WO2019223433A1 - 一种非达霉素基因工程菌及构建方法和应用 - Google Patents

一种非达霉素基因工程菌及构建方法和应用 Download PDF

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WO2019223433A1
WO2019223433A1 PCT/CN2019/081486 CN2019081486W WO2019223433A1 WO 2019223433 A1 WO2019223433 A1 WO 2019223433A1 CN 2019081486 W CN2019081486 W CN 2019081486W WO 2019223433 A1 WO2019223433 A1 WO 2019223433A1
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genetically engineered
fidaxomycin
plateau
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actinoplanes
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李永泉
毛旭明
俞品
郦月萍
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Zhejiang University
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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
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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
    • C12N15/67—General methods for enhancing the expression
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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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  • the invention belongs to the field of microbial pharmaceuticals, and relates to a high-yield fidamicin genetically engineered bacterium Degan Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) and a method for constructing the same and application thereof.
  • CDI Clostridium difficile infection
  • Vancomycin and metronidazole are the first choice drugs for the treatment of CDI, but with the emergence of drug-resistant strains, finding more effective treatments has become the main research target in recent years.
  • Fidaxomicin also known as Tiacumicin (also known as Lipiramycin A3, OPT-80, PAR-101), is a new macrolide antibiotic that can be administered orally.
  • Original research plant Optimer The company was approved by the FDA in May 2011, but so far no drug has been approved by the CFDA in China.
  • Clinical studies have shown that compared with vancomycin orally, fidaxomycin is more effective than vancomycin, and has a strong inhibitory effect on C. difficile after administration, and can effectively reduce the recurrence rate of C. difficile infection.
  • the object of the present invention is to provide a non-dacmycin engineering bacteria Degan Plateau zooplankton actinomycete YP-2, which is classified and named: Actinoplanes deccanensis YP-2, and has been deposited in the General Microbial Center of the China Microbial Species Collection Management Committee ( (CGMCC for short), deposit number: CGMCC No. 15743, deposit date: May 8, 2018, deposit address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • CGMCC General Microbial Center of the China Microbial Species Collection Management Committee
  • Another object of the present invention is to provide a method for constructing the actinomycete engineering bacteria YP-2 (Actinoplanes deccanensis YP-2), which includes the introduction of a gene for positive control of fidaxomycin biosynthesis.
  • the starting strain was screened to obtain a genetically engineered strain (Actinoplanes deccanensis YP-2) of Degan plateau zooplankton with high yield of fidaxomycin.
  • Upstream primer SEQ ID No. 3 aagatctgcccagcatatgaactgttgaaagttgttta
  • Downstream primer SEQ ID NO. 4 ccaagatctgcccagcatatgtcaggcggaatccgccatg;
  • step 2 The fragment recovered in step 1 is digested and inserted into the expression vector pIJ8630 to obtain the plasmid pIJ8630-ermE * -fadR1 and verified;
  • step 2 The plasmid obtained in step 2 is introduced into E. coli ET12567 / pUZ8002 through transformation;
  • steps 2-4 the sequence of SEQ ID NO. 1 is inserted into the plasmid pIJ8630 and introduced into the starting fungus Degan Plateau Actinomyces YP-1.
  • SEQ ID NO.1 in step 1 consists of 3063 nucleotides, positions 1-19 are NdeI recognition sites and protecting bases, positions 20-297 are erythromycin resistance gene promoters, and 297th -3042 is the coding sequence of the gene for positive regulation of fidaxomycin biosynthesis, positions 3043-3063 are the recognition site of NdeI and the protective base, and SEQ ID NO. 2 is the amino acid sequence of the gene for positive regulation of fidaxomycin biosynthesis.
  • step 2 inserts the SEQ ID NO.1 sequence fragment into NdI enzyme digestion and inserts it into the expression vector pIJ8630, the promoter is a erythromycin resistance gene-containing promoter;
  • step 3-4 uses the expression vector Escherichia coli was subjected to parental conjugation to obtain the high-yield fidaxomycin genetically engineered strain Dean Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2).
