WO2018177146A1 - 表达天维菌素b的重组微生物、制备方法及其用途 - Google Patents

表达天维菌素b的重组微生物、制备方法及其用途 Download PDF

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WO2018177146A1
WO2018177146A1 PCT/CN2018/079420 CN2018079420W WO2018177146A1 WO 2018177146 A1 WO2018177146 A1 WO 2018177146A1 CN 2018079420 W CN2018079420 W CN 2018079420W WO 2018177146 A1 WO2018177146 A1 WO 2018177146A1
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streptomyces
gene
acid sequence
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黄隽
余贞
林甲壇
李美红
常宗明
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浙江海正药业股份有限公司
台州市劢康生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
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    • 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
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    • 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
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    • C12R2001/465—Streptomyces

Definitions

  • the invention belongs to the field of genetic engineering and microbial fermentation, and particularly relates to a recombinant microorganism expressing avermectin B, a preparation method thereof, and a method for producing avermectin B by using the recombinant microorganism.
  • the avermectin compound is genetically engineered from the avermectin-producing strain Streptomyces avermitilis MA-4680, combined with the chemical structure and biological characteristics of avermectin, ivermectin and milbemycin (A group of 16-membered macrolide new compounds designed to overcome the advantages of insect resistance, insect resistance and biological toxicity (Jun Huang, An-Liang Chen, Hui Zhang, et al. Gene Replacement for the Generation) Of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities. Applied and Environmental Microbiology, August 2015, Volume 81, Number 16.).
  • the compounds are broad-spectrum, highly efficient, non-residual, and environmentally friendly.
  • the preliminary activity test results also showed that avermectin has much higher insect resistance to many common pests such as red spider, whitefly, rice planthopper, thrips, swine worm, etc.
  • Mainly used pesticides and veterinary drugs such as ivermectin, milbemycin, and methicillin, and the toxicity is lower than the commonly used pesticides and veterinary drugs on the market, and has the potential to be further developed into new high-efficiency pesticides and veterinary drugs.
  • the avermectin compound generally includes avermectin A (TEVA) and avermectin B (TEVB), and the specific structural formula is as follows:
  • R When R is CH 3 , it is avermectin A; when R is C 2 H 5 , it is avermectin B.
  • Patent PCT/CN2015/073960 discloses a process for the preparation of a avermectin compound by inactivating the aveD gene in S. avermitilis MA4680 and replacing the aveA1 gene in the Streptomyces genome with a functional milA1 gene.
  • a genetically engineered strain MA220 which can produce avermectin.
  • the content of veuridin B is low, and according to the published technical content, the ratio (mass ratio) of avermectin A and avermectin B is 7:3, 8 : 2 or 9:1.
  • avermectin component B against cinnabarin, diamondback moth, cotton bollworm, pine wood nematode, rice aphid and other agricultural and forestry crops is better than component A, so it is necessary to develop a high content of Tianwei. Engineering strain of bacteriocin B.
  • a first aspect of the present invention provides a recombinant Streptomyces expressing avermectin B, wherein the recombinant Streptomyces does not substantially express avermectin A.
  • the gene coding sequence of the milA1 AT0 domain in the recombinant Streptomyces is replaced with the gene coding sequence of the eryA1 AT0 domain.
  • the Streptomyces is Streptomyces avermitilis
  • the Streptomyces avermitilis is Streptomyces avermitilis MA220
  • the eryA1 gene is derived from a microorganism capable of producing erythromycin
  • a microorganism carrying an erythromycin biosynthetic gene cluster preferably, the eryA1 gene is derived from a bacterium of the genus Saccharopolysaccharide, more preferably, the saccharolytic bacterium is a genus Saccharopolyspora, most preferably, the red Saccharopolyspora erythraea NRRL23338.
  • the S. avermitilis MA220 can be prepared by the related method disclosed in Patent PCT/CN2015/073960.
  • the gene coding sequence of the milA1 AT0 domain comprises at least 70% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1, preferably 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% a nucleic acid sequence of 96%, 97%, 98% or 99% sequence identity; or
  • the coding has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, preferably 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity Sequence; and/or
  • the gene coding sequence of the eryA1 AT0 domain comprises at least 70% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3, preferably 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity nucleic acid sequence; or
  • the coding has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, preferably 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity sequence.
  • a second aspect of the present invention provides a recombinant Streptomyces expressing avermectin B, wherein a gene coding sequence of the milA1 AT0 domain in the recombinant Streptomyces is substituted with a gene coding sequence of the eryA1 AT0 domain.
  • the Streptomyces is S. avermitilis, preferably, the S. avermitilis is S. avermitilis MA220; and/or the eryA1 gene is derived from a microorganism capable of producing erythromycin
  • a microorganism carrying an erythromycin biosynthesis gene cluster preferably, the eryA1 gene is derived from a bacterium of the genus Saccharopolysaccharide, more preferably, the saccharospora bacterium is a Saccharopolyspora erythraea, and most preferably, the Saccharopolyspora erythraea NRRL23338.
  • the recombinant Streptomyces is the eryA1 AT0 domain of the milA1 AT0 domain of S. avermitilis MA220 is replaced with the eryA1 AT0 domain of Saccharopolyspora erythraea (preferably Saccharopolyspora erythraea NRRL23338) Gene coding sequence.
  • the S. avermitilis MA220 can be prepared by the related method disclosed in Patent PCT/CN2015/073960.
  • a third aspect of the invention provides the use of the recombinant Streptomyces of the invention for the production of avermectin B.
  • a fourth aspect of the present invention provides a method of producing avermectin B which comprises culturing the recombinant Streptomyces and recovering avermectin B from the culture.
  • a fifth aspect of the invention provides a method of constructing the recombinant Streptomyces, the method comprising:
  • the Streptomyces to be engineered is Streptomyces avermitilis, preferably Streptomyces faecalis MA220.
