技术领域technical field
本发明涉及的是生物医药领域，具体说是一种增强转录水平的高产安丝菌素菌株及其制备方法，主要是通过在珍贵束丝放线菌ATCC31280的糖基转移酶表达基因中断缺失突变株NXJ-22中，利用强启动子kasOp*过量表达后修饰基因asm10，增强安丝菌素甲基转移效率，提高安丝菌素生物合成能力，进而提高安丝菌素产量。The present invention relates to the field of biomedicine, specifically a high-yield ansamitocin strain with enhanced transcription level and its preparation method, mainly through the interruption deletion mutation of the expression gene of glycosyltransferase in Actinomyces spp. precious ATCC31280 In strain NXJ-22, the gene asm10 was modified after overexpression of the strong promoter kasOp* to enhance the methyl transfer efficiency of ansamitocin, improve the biosynthesis ability of ansamitocin, and then increase the production of ansamitocin.
背景技术Background technique
安丝菌素是由珍贵束丝放线菌（Actinosynnema pretiosum）产生的一种大环内酰胺类抗生素，它能够与微管蛋白的β亚基结合，阻碍微管组装，从而抑制肿瘤细胞分裂。来自ImmunoGen，Inc.公司的Chari等人通过在C-3位酯链上连接二硫键形成的DM1分子，经DTT还原后可与不同的抗体连接形成抗体结合分子。目前，安丝菌素多种DM1抗体结合分子已经进入不同的临床实验阶段，其中由罗氏公司开发的用于治疗人类乳腺癌的TrastuzumabEmtansine（即T-DM1）已经成药上市。除了抗肿瘤活性外，安丝菌素还能抑制真核生物，如真菌，酵母，昆虫等。Ansamitocin is a macrocyclic lactam antibiotic produced by Actinosynnema pretiosum, which can bind to the β subunit of tubulin to hinder the assembly of microtubules, thereby inhibiting tumor cell division. Chari et al. from ImmunoGen, Inc., formed a DM1 molecule formed by linking a disulfide bond on the C-3 ester chain, which can be linked with different antibodies to form antibody-binding molecules after DTT reduction. At present, a variety of DM1 antibody-binding molecules of Ansamectin have entered different stages of clinical trials, among which Trastuzumab Emtansine (T-DM1) developed by Roche for the treatment of human breast cancer has been marketed as a drug. In addition to antitumor activity, ansamitocin can also inhibit eukaryotic organisms, such as fungi, yeast, insects, etc.
安丝菌素生物合成主要包括三部分：起始单元3-氨基-5-羟基苯甲酸（AHBA）的合成，聚酮链的延伸（I型聚酮合酶催化）和复杂的后修饰途径，包括：氯代、氧甲基化、氨甲酰化、酰化、环氧化、N-甲基化（SAM依赖）或者糖基化形成安丝菌素，其中由糖基转移酶Asm25催化的N-糖基化反应为N-甲基化反应的竞争途径，生成分支产物-糖基化安丝菌素。虽然可通过糖基转移酶表达基因进行中断缺失，得到的突变株NXJ-22，使N位糖基化对N位甲基化途径的竞争被消除，但是，N位甲基化效率仍旧较低。通过现有技术公开的内容可知，后修饰途径的效率对于终产物的产量和活性都有着至关重要的作用。Ansamitocin biosynthesis mainly includes three parts: the synthesis of the initial unit 3-amino-5-hydroxybenzoic acid (AHBA), the elongation of the polyketide chain (catalyzed by type I polyketide synthase) and the complex post-modification pathway, Including: chlorination, oxymethylation, carbamylation, acylation, epoxidation, N-methylation (SAM-dependent) or glycosylation to form ansamicin, which is catalyzed by the glycosyltransferase Asm25 The N-glycosylation reaction is a competitive pathway of the N-methylation reaction, and a branch product-glycosylated ansamicin is generated. Although the glycosyltransferase expression gene can be interrupted and deleted, the obtained mutant strain NXJ-22 can eliminate the competition between N-glycosylation and N-methylation pathway, but the efficiency of N-methylation is still low . It can be seen from the content disclosed in the prior art that the efficiency of the post-modification pathway plays a crucial role in the yield and activity of the final product.
