm was first cloned into the Ase I-Hind III site of the vector pUAmT14, and the restriction enzyme digestion ensured that the orientation of the insert was correct, and the Xba I site was located upstream of the downstream homologous arm.
  • Reaction system fragment 5 (or vector pUAmT14), 20 ⁇ l; 10 ⁇ Tango buffer, 5 ⁇ l; ddH 2 O, 23 ⁇ l; Ase I (Fermentas, Cat. No. ER0911), 1 ⁇ l; EcoR I (Fermentas, Cat. No. ER0501), 1 ⁇ l.
  • primer 007UF (SEQ ID NO. 36, introduced Xba I site affected by methylation at the 5' end) /008UR (SEQ ID NO. 37, introduced at the 5' end PCR amplification was performed on the Xba I site affected by methylation to obtain an upstream homologous arm fragment 6: U PCR (SEQ ID NO. 49). Fragment 6 was digested with Xba I, ligated into and transformed with plasmid pAT-D digested with Xba I and dephosphorylated, and the transformant was plasmid-plucked with primer 009F (SEQ ID NO. 44) / 010R (SEQ ID NO.
  • Forward primer 009F is located on the upstream homology arm and reverse primer 010R is located on the downstream homology arm. If the insertion direction of the upstream homology arm is correct, the PCR product should be 170 bp, and if the insertion direction is wrong, the PCR product will not be obtained.
  • the recombinant plasmid pAT-DU was obtained by screening.
  • primer gttF (SEQ ID NO. 38, introduced Xba I site not affected by methylation at the 5' end) /gttR (SEQ ID NO. 39, at 5 PCR amplification was carried out by introducing a Xba I site affected by methylation to obtain a fragment 7 containing the gtt gene: gttPCR (SEQ ID NO. 50). Fragment 7 was digested with Xba I and ligated with plasmid pAT-DU digested with Xba I and dephosphorylated to obtain recombinant plasmid pAT-DgU.
  • the total DNA of S. spinosa was used as a template, and the primer epiF (SEQ ID NO. 40, introduced at the 5' end of the Xba I site not affected by methylation) / epiR (SEQ ID NO. 41, at 5) The end of the 'Xba I site affected by methylation was introduced for PCR amplification to obtain fragment 8 containing the epi gene: epi PCR (SEQ ID NO. 51). Fragment 8 was digested with Xba I and ligated with plasmid pAT-DgU digested with Xba I and dephosphorylated to obtain recombinant plasmid pAT-DgeU.
  • the total DNA of S. spinosa was used as a template, and the primer gdhF (SEQ ID NO. 42, introduced at the 5' end of the Xba I site not affected by methylation) / gdhR (SEQ ID NO. 43, in 5 PCR amplification was carried out by introducing an Xba I site not affected by methylation to obtain a fragment 9 containing the gdh+kre gene: gdh PCR (SEQ ID NO. 52).
  • Fragment 9 was digested with Xba I and ligated with the plasmid pAT-DgeU digested with Xba I and dephosphorylated to obtain a recombinant plasmid pAT-DgegU, and the plasmid map is shown in Fig. 14.
  • steps (3)-(5) need only confirm that they have been inserted, and it is not necessary to judge the direction and order of insertion.
  • the plasmid pAT-DgegU includes four rhamnose synthesis genes.
  • Example 13 Transfer of the spine synthase gene cluster of S. spinosa to Saccharopolyspora erythraea
  • the recombinant plasmid pAT-DgegU was transformed into the genetically engineered strain ES04 obtained in Example 11 by conjugative transfer. After the transformants were passaged in two relaxation cultures, the apramycin-sensitive colonies were screened. Total DNA was extracted and PCR assay was performed with primer 009F (SEQ ID NO. 44) / 010R (SEQ ID NO. 45). Primers 009F and 010R are located in the upstream homologous arm and the downstream homologous arm, respectively; if the four rhamnose synthesis genes have been successfully inserted, the PCR product should be 4931 bp; if not inserted, it is 1322 bp. The genetically engineered strain ES05 was screened.