  • the eryA1 gene is derived from a microorganism capable of producing erythromycin, such as a microorganism carrying an erythromycin biosynthetic gene cluster, preferably, the eryA1 gene is derived from a microorganism, more preferably, The Saccharopolysaccharococcus is Saccharopolyspora erythraea, and most preferably, the S. erythraea is Saccharopolyspora erythraea NRRL23338.
  • substitution is accomplished by introducing the DNA fragment S into Streptomyces, homologous recombination of the DNA fragment S with the milA1 gene in the Streptomyces.
  • the gene coding sequence of the milA1 AT0 domain comprises at least 70% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98% or 99% sequence identity nucleic acid sequence; or
  • amino acid sequence encoding at least 70% sequence identity, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: ;and / or
  • the gene coding sequence of the eryA1 AT0 domain comprises at least 70% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98 a nucleic acid sequence of % or 99% sequence identity; or
  • Amino acid sequence encoding at least 70% sequence identity, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 .
  • the DNA fragment S comprises a gene coding fragment of the homology arm T1, the eryA1 AT0 domain and a homology arm T2 from the 5' end to the 3' end, respectively; the homologous arm T1 and homology Arm T2 contains homologous sequences of the upstream and downstream genomic sequences of the milA1 gene, respectively.
  • the homology arm T1 is amplified by primer pairs SEQ ID NOs: 6 and 7; and/or the homology arm T2 is amplified by primer pairs SEQ ID NO: 8 and 9.
  • sequence of fragment S is set forth in SEQ ID NO: 5.
  • the DNA fragment S includes upstream and downstream homologous sequences of the milA1 AT0 gene coding sequence in the homology arm T1 and the homology arm T2, respectively.
  • T1 and T2 are homologously recombined with the upstream and downstream of milA1 AT0 of S. avermitilis MA220, the DNA fragment S will be recombined into the genome of S. avermitilis MA220, thereby allowing milA1 AT0 to be DNA fragment in the starting bacterium.
  • the eryA1 AT0 replacement carried by S causes eryA1 AT0 to be expressed in the constructed genetically engineered bacteria.
  • a sixth aspect of the invention provides the use of the recombinant Streptomyces of the invention in the preparation of a pesticide or veterinary drug.
  • the recombinant Streptomyces of the present invention can efficiently produce avermectin B, and the recombinant Streptomyces does not express avermectin A.
  • the technical problem that the existing Streptomyces strains in the prior art express the avermectin B content is too low and the production is difficult is solved.
  • the recombinant Streptomyces sp. fermented by the present invention has good stability and high yield, is more environmentally friendly and simpler than the chemical synthesis method, and greatly saves production cost.
  • Figure 1 Schematic diagram of the construction process of recombinant plasmid SAT-TUeatTD for double exchange of milA1 gene
  • Figure 2 Schematic diagram of the structure and restriction sites of the recombinant plasmid SAT-TAT0U;
  • Figure 3 The restriction enzyme electrophoresis pattern of the constructed plasmid SAT-TAT0U; wherein M is DNA Ladder (Fermentas #SM0333), and lane 1 is an electrophoresis pattern of PvuII and Xho I double-digestion plasmid SAT-TAT0U;
  • Figure 4 Schematic diagram of the structure and restriction sites of the recombinant plasmid SAT-TAT0UD;
  • Figure 5 Schematic diagram of the structure and restriction sites of the recombinant plasmid SAT-TUeatTD;
  • Figure 6 Enzyme digestion electrophoresis map of the constructed plasmids SAT-TAT0UD and SAT-TUeatTD; wherein M is DNA Ladder, and lanes 5, 10 and 15 are electrophoresis patterns of plasmid SAT-TAT0UD digested with BamHI, SmaI and SphI, respectively.
  • Lanes 1-4 are the electrophoresis patterns of the four SAT-TUeatTD plasmids constructed by BamHI digestion, and the lanes 6 to 9 are the electrophoresis patterns of the four SAT-TUeatTD plasmids after SmaI digestion, lane 11 ⁇ 14 are the electrophoresis patterns of the four SAT-TUeatTD plasmids constructed by SphI digestion;
  • Figure 7 Schematic diagram of the double exchange principle of milA1 gene and AT0 gene replacement
  • Figure 8 Electropherogram of the AUA1 gene AT0 domain screened by eryA1 AT0 replacement recombinant PCR; the lanes 1-15 are the strains to be tested TWB-1# ⁇ TWB-15#; the lane 16 is the starting strain MA220 (negative control); 17 is the plasmid SAT-TUeatTD (positive control); M is the DNA ladder (TaRaKa, Cat. No. D501A).
  • Figure 9 HPLC chromatogram of the fermentation product of avermectin MA220 (peak time 6.7-7.3 min) and avermectin B (peak time 8.2-8.7 min).
  • Figure 10 HPLC chromatogram of the fermentation product avermectin B (peak time 8.2-8.7 min) of the engineered strain of S. avermitilis TWB-13#.
  • substantially non-expressed means that the recombinant Streptomyces sp. of the present invention does not express avermectin A at all, or that expression alone does not have a detectable amount, that is, only a very small amount is expressed, and the result is not detected (for example, by HPLC detection). Has a significant significance of ivermectin A.
  • milA1 AT0 domain refers to the AT (acyl transferase) domain located in the milA1 gene loading module (LD module).
  • the milA1 gene type I polyketide synthase, contains three modules from upstream to downstream, namely LD (Loading Module), M1 (module 1) and M2 (module 2), each of which A module contains multiple domains.
  • LD Loading Module
  • M1 module 1
  • M2 module 2
  • a module contains multiple domains.
  • the LD module contains AT and ACP (acyl carrier protein) domains
  • M1 module contains KS (ketosynthase), AT, KR (ketoreductase) and ACP domains, which also contain the AT domain, but differ from this The AT0 domain described in the publication.
  • eryA1 AT0 domain the AT0 domain of the eryA1 gene, refers to the AT (acyl transferase) domain located in the eryA1 gene Loading module (LD module).