发明内容Contents of the invention
本发明的目的是提供一种高产安丝菌素的菌株、该突变株的制备方法以及通过该突变株高产安丝菌素的方法。该突变株基于过量表达N位甲基转移酶表达基因asm10，通过加倍安丝菌素生物合成途径中的限速步骤基因，增强甲基转移酶基因表达水平，从而提高安丝菌素的产量。The object of the present invention is to provide a strain with high ansamitocin production, a method for preparing the mutant strain and a method for high ansamitocin production through the mutant strain. The mutant strain is based on the overexpression of the N-position methyltransferase expression gene asm10, and by doubling the rate-limiting step gene in the ansamitocin biosynthesis pathway, the expression level of the methyltransferase gene is enhanced, thereby increasing the production of ansamitocin.
为实现上述目的，本发明提供了以下技术方案：To achieve the above object, the present invention provides the following technical solutions:
一方面，本发明提供了一种增强转录水平的高产安丝菌素菌株，其技术要点是：所述菌株的N位甲基转移酶过量表达。On the one hand, the present invention provides a highly ansamitocin-producing strain with enhanced transcription level, the technical gist of which is: the N-position methyltransferase of the strain is overexpressed.
进一步的，突变株由菌株ATCC 31280过量表达后获得。Further, the mutant strain was obtained after overexpressing strain ATCC 31280.
另一方面，本发明提供了该增强转录水平的高产安丝菌素菌株的制备方法，其技术要点是，包括以下步骤：In another aspect, the present invention provides a method for preparing the highly ansamitocin-producing strain with enhanced transcription level, the technical gist of which includes the following steps:
步骤1），构建用于加倍甲基转移酶基因asm10的整合型质粒载体；Step 1), constructing an integrated plasmid vector for doubling the methyltransferase gene asm10;
步骤2），将步骤1）构建得到的质粒载体通过接合转移导入受体菌NXJ-22中进行同源重组，并筛选基因过量表达的突变株。In step 2), the plasmid vector constructed in step 1) was introduced into the recipient strain NXJ-22 by conjugative transfer for homologous recombination, and mutant strains with gene overexpression were screened.
进一步的，步骤1）中，质粒载体由质粒pDR3-K*重组获得。Further, in step 1), the plasmid vector is obtained by recombination of the plasmid pDR3-K*.
进一步的，基因asm10来源于珍贵束丝放线菌ATCC 31565。Further, the gene asm10 is derived from Actinomyces spp. ATCC 31565.
进一步的，在起始密码子前插入RBS位点序列ATCTGAGTTGAAGAGGTGACGTC。Further, the RBS site sequence ATCTGAGTTGAAGAGGTGACGTC was inserted before the initiation codon.
进一步的，步骤3)中，通过抗性筛选和PCR验证获得过量表达asm10基因的突变株。Further, in step 3), a mutant strain overexpressing the asm10 gene is obtained through resistance screening and PCR verification.
进一步的，质粒载体的构建通过在质粒的SpeI/EcoRI位点插入asm10的PCR片段（SpeI/EcoRI）。Further, the plasmid vector was constructed by inserting the PCR fragment of asm10 (SpeI/EcoRI) at the SpeI/EcoRI site of the plasmid.
此外，本发明还提供了一种采用上述突变株制备安丝菌素的方法，其技术要点是，包括以下步骤：将活化后的asm10基因过量表达突变株的菌丝体在一级种子培养基中，30℃、220 r/min条件下培养24 h；按4%接种量转接至二级种子培养基中，30℃、220 r/min的转速下培养24 h；按10%的接种量转接至发酵培养基中，25℃、220 r/min的转速下发酵7 d后收集发酵液并进行萃取。In addition, the present invention also provides a method for preparing ansamectin by using the above-mentioned mutant strain, the technical gist of which includes the following steps: putting the mycelia of the activated asm10 gene overexpression mutant strain in the primary seed medium medium, cultured at 30°C and 220 r/min for 24 h; transferred to secondary seed medium at 4% inoculum size, and cultured at 30°C at 220 r/min for 24 h; at 10% inoculum size Transfer to the fermentation medium, ferment for 7 days at 25°C and 220 r/min, and then collect the fermentation broth and extract it.