  • the starting bacteria described in step 4 are Actinoplanes deccanensis YP-1, and the E. coli described in step 3 is E.coil ET12567 (pUZ8002).
  • the yield of the genetically engineered strain of non-dacomycin obtained from Degan Plateau Actinoplanes YP-2 (Actinoplanes deccanensis YP-2) was 400% to 500% higher than the starting bacteria and up to 130mg / L. .
  • Yet another object of the present invention is to provide the application of the fidaxomycin genetically engineered bacteria Dean Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) in the preparation of a medicament for treating diarrhea caused by Clostridium difficile infection.
  • the present inventor purchased a strain of zooxactinomycetes that produces fidaxomycin from the Common Microbial Center of the China Microbial Species Collection Management Committee (CGMCC for short), but this strain has low fidamycin production and is not suitable for industrial applications .
  • CGMCC Common Microbial Center of the China Microbial Species Collection Management Committee
  • the high-yield Fidaxomycin genetically engineered strain Dean Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) can produce a large amount of Fidaxomycin, a drug used to treat diarrhea caused by Clostridium difficile, through fermentation
  • the yield is increased by 400% to 500% compared to the starting bacteria, up to 130mg / L, which has a good application prospect and can promote the industrial development of fidaxomycin.
  • the present invention obtains high-yield fidamycin genetically engineered bacteria Degan Plateau Actinomycete YP-2 (Actinoplanes deccanensis YP-2) by over-expressing fidaxomycin biosynthesis positive regulatory genes in vivo, without the need for Perform in vitro expression, thereby overcoming the problems of difficult expression of some proteins in vitro, inactivity or low activity after expression, and fast and convenient operation; no need to prepare substrates for in vitro reactions, avoiding the difficulty of preparing or purchasing some substrates; The conditions of in vitro reactions need to be explored to simplify the process.
  • the high-yield non-dacmycin genetically engineered strain Depth plateau zooplankton actinomycete YP-2 (Actinoplanes deccanensis YP-2) produced by the present invention has an increase in yield of 400% to 500%, and up to 130mg / L compared with the starting bacteria. It greatly reduces the production cost of fidaxomycin and provides technical support for industrial production to increase the yield of fidaxomycin.
  • the high-yield fidaxomycin genetic engineering strain Degan Plateau Actinomyces YP-2 (Actinoplanes deccanensis YP-2) is obtained, and the method is highly efficient It is accurate, convenient and easy to operate, and provides a new research method for the construction of an efficient biosynthetic pathway for actinomycetes.
  • the genetically engineered strain of Fidaxomycin Degan Plateau Actinoplanes YP-2 (Actinoplanes deccanensis YP-2) can be used in the preparation of medicines for treating diarrhea caused by Clostridium difficile infection.
  • Figure 1 is a diagram of the construction of a high expression plasmid.
  • Fig. 2 is the biomass curve of the Dekan Plateau Actinomyces YP-1 and the Dekan Plateau Actinomyces YP-2 during fermentation.
  • Figure 3 shows the yield of fidaxomycin during the fermentation of Dekang Plateau S. actinomycetes YP-1 and Dekang Plateau S. actinomycetes YP-2.
  • Figure 4 shows the growth phenotype of the actinomycete YP-2 on the Degan Plateau.
  • ISP4 solid medium soluble starch 1%, MgSO 4 .7H 2 O 0.1% K 2 HPO 4 0.1%, NaCl 0.1%, (NH 4 ) 2 SO 4 0.2%, CaCO 3 0.2%, FeSO 4 . 7H 2 O 0.0001%, MnCl 0.0001% agar 2%, pH 7.0.
  • Seed medium glucose 1.75%, peptone 1.5%, NaCl 1.0%, and the rest is water. The percentages are all mass percentages, and the pH is natural.
  • Fermentation medium B yeast extract 0.3%, peptone 0.5%, malt extract 0.3%, glucose 1%, and the rest is water. The percentages are all mass percentages and the pH is natural.
  • MS solid medium 2% mannitol, 2% soybean powder, 2% agarose, and the rest is water. The percentages are all mass percentages, 10 mM MgCl 2 , and the pH is natural.