  • the eryA1 gene is located in the erythromycin biosynthesis gene cluster and is present in microorganisms having erythromycin synthesis function, such as Saccharopolyspora. It is structurally similar to the milA1 gene, and contains three modules from upstream to downstream, namely LD (Loading Module), M1 (module 1), and M2 (module 2), each of which contains multiple domains.
  • LD Loading Module
  • the LD module includes an AT and an ACP (acyl carrier protein) domain
  • the M1 module includes a KS (ketosynthase), an AT, a KR (ketoreductase), and an ACP domain, which also includes an AT domain, but is different from the present The AT0 domain mentioned in the publication.
  • the "gene coding sequence of the milA1 AT0 domain” and the “gene coding sequence of the AT0 domain of the eryA1 gene” may represent, in addition to the functional sequence naturally present in the living body, the functional sequence A variant formed by mutation, insertion or deletion of one or more nucleotides having the same or similar function as the above-described gene coding sequence.
  • avermectin components A and B are determined by the AT domain (milA1 AT0) of the milA1 gene Loading module (LD module).
  • the domain can start with acetyl-CoA and propionyl-CoA, and finally get avermectin A and avermectin B, respectively.
  • the LD module of the eryA1 gene in the erythromycin synthesis gene cluster (labeled as m00 module in the database) and the LD of the milA1 gene were found by the CSDB (http://csdb.bioserv.pbf.hr/csdb/ClustScanWeb.html) database query.
  • the module structure is consistent, both are AT-ACP domains, and the eryA1 AT0 domain in the erythromycin synthesis gene cluster is initiated by propionyl-CoA.
  • the inventors succeeded in obtaining a stable expression of Tianwei by replacing the milA1 AT0 domain gene coding sequence with the eryA1 AT0 domain gene coding sequence (for example, the eryA1 gene derived from Saccharopolyspora erythraea NRRL23338). Recombinant Streptomyces of bacteriocin B.
  • the PCR product was recovered by PCR product recovery kit (Corning Biotechnology Co., Ltd.), digested with XbaI, and the recovered product was ligated with the vector SupAmT digested with XbaI to obtain a recombinant plasmid SAT-TAT0U.
  • the structure and restriction sites of the plasmid SAT-TAT0U are shown in Figure 2.
  • the plasmid constructed by double digestion with PvuII and XhoI was used to detect whether it was correct.
  • the electrophoresis pattern of the plasmid SAT-TAT0U is shown in Figure 3.
  • the PCR product was recovered by PCR product recovery kit (Corning Biotechnology Co., Ltd.), digested with XbaI, and the recovered product was ligated with the plasmid SAT-TAT0U digested with XbaI to obtain a recombinant plasmid SAT-TAT0UD.
  • the PCR product was recovered by PCR product recovery kit (Corning Biotechnology Co., Ltd.), and the product was recovered with the plasmid SAT-TAT0UD digested with XbaI.
  • the PCR cloning kit (Nanjing Kingsray Biotechnology Co., Ltd., item number L00339) was ligated to obtain the recombinant plasmid SAT-TUeatTD.
  • the structures and restriction sites of the plasmids SAT-TAT0UD and SAT-TUeatTD are shown in Figures 4 and 5, respectively.
  • the constructed plasmid was digested with BamHI, SmaI, and SphI, respectively, to check whether the plasmid was correct.
  • the electrophoresis patterns of the plasmids SAT-TAT0UD and SAT-TUeatTD are shown in Fig. 6.
  • primers 266TUF and 269TDR in the SAT-TUeatTD plasmid were further sequenced by sequencing, wherein the sequence from TGGTGCACATCGACGAGTACGCCGGAATGATC (primer 266TUF partial sequence) to CAGCGG TCCATCACAGCAACAGGTCATCCGG (partial sequence of the reverse complement of primer 269TDR) is SEQ ID NO: The sequence of the connected fragment S shown by 5.
  • recombinant plasmid SAT-TUeatTD was transformed into E. coli ET12567 (pUZ8002): 1 ⁇ l of recombinant plasmid SAT-TUeatTD was added to 100 ⁇ l of E. coli ET12567 (pUZ8002) competent cells (prepared by CaCl 2 method) and placed on ice for 30 minutes. After that, heat was excited at 42 ° C for 90 seconds, and then rapidly placed on ice for 1 minute, added to 900 ⁇ l of LB, and incubated at 37 ° C for 50 minutes.
  • E. coli ET12567 (pUZ8002, SAT-TUeatTD): Pick a single transformant single colony in 3 ml of liquid LB medium containing 25 ⁇ g/ml Cm, 50 ⁇ g/ml Km and 50 ⁇ g/ml Am, and incubate at 37 ° C, 220 rpm. Overnight, 300 ⁇ l of the bacterial solution was inoculated into 30 ml of liquid LB medium containing Cm, Km, Am, and cultured at 37 ° C, 220 rpm for 4-6 h, to an OD600 of 0.4-0.6. The bacterial solution was collected, centrifuged, washed twice with LB medium, and finally suspended in 3 ml of LB medium for use.
  • Example 3 Screening, culture and identification of engineered bacteria of Streptomyces avermitilis replaced by eryA1-AT0 gene
  • the amplified cultured strain was screened by a PCR method using primers: 273 EAR: CGCCGCCCGATGGTCGACGGCCACGC CGCCGGTGAACGCCTGGG (SEQ ID NO: 11); 270 TCF: AGAACGAGTTCGCAGTGGCCGGTCATCCGTGGATC (SEQ ID NO: 12).
  • the PCR product size of 1238 bp is an engineered milA1-AT0 successfully replaced by eryA1-AT0 gene; no PCR product or PCR product size is not 1238 bp are back mutation, ie its genotype is the same as the starting bacteria MA220, milA1-AT0 not Replaced by eryA1-AT0.