进一步的，上述安丝菌素的制备方法中：Further, in the preparation method of the above-mentioned ansamitocin:
一级种子培养基包括TSB 3w/v%，酵母提取物0.5w/v%，蔗糖5w/v%；Primary seed medium includes TSB 3w/v%, yeast extract 0.5w/v%, sucrose 5w/v%;
或者二级种子培养基包括TSB 3w/v%，酵母提取物0.5w/v%，蔗糖2.5w/v%，异丁醇0.05v/v%，异丙醇0.05v/v%，淀粉1w/v%；Or secondary seed medium including TSB 3w/v%, yeast extract 0.5w/v%, sucrose 2.5w/v%, isobutanol 0.05v/v%, isopropanol 0.05v/v%, starch 1w/ v%;
或者发酵培养基包括酵母提取物0.8w/v%，麦芽提取物1w/v%，蔗糖1.5w/v%，异丁醇0.5v/v%，异丙醇1.2v/v%，淀粉2.5w/v%，MgCl22 mmol/L。Or the fermentation medium includes yeast extract 0.8w/v%, malt extract 1w/v%, sucrose 1.5w/v%, isobutanol 0.5v/v%, isopropanol 1.2v/v%, starch 2.5w /v%, MgCl2 2 mmol/L.
本发明的有益效果：现有技术中，通常选用的受体菌为A. pretiosum ATCC31565，以asm10基因回补所用载体为pIB139，所用启动子为permE*，而本发明所用受体菌为ATCC 31280衍生的突变株，asm10基因过量表达所用载体为pDR3-K*，所用启动子为kasOp*。通过上述方法，相较于现有技术asm10基因回补到突变株中时，AP-3产量恢复到与野生型相近水平；而通过本发明的方法在asm10基因过量表达后，AP-3产量对比对照菌株提高至少173%。Beneficial effects of the present invention: in the prior art, the usually selected recipient bacterium is A. pretiosum ATCC31565, the vector used for complementing asm10 gene is pIB139, and the used promoter is permE*, while the recipient bacterium used in the present invention is ATCC 31280 In the derived mutant strain, the vector used for overexpression of asm10 gene is pDR3-K*, and the promoter used is kasOp*. Through the above method, compared with the prior art when the asm10 gene is recomplemented into the mutant strain, the AP-3 production is restored to a level similar to that of the wild type; and after the asm10 gene is overexpressed by the method of the present invention, the AP-3 production is compared The control strain improved by at least 173%.
综上所述，在珍贵束丝放线菌ATCC 31280的糖基转移酶基因中断缺失突变株NXJ-22中，利用整合型载体pDR3-K*(ΦC31整合位点，pSET152衍生，带有kasOp*强启动子），在NXJ-22染色体上插入一个拷贝的甲基转移酶表达基因asm10 (来源于珍贵束丝放线菌ATCC31565），增强asm10的转录水平，较对照菌株（空白载体整合菌株）提高173%以上，最终产量达到141.8mg/L，通过本发明可显著提高安丝菌素的发酵产量。To sum up, in the glycosyltransferase gene interruption deletion mutant strain NXJ-22 of Actinomyces spp. precious ATCC 31280, the integrated vector pDR3-K* (ΦC31 integration site, derived from pSET152, carrying kasOp* strong promoter), insert a copy of the methyltransferase expression gene asm10 (derived from Actinomyces spp. ATCC31565) on the NXJ-22 chromosome, and enhance the transcription level of asm10, which is higher than that of the control strain (blank vector integrated strain) More than 173%, the final yield reaches 141.8mg/L, and the fermentation yield of ansamitocin can be significantly improved through the present invention.
附图说明Description of drawings
图1为asm10基因加倍质粒构建示意图；Figure 1 is a schematic diagram of the construction of asm10 gene doubling plasmid;
图2为asm10基因过表达突变株与对照菌株转录水平对比示意图；Figure 2 is a schematic diagram of the comparison of transcription levels between asm10 gene overexpression mutants and control strains;
图3为asm10基因加倍突变株与对照菌株安丝菌素发酵产量示意图。Figure 3 is a schematic diagram of the ansamitocin fermentation yields of the asm10 gene doubling mutant strain and the control strain.
具体实施方式Detailed ways
以下实例将结合附图对本发明作进一步说明。虽然以下给出了本发明优化的实施方式和过程，但本发明的保护范围不限于下述的实施例。下列实施例中未注明具体条件的实验方法的，按照常规条件或制造厂商的建议条件。The following examples will further illustrate the present invention in conjunction with the accompanying drawings. Although the optimized implementation and process of the present invention are given below, the protection scope of the present invention is not limited to the following examples. For the experimental methods that do not indicate the specific conditions in the following examples, follow the conventional conditions or the manufacturer's suggested conditions.