  • Example 1 Construction of a genetically engineered bacterium with high yield of Fidaxomycin Actinoplanes Deccanensis YP-2 (Actinoplanes deccanensis YP-2)
  • the expression vector for the positive regulation gene of fidaxomycin biosynthesis constructed in this embodiment is named pIJ8630-ermE * -fadR1, and this vector contains the positive regulation gene fadR1 of fidaxomycin biosynthesis.
  • the positive regulation gene of fidaxomycin biosynthesis The sequence of fadR1 is shown in SEQ ID NO.1.
  • SEQ ID NO.1 consists of 3063 nucleotides, positions 1-19 are NdeI recognition sites and protective bases, positions 20-297 are erythromycin resistance gene promoters, and fate 297-3042 are The coding sequence of the gene for positive control of biomycin biosynthesis. Positions 3043-3063 are NdeI recognition sites and protective bases, and SEQ ID NO. 2 is the amino acid sequence of the gene for positive control of fidaxomycin biosynthesis.
  • Upstream primer SEQ ID No. 3 aagatctgcccagcatatgaactgttgaaagttgttta
  • Downstream primer SEQ ID No. 4 ccaagatctgcccagcatatgtcaggcggaatccgccatg.
  • step (1) The SEQ ID No.1 fragment with NdeI digestion sites at both ends recovered in step (1) was digested with NdeI and the expression vector pIJ8630, which was cut by the same enzyme, was ligated to obtain the recombinant expression vector pIJ8630- ermE * -fadR1 ( Figure 1).
  • Actinoplanes deccanensis YP-1 2.Introduce the pIJ8630-ermE * -fadR1 expression vector into the starting bacteria Actinoplanes deccanensis YP-1 to obtain genetically engineered bacteria with high yields of fidaxomycin. (Actinoplanes deccanensis YP-2), the specific method is as follows.
  • the pIJ8630-ermE * -fadR1 vector was transformed into the demethylated starting E. coli ET1256 / (pUZ8002 by heat shock method to obtain recombinant E.coil ET12567 / pUZ8002 / pIJ8630-ermE * -fadR1 .
  • Actinoplanes YP-2 (Actinoplanes deccanensis YP-2) provided by the present invention has been deposited in the General Microbiology Center of the China Microbial Species Collection Management Committee (CGMCC), and is named and named: Actinoplanes deccanensis YP-2, deposit number: CGMCC No. 15743, deposit date: May 8, 2018, deposit address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. [0034] Example 2 Synthesis of Fidaxomycin fermentation by starting bacteria and genetically engineered strains.
  • CGMCC General Microbiology Center of the China Microbial Species Collection Management Committee
  • Bacterial biomass Take 1ml of bacterial solution fermented by fermentation medium B, collect the bacterial cells after centrifugation, wash with 1ml of sterile water, and then collect the bacterial cells after centrifugation. Dry at 50 ° C for 3 days and weigh. 2 is the biomass curve of zooplankton YP-1 and zooplankton YP-2.
  • Fidaxomycin standard curve Fidaxomycin standard was prepared with methanol to a certain concentration gradient solution and measured by HPLC.
  • HPLC conditions Chromatographic column: C18 column (Aglient, Eclipse Plus XDB, 5um, 4.6mm * 250mm); detection wavelength: 254nm; flow rate: 1.00mL / min; injection volume: 10ul; experimental mobile phase: mobile phase A phase is 10% acetonitrile, containing 0.08% trifluoroacetic acid (TFA), mobile phase B phase is 90% acetonitrile; HPLC sampling procedure: 0-20min, 30% -100% B phase; 20-25min, 100% B phase; 25- 30min, 100% -30% B phase.
  • TFA trifluoroacetic acid
  • the strain Deinoplankton actinomycete YP-2 was cultured in ISP4 agar medium for 10 days, and the basal hyphae and aerial hyphae developed well (Figure 4). Apparent characteristics in various different media, the results are shown in Table 1; physiological and biochemical characteristics of YP-2 in Degan Plateau, see Table 2.

