WO2021238832A1 - 一种达托霉素高产菌株及其应用 - Google Patents

一种达托霉素高产菌株及其应用 Download PDF

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WO2021238832A1
WO2021238832A1 PCT/CN2021/095417 CN2021095417W WO2021238832A1 WO 2021238832 A1 WO2021238832 A1 WO 2021238832A1 CN 2021095417 W CN2021095417 W CN 2021095417W WO 2021238832 A1 WO2021238832 A1 WO 2021238832A1
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daptomycin
strain
recombinant strain
recombinant
streptomyces roseosporus
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French (fr)
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吴杰群
张薇
平丽英
沈建宇
杨永梅
方丽纳
徐金勇
吴金荣
范萍
吴骏
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杭州中美华东制药有限公司
浙江工业大学
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    • C—CHEMISTRY; METALLURGY
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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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
    • C12N15/74—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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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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  • the present invention relates to the technical field of genetic engineering, in particular to a high-yield daptomycin strain and a preparation method thereof.
  • Daptomycin is a new type of cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. It interacts with cell membranes in a calcium ion-dependent manner and exerts bactericidal activity.
  • the U.S. FDA approved a new dosing regimen for Cubicin, a daptomycin injection of Cubist Pharmaceutical Co., Ltd., as a 2min intravenous bolus once a day.
  • Cubicin was first approved in the United States in 2003 for the treatment of complicated skin and skin tissue infections caused by certain gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, by intravenous infusion for 30 minutes once a day
  • the Food and Drug Administration of my country approved daptomycin for injection produced by Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd., Zhejiang Hisun Pharmaceutical Co., Ltd. and Jiangsu Hengrui Pharmaceutical Co., Ltd. in 2015 and 2016, respectively.
  • a number of studies have shown that the proportion of drug-resistant daptomycin has not increased significantly over the past 10 years after it has been on the market, demonstrating the unique advantages of daptomycin.
  • Daptomycin is a microbial secondary metabolite with a complex structure and very low yield. Genetic engineering is an important strategy to increase the yield of daptomycin and reduce production costs.
  • Decanoic acid is an exogenous precursor in the synthesis of daptomycin, which can change the direction of secondary metabolism of the bacteria.
  • decanoic acid is an essential substance, but excessive decanoic acid will be toxic to Streptomyces roseosporus; on the contrary, if the concentration of decanoic acid is too low, it will lead to insufficient precursor supply and make the fermentation broth The potency of the product is reduced.
  • the purpose of the present invention is to provide a daptomycin strain with improved utilization and tolerance of decanoic acid and a construction method thereof, so as to solve the problem of the low tolerance of Streptomyces roseosporus to precursors and daptomyces in the prior art.
  • the deposit number of the strain of the present invention is CGMCC No. 18297.
  • the purpose of the present invention is also to provide a method for constructing a recombinant plasmid containing a regulatory gene.
  • the third object of the present invention is to provide the application of the plasmid in genetic modification of daptomycin producing bacteria.
  • the classification of the recombinant strain is named Streptomyces roseosporus (Streptomyces roseosporus), and the deposit number of the recombinant strain is CGMCC No. 18297.
  • the preservation information of the recombinant strain of the present invention is as follows:
  • Streptomyces roseosporus Streptomyces roseosporus
  • the recombinant strain is a capric acid-tolerant strain.
  • the decanoic acid tolerance of the recombinant strain is twice that of the original strain.
  • the recombinant strain is used to improve the tolerance of Streptomyces roseosporus to capric acid, increase the utilization rate of capric acid, and increase the fermentation yield of daptomycin.
  • nucleotide sequence of the subcloned fragment in step (1) is shown in SEQ ID NO.1.
  • the vector in step (1) contains an erme promoter.
  • the recombinant plasmid in step (1) is pSET152CRP.
  • the advantage of the present invention is that by using the global regulatory factor CRP, the problems of high workload and high blindness of traditional Streptomyces breeding are solved, and a high-yield daptomycin strain cultivated by genetic engineering is obtained, which has broad application prospects; using this method
  • the starting bacterium Streptomyces roseosporus is modified, and the obtained recombinant strain can improve the tolerance and utilization of decanoic acid of Streptomyces roseosporus, and increase the yield of daptomycin.