  • Figure 8 is an electropherogram of PCR screening. The 13th strain was selected and named as TWB-13#, and the strain was identified by sequencing, and the sequencing result was as shown in SEQ ID NO: 13. The results of PCR electrophoresis and sequencing results indicate that the strain is the strain expected in the present invention.
  • the avermectin engineered strain TWB-13# which was replaced with the eryA1-AT0 gene, was cultured on YMS medium at 28 ° C for 5-6 days, and the colony having an area of about 1 cm 2 was excavated into 30 ml of seed medium (corn starch 2.5%, yellow).
  • Figure 9 HPLC chromatogram of the fermentation broth of the starting strain MA220
  • Figure 10 HPLC chromatogram of the fermentation broth of the genetically engineered strain TWB-13#.
  • the retention times of the avermectin A and B components were 6.95 and 8.39 minutes, respectively.
  • the results showed that compared with the starting strain MA220, the avermectin-engineered strain TWB-13#, which replaced milA1-AT0 with eryA1-AT0, produced only a single component of avermectin B, which no longer produced avermectin A component. .

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Abstract

提供了一种单组分天维菌素B基因工程菌及其制备方法。该基因工程菌是在天维菌素产生菌Streptomyces avermitilis MA220的基因组中将milA1基因的AT0结构域置换成红霉素生物合成基因簇中eryA1基因的AT0结构域而获得。该基因工程菌发酵后，只产生单一组分的天维菌素B。

Description

表达天维菌素B的重组微生物、制备方法及其用途
本申请要求于2017年4月1日提交中国专利局、申请号为201710216023.X、发明名称为“表达天维菌素B的重组微生物、制备方法及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于基因工程及微生物发酵领域，具体涉及表达天维菌素B的重组微生物及其制备方法，以及利用所述重组微生物生产天维菌素B的方法。
背景技术
天维菌素类化合物是从阿维菌素产生菌除虫链霉菌MA-4680出发进行基因改造，结合阿维菌素、伊维菌素和米尔贝霉素的化学结构和各自生物学特性(抗虫活性、抗虫谱以及生物毒性)上的优势，设计得到的一组十六元大环内酯类新化合物(Jun Huang,An-Liang Chen,Hui Zhang,et al.Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities.Applied and Environmental Microbiology,August 2015,Volume 81,Number 16.)。该类化合物具有广谱、高效、无残留毒性，对环境友好等特征。初步的活性试验结果也表明，天维菌素对多种常见害虫，如红蜘蛛、烟粉虱、稻飞虱、蓟马、猪鞭虫等等的抗虫活性 要远远高于阿维菌素、伊维菌素、米尔贝霉素、甲维盐等主流农药和兽药，而毒性则低于目前市面上常用的农药和兽药，具有进一步开发成新型高效农药和兽药的潜力。
天维菌素类化合物一般包括天维菌素A(TEVA)和天维菌素B(TEVB)，具体结构通式如下：
Figure PCTCN2018079420-appb-000001
R为CH 3时为天维菌素A；R为C 2H 5时为天维菌素B。
专利PCT/CN2015/073960公开了一种天维菌素类化合物的制备方法，其通过失活除虫链霉菌MA4680中的aveD基因，并且用功能性milA1基因替换链霉菌基因组中的aveA1基因从而获得了一种可以生产天维菌素的基因工程菌MA220。但是在其发酵代谢产物中，天维菌素B的含量较低，根据其公开的技术内容来看，天维菌素A和天维菌素B的比例(质量比)为7:3、8:2或9:1。而天维菌素B组分抗朱砂叶螨、小菜蛾、棉铃虫，松材线虫病，水稻螟虫等农林作物害虫活性要优于组分A，因此有必要开发一种可生产高含量天维菌素B的工程菌种。
发明内容
本发明的第一方面是提供一种表达天维菌素B的重组链霉菌，其中所述重组链霉菌基本不表达天维菌素A。
在一个实施方案中，所述重组链霉菌中的milA1 AT0结构域的基因编码序列被置换成eryA1 AT0结构域的基因编码序列。
在一个实施方案中，其中所述链霉菌是除虫链霉菌，优选地，所述除虫链霉菌是除虫链霉菌MA220，和/或所述eryA1基因来源于可产生红霉素的微生物，例如携带红霉素生物合成基因簇的微生物，优选地，所述eryA1基因来源于糖多孢菌，更优选地，所述糖多孢菌为红色糖多孢菌，最优选地，所述红色糖多孢菌是Saccharopolyspora erythraea NRRL23338。
所述除虫链霉菌MA220可通过专利PCT/CN2015/073960公开的相关方法进行制备。