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Abstract

提供了一种非达霉素基因工程菌及构建方法和应用，所述基因工程菌为德干高原游动放线菌 (Actinoplanes deccanensis) YP-2，保藏号：CGMCC No：15743。其构建方法是通过将非达霉素生物合成正调控基因导入德干高原游动放线菌YP-1，经筛选得到高产非达霉素的基因工程菌德干高原游动放线菌YP-2，产量比出发菌提高了400％～500％，达到130mg/L。还提供了所述非达霉素基因工程菌德干高原游动放线菌YP-2在制备治疗艰难梭菌感染引起的腹泻药物中应用。

Description

一种非达霉素基因工程菌及构建方法和应用 技术领域
本发明属于微生物制药领域，涉及一种高产非达霉素基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)及其构建方法与应用。
背景技术
临床上抗生素的大量使用，导致病原菌产生耐药性，更为严重的是细菌还能有效扩散耐药性基因信息，多种类别抗生素耐药的病原菌比例不断增加，对临床治疗产生了较大负面影响。艰难梭菌感染(Clostridium difficile infection，CDI)是由一种革兰氏阳性芽孢杆菌难辨梭状芽孢杆菌(Clostridium difficile)过度生长并释放毒素引起的，可导致结肠炎症、严重腹泻甚至死亡。以往资料显示，CDI主要影响老年人、长期住院和重症监护室的患者，近年来健康人群及儿童感染的病例报道逐渐增多。治疗CDI的首选药物是万古霉素和甲硝唑，但随着耐药菌株逐渐出现，寻找更加有效的治疗药物成为近年来的主要研究目标。
非达霉素(fidaxomicin)，又称为台勾霉素(Tiacumicin又称Lipiarmycin A3，OPT-80，PAR-101)，是一种可口服给药的新型大环内酯类抗生素，原研厂Optimer公司于2011年5月获FDA批准上市，但中国至今未有该药品获CFDA批准上市。临床研究表明：与口服万古霉素相比，非达霉素的疗效优于万古霉素，用药后对艰难梭菌有很强的抑制作用，并能有效降低艰难梭菌感染的复发率。
发明内容
本发明的目的是提供一种非达霉素工程菌德干高原游动放线菌YP-2，分类命名为：Actinoplanes deccanensis YP-2，已保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，保藏号：CGMCC No.15743，保藏日：2018年5月8日，保藏地址：北京市朝阳区北辰西路1号院3号。
本发明的另一个目的是提供所述非达霉素工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)的构建方法，包括将非达霉素生物合成正调控基因导入出发菌，筛选得到高产非达霉素的德干高原游动放线菌基因工程菌株(Actinoplanes deccanensis YP-2)。
本发明构建方法具体步骤如下：
(1)设计特异性引物扩增SEQ ID NO.1序列并回收，
上游引物SEQ ID NO.3：aagatctgcccagcatatgaactgttgaaagttgttta
下游引物SEQ ID NO.4：ccaagatctgcccagcatatgtcaggcggaatccgccatg；
(2)步骤1中回收的片段酶切插入表达载体pIJ8630上得到质粒pIJ8630-ermE*-fadR1，并验 证；
(3)步骤2中获得的质粒通过化转导入大肠杆菌E.coil ET12567/pUZ8002中；
(4)通过双亲本接合转导将步骤2中的质粒转入出发菌德干高原游动放线菌(Actinoplanes deccanensis)YP-1中；德干高原游动放线菌YP-1购自中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，其保藏号为：CGMCC No.4.2098，分类命名：德干高原游动放线菌(Actinoplanes deccanensis)，保藏日期：2001年8月16日；
(5)通过发酵获筛选得到高产非达霉素基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，产量高达130mg/L，比出发菌高400％～500％，所述菌株已保藏于中国微生物菌种保藏管理委员会普通微生物中心，分类命名：Actinoplanes deccanensis YP-2，保藏号：CGMCC No.15743，保藏日：2018年5月8日，保藏地址：北京市朝阳区北辰西路1号院3号。