  • the interface between the plasmids of the spinosyn synthetic gene clusters in the genetically engineered strain ES05 was sequenced, and the relative positions of the interfaces were as shown in Fig. 16.
  • the results are shown in Table 3.
  • the sequencing results were consistent with the expected results, indicating that the spinosyn synthetic gene cluster has been transferred into the genetically engineered bacteria, and the sequence sequence is consistent with the sequence sequence of the spinosyn synthetic gene cluster in S. spinosa.
  • Porition refers to the relative position on the spinosyn biosynthetic gene cluster. Since there is no overlap between EU-10G3 and 15D1-ED, only the position linked to the chromosome of S. erythraea is displayed;
  • the sequence of the spinosyn synthetic gene cluster and the rhamnose synthetic gene cluster inserted in the genetic engineering strain ES05 was further sequenced, and the results were completely consistent with the expected sequence, indicating that the method can obtain genetically engineered bacteria with clear genetic background.
  • Example 14 Fermentation test of genetically engineered strain ES05
  • the colony of ES05 was cultured in seed medium (starch 3.0%, soybean cake powder 2.5%, peptone 0.5%, dextrin 3.0%, glucose 1.0%, sodium chloride 0.4%, pH 7.5), 34 ° C ⁇ 200 rpm After 48 hours of culture, transfer to the fermentation medium with 10% inoculum (soybean cake powder 3.0%, corn starch 4.0%, dextrin 3.0%, ammonium sulfate 0.2%, calcium sulfate 0.6%, glucose 1.0%, dihydrogen phosphate) Potassium 0.04%, pH 6.8), cultured at 34 ° C ⁇ 200 rpm for 7-8 days.

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Abstract

提供了一种多杀菌素异源表达菌株的构建方法，通过该方法获得的多杀菌素异源表达菌株及其在制备多杀菌素中的应用。方法利用多次同源重组，将红色糖多孢菌的红霉素合成基因簇替换为多杀菌素合成基因簇和鼠李糖合成基因簇，使红色糖多孢菌表达多杀菌素。

Description

多杀菌素异源表达菌株及其构建方法和应用 技术领域
本发明属于生物技术领域，涉及多杀菌素的异源表达；更具体地，涉及一种多杀菌素异源表达菌株、其构建方法和应用。
背景技术
多杀菌素是一种大环内酯类化合物，兼有生物农药的安全性和化学农药的速效性，并于1999年6月获得美国“总统绿色化学品挑战奖”。在自然界中，多杀菌素由刺糖多孢菌(Saccharopolyspora spinosa)发酵而来，其生物合成主要包括两方面：一是糖苷配体，由聚酮合酶(PKS)途径合成；二是鼠李糖和福乐糖胺的加入和修饰，分别由相关的合成酶和修饰酶负责(参见苏建亚等，多杀菌素的生物合成，中国生物工程杂志，第23卷第5期)。在刺糖多胞菌中，除鼠李糖合成基因外，其它相关的多杀菌素生物合成基因成簇地集中在一个约80kb的DNA片段(GenBank AY007564)上。
刺糖多胞菌虽为多杀菌素的天然产生菌，但是该菌产量并不高。且刺糖多孢菌的遗传操作比较困难，改造空间有限，难以获得具有良好生产性能的菌株。现有技术中，有人通过双亲灭活的方法对刺糖多孢菌及红色糖多孢菌进行种间融合以选育多杀菌素高产菌株(吴萍等，原生质体融合选育多杀菌素高产菌株，粮油食品科技，2012，20(3)：46-49)。但是通过原生质体融合后，两个亲本的基因组之间发生了错综复杂的重组，导致最后筛选到的菌株遗传背景复杂，遗传性状不稳定，不利于进一步的基因改造。
发明内容
本发明的目的是提供一种构建多杀菌素表达菌的方法并获得多杀菌素异源表达菌，以解决上述的重组表达菌遗传背景不清楚和遗传性状不稳定的问题。
在第一个方面，本发明提供一种构建多杀菌素异源表达菌的方法，所述方法将红色糖多胞菌中的红霉素合成基因簇替换为刺糖多胞菌的多杀菌素合成基因簇和鼠李糖合成基因簇；优选地，所述方法是利用多次同源重组的方式，将红色糖多胞菌中的红霉素合成基因簇替换为刺糖多胞菌的多杀菌素合成基因簇和鼠李糖合成基因簇。
进一步优选地，该方法过程包括：
(1)获得覆盖刺糖多胞菌的多杀菌素合成基因簇序列及其上下游序列的多个核酸片段，相邻的核酸片段之间有重叠序列；(2)利用同源重组的方式，将步骤(1)获得的多个核酸片段依次连入红色糖多胞菌基因组中，使红色糖多胞菌中的红霉素合成基因簇被刺糖多胞菌的多杀菌素合成基因簇序列及其上下游序列替换，获得重组菌；(3)获得刺糖多胞菌的鼠李糖合成基因簇的核酸片段，利用同源重组的方式，使鼠李糖合成基因簇的核酸片段替换步骤(2)获得的重组菌的多杀菌素合成基因簇的下游序列，获得多杀菌素异源表达菌。
优选地，步骤(2)中，将多个核酸片段分别构建成质粒，所述质粒与红色糖多胞菌完成同源重组，其中，按照多个核酸片段从5’-3’的序列顺序，除包含最后一个核酸片段的质粒外，其它质粒均包括顺次连接的5’同源臂、步骤(1)获得的核酸片段和接合转移必需的抗性基因 盒，其中每个质粒的5’同源臂与红色糖多胞菌的红霉素合成基因簇上游序列同源；包含最后一个核酸片段的质粒包括顺次连接的抗性基因盒、5’同源臂、最后一个核酸片段和3’同源臂，其中所述3’同源臂与红色糖多胞菌的红霉素合成基因簇的下游序列同源。进一步优选地，首先将包含最后一个核酸片段的质粒与起始红色糖多胞菌进行同源重组，再按顺序依次将包含其它核酸片段的质粒与前一步同源重组获得的红色糖多胞菌进行同源重组。优选地，所述起始红色糖多胞菌是ATCC11635。
进一步优选地，步骤(2)中以柯斯质粒supercos-1为起始质粒；优选地，所述抗性基因盒包括aac(3)IV+oriT序列。
进一步优选地，上述步骤(2)的质粒构建过程为：首先在柯斯质粒supercos-1中插入红色糖多孢菌红霉素合成基因簇的上、下游核酸片段(分别大约3kb)，分别作为5’同源臂和3’同源臂，得到改造的柯斯质粒eryUD-cos2；再将步骤(1)获得的核酸片段连入至柯斯质粒eryUD-cos2的两条同源臂之间；在包含最后一个核酸片段的质粒的5’同源臂的上游接入抗性基因盒；在其它质粒中，用抗性基因盒替换其它质粒的3’同源臂；进一步优选地，通过同源重组的方法用抗性基因盒替换其它质粒的3’同源臂。
进一步优选地，所述5’同源臂的序列如SEQ ID NO.46所示，所述3’同源臂的序列如SEQ ID NO.47所示。
优选地，步骤(3)中，将鼠李糖合成基因簇构建成用于同源重组的质粒，所述质粒与步骤(2)获得的重组菌进行同源重组，所述质粒包括两个同源臂和位于同源臂之间的鼠李糖合成基因簇，所述两个同源臂均与多杀菌素合成基因簇的下游序列分别同源。优选地，所述两个同源臂的序列分别为SEQ ID NO.49和SEQ ID NO.48。