  • Fig. 1 is a comparison diagram of the normalized fermentation yield of Streptomyces roseosporus and the existing Streptomyces roseosporus under different precursor concentrations in the culture medium;
  • Figure 2 is the physical map of pSET152CRP
  • Figure 3 shows the integration method of int ⁇ C31
  • Figure 4 shows the two feeding processes of the constructed bacteria and the starting bacteria in the fermentation process, the daily supplement of the amount of capric acid in the normal production process and the daily supplement of twice the normal amount of capric acid, each Comparison chart after normalization of the titer of Zidatomycin;
  • Figure 5 shows the two feeding processes of the constructed bacteria and the starting bacteria in the fermentation process, the daily supplement of the amount of capric acid in the normal production process and the daily supplement of twice the normal amount of capric acid, each Contrast chart of daily decanoic acid utilization (titer/accumulative added amount of decanoic acid).
  • Example 1 Construction of pSET152CRP containing the recombinant plasmid of CRP gene:
  • CRPS ATTTCTAGAAATACCTGACCGAGCACG;
  • CRPA ATTGGATCCATCGCACTGTTTTACCGT.
  • the amplified CRP gene was ligated with the pSET152 plasmid fragment recovered by restriction enzyme digestion.
  • the restriction enzyme digestion ligation reaction system vector 0.03pmol, fragment 0.03-0.3pmol, T4DNA ligase 1200U, buffer 5ul, and the remaining volume is made up with distilled water. Connect at 16°C for 16h to obtain the ligation product.
  • E. coli competent cells Take 200ul of E. coli competent cells, add the above ligation product, rotate gently to mix the contents, and place in ice for 30 minutes. Place the tube in a circulating water bath preheated to 42°C for 90 seconds. Quickly transfer the tube to a water bath and allow the cells to cool for 1-2 minutes. Add 800ul of pre-warmed LB medium at 37°C to each tube, and then transfer to a 37°C water bath for 45 minutes. Transfer an appropriate volume of transformed competent cells to the LB medium containing the corresponding resistance. Colonies appeared after 12-16h inverted culture at 37°C. Pick the colonies for restriction enzyme digestion verification, and take the verified colonies for storage and sequencing verification.
  • the recombinant plasmid pSET152CRP is 7033 bp in size and carries an Ampra resistance gene. It can be screened in E. coli and Streptomyces roseosporus. Int ⁇ C31 is the integrase gene, attP is the integration site, and p*erme is the erythromycin promoter.
  • LB medium formula tryptone 10g, yeast extract 10g, NaCl 10g, add deionized water to 1000ml, pH 7.0. Sterilize at 121°C for 30 minutes for culturing Escherichia coli. Add 2% agar powder to the solid medium.
  • the Escherichia coli ET12567 containing the recombinant plasmid PSET152CRP was conjugated and transferred with Streptomyces roseosporus NO.CGMCC4.7231, and the conjugant containing the PSET152CRP plasmid was obtained by screening with ampramycin and nalidixic acid. At the same time, an empty vector PSET152 was introduced. The vector of the plasmid is used as a control.
  • the cells were resuspended twice with the same volume of LB medium, and finally resuspended in 0.1 times the volume of LB medium. While washing the cells, 10-8 spores required for each conjugation transfer are suspended in 500ul 2 ⁇ YT medium, heat shocked at 50°C for 10 minutes, and cooled to room temperature. Take 500ul of E. coli solution and Streptomyces roseosporus spore solution and mix thoroughly, centrifuge to remove most of the supernatant, and resuspend with the remaining liquid. Spread the mixed bacterial liquid on MS solid medium containing 10mM MgCl 2 and incubate at 29°C for 16-20h.
  • Example 3 Fermentation process of recombinant bacteria
  • the high antibiotic production Streptomyces roseosporus prepared in the present invention has a higher fermentation yield, that is, a better Dato The yield of mycin, the fermentation yield is increased by more than 200%.