上述方法中，步骤2-4将SEQ ID NO.1的序列插入质粒pIJ8630导入到出发菌德干高原游动放线菌YP-1。
其中，步骤1中SEQ ID NO.1由3063个核苷酸组成，第1-19位为NdeI识别位点和保护碱基，第20-297位为红霉素抗性基因启动子，第297-3042为非达霉素生物合成正调控基因的编码序列，第3043-3063位为NdeI识别位点和保护碱基，SEQ ID NO.2为非达霉素生物合成正调控基因的氨基酸序列。
上述方法中，步骤2将所述的SEQ ID NO.1序列片段经过NdeI酶切后插入表达载体上pIJ8630上，启动子为含红霉素抗性基因启动子；步骤3-4将表达载体利用大肠杆菌进行双亲接合获得所述的高产非达霉素基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)。
上述方法中，步骤4所述作为出发菌为德干高原游动放线菌(Actinoplanes deccanensis)YP-1，步骤3所述大肠杆菌为E.coil ET12567(pUZ8002)。
上述方法中，经筛选得到的高产非达霉素基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)产量比出发出发菌高400％～500％，高达130mg/L。
本发明的再一个目的是提供所述非达霉素基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)在制备治疗艰难梭菌感染引起的腹泻药物中的应用。
本发明人从中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)购买了一株产非达霉素的出发菌游动放线菌，但该菌株产非达霉素低，不适合工业应用。本发明所述的高产非达霉素基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，经过发酵可大量生产治疗艰难梭菌感染引起腹泻的药物非达霉素，其产量比出发菌提高了 400％～500％，高达130mg/L，具有很好的应用前景，可推进非达霉素产业化开发。
本发明的优点是：
(1)本发明通过体内过表达非达霉素生物合成正调控基因，获得了高产非达霉素基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，不需进行体外表达，从而克服了有些蛋白在体外难以表达，表达后无活性或活性较低等问题，操作快捷方便；不需准备体外反应底物，避免了有些底物难以制备或购买的问题；不需摸索体外反应条件，简化流程。
(2)本发明所构建的高产非达霉素基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)产量比出发菌提高了400％～500％，高达130mg/L，大大降低了非达霉素的生成成本，为工业生产提高非达霉素发酵产量提供技术支持。
本发明通过体内基因工程改造，理性优化非达霉素的生物合成，获得了高产非达霉素基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，该方法高效、准确、操作方便，为放线菌药物高效生物合成途径的构建提供了一种新的研究手段。所述的非达霉素基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)可在制备治疗艰难梭菌感染引起的腹泻药物中的应用。
附图说明
图1是高表达质粒构建图。
图2是德干高原游动放线菌YP-1和德干高原游动放线菌YP-2在发酵过程中生物量曲线。
图3是德干高原游动放线菌YP-1和德干高原游动放线菌YP-2在发酵过程中非达霉素产量。
图4是德干高原游动放线菌YP-2的生长表型。
具体实施方式
本发明结合附图和实施例作进一步的说明。以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例中所使用的培养基：
(1)ISP4固体培养基：可溶性淀粉1％、MgSO 4.7H 2O 0.1％K 2HPO 4 0.1％、NaCl 0.1％、(NH 4) 2SO 4 0.2％、CaCO 3 0.2％、FeSO 4.7H 2O 0.0001％、MnCl 0.0001％琼脂2％，pH7.0。
(2)2×YT液体培养基：蛋白胨1.6％，酵母提取物1％，NaCl 0.5％。
(3)种子培养基：葡萄糖1.75％，蛋白胨1.5％，NaCl 1.0％，其余为水，所述百分含量均为质量百分含量，pH自然。
(4)发酵培养基B：酵母提取物0.3％，蛋白胨0.5％，麦芽提取物0.3％，葡萄糖1％，其余为水，所述百分含量均为质量百分含量，pH自然。