实施例1Example 1
本实施例为制备基因asm10过量表达的突变株的具体过程。具体包括以下步骤：This example is a specific process for preparing a mutant strain with overexpression of the gene asm10. Specifically include the following steps:
步骤1），构建质粒pLQ586：以珍贵束丝放线菌ATCC 31565基因组DNA作为模板，使用引物asm10-F/R，通过PCR扩增得到asm10片段（885 bp），在序列两端引入SpeI/EcoRI酶切位点，并在起始密码子前插入RBS序列（ATCTGAGTTGAAGAGGTGACGTC）。在质粒pDR3-K*的SpeI/EcoRI位点插入asm10 (SpeI/EcoRI))，得到质粒pLQ586。Step 1) Plasmid pLQ586 was constructed: Genomic DNA of Actinomyces spp. precious ATCC 31565 was used as a template, and the asm10 fragment (885 bp) was amplified by PCR using primers asm10-F/R, and SpeI/EcoRI were introduced at both ends of the sequence site, and insert the RBS sequence (ATCTGAGTTGAAGAGGTGACGTC) before the start codon. Asm10 (SpeI/EcoRI)) was inserted at the SpeI/EcoRI site of plasmid pDR3-K* to obtain plasmid pLQ586.
*本发明涉及的各内切酶识别位点（酶切位点）如下：* Each endonuclease recognition site (enzyme cutting site) involved in the present invention is as follows:
步骤1)所用到的引物序列为：The primer sequence used in step 1) is:
*步骤1）中基因片段制备所采用的PCR体系及条件：*PCR system and conditions used in gene fragment preparation in step 1):
PCR反应体系：DNA模板30 ng，引物30 pmol，50%DMSO 3 mL，25 mM Mg2+ 2 mL，缓冲液3mL，KOD聚合酶1个单位，加纯水补齐至30 mL；PCR reaction system: DNA template 30 ng, primer 30 pmol, 50% DMSO 3 mL, 25 mM Mg2+ 2 mL, buffer 3 mL, KOD polymerase 1 unit, add pure water to make up to 30 mL;
PCR条件：95℃ 5 min；95℃ 30 s；60℃ 30 s；68℃ 2 min；循环30次；68℃ 10 min。PCR conditions: 95°C for 5 min; 95°C for 30 s; 60°C for 30 s; 68°C for 2 min; cycle 30 times; 68°C for 10 min.
步骤2），将步骤1）构建得到的过量表达（基因加倍）的质粒载体pLQ586通过接合转移导入受体菌NXJ-22中进行同源重组，并通过PCR验证筛选正确的接合子，从而得到asm10基因加倍（过量表达）的突变株。具体包括以下步骤：Step 2), the overexpression (gene doubling) plasmid vector pLQ586 obtained in step 1) was introduced into the recipient strain NXJ-22 by conjugative transfer for homologous recombination, and the correct zygote was screened by PCR verification to obtain asm10 Gene doubling (overexpression) mutants. Specifically include the following steps:
将基因asm10过量表达的质粒pLQ586转化进入宿主ET12567（含有pUZ8002质粒）中。取ET12567于含有Apr、Kan和Chl三种抗生素的LB中，37°C过夜培养，用相同的培养基，将过夜培养物按10%的比例转接一次并培养2.5 h，然后用新鲜的LB溶液漂洗菌体以除去培养物中的抗生素。同时制备突变株NXJ-22的新鲜菌丝体（约16 h培养物），用LB溶液漂洗2~3次之后，将其与之前制备的宿主菌ET12567混合(菌丝体细胞和宿主菌的比例约为1：10)均匀后涂布于含有10mM镁离子的YMG平板，待平板吹干后转移至37°C培养箱倒置培养12 h后取出平板，分别取阿泊拉霉素和萘啶酮酸两种抗生素的储存液各40mL加入1.5 mL无菌水中混匀后覆盖在YMG平板上，将平板晾干后转移至30°C培养箱中倒置培养。一般3~5 d后可见平板上有单菌落接合子长出，将其通过平板涂布于含有阿泊拉霉素和萘啶酮酸两种抗生素的YMG平板扩大培养，通过菌丝体PCR验证筛选得到asm10基因加倍的突变株。The plasmid pLQ586 overexpressing the gene asm10 was transformed into host ET12567 (containing the pUZ8002 plasmid). Take ET12567 in LB containing three antibiotics of Apr, Kan and Chl, culture overnight at 37°C, use the same medium, transfer the overnight culture once at a ratio of 10% and culture for 2.5 h, then use fresh LB The solution rinses the cells to remove antibiotics from the culture. At the same time, prepare fresh mycelium of mutant NXJ-22 (about 16 h culture), rinse it with LB solution for 2-3 times, and mix it with the previously prepared host strain ET12567 (the ratio of mycelium cells to host strain About 1:10) evenly spread on the YMG plate containing 10mM magnesium ions, after the plate is blown dry, transfer to 37°C incubator and incubate upside down for 12 hours, take out the plate, take apramycin and nalidinone respectively Add 40 mL of each of the stock solutions of the two antibiotics to 1.5 mL of sterile water and mix well, then cover the YMG plate, dry the plate and transfer it to a 30°C incubator for upside-down cultivation. Generally, after 3 to 5 days, a single colony of zygotes can be seen growing on the plate, which is spread on a YMG plate containing two antibiotics, apramycin and nalidixic acid, for expansion culture, and verified by mycelium PCR Mutants with doubling of the asm10 gene were screened.