  • Trace element solution (per liter): ZnCl 2 40mg, FeCl 2 ⁇ 6H 2 O 200mg, CuCl 2 ⁇ 2H 2 O 10mg, MnCl 2 ⁇ 2H 2 O 10mg, Na 2 B 4 O 7 ⁇ 10H 2 O 10mg, (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 10mg
  • Yeast extract 1.5g, tryptone 5g, malt extract 3g, glucose 10g, sucrose 250g, add deionized water to 1000ml, and steam sterilize at 115°C for 15min.
  • Chromatographic conditions Chromatographic column: Phenomenex IB-SIL C8 4.6 ⁇ 250mm 5um, flow rate: 1.0ml/min, detection wavelength: 223nm, injection volume: 25ul, column temperature: 25°C, gradient elution mobile phase A: weigh 3.4 g Ammonium dihydrogen phosphate was dissolved in 1000ml of distilled water, adjusted to pH 3.1 with phosphoric acid, mobile phase B: acetonitrile.
  • Example 5 Tolerance test of the constructed strain to the concentration of precursor
  • Example 3 Using the fermentation process of Example 3, the tolerance range of the starting strain and the constructed strain NO.CGMCC4.7231 to the concentration of the fermentation precursor in the medium was compared in a shake flask. The results of the experiment are shown in Figure 1 of the specification. The production of daptomycin was the highest when the precursor concentration of the constructed bacteria was 3% to 5% in the shake flask, and the yield of daptomycin was the highest when the concentration exceeded 5%; The body concentration is between 2% and 3%. After 3%, the yield will drop significantly. It shows that the CRP gene can improve the tolerance of Streptomyces roseosporus to the precursors in the culture medium, and can increase the amount of capric acid added in the fermentor during industrial production to obtain a higher yield per unit volume.
  • Example 6 Comparison of pilot fermentation process with different starting bacteria and constructed bacteria
  • the constructed Streptomyces roseosporus strain was cultured in the seed tank for 22-26 hours, then transferred to the fermentation tank, cultured at about 30°C for 25 hours, and continuously fed during fermentation.
  • Capric acid is used as a fermentation precursor.
  • the amount of decanoic acid added to the tank is twice that of the starting bacteria's normal process.