(5)MS固体培养基：甘露醇2％，黄豆粉2％，琼脂糖2％，其余为水，所述百分含量均为质量百分含量，10mM MgCl 2，pH自然。
实施例1 构建高产非达霉素的基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)
一、途径特异性基因表达载体pIJ8630-ermE*-fadR1的构建
本实施例构建的非达霉素生物合成正调控基因的表达载体名称为pIJ8630-ermE*-fadR1，该载体含有非达霉素生物合成的正调控基因fadR1.非达霉素生物合成正调控基因fadR1的序列如SEQ ID NO.1所示。SEQ ID NO.1由3063个核苷酸组成，第1-19位为NdeI识别位点和保护碱基，第20-297位为红霉素抗性基因启动子，第297-3042为非达霉素生物合成正调控基因的编码序列，第3043-3063位为NdeI识别位点和保护碱基，SEQ ID NO.2为非达霉素生物合成正调控基因的氨基酸序列。
pIJ8630-ermE*-fadR1的构建方法如下：
(1)设计特异性引物扩增SEQ ID NO.1序列并回收；
上游引物SEQ ID NO.3：aagatctgcccagcatatgaactgttgaaagttgttta
下游引物SEQ ID NO.4：ccaagatctgcccagcatatgtcaggcggaatccgccatg。
(2)将步骤(1)中回收的两端带有NdeI酶切位点的SEQ ID NO.1片段经过NdeI单酶切后与经过相同酶切的表达载体pIJ8630连接，得到重组表达载体pIJ8630-ermE*-fadR1(酶切验证图1)。
二、将pIJ8630-ermE*-fadR1表达载体导入出发菌德干高原游动放线菌(Actinoplanes deccanensis)YP-1得到高产非达霉素的基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，具体方法如下。
(1)通过热激法将pIJ8630-ermE*-fadR1载体转化进入去甲基化的出发大肠杆菌E.coil ET1256/(pUZ8002中，得到重组菌E.coil ET12567/pUZ8002/pIJ8630-ermE*-fadR1。
(2)将E.coil ET1256/(pUZ8002/pIJ8630-ermE*-fadR1接种到5ml LB液体培养基(含氯霉素、卡那霉素和阿泊拉霉素)中，37℃培养至OD 600为0.6，离心收集菌体，用10ml LB液体培养基洗涤两次，用0.5ml的2×YT液体培养基重悬；将德干高原游动放线菌YP-1(Actinoplanes deccanensis YP-1)在2×YT液体培养中培养36h，取200μl菌丝体用10ml LB 液体培养基洗涤两次，用0.5ml的2×YT液体培养基重悬；将上述的大肠杆菌和游动放线菌菌丝体混合后均匀涂在MS固体培养基上，30℃培养16小时后，加入20μg/ml的萘啶酸和阿泊拉霉素，30℃继续培养4-5天；将上述得到的转化子在含有阿泊拉霉素的斜面培养基上培养4-5天，通过PCR方法确证目标DNA序列已经融合在德干高原游动放线菌(Actinoplanes deccanensis)YP-1中，并通过发酵筛选得到高产非达霉素的基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)。
(3)本发明所提供的德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，已保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，分类命名：Actinoplanes deccanensis YP-2，保藏号：CGMCC No.15743，保藏日：2018年5月8日，保藏地址：北京市朝阳区北辰西路1号院3号。[0034]实施例2 出发菌和基因工程菌株合成非达霉素发酵验证。
(1)将出发菌德干高原游动放线菌(Actinoplanes deccanensis)YP-1和基因工程菌株德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)在ISP4固体培养基上培养10天。
(2)在德干高原游动放线菌YP-1和德干高原游动放线菌YP-2培养的ISP4固体培养基上，各取1cm×1cm左右的菌块，接种到种子培养基中，30℃培养16小时，转速200rpm；将种子培养基中的菌丝体接种至发酵培养基B中至OD 600为0.15，30℃培养168小时，在培养12、24、48、72、96、120小时后分别取样测定菌体生物量、非达霉素产量。实验重复三次。