在另一个实施方案中，所述milA1 AT0结构域的基因编码序列包含与SEQ ID NO:1所示的核酸序列具有至少70％序列同一性，优选75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的核酸序列；或者
编码与SEQ ID NO:2所示的氨基酸序列具有至少70％序列同一性，优选75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和/或
所述eryA1 AT0结构域的基因编码序列包含与SEQ ID NO:3所示的核酸序列具有至少70％序列同一性，优选75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的核酸序列；或者
编码与SEQ ID NO:4所示的氨基酸序列具有至少70％序列同一性，优选75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
本发明的第二方面是提供一种表达天维菌素B的重组链霉菌，其中所述重组链霉菌中的milA1 AT0结构域的基因编码序列置换成eryA1 AT0结构域的基因编码序列。
在一个实施方案中，所述链菌霉是除虫链菌霉，优选地，所述除虫链霉菌是除虫链霉菌MA220；和/或所述eryA1基因来源于可产生红霉素的微生物，例如携带红霉素生物合成基因簇的微生物，优选地，所述eryA1基因来源于糖多孢菌，更优选地，所述糖多孢菌为红色糖多孢菌，最优选地，所述红色糖多孢菌是Saccharopolyspora erythraea NRRL23338。
在一个具体的实施方案中，所述重组链霉菌是将除虫链霉菌MA220中的milA1 AT0结构域的基因编码序列置换成红色糖多孢菌(优选为Saccharopolyspora erythraea NRRL23338)的eryA1 AT0结构域的基因编码序列。
所述除虫链霉菌MA220可通过专利PCT/CN2015/073960公开的相关方法进行制备。
本发明的第三方面是提供本发明的重组链霉菌用于生产天维菌素B的用途。
本发明的第四个方面是提供一种生产天维菌素B的方法，其包括培养所述重组链霉菌，以及从培养物中回收天维菌素B。
本发明的第五个方面是提供一种构建所述重组链霉菌的方法，所述方法包括：
(1)提供待改造的链霉菌；
(2)将所述待改造链霉菌中的milA1 AT0结构域基因编码序列置换成eryA1 AT0结构域基因序列。
在一个实施方案中，所述待改造的链霉菌是除虫链霉菌，优选地是除虫链霉菌MA220。
在一个实施方案中，所述eryA1基因来源于可产生红霉素的微生物，例如携带红霉素生物合成基因簇的微生物，优选地，所述eryA1基因来源于糖多孢菌，更优选地，所述糖多孢菌为红色糖多孢菌，最优选地，所述红色糖多孢菌是Saccharopolyspora erythraea NRRL23338。
在另一个实施方案中，其中所述置换是通过将DNA片段S导入到链霉菌中，使DNA片段S与所述链霉菌中的milA1基因发生同源重组而完成。
在一个具体实施方案中，所述milA1 AT0结构域的基因编码序列 包含与SEQ ID NO:1所示的核酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的核酸序列；或者
编码与SEQ ID NO:2所示的氨基酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的氨基酸序列；和/或
所述eryA1 AT0结构域的基因编码序列包含与SEQ ID NO:3所示的核酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的核酸序列；或者
编码与SEQ ID NO:4所示的氨基酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的氨基酸序列。
在又一个实施方案中，所述DNA片段S自5’端到3’端分别包含同源臂T1、eryA1 AT0结构域的基因编码片段和同源臂T2；所述同源臂T1和同源臂T2分别包含milA1基因上游和下游基因组序列的同源序列。
在一个具体的实施方案中，所述同源臂T1由引物对SEQ ID NO:6和7扩增得到；和/或同源臂T2由引物对SEQ ID NO:8和9扩增得到。
在一个优选的实施方案中，所述片段S的序列如SEQ ID NO:5所示。
同源重组的原理是本领域技术人员已知的。
所述DNA片段S分别在同源臂T1和同源臂T2包括milA1 AT0 基因编码序列的上游和下游同源序列。当T1、T2与除虫链霉菌MA220的milA1 AT0的上下游发生同源重组时，所述DNA片段S将被重组至除虫链霉菌MA220的基因组中，从而使出发菌中milA1 AT0被DNA片段S所携带的eryA1 AT0置换，使eryA1 AT0在构建的基因工程菌中表达。本领域技术人员可以理解，当milA1 AT0基因的上游和下游分别被扩增后，需要将二者连接起来以形成完整的DNA片段。通常情况下，本领域技术人员将使用限制性内切酶识别序列来连接两个片段。限制性内切酶的选择、包含限制性内切酶识别序列的引物设计、PCR扩增以及扩增片段的回收和连接等都是本领域技术人员根据所使用的质粒、菌株和实验条件可以选择和判断的。
本发明的第六个方面提供了本发明的重组链霉菌在农药或兽药制备中的用途。
本公开内容仅仅举例说明了要求保护的一些具体实施方案，其中一个或更多个实施方案中所记载的一个或更多个技术特征可以与任意的一个或更多个其它实施方案相组合，这些经组合而得到的技术方案也在本申请保护范围内，就像这些经组合而得到的技术方案已经在本公开内容中具体记载一样。
通过以上至少一个方面，本发明的重组链霉菌可以高效地生产天维菌素B，并且该重组链霉菌并不表达天维菌素A。从而解决了现有技术中现有链霉菌株表达天维菌素B含量过低，生产困难的技术问题。本发明的重组链霉菌发酵生产天维菌素B的稳定性好，得率高，较化学合成方法更为环境友好和简单，还极大地节省了生产成本。
附图说明
下面将结合附图以及进一步的详细说明来举例说明本发明。需要指出的是，以下说明仅仅是对本发明要求保护的技术方案的举例说明，并非对这些技术方案的任何限制。本发明的保护范围以所附权利要求书记载的内容为准。
图1：用于milA1基因双交换的重组质粒SAT-TUeatTD构建过程示意图；
图2：重组质粒SAT-TAT0U的结构及酶切位点示意图；
图3：构建的质粒SAT-TAT0U的酶切电泳检测图；其中M是DNA Ladder(Fermentas#SM0333)，泳道1是PvuII和Xho I双酶切质粒SAT-TAT0U后的电泳图；