优选地，步骤(1)中，所述多个核酸片段为至少3个核酸片段，优选地，为3、4、5、6、7个核酸片段，每个核酸片段的大小为25-40kb；进一步优选地，所述多个核酸片段为4个核酸片段，其序列分别如SEQ ID NO.17-20所示。
优选地，步骤(1)中，通过将刺糖多胞菌的基因组DNA以Sau3AI进行酶切，构建基因组文库并利用PCR方法筛选覆盖多杀菌素合成基因簇序列及其上下游序列的多个核酸片段。
在第二个方面，本发明提供一种通过本发明的方法构建的多杀菌素异源表达菌，所述表达菌为红色糖多胞菌，其中的红霉素合成基因簇被刺糖多胞菌的多杀菌素合成基因簇和鼠李糖合成基因簇取代。优选地，所述表达菌是基因工程菌ES05。
在第三个方面，本发明提供本发明的多杀菌素异源表达菌在制备多杀菌素中的应用。
在第四个方面，本发明提供一种制备多杀菌素的方法，所述方法使用本发明构建的多杀菌素异源表达菌。
本发明以刺糖多孢菌的多杀菌素生物合成基因簇(80kb，GenBank AY007564)置换红霉素产生菌红色糖多孢菌的红霉素生物合成基因簇(32kb，GenBank AY661566.1)，并加入鼠李糖生物合成基因簇(共四个基因：gdh+kre，GenBank AF355468.1；gtt，GenBank AF355467.1；epi，GenBank AF355466.1)，使红色糖多孢菌表达多杀菌素，得到的基因工程菌遗传背景清楚，其整个基因组的序列信息相当明确，因此可以对任何位置的基因进行精确的改造，便于进行基因操作和菌种选育。由于红色糖多胞菌本身具有改造潜力，因而得到的重组菌同样具有改造潜力，优于原多杀菌素生成菌刺糖多胞菌；且重组菌发酵时间短，不容易染菌，更利 于生产多杀菌素，降低成本，保证质量，易于工业化大规模生产。
附图说明：
图1：质粒pBS-eryUD的构建过程；
图2：质粒Supercos-1(A)和eryUD-cos2(B)的质粒图谱；
图3：多杀菌素产生菌总DNA的酶切结果；各泳道分别表示：1、未经酶切的总DNA；2、酶切5min；3、酶切8min；4、酶切11min；5、酶切14min；6、酶切17min；7、酶切20min；M、λ/Hind III；
图4：多杀菌素产生菌总DNA的酶切结果；各泳道分别表示：1、酶切5min；2、酶切6min；3、酶切7min；M、λ/Hind III；
图5：利用96孔板筛选基因组质粒文库的流程示意图；
图6：多杀菌素合成基因簇中各基因位置和基因组质粒文库位置示意图；
图7A-7F：筛选包含spnR、spnF和spnB的质粒文库的PCR电泳结果；
图8A-8F：对图7A-7F获得的阳性结果进一步筛选的PCR电泳结果；
图9A-9D：筛选包含spnD和spnE的质粒文库的PCR电泳结果；
图10A-10D：对图9A-9D获得的阳性结果进一步筛选的PCR电泳结果；
图11：多杀菌素合成基因簇的片段改造和同源重组过程示意图；
图12：质粒15 D1-AmT的构建过程；
图13：质粒pUAmT14的图谱；
图14：质粒pAT-DgegU的图谱；
图15A-15C：3种菌发酵的HPLC图谱；
图16：各片段重叠部分的PCR检测示意图。
具体实施方式
下面将通过具体实施例说明本发明，但本发明的内容不限于此。
本发明的构建多杀菌素异源表达菌的方法可以描述为以下步骤：
(1)对原有的柯斯质粒进行改造：即在柯斯质粒supercos-1的克隆位点BamH I两侧分别插入红色糖多孢菌红霉素合成基因簇的上下游各约3kb大小的片段，得到改造的柯斯质粒eryUD-cos2。克隆到的目的片段可通过这两个同源片段直接置换掉红色糖多孢菌的染色体上的红霉素合成基因簇。
(2)克隆刺糖多孢菌的多杀菌素合成基因簇片段：将刺糖多孢菌的基因组DNA以Sau3A I进行部分酶切，使其酶切后的片段大小较多地分布在25-40kb的范围内，然后以改造好的柯斯质粒eryUD-cos2为载体，利用包装试剂盒
Figure PCTCN2014091118-appb-000001
III XL Packaging Extract进行包装，构建刺糖多孢菌基因组文库，并利用PCR方法筛选包含多杀菌素合成基因簇片段的文库质粒。
(3)对筛选到的基因组文库进行测序，以确定其所包含的DNA片段在多杀菌素生物合成基因簇的相对位置：选择其中4个外源片段的插入方向相同、能完全覆盖多杀菌素生物合成基因簇并且彼此之间有足够长的重叠部分的文库质粒用于基因簇的转移。
(4)文库质粒的修剪：利用PCR targeting的方法，以阿泊拉霉素抗性基因盒aac(3)IV+ oriT来置换多杀菌素合成基因簇片段上多余的同源臂片段，使其同源臂与eryUD-cos2中设计的同源臂长度相当，并具有接合转移必需元件。由于经过PCR targeting置换后的抗性基因盒在两个同源臂的外部，当发生同源双交换而把目的片段转移到红色糖多孢菌染色体上后便会丢失，因此在下一轮的修剪和DNA片段转移中可重复使用。
(5)将修剪好的4个文库质粒按所包含DNA片段的顺序依次通过接合转移的方法转化到红色糖多孢菌，并通过筛选双交换子实现基因片段的转移。经过4步转移，80kb的多杀菌素合成基因簇被成功地转移到红色糖多孢菌染色体上红霉素合成基因簇原本所在的位置。同时转移的还有多杀菌素合成基因簇上下游各约10kb的片段，这些片段可作为后续基因操作的靶位置，分别称作“可操作区1”和“可操作区2”。
(6)将包括gtt、epi、gdh和kre基因的鼠李糖合成基因簇克隆并连接到载体上，利用同源双交换的方法插入到上述“可操作区2”，完成整个重组过程，获得多杀菌素异源表达菌。
以下实施例中将使用下列方法，具体为：
方法1：DNA的连接反应
外源片段与线性化载体按摩尔比3∶1-9∶1的比例配制，总体积为3μl。加入溶液I(TaKaRa，货号D6020A)3μl，16℃水浴30min以上。
方法2：大肠杆菌转化(CaCl2法)(分子克隆实验指南，第三版；北京：科学出版社，2002：93-99)
1)大肠杆菌单菌落接种到3ml LB培养基(如该大肠杆菌带有抗性基因，则加入相应的抗生素)，37℃(BW25113为30℃)，220rpm培养14-18h；
2)以1％的接种量转接到LB培养基(如有需要，则加入相应的抗生素。各种抗生素的终浓度如下：氨苄青霉素(Ap)终浓度为100μg/mL，卡那霉素(Km)和阿泊拉霉素(Am)的终浓度均为50μg/mL，氯霉素(Cm)的终浓度为25μg/mL)，37℃(BW25113为30℃)，220rpm培养至OD600在0.4-0.6之间；
3)离心收集菌体，以等体积的无菌的100mM CaCl2悬浮，冰上放置20min；
4)离心收集菌体，以1/10体积的100mM CaCl2/15％(W/V)甘油悬浮，并以100μl/管分装于1.5ml离心管，得到感受态细胞；
5)取1管感受态细胞，加入DNA(质粒或连接产物，体积不超过10μl)，轻轻混匀后，冰上放置30min；
6)42℃水浴90sec后，立即置冰上1-2min；
7)加入900μl LB培养基，37℃水浴45-60min；
8)菌液(若是质粒转化，取50μl；若是连接产物转化，则离心后去上大部分上清，以残留的液体悬浮菌体)涂布于含相应筛选抗生素的LB平板上，37℃(BW25113为30℃)培养14-18h，长出转化子。
方法3：PCR扩增
按以下比例配制反应液：2×PrimeSTAR GC缓冲液(Mg2+Plus)，25μl；dNTP混合物(各2.5mM)，4μl；上游引物(25μM)，1μl；下游引物(25μM)，1μl；ddH2O，18μl；模板DNA，0.5μl；
Figure PCTCN2014091118-appb-000002
HS DNA聚合酶，0.5μl；
反应程序：95℃×5min；(98℃×10sec，68℃×1min/1kb目标片段长度)×25循环；72℃ ×2min，16℃×1min。
方法4：DNA的回收
回收所用的是上海华舜生物技术有限公司的PCR产物回收试剂盒(货号：W5202)和胶回收试剂盒(货号：W5203)。
1)若是从溶液中回收DNA，在溶液中加入5倍体积的PB缓冲液，混匀后吸到吸附柱；若是从电泳胶中回收，则在切下的胶中加入3倍重量(按100μl相当于100mg计算)的S1缓冲液，于50℃水浴，将胶化开后，吸到吸附柱；
2)9000rcf离心30sec；
3)倒掉回收管中的液体，加入500μl W1缓冲液，9000rcf离心30sec；
4)重复步骤3)，倒掉回收管中的液体；
5)将吸附柱转移到干净的1.5ml离心管，向柱中央加入30μl T1缓冲液，室温放置5min；
6)9000rcf离心1min，得到回收的DNA。
方法5：DNA片段粘性末端的补平与5’的磷酸化
配制以下反应液：纯化的DNA片段，4.2μl；BKL缓冲液，0.5μl；BKL酶混合物，0.25μl；37℃水浴30min以上，然后转移到70℃水浴5min以灭活酶混合物。
方法6：经核酸内切酶酶切线性化后DNA片段的去磷酸化