实施例3 基因工程菌株和出发菌生物量、非达霉素产量对比验证
(1)菌体生物量：取1ml通过发酵培养基B发酵的菌液，离心后收集菌体，用1ml无菌水洗涤后再次离心后收集菌体，50℃烘干3天后称重，图2为游动放线菌YP-1和游动放线菌YP-2生物量曲线。
(2)非达霉素标准曲线：将非达霉素标准品用甲醇配成一定浓度梯度的溶液，通过HPLC进行测定。HPLC条件：色谱柱：C18柱子(Aglient，Eclipse Plus XDB，5um，4.6mm*250mm)；检测波长：254nm；流速：1.00mL/min；进样量：10ul；实验流动相：流动相A相为10％乙腈，含0.08％三氟乙酸(TFA)，流动相B相为90％乙腈；HPLC走样程序：0-20min，30％-100％B相；20-25min，100％B相；25-30min，100％-30％B相。
(3)非达霉素产量分析：将通过发酵获得的1ml发酵液加入9mL甲醇，充分振荡后，12000rpm/min离心10min沉降菌丝体及固形物，上清液稀释10倍后用0.45um的无菌微孔滤膜过滤，收集滤液，所得的样品用于HPLC检测。检测条件同上述测定非达霉素标准曲线条件。图3为德干高原游动放线菌YP-1和德干高原游动放线菌YP-2非达霉素产量曲线。
(4)发酵结果显示，基因工程菌株和出发菌的生长曲线没有太大差异，但基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)在发酵中的非达霉素产量比出发菌提高了400％～500％，高达130mg/L，而非达霉素能够很好地治疗艰难梭菌感染引起的腹泻，因此本发明所述的基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)具有很好的应用前景。
实施例4 基因工程菌德干高原游动放线菌YP-2菌体形态特征
菌株德干高原游动放线菌YP-2在ISP4琼脂培养基培养10天，基内菌丝和气生菌丝发育良好(图4)。在各种不同的培养基中的表观特征，结果见表1；德干高原游动放线菌YP-2的生理生化特性见表2。
表1：德干高原游动放线菌YP-2的培养特性：
培养基 生长情况 基内菌丝 气生菌丝 可溶性色素
酵母精麦芽精琼脂 极丰富 浅橙色 无 无
燕麦粉琼脂 稀疏 浅琥珀色 无 无
无机盐淀粉琼脂 丰富 橙色 无 无
苹果酸钙琼脂 稀疏 浅橙色 无 无
甘油天冬素琼脂 丰富 橙色 无 无
葡糖天冬素琼脂 稀疏 浅橙色 无 无
酪氨酸琼脂 浅橙色 无 无 褐色
表2：德干高原游动放线菌YP-2的生理生化特性：
特性 结果 特性 结果
生长pH范围   碳源利用  
pH<4 － D-葡萄糖 +
pH＝5～11 + D-果糖 —
pH＝12 W 甘露糖 +
NaCl耐受性   蔗糖 +
3％NaCl + 乳糖 +
5％NaCl W 肌醇 —
7％NaCl － D-甘露醇 —
生长温度   阿拉伯糖 +
37℃ + 木糖 +
45℃ － 鼠李糖 +
黑色素产生 － 醋酸钠 －
硝酸盐还原 + 棉子糖 －
淀粉水解 +   －
明胶液化 +    
纤维素分解 —    
注：“W”表示弱阳性结果，“+”表示阳性结果，“－”表示阴性结果。

Claims (7)

  1. 一种非达霉素基因工程菌，其特征在于，所述达霉素基因工程菌为德干高原游动放线菌，分类命名为：德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏号：CGMCC No.15743，保藏日：2018年5月8日，保藏地址：北京市朝阳区北辰西路1号院3号。
  2. 一种构建非达霉素基因工程菌的方法，其特征在于，通过以下步骤实现：将非达霉素生物合成的正调控基因导入出发菌德干高原游动放线菌YP-1中，筛选得到非达霉素产量高于出发菌的基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)；德干高原游动放线菌YP-1购自中国微生物菌种保藏管理委员会普通微生物中心，其保藏号为：CGMCC No.4.2098，分类命名：德干高原游动放线菌(Actinoplanes deccanensis)，保藏日期：2001年8月16日。
  3. 根据权利要求2所述的一种构建非达霉素基因工程菌的方法，其特征在于，通过以下步骤实现：
    (1)设计特异性引物扩增SEQ ID NO.1序列并回收；
    上游引物SEQ ID NO.3：aagatctgcccagcatatgaactgttgaaagttgttta
    下游引物SEQ ID NO.4：ccaagatctgcccagcatatgtcaggcggaatccgccatg