图4：重组质粒SAT-TAT0UD的结构及酶切位点示意图；
图5：重组质粒SAT-TUeatTD的结构及酶切位点示意图；
图6：构建的质粒SAT-TAT0UD、SAT-TUeatTD的酶切电泳检测图；其中M是DNA Ladder，泳道5、10、15是质粒SAT-TAT0UD分别以BamHI、SmaI、SphI酶切后的电泳图，泳道1～4分别是构建的4个SAT-TUeatTD质粒以BamHI酶切后的电泳图，泳道6～9分别是构建的4个SAT-TUeatTD质粒以SmaI酶切后的电泳图，泳道11～14分别是构建的4个SAT-TUeatTD质粒以SphI酶切后的电泳图；
图7：milA1基因双交换原理及AT0基因置换示意图；
图8：milA1基因AT0结构域被eryA1 AT0置换重组子PCR筛选的电泳图；其中1～15泳道是待检测菌株TWB-1#～TWB-15#；16泳道是出发菌株MA220(负对照)；17是质粒SAT-TUeatTD(正对 照)；M是DNA ladder(TaRaKa，货号D501A)。
图9：出发菌MA220的发酵产物天维菌素A(出峰时间为6.7-7.3min)以及天维菌素B(出峰时间为8.2-8.7min)的HPLC图谱。
图10：除虫链霉菌TWB-13#工程菌的发酵产物天维菌素B(出峰时间为8.2-8.7min)的HPLC图谱。
具体实施方式
本申请所用术语具有与现有技术中该术语相同的含义。为了清楚地表明所用术语的含义，以下给出一些术语在本申请中的具体含义。当以下定义与该术语的常规含义有冲突时，以以下定义为准。
术语“基本不表达”是指本发明的重组链霉菌完全不表达天维菌素A，或者只表达不具有可检测量的，即仅表达非常少量的、在检测(如HPLC检测)结果上不具有显著意义的天维菌素A。
术语“milA1 AT0结构域”即milA1基因的AT0结构域，是指位于milA1基因Loading module(LD模块)中的AT(acyl transferase)结构域。其中milA1基因即I型聚酮合成酶(Type I polyketide synthase)，从上游到下游依次包含有3个模块，分别为LD(Loading module)、M1(module 1)和M2(module 2)，其中每一个模块包含有多个结构域。例如LD模块包含有AT和ACP(acyl carrier protein)结构域，而M1模块中则包含KS(ketosynthase)、AT、KR(ketoreductase)和ACP结构域，其中也包含有AT结构域，但区别于本公开中所述的AT0结构域。
术语“eryA1 AT0结构域”即eryA1基因的AT0结构域，是指位 于eryA1基因Loading module(LD模块)中的AT(acyl transferase)结构域。其中eryA1基因位于红霉素生物合成基因簇中，存在于具有红霉素合成功能的微生物中，例如红色糖多孢菌。结构上与milA1基因类似，与从上游到下游依次包含有3个模块，分别为LD(Loading module)、M1(module 1)和M2(module 2)，其中每一个模块包含有多个结构域，并且LD模块包含有AT和ACP(acyl carrier protein)结构域，而M1模块中则包含KS(ketosynthase)、AT、KR(ketoreductase)和ACP结构域，其中也包含有AT结构域，但区别于本公开中所提到的AT0结构域。
在本公开中，“milA1 AT0结构域的基因编码序列”以及“eryA1基因的AT0结构域的基因编码序列”除了可以表示自然存在于生物体内的功能序列外，还可以表示对所述功能序列进行一个或更多个核苷酸的突变、插入或缺失而形成的与上述基因编码序列具有相同或相近功能的变体。
通过大量研究努力，发明人发现，天维菌素组分A和B是由milA1基因Loading module(LD模块)的AT结构域(milA1 AT0)决定。该结构域能以乙酰辅酶A和丙酰辅酶A为启始单元，最后分别得到天维菌素A和天维菌素B。经CSDB(http://csdb.bioserv.pbf.hr/csdb/ClustScanWeb.html)数据库查询，发现红霉素合成基因簇中eryA1基因的LD模块(数据库中标记为m00模块)与milA1基因的LD模块结构一致，均为AT-ACP结构域，而红霉素合成基因簇中的eryA1 AT0结构域是以丙酰辅酶A为启始单元。发明人通过将milA1 AT0结构域基因编码序列置换成eryA1 AT0 结构域基因编码序列(例如来源于红色糖多孢菌(优选为Saccharopolyspora erythraea NRRL23338)的eryA1基因)，成功获得了一种稳定表达天维菌素B的重组链霉菌。
实施例1用于置换milA1-AT0基因的重组质粒SAT-TUeatTD的构建
构建过程如图1所示，具体步骤如下：
a)放线菌基因组的分离：取放线菌冻存管菌液200μl接种于30ml TSB培养基(Bacto TM Tryptic Soy Broth.BD公司，货号211825)，28℃，220rpm培养48hr后，于50ml离心管中，4000rpm离心10分钟，去上清，沉淀用30ml蔗糖-Tris缓冲液(10.3％蔗糖，10mM Tris-HCl，pH 8.0)洗涤2遍后，以5ml蔗糖-Tris缓冲液悬浮。加入100mg/ml溶菌酶溶液20μl，37℃水浴2hr。加入10％SDS溶液500μl，温和颠倒直至基本澄清。加酚-氯仿-异戊醇(25:24:1，pH8.0)溶液5ml，温和颠倒数次后，4000rpm离心10分钟。取上层溶液4ml，加酚-氯仿-异戊醇(25:24:1，pH8.0)溶液4ml，温和颠倒数次后4000rpm离心10分钟。取上层溶液3ml，加入3mol/L的HAc/NaAc缓冲液(pH 5.3)300μl，异丙醇3ml，温和颠倒数次后，将结团的沉淀用吸头挑到1.5ml离心管中。沉淀用70％乙醇洗涤2遍后，室温干燥。加入500μl Tris-HCl(pH 8.0)溶解，得到放线菌的总DNA。
b)milA1-AT0结构域编码基因上游片段的扩增和克隆：引物为：266TUF:AAATCTAGATCTGGTGCACATCGACGAGTACGCCGGAATGATCG(SEQ ID NO:6)；267TUR:AAATCTAGAGAACGCGACCCCGTCGCCGCCCGC(SEQ ID NO:7)。
按以下配比配制反应液：
Figure PCTCN2018079420-appb-000002