  • the starting bacteria tried to use the same feeding process as the constructed bacteria that is, the supplementary amount of capric acid was twice that of the original process). Cultivation to the end of fermentation, HPLC determination of fermentation units, comparison.
  • Seed pot formula potato starch 6%, glucose 1.5%, sugar cane molasses 0.72%, ferrous ammonium sulfate 0.08%. Soak the enemy 0.05%.
  • Fermentation tank formula potato starch 7.2%, glucose 1%, sugar cane molasses 0.72%, yeast powder 1.2%, ferrous ammonium sulfate 0.086%, foam enemy 0.05%.
  • the constructed bacteria significantly increased the expression of daptomycin on the tank and increased the yield of a single fermentation; secondly, the constructed bacteria significantly improved the utilization rate of capric acid, so that the same amount of daptomycin was added. After the capric acid, more daptomycin is produced.

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Abstract

提供了一种达托霉素高产菌株及其制备方法，重组菌株的分类命名为玫瑰孢链霉菌（Streptomyces roseosporus），保藏号为NO.CGMCC18297。通过使用全局调控因子CRP，解决了传统链霉菌育种的高工作量，高盲目性的问题，得到了利用基因工程培育的达托霉素高产菌株，应用前景广阔；利用该方法对出发菌玫瑰孢链霉菌进行改造，得到的重组菌株能提高玫瑰孢链霉菌对癸酸的耐受度、癸酸利用率，以及提高达托霉素的产量。

Description

一种达托霉素高产菌株及其应用 技术领域
本发明涉及基因工程技术领域，具体涉及一种达托霉素高产菌株及其制备方法。
背景技术
达托霉素是由玫瑰孢链霉菌(Streptomyces roseosporus)产生的一种新型的环脂肽抗生素，是以钙离子依赖的方式与细胞膜相互作用并发挥杀菌活性。2010年12月,美国FDA批准了Cubist制药有限公司的达托霉素(daptomycin)注射剂Cubicin以一日1次2min静脉内推注用新给药方案。Cubicin在美最早于2003年获得批准,用于一日1次30min静脉内输注给药治疗由某些革兰阳性菌、包括耐甲氧西林金黄色葡萄球菌引起的复杂性皮肤和皮肤组织感染,2006年又获得了治疗由对甲氧西林敏感和耐药的金黄色葡萄球菌引起的菌血症、包括右心感染性心内膜炎的新适应证，被视为万古霉素的最佳替代品。我国食品药品监督管理总局分别于2015年和2016年批准了杭州中美华东制药有限公司，浙江海正药业股份有限公司和江苏恒瑞医药股份有限公司生产的注射用达托霉素。多项研究表明达托霉素上市10多年来耐药比例并未显著提高，彰显出达托霉素的独特优势。
达托霉素是一种微生物次级代谢产物，结构复杂，产量非常低，通过基因工程改造是提高达托霉素产量、降低生产成本的重要策略。
癸酸是达托霉素合成中的外源性前体，可改变菌体的次级代谢方向。在达托霉素的生产中，癸酸是必需物质，但过量的癸酸会对玫瑰孢链霉菌产生毒性；相反，若癸酸浓度过低，则会导致前体供应不足而使发酵液中的产品的效价降低。
目前国际上达托霉素发酵单位接近2g/L，而国内整体发酵单位远低于这一水平，针对发酵产量低的现状，国内已经有部分相关研究来提高其产量，但在基因水平上进行定向改造来提高达托霉素的产量报道不多。
发明内容
本发明的目的在于提供一种提高癸酸利用率及耐受度的达托霉素菌株及其构建方法，以解决现有技术中的玫瑰孢链霉菌对前体耐受度低以及达托霉素产量低的技术问题。本发明的菌株的保藏号为CGMCC No.18297。
本发明的目的还在于提供含调控基因的重组质粒的构建方法。
本发明的第三个目在于提供该质粒在达托霉素产生菌遗传改造上的应用。
作为一种具体的实施方式，所述重组菌株的分类命名为玫瑰孢链霉菌(Streptomyces roseosporus)，所述重组菌株的保藏号为CGMCC No.18297。