*步骤2）中通过PCR验证筛选时所采用的PCR体系及条件：*PCR system and conditions used when passing PCR verification and screening in step 2):
PCR体系：DNA模板10~100 ng，引物30 pmol，50%DMSO 3 mL，缓冲液3 mL，Taq聚合酶0.5个单位，加纯水补齐至30 mL；PCR system: DNA template 10~100 ng, primer 30 pmol, 50% DMSO 3 mL, buffer 3 mL, Taq polymerase 0.5 unit, add pure water to make up to 30 mL;
PCR条件：95℃ 5 min；95℃ 30 秒；58℃ 30 s；72℃ 1 min；循环30次；72℃ 10 min。PCR conditions: 95°C for 5 min; 95°C for 30 seconds; 58°C for 30 s; 72°C for 1 min; cycle 30 times; 72°C for 10 min.
实施例2Example 2
本实施例为通过基因asm10过量表达的突变株生物合成安丝菌素的发酵过程。具体步骤如下：将asm10过量表达的菌株涂布于固体YMG培养基上活化，30℃培养2 d后，挑取少量菌丝体接种至一级种子培养基中，30℃、220 r/min条件下培养24 h；按4%接种量转接至二级种子培养基中，30℃、220 r/min条件下培养24 h；按10%的接种量转接至发酵培养基，25℃、220 r/min条件下发酵7 d后收集发酵液进行萃取和化合物检测。This example is a fermentation process for biosynthesizing ansamitocin by a mutant strain overexpressing the gene asm10. The specific steps are as follows: spread the asm10 overexpressed strain on solid YMG medium for activation, and after culturing at 30°C for 2 days, pick a small amount of mycelium and inoculate it into the primary seed medium, at 30°C, 220 r/min 24 h under high temperature; transfer to secondary seed medium at 4% inoculum size, and cultivate at 30°C, 220 r/min for 24 h; transfer to fermentation medium at 10% inoculum amount, at 25°C, 220 After 7 days of fermentation under the condition of r/min, the fermentation broth was collected for extraction and compound detection.
表1种子培养基及发酵培养基的成分构成Table 1 Composition of seed medium and fermentation medium
实施例3Example 3
本实施例为通过荧光定量PCR测定基因加倍菌株中加倍基因转录水平的方法。（用于RNA提取的样品一般都保存在Redzol溶液中。RNA提取过程要求低温，离心过程除特殊说明，均在4°C、12000 r/min的条件下进行。）This example is a method for measuring the transcription level of the doubled gene in the gene doubled strain by fluorescent quantitative PCR. (The samples used for RNA extraction are generally stored in Redzol solution. The RNA extraction process requires low temperature, and the centrifugation process is carried out at 4°C and 12000 r/min unless otherwise specified.)