向核酸内切酶酶切反应液中直接加入1μl FastAP(Fermentas，货号EF0651)，37℃水浴30min。
方法7：大肠杆菌-红色糖多孢菌接合转移方法
1)大肠杆菌ET12567(pUZ8002)(Gust B，Kieser T，Chater KF.REDIRECT Technology：PCR-targeting system in Streptomyces coelicolor.Norwich：John Innes Centre.2002：13-35)按方法2制备感受态细胞，培养时在培养基中加入Km、Cm；
2)取5μl用于接合转移的质粒按方法2转化到ET12567(pUZ8002)感受态细胞，转化后涂布于含Km、Cm和Am的LB平板；
3)挑1个转化子，在含Km、Cm和Am的3ml LB液体培养基中，37℃，220rpm培养14-18h；
4)以1％接种量转移到含Km、Cm和Am的30ml LB液体培养基中，37℃，220rpm培养至OD600在0.4-0.6之间；
5)离心收集菌体，以LB液体培养基洗涤两遍后，以2ml LB液体培养基悬浮，室温放置待用；
6)在步骤5)的同时，将含有106-108个细胞的悬浮于500μl 2×YT培养基(胰蛋白胨1.6％，酵母抽提物1.0％，NaCl 0.5％)的红色糖多孢菌孢子液于50℃热击10min，室温冷却；
7)取500μl步骤5)的菌液与步骤6)的经过热击的孢子液混合，9000rcf离心1min后，弃掉约800μl上清，以残留液体悬浮菌体，涂布于的MS(黄豆饼粉2％，甘露醇2％，琼脂粉1.5％)平板(该平板事先在无菌的环境下吹风1h，以使其部分脱水)，34℃培养14-18h；
8)将含1.25mgAm和0.5mg萘啶酮酸(Nal)的1ml无菌水覆盖在平板上，继续34℃培养；
9)6-7d后，长出转化子。
方法8：碱裂解法制备质粒DNA
1)接种一单菌落于3ml(如有必要，可使用更大体积)LB液体培养基中，加适当的抗生素，37℃摇床培养14-18h；
2)取1.5ml(制备低拷贝质粒取5ml)，12000r/min，离心30sec；
3)吸尽上清液，悬浮菌体于100μl预冷的溶液I(葡萄糖50mmol/L，Tris-HCl 25mmol/L，EDTA 10mmol/L，pH8.0)中；
4)加入200μl新鲜配制的溶液II(NaOH 0.2mol/L，SDS 1％)，快速颠倒5次，冰上放置3min以内；
5)加入150μl预冷的溶液III(5mol/L乙酸钾60ml，冰乙酸11.5ml，水28.5ml)快速上下混匀后，冰上放置3min；
6)12000r/min，离心5min；上清液转移至另一离心管，加入等量的酚-氯仿-异戊醇(BioFlux，货号：BSA03M1)振荡混匀；
7)12000r/min，离心5min，小心将上清转移至另一离心管，加入冰冷的两倍体积无水乙醇，室温放置2min；
8)12000r/min，离心5min，倒去上清液，沉淀用70％的冰预冷乙醇洗两遍；
9)干燥沉淀，溶于20-50μl TE(pH8.0)，储存于-20℃。
方法9：PCR检验方法
配制以下反应液：2×GC I缓冲液(TaKaRa，货号DRR20GCI)，25μl；2.5mM dNTP，4μl；引物1(25μM)，1μl；引物2(25μM)，1μl；模板DNA，1μl(10-100ng)；H2O，32.5μl。
PCR产物小于3kb的，加rTaq DNA聚合酶(TaKaRa，货号R001)，0.5μl；
PCR产物大于3kb的，加LATaq DNA聚合酶(TaKaRa，货号DRR002B)，0.5μl。
程序：94℃×5min，(95℃×30sec，(Tm-5)℃×15sec，72℃×1min/kbp)×30循环，72℃×2min，16℃×1sec。
方法10：放线菌总DNA的提取
1)取放线菌孢子或菌丝接种于30ml TSB培养基(货号211825，BD，美国)，28℃，200rpm培养40h；
2)离心收集菌体，以无菌水洗涤2遍后，以4ml溶菌酶溶液(10mM Tris-HCl，pH7.0，10.3％蔗糖，4mg/ml溶菌酶)悬浮后，于37℃水浴4hr；
3)加入400μl10％SDS溶液和15μl 20mg/ml的蛋白酶K(TaKaRa，货号D9033)溶液，37℃继续水浴30min；
4)用等体积的酚-氯仿-异戊醇(BioFlux，货号BSA03M1)抽提2遍，取上清2.5ml；
5)加入250μl 3M NaAc溶液(pH5.3)和3ml异丙醇，颠倒数次后，挑出白色絮状沉淀于新的2.5ml离心管；以70％乙醇洗2遍后，室温干燥1h；
6)用2ml的2mM Tris-HCl溶液(pH8.0)溶解沉淀，并加入2μl 20mg/ml RNA酶。
若无特别说明，本发明所用的具体实验方法均为本研究领域公知的常规方法，比如：
1、核酸内切酶的单酶切按产品说明书操作；
2、质粒的提取为碱裂解法，具体见方法8；
3、PCR检验用的是普通的Taq DNA聚合酶，具体见方法9；
此外，为了便于描述，以下的表述实际包含的内容为：
1、构建质粒时，所述的“连接得到质粒”是一个连续的操作过程，包括以下几方面的操作内容：(1)DNA片段的连接反应(见方法1)；(2)连接反应产物转化到大肠杆菌DH5α(见方法2)；(3)挑转化子提质粒；(4)质粒以核酸内切酶酶切，检验其酶切产物的片段大小与预期的是否一致；
2、“PCR扩增”：若非特别说明，是指利用
Figure PCTCN2014091118-appb-000003
HS DNA聚合酶及GC缓冲液进行扩增，具体见方法3；
3、“回收”：从溶液中回收使用PCR产物回收试剂盒，从电泳胶中回收DNA则用胶回收试剂盒；具体操作见方法4。
若无特别说明，以下实施例中使用的试剂均是可以从化学或生物试剂商店或供应商处购买到的；所使用的仪器也是本领域常规仪器。
实施例1：cosmid载体的改造
步骤(1)和(2)的目的是将红霉素生物合成基因簇的上游片段和下游片段(即用于双交换的两个同源臂，5’同源臂和3’同源臂)按顺序克隆到载体pBluscript KS(+)(GenBank：X52331.1)上，并在两个同源臂之间引入BamH I位点，在上游片段的前端和下游片段的末端各引入一个Not I位点，以便用Not I切下包含两个同源臂的片段。构建过程如图1所示：
(1)以红色糖多胞菌(ATCC 11635)的基因组(基因组DNA的提取见方法10)为模板，以euF(SEQ ID NO.1，在5’端引入了Not I位点)/euR(SEQ ID NO.2，在5’端引入了BamH I位点)为引物，PCR扩增红霉素生物合成基因簇上游2511bp的片段1：eryU(SEQ ID NO.46，包含酶切位点及保护碱基序列，因此大小为2531bp)。PCR产物回收后，先以Not I(TaKaRa)酶切1h，酶切产物回收后，再以BamH I(TaKaRa)酶切1h，回收得到双酶切产物；载体pBluscript KS(+)同样先后以Not I和BamH I酶切并回收后，与上述经过双酶切的PCR产物连接，经筛选和检验，得到重组质粒1：pBS-eryU；
(2)再以红霉素产生菌的基因组为模板，以引物edF(SEQ ID NO.3，在5’端引入了Pst I位点)/edR(SEQ ID NO.4，在3’端引入了Not I和Xho I位点)PCR扩增红霉素生物合成基因簇下游2642bp的片段2：eryD(SEQ ID NO.47，包含酶切位点及保护碱基序列，因此大小为2667bp)。PCR产物回收后，与重组质粒1：pBS-eryU分别以Xho I和Pst I进行双酶切：10×H缓冲液，5μl；PCR产物(或质粒1：pBS-eryU)，25μl；ddH2O，18μl；Pst I(TaKaRa，货号1073A)，1μl；Xho I(TaKaRa，货号1094A)，1μl。37℃水浴1h，分别回收后，连接，通过筛选和鉴定，得到重组质粒2：pBS-eryUD。
(3)将重组质粒2pBS-eryUD以Not I酶切，切胶回收5179bp的含有两个同源臂(即片段1：eryU和片段2：eryD)的片段，与经Not I酶切并去磷酸化的柯斯质粒supercos-1(Stratagene，货号251301，图谱见图2A)连接。通过PCR和酶切检验，得到柯斯质粒EryUD-Cos2(图谱见图2B)。
实施例2：多杀菌素产生菌总DNA的提取
取多杀菌素产生菌刺糖多胞菌(NRRL 18538)冻存管的孢子液接种于30ml TSB培养基，按方法10提取总DNA。
实施例3：多杀菌素产生菌基因组文库的构建
(1)多杀菌素产生菌总DNA的部分酶切条件试验：
配制以下反应液：实施例2制备的多杀菌素产生菌总DNA，100μl；10×H缓冲液，15μl；ddH2O，35μl。