(PrimeSTAR试剂盒，TaKaRa)
进行PCR反应，程序为：
95℃×5分钟，
(98℃×15秒、68℃×1分钟20秒)×25个循环，
72℃×5分钟，
16℃×1分钟。
PCR产物以PCR产物回收试剂盒(康宁生物技术有限公司)回收后，以XbaI酶切，回收产物与以XbaI酶切的载体SupAmT连接，得到重组质粒SAT-TAT0U。
质粒SAT-TAT0U的结构及酶切位点如图2所示。利用PvuII和XhoI双酶切构建的质粒以检测是否正确。质粒SAT-TAT0U的酶切电泳图如图3所示。
c)milA1-AT0结构域编码基因下游片段的扩增和克隆：引物为：268TDF:AAATCTAGAGACCATCGGGCGGCGTTCTCGGTG(SEQ ID NO:8)269TDR:AAATCTAGATCCGGATGACCTGTTGCTGTGATGGACCGCTG(SEQ ID NO:9)
按以下配比配制反应液：
Figure PCTCN2018079420-appb-000003
进行PCR反应，程序为：
95℃×5分钟，
(98℃×15秒、68℃×1分钟20秒)×25个循环，
72℃×5分钟，
16℃×1分钟。
PCR产物以PCR产物回收试剂盒(康宁生物技术有限公司)回收后，以XbaI酶切，回收产物与以XbaI酶切的质粒SAT-TAT0U连接，得到重组质粒SAT-TAT0UD。
d)eryA1-AT0结构域编码基因片段的扩增和克隆：引物为：272EAF:GACGGGGTCGCGTTCGTCTTCCCGGGCCAGGGCGCGCAATGG(SEQ ID NO:10)；273EAR:CGCCGCCCGATGGTCGACGGCCACGCCGCCGGTGAACGCCTGGG(SEQ ID NO:11)
按以下配比配制反应液：
Figure PCTCN2018079420-appb-000004
Figure PCTCN2018079420-appb-000005
进行PCR反应，程序为：
95℃×5分钟，
(98℃×15秒、68℃×1分钟)×25个循环，
72℃×5分钟，
16℃×1分钟。
PCR产物以PCR产物回收试剂盒(康宁生物技术有限公司)回收后，回收产物与以XbaI酶切的质粒SAT-TAT0UD以
Figure PCTCN2018079420-appb-000006
PCR克隆试剂盒(南京金斯瑞生物科技有限公司，货号L00339)连接，得到重组质粒SAT-TUeatTD。
质粒SAT-TAT0UD、SAT-TUeatTD的结构及酶切位点分别如图4、5所示。分别用BamHI、SmaI、SphI酶切构建的质粒以检测质粒是否正确。质粒SAT-TAT0UD、SAT-TUeatTD的酶切电泳图如图6所示。
进一步通过测序检验SAT-TUeatTD质粒中引物266TUF和269TDR之间的序列，其中从TGGTGCACATCGACGAGTACGCCGGAATGATC(引物266TUF部分序列)开始至CAGCGG TCCATCACAGCAACAGGTCATCCGG(引物269TDR的反向互补序列的部分序列)之间的序列即为SEQ ID NO:5所示的连接片段S的序列。
实施例2将milA1-AT0基因置换的重组质粒SAT-TUeatTD转化到宿 主菌天维菌素生产菌MA220
a)将重组质粒SAT-TUeatTD转化到大肠杆菌ET12567(pUZ8002)：取1μl重组质粒SAT-TUeatTD加入到100μl大肠杆菌ET12567(pUZ8002)感受态细胞(以CaCl 2法制备)中，冰上放置30分钟后，42℃热激90秒，再迅速放到冰上冷却1分钟，加入900μl LB培养，37℃水浴50分钟。取100μl涂布于含25μg/ml氯霉素(Cm)、50μg/ml卡那霉素(Km)、50μg/ml安普霉素(Am)的固体LB培养上，37℃培养过夜，长出转化子，挑选其中一个转化子并鉴定已转入质粒SAT-TUeatTD，命名为ET12567(pUZ8002,SAT-TUeatTD)。
b)大肠杆菌ET12567(pUZ8002,SAT-TUeatTD)的培养：挑一个转化子单菌落于3ml含25μg/ml Cm、50μg/ml Km和50μg/ml Am的液体LB培养基中，37℃，220rpm培养过夜，菌液300μl接种于30ml含Cm、Km、Am的液体LB培养基中，37℃，220rpm培养4-6h，至OD600为0.4-0.6之间。收集菌液，离心后，以LB培养基洗涤2遍，最后以3ml LB培养基悬浮，备用。
c)宿主菌MA220的准备：从平板上刮下除虫链霉菌MA220的孢子到1000μl 2×YT培养基中悬浮，50℃热激10min，自然冷却，备用。
d)接合转移：取500μl b)的菌液加入到1000μl c)的孢子悬液中，混匀后离心去掉800μl上清。以剩余的上清悬浮菌体，并涂布于MS培养基。28℃培养16-20h后，以含有500μg Am和500μg萘啶酮酸(Nal)的1ml无菌水覆盖，28℃培养6-7d，长出转化子。
双交换过程的原理图如图7所示。
实施例3 milA1-AT0被eryA1-AT0基因置换的除虫链霉菌工程菌的筛选培养及鉴定
a)挑一个转化子，在含有25μg/ml Am和25μg/ml Nal的YMS培养基上划线，28℃培养5-6d。将生长的菌落在不含抗生素的YMS培养基上28℃连续培养2代后，再在不含抗生素的YMS培养基上划线分离单菌落，28℃培养5-6d。
b)用牙签将a)得到的单菌落分别于含和不含25μg/ml Am的YMS培养基上点种，28℃培养5-6d。挑选在含25μg/ml Am的YMS培养基上不生长而在不含Am的YMS培养基上生长的菌落，在不含抗生素的YMS培养基上进行放大培养。
c)利用PCR方法对放大培养的菌株进行筛选，所用的引物为：273EAR:CGCCGCCCGATGGTCGACGGCCACGC CGCCGGTGAACGCCTGGG(SEQ ID NO:11)；270TCF:AGAACGAGTTCGCAGTGGCCGGTCATCCGTGGATC(SEQ ID NO:12)。
按以下配比配制反应液：
Figure PCTCN2018079420-appb-000007
15μl/管分装，分别用牙签挑取步骤b)所筛选的菌落为模版进行PCR反应，并以1.0μl MA220总DNA和重组质粒SAT-TUeatTD分别为负对照和正对照。PCR反应程序为：
95℃×5分钟，
(94℃×30秒、68℃×1分钟20秒)×25个循环，
72℃×5分钟，
16℃×1分钟。