本发明所述重组菌株的保藏信息如下：
保藏时间：2019年7月26日；
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心；
保藏编号：CGMCC No.18297；
保藏单位地址：北京市朝阳区北辰西路1号院3号；
分类命名：玫瑰孢链霉菌(Streptomyces roseosporus)。
作为一种具体的实施方式，所述重组菌株为癸酸耐受菌株。
作为一种具体的实施方式，所述重组菌株的癸酸耐受度为出发菌株的两倍。
作为一种具体的实施方式，所述的重组菌株在发酵生产达托霉素中的应用。
作为一种具体的实施方式，所述的重组菌株在提高玫瑰孢链霉菌对癸酸的耐受、提高癸酸利用率及提高达托霉素发酵产量中的应用。
本发明提供的提高达托霉素产量的步骤如下：
(1)通过NCBI数据库得到CRP在玫瑰孢链霉菌中的同源序列，从玫瑰孢链霉菌的基因组中获得CRP的亚克隆片段，将该片段克隆到载体上，得到含有CRP基因片段的重组质粒。
(2)通过链霉菌结合转移的方法重组质粒导入到玫瑰孢链霉菌NO.CGMCC4.7231中，通过抗生素筛选得到包含CRP基因的重组菌株。
(3)针对重组菌株进行发酵验证，得到高产菌株。
作为一种具体的实施方式，步骤(1)中的亚克隆片段的核苷酸序列如SEQ ID NO.1所示。
作为一种具体的实施方式，步骤(1)中的载体含有erme启动子。
作为一种具体的实施方式，步骤(1)中的重组质粒为pSET152CRP。
本发明的优点是通过使用全局调控因子CRP，解决了传统链霉菌育种的高工作量，高盲目性的问题，得到了利用基因工程培育的达托霉素高产菌株，应用前景广阔；利用该方法对出发菌玫瑰孢链霉菌进行改造，得到的重组菌株能提高玫瑰孢链霉菌对癸酸的耐受度和利用率，以及提高达托霉素的产量。
附图说明
图1为在培养基中不同前体浓度下，本发明中高抗生素产量玫瑰孢链霉菌与现有玫瑰孢链霉菌发酵产量归一化处理后的对比图；
图2为pSET152CRP的物理图谱；
图3为intΦC31整合方式；
图4为本发明中的构建菌与出发菌在发酵过程中，每日补入正常生产过程中癸酸的量和每日补入两倍正常量的癸酸的两种补料工艺下，每日达托霉素效价归一化处理后的对比图；
图5为本发明中的构建菌与出发菌在发酵过程中，每日补入正常生产过程中癸酸的量和每日补入两倍正常量的癸酸的两种补料工艺下，每日癸酸利用率(效价/癸酸累计加入量)的对比图。
具体实施方式
下面通过实施例对本发明进行详细说明。
在本发明中，若非特指，所有的设备和原料均可从市场上购得或是本行业常用的，下述实施例中的方法，如无特别说明，均为本领域常规方法。
实施例1：包含CRP基因的重组质粒的pSET152CRP的构建：
通过天蓝色链霉菌中报道的同源基因CRP的核苷酸序列，与NCBI(网站：www.ncbi.nlm.nih.gov)的玫瑰孢链霉菌的基因组序列进行Blast，获得玫瑰孢链霉菌中的目的基因CRP，其大小为672bp。
用primer 5.0设计引物如下：
CRPS:ATTTCTAGAAATACCTGACCGAGCACG；
CRPA:ATTGGATCCATCGCACTGTTTTACCGT。
提取玫瑰孢链霉菌的总DNA，扩增CRP基因，得到目的基因片段。
使用SanPerp柱式质粒DNA小量抽提试剂盒，参照试剂盒说明书方法提取pSET152质粒。将所得到的质粒DNA溶液置于-20℃保存或用于后续试验。
使用限制性内切酶处理pSET152质粒，酶切后进行DNA琼脂糖凝胶电泳，使用SanPerp柱式DNA胶回收试剂盒，根据说明书操作，回收大小为858bp的片段将所得的DNA溶液置于-20℃保存或用于后续实验。
将扩增后的CRP基因与酶切回收后的pSET152质粒片段进行连接，酶切连接反应体系：载体0.03pmol，片段0.03-0.3pmol，T4DNA连接酶1200U，缓冲液5ul，剩余体积用蒸馏水补足，16℃连接16h，得到连接产物。
取大肠杆菌感受态细胞200ul，加入上述连接产物，轻轻旋转以混匀内容物，在冰中放置30min。将管放入预加温至42℃的循环水浴中放置90s。快速将管转移到水浴中，使细胞冷却1-2min。每管加入37℃预热的LB培养基800ul，然后转移至37℃水浴45min。将适当体积已转化的感受态细胞转移到含相应抗性的LB培养基上。37℃倒置培养12-16h后出现菌落。挑取菌落进行酶切验证，取验证成功菌落进行保存及测序验证。
重组质粒pSET152CRP大小为7033bp，带有安普拉抗性基因，可在大肠杆菌和玫瑰孢链霉菌中筛选，intΦC31为整合酶基因，attP为整合位点，p*erme为红霉素启动子。
LB培养基配方：胰蛋白胨10g，酵母提取物10g，NaCl 10g，加去离子水至1000ml，PH7.0。在121℃下灭菌30min，用于培养大肠杆菌。固体培养基加入2％的琼脂粉。
实施例2：将重组质粒通过属间接合转移至玫瑰孢链霉菌
将包含重组质粒PSET152CRP的大肠杆菌ET12567与玫瑰孢链霉菌NO.CGMCC4.7231进行接合转移，用安普拉霉素和萘啶酮酸筛选得到含有PSET152CRP质粒的接合子，同时用导入空载体PSET152空质粒的载体做对照。