37℃水浴5min后，加入0.25u Sau3A I(宝生物工程(大连)有限公司，货号：D1082A，以酶贮存液稀释到目标浓度)，继续37℃水浴。从第5分钟起，每隔3min取25μl反应物于5μl 6×上样缓冲液(宝生物工程(大连)有限公司，货号：D604)中终止反应。电泳结果(图3)表明，反应时间为5-8min时，多杀菌素产生菌总DNA的酶切片段在30kb附近的分布较为集中。
(2)多杀菌素产生菌总DNA的部分酶切：
配制以下反应液：多杀菌素产生菌总DNA，1000μl；10×H缓冲液，150μl；ddH2O，350μl。
37℃水浴5min后，加入0.25u Sau3A I，继续37℃水浴。分别在反应5min，6min和7min时，取500μl反应液，加入等体积苯酚-氯仿-异戊醇抽提后，取5μl电泳检验。结果表明反应时间为5min和6min时，多杀菌素产生菌总DNA的酶切片段在25-40kb附近的分布较为集中，结果如图4所示。
将反应时间分别为5min和6min的两管合并，加入100μl 3mol/L NaAc溶液(pH5.3)和1.1ml异丙醇，颠倒数次后，挑出白色絮状沉淀于新的2.5ml离心管。以70％乙醇洗2遍后，室温干燥30min，以100μl 2mmol/L Tris-HCl溶液(pH8.0)溶解沉淀。
(3)多杀菌素产生菌总DNA的部分酶切产物的去磷酸化：
配制以下反应液：多杀菌素产生菌总DNA的部分酶切产物，100μl；FastAP缓冲液，11.5μl；FastAP，3μl。
37℃水浴50min后，加入等体积苯酚-氯仿-异戊醇抽提；取上清90μl，加入9μl 3mol/L NaAc溶液(pH5.3)和200μl无水乙醇，离心去上清，以70％乙醇洗2遍后，室温干燥30min；以50μl 2mmol/L Tris-HCl溶液(pH8.0)溶解沉淀，得到去磷酸化的多杀菌素产生菌总DNA的部分酶切产物。
(4)柯斯质粒EryUD-Cos2的酶切和去磷酸化：
配制以下反应液：柯斯质粒EryUD-Cos2，50μl；10×H缓冲液，10μl；ddH2O，37μl；Xho I，3μl。
37℃水浴2h后，加入FastAP 1μl，37℃×30min；加入等体积苯酚-氯仿-异戊醇抽提；取上清80μl，加入8μl 3M NaAc溶液(pH5.3)和180μl无水乙醇，离心去上清；以70％乙醇洗2遍后，室温干燥30min；以50μl 2mM Tris-HCl溶液(pH8.0)溶解沉淀，得到5’去磷酸化的线性化柯斯质粒EryUD-Cos2。
配制以下反应液：5’去磷酸化的线性化柯斯质粒EryUD-Cos2，50μl；10×BamH I缓冲液，10μl；ddH2O，37μl；BamH I，3μl。
37℃水浴2h后，加入等体积苯酚-氯仿-异戊醇抽提；取上清80μl，加入8μl 3mol/L NaAc溶液(pH5.3)和180μl无水乙醇，离心去上清；以70％乙醇洗2遍后，室温干燥30min；以30μl 2mM Tris-HCl溶液(pH8.0)溶解沉淀，得到Xho I和BamH I双酶切的柯斯质粒 EryUD-Cos2。
(5)多杀菌素产生菌基因组文库的连接：
配制以下反应液：去磷酸化的多杀菌素产生菌总DNA的部分酶切产物，3μl；经Xho I和BamH I双酶切的柯斯质粒EryUD-Cos2，1μl；10×T4连接酶缓冲液，3μl；ddH2O，21μl；T4连接酶(TaKaRa，货号D2011A)，2μl。
16℃水浴过夜后，加3μl 3mol/LNaAc溶液(pH5.3)和180μl无水乙醇，离心去上清，以70％乙醇洗2遍后，室温干燥30min，以5μl ddH2O溶解沉淀，得到连接产物。
(6)多杀菌素产生菌基因组文库的包装和转染
包装试剂盒为
Figure PCTCN2014091118-appb-000004
III XL包装系统(Stratagene Inc.，货号：200201)，按以下步骤操作：
a)取出试剂盒的一个包装(一个离心管)，握在手中至管内的试剂融化；
b)加入4μl从第(5)步得到的连接产物，轻轻搅拌混合后，在离心机上快速离心3-5s，以保证所有的溶液都在离心管的底部；
c)22℃反应2h后，加入500μl SM缓冲液(每1000ml含以下物质：5.8g NaCl，2.0g MgSO4·7H2O，50ml1M Tris-HCl(pH7.5)，5.0ml 2％(W/V)明胶)；
d)加入20μl氯仿，温和混匀，并快速离心5-10s后，4℃冰箱保存待用；
e)从试剂盒中的甘油并存管中挑取大肠杆菌DH10B于LB培养基上划线，37℃培养14-18h，长出单菌落；
f)挑一个单菌接种于含10mM MgSO4和0.2％(w/v)麦芽糖的3ml LB培养基中，37℃，220rpm振荡培养4-6h，至OD600在0.6到0.8之间；
g)取1.5ml菌液，500×g离心10min收集菌体，并以无菌的10mM MgSO4悬浮沉淀，并稀释到OD600＝0.5，得到感受态细胞；
h)取d)得到的包装产物5μl，以SM缓冲液稀释到50μl后，取1μl加入到200μl感受态细胞中，37℃培养15min；
i)加入1ml LB培养基，混匀后，平均涂布在10个含100μg/ml氨苄青霉素的LB平板上，37℃培养约16h后，每个平板的转化子个数为300-400个。
(7)多杀菌素产生菌基因组文库质粒的筛选：
将含有100μg/ml Cb的LB培养基以150μl/孔分装到15块96孔板中，15块板的编号分别为1#到15#，每个孔分别用牙签挑一个上述转化子接种，于37℃×220rpm培养16h左右。
将上述96孔板中每12个孔中的菌液各10μl混合到新的96孔板(编号分别为16#和17#)中，规则如下：
16#96孔板的纵向：1#96孔板中的A1-A12孔混合到16#96孔板的A1孔，B1-B12孔混合到16#96孔板的B1孔，依此类推，H1-H12孔混合到16#96孔板的H1孔。
16#96孔板的横向：1#96孔板中的A1-A12孔混合到16#96孔板的A1孔，2#96孔板中的A1-A12孔混合到16#96孔板的A2孔，依此类推，12#96孔板中的A1-A12孔混合到16#96孔板的A12孔。具体可见附图5。
同理，17#96孔板的纵向：13#96孔板中的A1-A12孔混合到17#96孔板的A1孔，B1-B12孔混合到17#96孔板的B1孔，H1-H12孔混合到17#96孔板的H1孔。
17#96孔板的横向：13#96孔板中的A1-A12孔混合到17#96孔板的A1孔(重新编号为A13)，14#96孔板中的A1-A12孔混合到17#96孔板的A2孔(重新编号为A14)，15#96孔板中的A1-A12孔混合到17#96孔板的A3孔(重新编号为A15)。
(8)多杀菌素产生菌特殊基因组文库的PCR筛选
a)利用PCR检测文库质粒中是否含有spnR、spnF、spnB这3个基因所包含的部分片段(在多杀菌素生物合成基因簇上的相对位置分别是4168-5330，20151-21020，34049-34639)，这3个片段在多杀菌素生物合成基因簇中的相对位置如图6所示(多杀菌素生物合成基因簇上的箭头即表示片段所在位置)。这三个基因的部分片段的PCR产物大小分别是1163bp(spnR)，870bp(spnF)和591bp(spnB)。PCR扩增这三个基因所使用的引物序列分别为：
spnR引物：spnRF(SEQ ID NO.5)和spnRR(SEQ ID NO.6)；
spnF引物：spnFF(SEQ ID NO.7)和spnFR(SEQ ID NO.8)；
spnB引物：spnBF(SEQ ID NO.9)和spnBR(SEQ ID NO.10)。
配制以下反应液：
2×GC I缓冲液，750μl；2.5mM dNTP，120μl；spnRF(25μM)，15μl；spnRR(25μM)，15μl；spnFF(25μM)，15μl；spnFR(25μM)，15μl；spnBF(25μM)，15μl；spnBR(25μM)，15μl；ddH2O，535μl；rTaq(TaKaRa，货号DR001A)，7.5μl。
10μl/管分装，分别加16#和17#96孔板的各孔菌液0.5μl，以多杀菌素产生菌总DNA 0.2μl为对照。PCR反应程序为：95℃×10min，(94℃×30sec，55℃×30sec，72℃×1min10sec)×35个循环，72℃×1min，16℃×1sec。
通过电泳检验PCR结果，结果如图7A-7F所示。
图7A-7C的结果表明：B9、D9、E4、F1和F4有目的条带，即1#96孔板的F1-F12(记作1F1-1F12)、4#96孔板的E1-E12(记作4E1-4E12)和F1-F12(记作4F1-4F12)、9#96孔板的B1-B12(记作9B1-9B12)和D1-D12(记作9D1-9D12)的菌液中有相应的目的基因，以上5个记作第一组。图7D-7F的结果表明：H5、E7、H9、A10、D10、G10和H10有目的条带，即：5#96孔板的H1-H12(记作5H1-5H12)、7#96孔板的E1-E12(记作7E1-7E12)、9#96孔板的H1-H12(记作9H1-9H12)、10#96孔板的A1-A12(记作10A1-10A12)、D1-D12(记作10D1-10D12)、G1-G12(记作10G1-10G12)和H1-H12(记作10H1-10H12)菌液中有相应的目的基因，以上7个记作第二组。