PCR产物大小为1238bp的是milA1-AT0被eryA1-AT0基因成功置换的工程菌；无PCR产物或者PCR产物大小非1238bp的均为回复突变，即其基因型与出发菌MA220相同，milA1-AT0未被eryA1-AT0置换。图8是PCR筛选的电泳图。选择第13#菌，命名为TWB-13#，对该菌进行测序鉴定，测序结果如SEQ ID NO:13所示。PCR电泳结果与测序结果说明该菌株即是本发明预期的菌株。
实施例4基因工程菌TWB-13#的发酵检验
将eryA1-AT0基因置换的天维菌素工程菌TWB-13#在YMS培养基上28℃培养5-6天后，将面积为1cm 2左右菌落挖到30ml种子培养基(玉米淀粉2.5％，黄豆饼粉0.8％，花生饼粉1％，酵母粉0.95％，CoCl 2 0.003％，pH 7.2-7.4)中，28℃，250rpm培养40hr，以6％的接种量转接于发醉培养基(玉米淀粉14％，淀粉酶0.003％，黄豆饼粉2.0％，酵母粉1％，沸石粉0.2％，MnSO 4 0.0024％,Na 2MoO 4 0.0024％,CoC1 2·6H 20 0.002％，pH 7.2-7.4)，28℃，250rpm，培养8d。取1ml发酵液，加入4ml无水甲醇浸泡，超声1h后，过滤。滤液直接用于HPLC分析。HPLC分析的条件为：色谱柱：C18 Hypersil ODS2  4.6×250×5(大连依利特)；流动相：甲醇：乙醇：水＝81:7:12；流速：1ml/min；吸收波长：240nm。
检测结果如图9、图10所示。图9：出发菌株MA220的发酵液HPLC图谱；图10：基因工程菌TWB-13#的发酵液HPLC图谱。从图谱可见，天维菌素A和B组分的保留时间分别是6.95和8.39分钟。结果表明，与出发菌株MA220相比，以eryA1-AT0置换milA1-AT0的天维菌素工程菌TWB-13#只产生天维菌素B单组分，不再产生天维菌素A组分。

Claims (13)

  1. 一种表达天维菌素B的重组链霉菌，其中所述重组链霉菌基本不表达天维菌素A。
  2. 根据权利要求1所述的重组链霉菌，其中所述重组链霉菌中的milA1 AT0结构域的基因编码序列置换成eryA1 AT0结构域的基因编码序列。
  3. 根据权利要求1或2的重组链霉菌，其中所述链霉菌是除虫链霉菌，优选地，所述除虫链霉菌是除虫链霉菌MA220，和/或所述eryA1基因来源于可产生红霉素的微生物，例如携带红霉素生物合成基因簇的微生物，优选地，所述eryA1基因来源于糖多孢菌，更优选地，所述糖多孢菌为红色糖多孢菌，最优选地，所述红色糖多孢菌是Saccharopolyspora erythraea NRRL23338。
  4. 根据权利要求1-3中任一项所述的重组链霉菌，其中所述milA1 AT0结构域的基因编码序列包含与SEQ ID NO:1所示的核酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的核酸序列；或者
    编码与SEQ ID NO:2所示的氨基酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的氨基酸序列；和/或
    所述eryA1 AT0结构域的基因编码序列包含与SEQ ID NO:3所示的核酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的核酸序列；或者
    编码与SEQ ID NO:4所示的氨基酸序列具有至少70％序列同一性， 优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的氨基酸序列。
  5. 权利要求1-4任一项所述的重组链霉菌用于生产天维菌素B的用途。
  6. 一种生产天维菌素B的方法，其包括培养所述权利要求1-4任一项所述重组链霉菌，以及从培养物中回收天维菌素B。
  7. 一种构建权利要求1-4中任一项所述重组链霉菌的方法，所述方法包括：
    (1)提供待改造的链霉菌；
    (2)将所述待改造链霉菌中的milA1 AT0结构域的基因编码序列置换成eryA1 AT0结构域基因编码序列。
  8. 根据权利要求7的方法，其中所述链霉菌是除虫链霉菌，优选地，所述除虫链霉菌是除虫链霉菌MA220，和/或所述eryA1基因来源于可产生红霉素的微生物，例如携带红霉素生物合成基因簇的微生物，优选地，所述eryA1基因来源于糖多孢菌，更优选地，所述糖多孢菌为红色糖多孢菌，最优选地，所述红色糖多孢菌是Saccharopolyspora erythraea NRRL23338。
  9. 根据权利要求7的方法，其中所述置换是通过将DNA片段S导入到链霉菌中，使DNA片段S与所述链霉菌中的milA1基因发生同源重组而完成。
  10. 根据权利要求7-9任一项的方法，其中所述milA1 AT0结构域的基因编码序列包含与SEQ ID NO:1所示的核酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％ 序列同一性的核酸序列；或者
    编码与SEQ ID NO:2所示的氨基酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的氨基酸序列；和/或
    所述eryA1 AT0结构域的基因编码序列包含与SEQ ID NO:3所示的核酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的核酸序列；或者
    编码与SEQ ID NO:4所示的氨基酸序列具有至少70％序列同一性，优选80％、85％、90％、93％、95％、97％、98％或99％序列同一性的氨基酸序列。
  11. 根据权利要求9或10的方法，所述DNA片段S自5’端到3’端分别包含同源臂T1、eryA1 AT0结构域的基因编码片段和同源臂T2；所述同源臂T1和同源臂T2分别包含milA1基因上游和下游基因组序列的同源序列，优选地，所述同源臂T1由引物对SEQ ID NO:6和7扩增得到；和/或同源臂T2由引物对SEQ ID NO:8和9扩增得到。
  12. 根据权利要求9-11中任一项所述的方法，所述片段S的序列如SEQ ID NO:5所示。
  13. 权利要求1-4任一项所述的重组链霉菌在农药或兽药制备中的用途。
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