大肠杆菌和玫瑰孢链霉菌NO.CGMCC4.7231属间接合转移的方法：
接种ET12567(PUZ8002/PSET152)和ET12567(PUZ8002/PSET152CRP)至LB(含卡那霉素/氯霉素/氨苄霉素)培养基，在37℃220rpm的摇床中过夜培养。按1:100的比例分别转接ET12567(PUZ8002/PSET152)和ET12567(PUZ8002/PSET152CRP)至新鲜的LB培养基(含卡那霉素/氯霉素/氨苄霉素)，37℃，220rpm的摇床中培养至OD600达到0.3～0.4。用相同体积的LB培养基重悬细胞2次，最后重悬于0.1倍体积的LB培养基中。在洗细胞的同时，每次接 合转移所需的10-8个孢子悬浮于500ul 2×YT培养基中，50℃热激10min，冷却至室温。各取500ul大肠杆菌液和玫瑰孢链霉菌孢子液充分混匀，离心去倒去大部分的上清，用剩余的液体重悬。将混合好的菌液涂布于含10mM MgCl 2的MS固体培养基，在29℃下培养16-20h。取1ml含0.5mg萘啶酮酸和1mg安普拉霉素的无菌水，在接合转移的平板上均匀覆盖。继续置于29℃培养箱中培养2-3天，即可观察到接合子。用划线的方法得到含PSET152CRP的玫瑰孢链霉菌单菌落，将单菌落接种至含75ug萘啶酮酸和250ug阿普拉霉素的5mlTSB液体试管中进行传代，验证接合子的基因型。
2×YT培养基配方：
胰蛋白胨16g，酵母提取物10g，NaCl 5g，加入去离子水至1000ml，用5N NaOH调PH至7.0，121℃高压蒸汽灭菌20min。
MS培养基配方：
甘露醇20g，黄豆饼粉20g，琼脂20g，加去离子水至1000ml，PH7.2-7.3，115℃、15min灭菌两次，使用时加入1M MgCl 2至终浓度为10mM/L。(1M MgCl 2：MgCl 2.7H 2O)
实施例3：重组菌发酵工艺
将构建完的玫瑰孢链霉菌菌株在R5斜面培养基30℃培养5d后，将其转移至含50ml YEME液体培养基的摇瓶中，30℃振荡(220rpm/min)培养25h，发酵时采用一次性补入料癸酸作为发酵前体，培养至发酵结束，选取最高产的菌株。由说明书附图1所示与现有的玫瑰孢链霉菌(出发菌)相比，本发明中制得的高抗生素产量玫瑰孢链霉菌具有更高的发酵产量，也就是具有更好的达托霉素产量，发酵产量提高200％以上。
TSB液体培养基配方：
胰酪蛋白胨17g，大豆蛋白胨3g，D-葡萄糖2.5g，氯化钠5g，磷酸氢二钾2.5g，加入去离子水至1000ml，121℃灭菌30min。
R5培养基配方：
蔗糖103.0g，K 2SO 4 0.25g，MgCl 2·6H 20 10.12g，葡萄糖10.0g，水解酪蛋白0.1g，微量元素溶液2.0g，酵母膏5.0g，TES缓冲液57.3ml，KH 2PO 4(0.5％)10ml，CaCl 2·H 2O(5M)4ml，L-脯氨酸(20％)150ml，NaOH(1N)7ml，琼脂20.0g，加去离子水至1000ml，PH 7.2。在115℃灭菌30min。
微量元素溶液(每升)：ZnCl 2 40mg，FeCl 2·6H 2O 200mg，CuCl 2·2H 2O 10mg，MnCl 2·2H 2O 10mg，Na 2B 4O 7·10H 2O 10mg，(NH 4) 6Mo 7O 24·4H 2O 10mg
YEME培养基配方：
酵母提取物1.5g，胰蛋白胨5g，麦芽提取物3g，葡萄糖10g，蔗糖250g，加入去离子水至1000ml，115℃蒸汽灭菌15min。
实施例4：HPLC测定达托霉素发酵单位：
色谱条件：色谱柱：Phenomenex IB-SIL C8 4.6×250mm 5um，流速：1.0ml/min，检测波长：223nm，进样量：25ul，柱温：25℃，梯度洗脱流动相A：称取3.4g磷酸二氢铵用1000ml蒸馏水溶解，用磷酸调PH至3.1，流动相B：乙腈。
实施例5：构建菌株对前体浓度的耐受测试
采用实施例3的发酵工艺，在摇瓶中对比出发菌株与构建菌株NO.CGMCC4.7231对于发酵前体在培养基中浓度的耐受范围。实验结果如说明书附图1所示，构建菌在摇瓶中前体浓度3％～5％时达托霉素的产量最高，超过5％以后产量有明显的下降；但出发菌产量最高的前体浓度在2％～3％，在3％以后产量就有明显的下降。说明CRP基因可以提高玫瑰孢链霉菌对于培养基中前体的耐受度，在工业生产时可以提高发酵罐中癸酸的加入量，获得单位体积更 高的产量。
实施例6：出发菌与构建菌不同的中试发酵工艺对比
按照达托霉素的中试发酵工艺，将构建完的玫瑰孢链霉菌菌株在种子罐中培养22-26小时后，将其转移至发酵罐中，30℃左右培养25h，发酵时连续补料癸酸作为发酵前体，根据构建菌对前体的耐受度，构建均在罐上补入的癸酸量是出发菌正常工艺的两倍。作为对比，出发菌尝试与构建菌使用相同的补料工艺(即癸酸补入量是原始工艺的两倍)。培养至发酵结束，HPLC测定发酵单位，对比。
种子罐配方：马铃薯淀粉6％，葡萄糖1.5％，甘蔗糖蜜0.72％，硫酸亚铁铵0.08％。泡敌0.05％。