将第一组的60个菌液和第二组的84个菌液分别单独进行PCR检验，配方同上。结果如图8A-8F(图8A-8C为第一组，图8D-8F为第二组)：
第一组结果表明：1F9、4E11、4F11、9B5和9D10分别有相应的目的片段；第二组结果表明：7E9、9H9、10A5、10D5，10D6和10G3分别有相应的目的片段。将上述菌液重新培养提质粒后，分别以上述编号命名，并送样测序。测序引物为：cosF：SEQ ID NO.11；cosR：SEQ ID NO.12。
b)通过PCR检测文库质粒是否含有spnD、spnE这2个基因的部分片段(在多杀菌素生物合成基因簇上的相对位置分别是50305-51725，69264-70076)，这2个片段在多杀菌素生物合成基因簇中的相对位置如图6所示，2个片段的PCR产物大小分别为1421bp和813bp；所用的引物序列为：
spnD引物：spnDF(SEQ ID NO.13)，spnDR(SEQ ID NO.14)；
spnE引物：spnEF(SEQ ID NO.15)，spnER(SEQ ID NO.16)。
配制以下反应液：
2×GC I缓冲液，750μl；2.5mM dNTP，120μl；spnDF(25μM)，15μl；spnDR(25μM)，15μl；spnEF(25μM)，15μl；spnER(25μM)，15μl；ddH2O，580μl；rTaq，7.5μl。
10μl/管分装，分别加入16#和17#96孔板的各孔菌液0.5μl，以多杀菌素产生菌总DNA0.2μl为对照。PCR反应程序为：95℃×10min，(94℃×30sec，55℃×30sec，72℃×1min30sec)×35个循环，72℃×1min，16℃×1sec。
通过电泳检验PCR产物，结果图9A-9D所示。
结果表明：B8、C2、D4、E4、E12、F4、A15和D15有相应的目的条带，即8#96孔板的B1-B12(记作8B1-8B12)、2#96孔板的C1-C12(记作2C1-2C12)、4#96孔板的D1-D12(记作4D1-4D12)、E1-E12(记作4E1-4E12)和F1-F12(记作4F1-4F12)、12#96孔板的E1-E12(记作12E1-12E12)、15#96孔板的A1-A12(记作15A1-15A12)和D1-D12(记作15D1-15D12)菌液中有相应的目的基因。挑上述96个菌液分别单独PCR检验，配方同上。结果如图10A-10D所示：
结果表明2C7、4D1、4E11、4F11、8B8、12E10、15A11和15D1的菌液中有相应的目的基因。将上述菌液重新培养提质粒后，分别以上述编号命名送样测序。测序引物同样为cosF和cosR。
测序结果表明：9D10和10G3具有完全相同的序列，2C7、4D1和12E10具有完全相同的序列，4E11和4F11具有完全相同的序列。2C7/4D1/12E10和9H9所携带的片段是反向的，而其余的则均为正向。
各文库质粒所包含的多杀菌素生物合成基因簇片段的相对位置如下表。设定GenBank AY007564.1公开的序列位置为1-80161，9D10、10G3、15A11和15D1都有部分序列位于这个范围之外。根据10G3(SEQ ID NO.17)和15D1(SEQ ID NO.20)额外的测序结果，位于位置1之前的序列设定为负值，位于位置80161之后的序列顺次计数。
表1：各质粒携带片段的起始位置和插入方向
Figure PCTCN2014091118-appb-000005
各质粒所携带的片段相对于多杀菌素生物合成基因簇的位置见图6所示。
其中10G3、9B5、8B8和15D1这4个质粒携带的片段能覆盖完整的多杀菌素生物合成 基因簇及其上下游序列，这4个质粒所携带的片段的序列如下：
10G3携带的DNA片段序列：SEQ ID NO.17；
9B5携带的DNA片段的序列：SEQ ID NO.18；
8B8携带的DNA片段的序列：SEQ ID NO.19；
15D1携带的DNA片段的序列：SEQ ID NO.20。
将文库质粒15D1、8B8、9B5和10G3经过修剪之后，通过同源双交换依次整合到红色糖多孢菌红霉素合成基因簇的位置。图11是整个过程的示意图，其中“US”是红霉素合成基因簇上游片段eryU，“DS”即红霉素合成基因簇下游片段eryD。下面将详细介绍如何改造各个质粒和进行同源重组。
实施例4：文库质粒15D1的改造
本实施例的目的是将含有aac(3)IV基因(阿泊拉霉素抗性基因)和oriT(接合转移起始位点，接合转移的必需元件)的抗性片段插入到文库质粒15D1的Hind III位点，改造好的质粒可用于接合转移。该实施例分两步进行：首先从质粒pIJ773上以Cla I和EcoR I切下抗性基因盒，平末端化后(SEQ ID NO：65)，连接到载体pUC118(TaKaRa，货号D3322)的Hinc II位点。由于切下的抗性片段在靠近Cla I的末端有一个Hind III位点，而载体pUC118上也有一个Hind III位点，因此筛选正确插入方向的转化子，可以以Hind III切下抗性片段；第二步是将以Hind III切下抗性片段插入到15D1的Hind III位点。由于该抗性片段只起筛选和接合转移的作用，其插入方向不影响后续的实验的结果，因此不需要确定其插入方向。具体操作如下(图12所示)：
配制以下反应液：pIJ773，20μl；10×H缓冲液，5μl；ddH2O，23μl；Cla I(TaKaRa，货号D1034A)，1μl；EcoR I(TaKaRa，货号D1040A)，1μl。
37℃水浴1h，利用电泳回收包含aac(3)IV基因和oriT的1389bp片段。以BKL试剂盒平末端化后，与pUC118/Hinc II，BAP(TaKaRa，货号D3322)连接转化。挑转化子提质粒，以Hind III酶切，筛选酶切结果为3502bp+1402bp的重组质粒。通过电泳回收含aac(3)IV基因和oriT的1402bp片段，与经Hind III酶切并去磷酸化的质粒15D1连接，得到重组质粒15D1-AmT。
实施例5：多杀菌素生物合成基因簇第一个片段迁移到红色糖多孢菌
重组质粒15D1-AmT通过接合转移(见方法7)转化到红色糖多孢菌(ATCC 11635)。转化子经两次松弛培养传代后，筛选阿泊拉霉素敏感的菌落。提取基因组DNA(按方法10进行，但是TSB的体积改为3ml，除70％酒精洗涤时体积仍为500μl外，其它试剂的体积也相应缩小。下同)，分别以引物spnEF(SEQ ID NO.15)/spnER(SEQ ID NO.16)和引物ery1F(SEQ ID NO.21)/ery1R(SEQ ID NO.22)进行PCR检验。引物spnEF/spnER扩增的是文库质粒15D1所携带的DNA片段内部的序列，引物ery1F/ery1R扩增的是红霉素合成基因簇内部的序列。因此，spnEF/spnER能扩增出目的条带而ery1F/ery1R不能扩增出目的条带的，说明红霉素合成基因簇已被文库质粒15D1所携带的DNA片段替换，是目标菌株。筛选得到基因工程菌ES01。
以下实施例6、实施例8和实施例10的目的是利用抗性基因盒aac(3)IV+oriT(该抗 性基因盒从质粒pIJ773上PCR扩增而来，SEQ ID NO.65的第14-1382位)替换相应文库质粒上的DS和所携带基因组片段的3’端的部分序列。主要分为两个步骤：首先设计长度分别为59nt和58nt的正反向引物，引物的5’端的39nt分别作为同源臂，而3’的20nt(正向引物，SEQ ID NO.63)和19nt(反向引物，SEQ ID NO.64)则与抗性基因盒上的引物序列匹配。利用PCR从pIJ773上扩增抗性基因盒，PCR产物在抗性基因盒的两端分别引入了长度为39bp的同源臂，这两个同源臂分别位于文库质粒上将被替换的片段的两侧；然后将PCR产物转化到含文库质粒的大肠杆菌BW25113(pIJ790)，利用该菌内的重组系统，以抗性基因盒替换掉靶序列。各实施例所用的引物及被替换片段的相关信息见表2。
表2：实施例6、实施例8和实施例10所改造的文库质粒相关信息
Figure PCTCN2014091118-appb-000006
*指多杀菌素生物合成基因簇上的相对位置
实施例6：文库质粒8B8的改造
(1)将文库质粒8B8按方法2转化到BW25113(pIJ790)(见文献2)感受态细胞；
(2)挑1个转化子BW25113(pIJ790，8B8)接种于含Cm的3ml LB培养基，30℃，220rpm培养14-18h；