    (2)步骤(1)中回收的片段酶切插入表达载体pIJ8630上得到质粒pIJ8630-ermE*-fadR1，并验证；
    (3)步骤(2)中获得的质粒通过化转导入大肠杆菌E.coil ET12567/pUZ8002中；
    (4)通过双亲本接合转导将步骤(2)中的质粒转入出发菌德干高原游动放线菌YP-1中；
    (5)通过发酵筛选得到高产非达霉素基因工程菌株德干高原游动放线菌YP-2，产量达130mg/L。
  4. 根据权利要求3所述的一种构建非达霉素基因工程菌的方法，其特征在于，步骤(2)中将SEQ ID NO.1的序列插入质粒pIJ8630后导入到出发菌德干高原游动放线菌YP-1。
  5. 根据权利要求4所述的一种构建非达霉素基因工程菌的方法，其特征在于，SEQ ID NO.1由3063个核苷酸组成，第1-19位为NdeI识别位点和保护碱基，第20-297位为红霉素抗性基因启动子，第297-3042为非达霉素生物合成正调控基因的编码序列，第3043-3063位为NdeI识别位点和保护碱基，SEQ ID NO.2为非达霉素生物合成正调控基因的氨基酸序列。
  6. 根据权利要求4所述的一种构建非达霉素基因工程菌的方法，其特征在于，将所述的SEQ ID NO.1序列片段经过NdeI酶切后插入表达载体上pIJ8630上，启动子为含红霉素抗性基因启动子；将表达载体利用大肠杆菌进行双亲接合获得所述的高产非达霉素基因工程菌德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)。
  7. 根据权利要求1所述的非达霉素基因工程菌在制备治疗艰难梭菌感染引起的腹泻药物中的应用，所述基因工程菌为德干高原游动放线菌YP-2(Actinoplanes deccanensis YP-2)。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103320355A (zh) * 2013-05-29 2013-09-25 华北制药集团新药研究开发有限责任公司 一种游动放线菌菌株及其在制备非达霉素中的应用
CN104560766A (zh) * 2013-10-16 2015-04-29 浙江海正药业股份有限公司 一种游动放线菌菌株及其应用
CN104603279A (zh) * 2012-08-07 2015-05-06 欧伦股份公司 生产台勾霉素b的流程
CN108841769A (zh) * 2018-05-24 2018-11-20 浙江大学 一种非达霉素基因工程菌及构建方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104603279A (zh) * 2012-08-07 2015-05-06 欧伦股份公司 生产台勾霉素b的流程
CN103320355A (zh) * 2013-05-29 2013-09-25 华北制药集团新药研究开发有限责任公司 一种游动放线菌菌株及其在制备非达霉素中的应用
CN104560766A (zh) * 2013-10-16 2015-04-29 浙江海正药业股份有限公司 一种游动放线菌菌株及其应用
CN108841769A (zh) * 2018-05-24 2018-11-20 浙江大学 一种非达霉素基因工程菌及构建方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LV , WEIXUN: "Breeding of Fidaxomicin Production Strain and Optimization of Fermentation Conditions", SCIENCE -ENGINEERING (A) , CHINA MASTER S THESES FULL-TEXT DATABASE, 15 January 2018 (2018-01-15), ISSN: 1674-0246 *
LV , WEIXUN: "Breeding of Fidaxomicin Production Strain and Optimization of Fermentation Conditions", SCIENCE -ENGINEERING (A), CHINA MASTER S THESES FULL-TEXT DATABASE, 15 January 2018 (2018-01-15), ISSN: 1674-0246 *

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