发酵罐配方：马铃薯淀粉7.2％，葡萄糖1％，甘蔗糖蜜0.72％，酵母粉1.2％，硫酸亚铁铵0.086％，泡敌0.05％。
实验结果如说明书附图5所示，在罐上发酵第7天和第8天时，构建菌的效价为出发菌原始工艺的2.5～3倍，是出发菌相同工艺(即癸酸补入量是原始工艺的两倍)的2倍以上。结果显示，在发酵过程中，构建菌的癸酸利用率(以效价/癸酸累计补入量计算)是出发菌原始工艺的2倍以上，同时也比出发菌相同工艺(即癸酸补入量是原始工艺的两倍)明显提高。以上两幅图说明了，首先，构建菌明显提高了罐上达托霉素的表达量，增加了单次发酵的产量；其次，构建菌明显提高了对于癸酸的利用率，使得加入相同量的癸酸后产生更多的达托霉素。
应当理解的是，对于本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (8)

  1. 一种高产达托霉素的重组菌株，其特征在于，所述重组菌株的分类命名为玫瑰孢链霉菌(Streptomyces roseosporus)，所述重组菌株的保藏号为NO.CGMCC18297。
  2. 根据权利要求1所述的高产达托霉素的重组菌株，其特征在于，所述重组菌株为癸酸耐受菌株。
  3. 根据权利要求3所述的高产达托霉素的重组菌株，其特征在于，所述重组菌株的癸酸耐受度为出发菌株的两倍。
  4. 权利要求1-3任一项所述的高产达托霉素的重组菌株构建方法，其特征在于，包括以下步骤：
    1)获取SEQ ID NO.1所示的编码CRP蛋白的调控基因；
    2)将所述调控基因与表达载体连接，构建重组质粒；
    3)将所述重组质粒导入玫瑰孢链霉菌株，得到重组菌株。
  5. 根据权利要求4所述的高产达托霉素的重组菌株构建方法，其特征在于，所述步骤1)优选为从玫瑰孢链霉菌中获取SEQ ID NO.1所示的编码CRP蛋白的调控基因；
    所述步骤2)优选为将CRP蛋白的调控基因克隆到含强启动子的表达载体PSET152上，得到含CRP基因的重组质粒，所述重组质粒的核苷酸序列如SEQ ID NO.2所示；
    所述步骤3)优选为通过接合转移的方法，将步骤(2)得到的重组质粒转移到玫瑰孢链霉菌中，获得重组菌株。
  6. 根据权利要求4所述的高产达托霉素的重组菌株，其特征在于，通过将步骤3)得到的重组菌株进行抗生素筛选，发酵培养及达托霉素效价测定，获得达托霉素高产的链霉菌菌株。
  7. 权利要求1-3任一项所述的重组菌株在发酵生产达托霉素中的应用。
  8. 权利要求1-3任一项所述的重组菌株在提高玫瑰孢链霉菌对癸酸的耐受度、利用率及提高达托霉素发酵产量中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116731134A (zh) * 2023-04-28 2023-09-12 中国农业科学院植物保护研究所 一种糖转运蛋白tp6568及其在改造高产链霉菌中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014297A2 (en) * 2001-08-06 2003-02-20 Cubist Pharmaceuticals, Inc. Compositions and methods relating to the daptomycin biosynthetic gene cluster
CN102146414A (zh) * 2010-07-26 2011-08-10 天津大学 玫瑰孢链霉菌基因工程菌的构建方法及其应用
CN102586147A (zh) * 2012-02-17 2012-07-18 厦门大学 一种可高产达托霉素的玫瑰孢链霉菌的筛选方法
WO2019195655A1 (en) * 2018-04-06 2019-10-10 University Of Florida Research Foundation, Incorporated Engineered streptomyces albus strains

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1818398A1 (en) * 2006-02-14 2007-08-15 Universiteit Leiden Methods and means for metabolic engineering and improved product formation by micro-organisms
CN101302531B (zh) * 2008-06-20 2011-05-18 南京师范大学 一种在大肠杆菌-链霉菌-假单胞菌之间穿梭表达的bac载体及其构建方法
WO2017031399A1 (en) * 2015-08-20 2017-02-23 Genomatica, Inc. Compositions and multiplexed systems for coupled cell-free transcription-translation and protein synthesis and methods for using them