(3)以1％接种量转接到30ml含Km、Cm、Ap和300μl1M的L-阿拉伯糖的SOB培养基(胰蛋白胨2.0％，酵母抽提物0.5％，NaCl 0.05％，1M KCl 2.5ml/L；灭菌后加2.5M的MgCl24ml/L)，30℃，220rpm培养至OD600为0.4-0.6之间；
(4)4℃下离心收集菌体，并以10％甘油洗涤2遍后，以100μl 10％甘油悬浮，得到电转化感受态细胞；
(5)以质粒pIJ773为模板，PCR扩增aac(3)IV+oriT抗性基因盒，并在抗性基因盒两端加入用于同源交换的长度为39bp的同源臂：
反应体系：5×PrimeSTAR缓冲液(Mg2+Plus)，25μl；dNTP混合物(各2.5mM)，4μl；引物8BA-L(SEQ ID NO.23，25μM)，1μI；引物8BA-R(SEQ ID NO.24，25μM)，1μl；ddH2O，18μl；质粒pIJ773，0.5μl；
Figure PCTCN2014091118-appb-000007
HS DNA聚合酶，0.5μl。
反应程序：95℃×5min；(98℃×10sec，50℃×10sec，72℃×90sec)×10循环；72℃×2min，16℃×1min。(98℃×10sec，68℃×90sec)×15循环；72℃×1min，16℃×1min。
PCR产物电泳后，切胶回收约1.4kb的目标片段。
(6)取3μl第(5)步得到的抗性片段加入50μl第(4)步得到的BW25113(pIJ790/8B8) 电转化感受态细胞，全量转移到2mm电转化杯(BioRad)。电击参数为：2500V，25μF，200Ω。电击后迅速加入1ml SOC培养基(每100ml SOB培养基中加入2ml 1mol/L葡萄糖)，全量转移到1.5ml离心管。
(7)37℃水浴1h后，离心去掉900μl上清，以剩余的培养基悬浮沉淀，并全量涂布于含Am的LB固体培养基，37℃培养16h。
(8)挑较大的转化子于3ml含Am的LB液体培养基中，37℃×200rpm培养6h，提质粒，利用引物8BD-L(SEQ ID NO.25)/8BD-R(SEQ ID NO.26)进行PCR检验(PCR反应程序中的延伸时间为4min)。引物8BD-L和8BD-R分别位于文库质粒8B8上被替换序列的两侧，若替换成功，PCR产物应为1963bp的目的条带的质粒；若替换不成功，则PCR产物就为3957bp。经过筛选，得到重组质粒8B8-AmT。
实施例7：多杀菌素生物合成基因簇第二个片段迁移到红色糖多孢菌
重组质粒8B8-AmT通过接合转移转化到实施例5得到的基因工程菌ES01。转化子经两次松弛培养传代后，筛选阿泊拉霉素敏感的菌落。提取总DNA，以引物8BD-L(SEQ ID NO.25)/8BD-R(SEQ ID NO.26)进行PCR检验。原理同实施例6步骤(8)，PCR产物仅为一个1963bp的条带的是目标菌株。筛选得到基因工程菌ES02。
实施例8：文库质粒9B5的改造
目的和方法与步骤6的文库质粒8B8类似，扩增抗性基因盒的引物为9B5-L(SEQ ID NO.27)/9B5-R(SEQ ID NO.28)。质粒PCR检验利用引物95A-L(SEQ ID NO.29)/95A-R(SEQ ID NO.30)，筛选能扩增出1881bp的目的条带的质粒，得到重组质粒9B5-AmT。
实施例9：多杀菌素生物合成基因簇第三个片段迁移到红色糖多孢菌
重组质粒9B5-AmT通过接合转移转化到实施例7得到的基因工程菌ES02。转化子经两次松弛培养传代后，筛选阿泊拉霉素敏感的菌落。提取总DNA，以引物95A-L(SEQ ID NO.29)/95A-R(SEQ ID NO.30)进行PCR检验，PCR产物仅为一个1881bp的条带的是目标菌株。筛选得到基因工程菌ES03。
实施例10：文库质粒10G3的改造
目的和方法与实施例6的文库质粒8B8类似，扩增抗性基因盒的引物为10G3-L(SEQ ID NO.31)/9B5-R(SEQ ID NO.28)。PCR检验利用引物10G-L(SEQ ID NO.32)/10G-R(SEQ ID NO.33)进行，筛选能扩增出1676bp的目的条带的质粒，得到重组质粒10G3-AmT。
实施例11：多杀菌素生物合成基因簇第四个片段迁移到红色糖多孢菌
重组质粒10G3-AmT通过接合转移转化到实施例9得到的基因工程菌ES03。转化子经两次松弛培养传代后，筛选阿泊拉霉素敏感的菌落。提取总DNA，以引物10G-L/10g-R进行PCR检验，PCR产物仅为一个1676bp的条带的是目标菌株。筛选得到基因工程菌ES04。
实施例12：包含刺糖多孢菌鼠李糖合成基因簇的重组质粒的构建
本实施例的目的是将4个鼠李糖合成基因集中在一起克隆到上下两个同源臂之间，以用于利用同源双交换将这4个基因插入到实施例11得到的基因工程菌ES04的染色体上。在基因工程菌ES04的染色体上，通过前面的实施例已将80kb的多杀菌素生物合成基因簇插入到红霉素合成基因簇的位置，同时删除了红霉素合成基因簇，但同时也引入了来源于刺糖多孢菌的两段与多杀菌素生物合成不相关的序列，称之为“可操作区”(如图11所示，KCZ1、 KCZ2)。而本实施例将两个同源臂选择在其中的一个“可操作区”，这样插入的4个基因将不会破坏多杀菌素的生物合成基因簇。因此，本实施例包括以下几个步骤：
(1)从“可操作区2”克隆上下两个同源臂，并按顺序插入到载体pUAmT14；
(2)分别克隆4个鼠李糖合成基因，并依次插入到两个同源臂之间。
由于本实施例中涉及到一个载体和5个片段(2个同源臂，4个鼠李糖合成基因分布在3个片段里面)，为了便于操作，在第二个同源臂和3个基因片段的克隆时，只选择Xba I位点进行克隆。这是基于以下条件而设计的方案：
(1)2个同源臂和4个鼠李糖合成基因上都没有Xba I位点，而载体pUAmT14(图13)上虽然有2个Xba I位点，但经过第一步的酶切后仅余下1个Xba I位点；
(2)核酸内切酶Xba I是受甲基化影响的酶，当其识别位点TCTAGA后面的两个碱基是TC时，由具有甲基化功能的宿主菌如DH5α提取的质粒就不能被Xba I切开，而PCR产物却是没有甲基化的，因此PCR产物直接进行酶切时，不管识别位点后面的碱基是什么序列，都不会影响酶切。
因此，具体方案是：
(1)先将下游同源臂克隆到载体pUAmT14的Ase I-Hind III位点，双酶切保证插入片段的方向正确，Xba I位点位于下游同源臂的上游。
(2)在PCR扩增得到的上游同源臂的5’端引入受甲基化影响的Xba I位点，而在3’端引入不受甲基化影响的Xba I位点。当该片段经Xba I酶切并插入到上一步得到的质粒的Xba I位点后，从DH5α提取得到的质粒中，仅位于上游同源臂和下游同源臂之间的Xba I位点能够被切开，另一个Xba I位点由于甲基化影响而不能被切开。这样能保证以下的鼠李糖基因片段都插在两个同源臂之间。
(3)将3个鼠李糖基因片段也以相同的方法插入到Xba I位点。
以下是具体实施过程：
(1)下游同源臂的插入：
以文库质粒15D1为模板，以引物005DF(SEQ ID NO.34，5’端引入了Hind III位点)/006DR(SEQ ID NO.35，5’端引入了Ase I位点)进行PCR扩增，得到下游同源臂片段5：D PCR(SEQ ID NO.48)。回收片段5，将其与载体pUAmT14分别以Ase I+Hind III双酶切：
反应体系：片段5(或载体pUAmT14)，20μl；10×Tango缓冲液，5μl；ddH2O，23μl；Ase I(Fermentas，货号ER0911)，1μl；EcoR I(Fermentas，货号ER0501)，1μl。
37℃水浴1h后，分别直接回收，连接得到重组质粒pAT-D。
(2)上游同源臂的插入：
以文库质粒15D1为模板，以引物007UF(SEQ ID NO.36，在5’端引入了受甲基化影响的Xba I位点)/008UR(SEQ ID NO.37，在5’端引入了不受甲基化影响的Xba I位点)进行PCR扩增，得到上游同源臂片段6：U PCR(SEQ ID NO.49)。将片段6以Xba I酶切，与经Xba I酶切并去磷酸化的质粒pAT-D连接并转化，转化子提质粒，以引物009F(SEQ ID NO.44)/010R(SEQ ID NO.45)进行PCR检验。正向引物009F位于上游同源臂，反向引物010R位于下游同源臂。如果上游同源臂的插入方向正确，PCR产物应是170bp，而如果插入方向错误，将得不到PCR产物。经筛选得到重组质粒pAT-DU。
(3)gtt基因插入上游同源臂和下游同源臂之间：