CN113717259A (zh) * 2020-05-26 2021-11-30 浙江工业大学 一种玫瑰孢链霉菌中提高癸酸利用率及耐受度的调控基因及应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003014297A2 (en) * 2001-08-06 2003-02-20 Cubist Pharmaceuticals, Inc. Compositions and methods relating to the daptomycin biosynthetic gene cluster
CN102146414A (zh) * 2010-07-26 2011-08-10 天津大学 玫瑰孢链霉菌基因工程菌的构建方法及其应用
CN102586147A (zh) * 2012-02-17 2012-07-18 厦门大学 一种可高产达托霉素的玫瑰孢链霉菌的筛选方法
WO2019195655A1 (en) * 2018-04-06 2019-10-10 University Of Florida Research Foundation, Incorporated Engineered streptomyces albus strains

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GAO CHAN, HINDRA, MULDER DAVID, YIN CHARLES, ELLIOT MARIE A.: "Crp Is a Global Regulator of Antibiotic Production in Streptomyces", MBIO, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 3, no. 6, 11 December 2012 (2012-12-11), US , pages 1 - 12, XP055871565, ISSN: 2161-2129, DOI: 10.1128/mBio.00407-12 *
LIN CHUN-YAN, ZHANG YUE, WU JI-HUA, XIE RONG-HUI, QIAO JIANJUN, ZHAO GUANG-RONG: "Regulatory Patterns of Crp on Monensin Biosynthesis in Streptomyces cinnamonensis", MICROORGANISMS, vol. 8, no. 2, 17 February 2020 (2020-02-17), XP055872412, DOI: 10.3390/microorganisms8020271 *
MAO XU-MING, LUO SHUAI, ZHOU RI-CHENG, WANG FENG, YU PIN, SUN NING, CHEN XIAO-XIA, TANG YI, LI YONG-QUAN: "Transcriptional Regulation of the Daptomycin Gene Cluster in Streptomyces roseosporus by an Autoregulator, AtrA", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 290, no. 12, 3 January 2015 (2015-01-03), US , pages 7992 - 8001, XP055872409, ISSN: 0021-9258, DOI: 10.1074/jbc.M114.608273 *
PENG XI-XI, SAI-SAI WANG, CHEN FU, CHANG-HUA HU, LIAO GUO-JIAN: "Current status and future perspectives of daptomycin development", ACTA PHARMACEUTICA SINICA, YAOXUE XUEBAO, CN, vol. 53, no. 6, 30 June 2018 (2018-06-30), CN , pages 839 - 844, XP055872785, ISSN: 0513-4870, DOI: 10.16438/j.0513-4870.2018-0096 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116731134A (zh) * 2023-04-28 2023-09-12 中国农业科学院植物保护研究所 一种糖转运蛋白tp6568及其在改造高产链霉菌中的应用
CN116731134B (zh) * 2023-04-28 2024-05-31 中国农业科学院植物保护研究所 一种糖转运蛋白tp6568及其在改造高产链霉菌中的应用

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