以刺糖多孢菌总DNA为模板，以引物gttF(SEQ ID NO.38，在5’端引入了不受甲基化影响的Xba I位点)/gttR(SEQ ID NO.39，在5’端引入了受甲基化影响的Xba I位点)进行PCR扩增，得到包含gtt基因的片段7：gttPCR(SEQ ID NO.50)。将片段7以Xba I酶切，与经Xba I酶切并去磷酸化的质粒pAT-DU连接，得到重组质粒pAT-DgU。
(4)epi基因插入上游同源臂和下游同源臂之间：
以刺糖多孢菌总DNA为模板，以引物epiF(SEQ ID NO.40，在5’端引入了不受甲基化影响的Xba I位点)/epiR(SEQ ID NO.41，在5’端引入了受甲基化影响的Xba I位点)进行PCR扩增，得到包含epi基因的片段8：epi PCR(SEQ ID NO.51)。将片段8以Xba I酶切，与经Xba I酶切并去磷酸化的质粒pAT-DgU连接，得到重组质粒pAT-DgeU。
(5)gdh和kre基因插入上游同源臂和下游同源臂之间：
以刺糖多孢菌总DNA为模板，以引物gdhF(SEQ ID NO.42，在5’端引入了不受甲基化影响的Xba I位点)/gdhR(SEQ ID NO.43，在5’端引入了不受甲基化影响的Xba I位点)进行PCR扩增，得到包含gdh+kre基因的片段9：gdh PCR(SEQ ID NO.52)。将片段9以Xba I酶切，与经Xba I酶切并去磷酸化的质粒pAT-DgeU连接，得到重组质粒pAT-DgegU，质粒图谱如图14所示。
由于4个鼠李糖合成基因的顺序和方向都不影响鼠李糖的合成，因此步骤(3)-(5)只需确认已插入即可，不需要判断插入的方向和顺序。经测序检测，质粒pAT-DgegU包括4个鼠李糖合成基因。
实施例13：刺糖多孢菌鼠李糖合成基因簇转移到红色糖多孢菌
将重组质粒pAT-DgegU通过接合转移转化到实施例11得到的基因工程菌ES04。转化子经两次松弛培养传代后，筛选阿泊拉霉素敏感的菌落。提取总DNA，以引物009F(SEQ ID NO.44)/010R(SEQ ID NO.45)进行PCR检验。引物009F和010R分别位于上游同源臂和下游同源臂；如果4个鼠李糖合成基因已成功插入，PCR产物应为4931bp；若没有插入，则为1322bp。筛选得到基因工程菌ES05。
对基因工程菌ES05中多杀菌素合成基因簇各文库质粒之间的接口部分进行测序检验，各接口的相对位置如图16，结果见表3。测序结果与预期的一致，说明多杀菌素合成基因簇已转入至基因工程菌中，序列顺序与刺糖多胞菌中的多杀菌素合成基因簇序列顺序一致。
表3：文库质粒之间的接口部分测序结果
Figure PCTCN2014091118-appb-000008
Figure PCTCN2014091118-appb-000009
1“位置”是指多杀菌素生物合成基因簇上的相对位置。由于EU-10G3、15D1-ED之间没有重叠部分，因此仅显示与红色糖多孢菌染色体连接的位置；
2“一致”是指与预期的结果一致。
进一步对基因工程菌ES05中插入的多杀菌素合成基因簇和鼠李糖合成基因簇进行全序列测序，结果与预期序列完全一致，说明本方法能够获得遗传背景清楚的基因工程菌。
实施例14：基因工程菌ES05的发酵检验
取ES05的菌落挖块培养于种子培养基(淀粉3.0％，黄豆饼粉2.5％，蛋白胨0.5％，糊精3.0％，葡萄糖1.0％，氯化钠0.4％，pH7.5)，34℃×200rpm培养48h后，以10％的接种量转接到发酵培养基(黄豆饼粉3.0％，玉米淀粉4.0％，糊精3.0％，硫酸铵0.2％，硫酸钙0.6％，葡萄糖1.0％，磷酸二氢钾0.04％，pH6.8)，34℃×200rpm培养7-8d。取1ml发酵液，以4ml无水乙醇浸泡，超声1h后，过滤。滤液以HPLC检验，条件为：C18柱，流动相为甲醇∶乙腈∶0.05％醋酸铵水溶液＝1800∶1800∶400，流速1ml/min，检测波长250nm。同时以多杀菌素产生菌刺糖多胞菌作为正对照，以红色糖多胞菌作为负对照。结果如图15A-15C所示：正对照(图15A)和本发明获得的基因工程菌ES05(图15B)都能够产生多杀菌素A和D组分，而负对照(图15C)则不能产生多杀菌素A和D。

Claims (10)

  1. 一种构建多杀菌素异源表达菌的方法，所述方法将红色糖多胞菌中的红霉素合成基因簇替换为刺糖多胞菌的多杀菌素合成基因簇和鼠李糖合成基因簇；优选地，所述方法利用多次同源重组的方式，将红色糖多胞菌中的红霉素合成基因簇替换为刺糖多胞菌的多杀菌素合成基因簇和鼠李糖合成基因簇；进一步优选地，所述方法包括：(1)获得覆盖刺糖多胞菌的多杀菌素合成基因簇序列及其上下游序列的多个核酸片段，相邻的核酸片段之间有重叠序列；(2)利用同源重组的方式，将步骤(1)获得的多个核酸片段依次连入红色糖多胞菌基因组中，使红色糖多胞菌中的红霉素合成基因簇被刺糖多胞菌的多杀菌素合成基因簇序列及其上下游序列替换，获得重组菌；(3)获得刺糖多胞菌的鼠李糖合成基因簇的核酸片段，利用同源重组的方式，使鼠李糖合成基因簇的核酸片段替换步骤(2)获得的重组菌的多杀菌素合成基因簇的下游序列，获得多杀菌素异源表达菌。
  2. 根据权利要求1所述的方法，其中，步骤(1)中，所述多个核酸片段为至少3个核酸片段，优选地，为3、4、5、6、7个核酸片段，每个核酸片段的大小为25-40kb；进一步优选地，所述多个核酸片段为4个核酸片段，其序列分别如SEQ ID NO.17-20所示。
  3. 根据权利要求2所述的方法，其中，步骤(1)中，通过将刺糖多胞菌的基因组DNA以Sau3AI进行酶切，构建基因组文库并利用PCR方法筛选覆盖多杀菌素合成基因簇序列及其上下游序列的多个核酸片段。
  4. 根据权利要求1-3任一项所述的方法，其中，步骤(2)中，将多个核酸片段分别构建成质粒，所述质粒与红色糖多胞菌完成同源重组，其中，按照多个核酸片段从5’-3’的序列顺序，除包含最后一个核酸片段的质粒外，其它质粒均包括顺次连接的5’同源臂、步骤(1)获得的核酸片段和抗性基因盒，其中每个质粒的5’同源臂与红色糖多胞菌的红霉素合成基因簇上游序列同源；包含最后一个核酸片段的质粒包括顺次连接的抗性基因盒、5’同源臂、最后一个核酸片段和3，同源臂，其中所述3’同源臂与红色糖多胞菌的红霉素合成基因簇的下游序列同源；优选地，步骤(2)中以柯斯质粒supercos-1为起始质粒，所述抗性基因盒包括aac(3)IV+oriT序列。
  5. 根据权利要求4所述的方法，其中，步骤(2)中的质粒构建过程为：首先在柯斯质粒supercos-1中插入红色糖多孢菌红霉素合成基因簇的上、下游核酸片段，分别作为5’同源臂和3，同源臂，得到改造的柯斯质粒eryUD-cos2；再将步骤(1)获得的核酸片段连入至柯斯质粒eryUD-cos2的两条同源臂之间；在包含最后一个核酸片段的质粒的5’同源臂的上游接入抗性基因盒；在其它质粒中，用抗性基因盒替换其它质粒的3’同源臂；优选地，通过同源重组的方法用抗性基因盒替换其它质粒的3’同源臂；进一步优选地，所述5’同源臂的序列如SEQ ID NO.46所示，所述3’同源臂的序列如SEQ ID NO.47所示。
  6. 根据权利要求4或5所述的方法，其中，步骤(2)为：首先将包含最后一个核酸片段的质粒与起始红色糖多胞菌进行同源重组，再按顺序依次将包含其它核酸片段的质粒与前一步同源重组获得的红色糖多胞菌进行同源重组；优选地，所述起始红色糖多胞菌是ATCC11635。
  7. 根据权利要求1-6任一项所述的方法，其中，步骤(3)中，将鼠李糖合成基因簇构 建成用于同源重组的质粒，所述质粒与步骤(2)获得的重组菌进行同源重组，所述质粒包括两个同源臂和位于同源臂之间的鼠李糖合成基因簇，所述两个同源臂均与多杀菌素合成基因簇的下游序列分别同源；优选地，所述两个同源臂的序列分别为SEQ ID NO.49和SEQ ID NO.48。
  8. 根据权利要求1-7任一项所述的方法获得的多杀菌素异源表达菌，其中，所述表达菌为红色糖多胞菌，其中的红霉素合成基因簇被刺糖多胞菌的多杀菌素合成基因簇和鼠李糖合成基因簇取代；优选地，所述表达菌是基因工程菌ES05。
  9. 根据权利要求8所述的多杀菌素异源表达菌在制备多杀菌素中的应用。
  10. 一种制备多杀菌素的方法，所述方法使用权利要求8所述的多杀菌素异源表达菌。
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CN106167815A (zh) * 2016-05-30 2016-11-30 浙江农林大学 天维菌素衍生物的制备方法及用途
CN107523598A (zh) * 2017-06-20 2017-12-29 上海农乐生物制品股份有限公司 一种提高多杀菌素产量的发酵方法
CN114717281A (zh) * 2021-01-06 2022-07-08 武汉大学 通过优化碳源提升异源多杀菌素表达菌株发酵产量的方法
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