具体步骤为：取已经破碎处理的样品500 mL加入100mL氯仿涡旋振荡混匀，离心15 min后吸取上清液，加入100 mL无水乙醇并混匀后将样品吸入离心柱（北京赛百盛基因技术有限公司）中，静置2 min，离心1 min，弃液体，用漂洗液（Washing Buffer，北京赛百盛基因技术有限公司)漂洗两次，弃液体，将离心柱置于收集管中继续离心2 min。换用新的收集管，往离心柱中加入60 mL DEPC处理过的水，离心2 min，将RNA样品从离心柱上洗脱下来。用Nanodrop 2000型核酸蛋白分析仪测定RNA的浓度和OD260/280，提取后的RNA样品-80°C保存。RNA样品的消化反应体系可参照下表配制：The specific steps are: take 500 mL of the crushed sample, add 100 mL of chloroform, vortex and mix well, centrifuge for 15 min, draw the supernatant, add 100 mL of absolute ethanol and mix well, then suck the sample into the spin column (Beijing Saibaisheng Gene Technology Co., Ltd. Technology Co., Ltd.), let stand for 2 min, centrifuge for 1 min, discard the liquid, rinse twice with washing buffer (Washing Buffer, Beijing Saibaisheng Gene Technology Co., Ltd.), discard the liquid, put the spin column in the collection tube and continue centrifuging 2 min. Replace with a new collection tube, add 60 mL of DEPC-treated water to the spin column, centrifuge for 2 min, and elute the RNA sample from the spin column. The RNA concentration and OD260/280 were measured with a Nanodrop 2000 nucleic acid and protein analyzer, and the extracted RNA samples were stored at -80°C. The digestion reaction system of RNA samples can be prepared according to the following table:
反应体系置于37°C孵育4 h后每个反应体系加入5 mL 50 mM EDTA后65°C加热10 min即可终止消化，消化好的RNA样品-80°C保存。RNA经过逆转录后就得到cDNA，可用于后续的基因转录分析。将得到的cDNA利用荧光定量PCR进行检测，试剂盒可选用Fermentas公司的MaximaSYBR Green/ROX q PCR Maxter Mix(2×)。After the reaction system was incubated at 37°C for 4 h, 5 mL of 50 mM EDTA was added to each reaction system, and then the digestion was terminated by heating at 65°C for 10 min, and the digested RNA samples were stored at -80°C. After reverse transcription of RNA, cDNA is obtained, which can be used for subsequent analysis of gene transcription. The obtained cDNA was detected by fluorescent quantitative PCR, and the kit could be MaximaSYBR Green/ROX q PCR Maxter Mix (2×) from Fermentas Company.
测定基因转录水平时使用的引物：Primers used to determine gene transcription levels:
图2为asm10基因加倍突变株与对照菌株的荧光定量PCR结果。结果表明突变株和对照相比，目的基因在加倍突变株中的转录水平对比对照提高大约22倍，说明asm10基因已经被成功加倍。Fig. 2 is the fluorescent quantitative PCR result of the asm10 gene doubling mutant strain and the control strain. The results showed that compared with the control, the transcript level of the target gene in the doubling mutant was increased by about 22 times compared with the control, indicating that the asm10 gene had been successfully doubled.
实施例4Example 4
本实施例为利用HPLC检测安丝菌素的发酵产量的方法。具体为：采用安捷伦公司的Agilent 1200系列HPLC进行色谱分析，并采用DAD二极管阵列检测器测定236 nm下的色谱吸收峰。This embodiment is a method for detecting the fermentation yield of ansamicin by HPLC. Specifically: Agilent 1200 series HPLC of Agilent was used for chromatographic analysis, and a DAD diode array detector was used to measure the chromatographic absorption peak at 236 nm.
其中，HPLC参数如下：Wherein, HPLC parameter is as follows:
色谱柱：Agilent ZORBAX SB-C18, 2.1´150 mm, 3.5 µm；Column: Agilent ZORBAX SB-C18, 2.1´150 mm, 3.5 µm;
流动相流速：0.1 mL/min；Mobile phase flow rate: 0.1 mL/min;
流动相：水溶液和HPLC级甲醇梯度洗脱。Mobile phase: aqueous solution and HPLC grade methanol gradient elution.
柱温：室温。Column temperature: room temperature.
图3为asm10基因加倍突变株和安丝菌素发酵水平检测。结果表明突变株的产量对比野生型菌株提高173%以上，最终产量达到141.8 mg/L。Figure 3 is the detection of asm10 gene doubling mutant strain and ansamectin fermentation level. The results showed that the yield of the mutant strain increased by more than 173% compared with the wild-type strain, and the final yield reached 141.8 mg/L.
本发明所涉及的菌株ATCC 31280已在文献《PAN WenQin, KANG QianJin, WANGLei, BAI LinQuan* & DENG ZiXin : Asm8, a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production. Science ChinaLife Sciences 2013（7）：601-608》中记载。The bacterial strain ATCC 31280 involved in the present invention has been described in the literature "PAN WenQin, KANG QianJin, WANGLei, BAI LinQuan* & DENG ZiXin: Asm8, a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production. Science ChinaLife Recorded in Sciences 2013 (7): 601-608.
本发明所涉及的菌株ATCC 31565已在申请公开号CN101103120A的发明专利申请中公开。The bacterial strain ATCC 31565 involved in the present invention has been disclosed in the invention patent application with application publication number CN101103120A.
序列表sequence listing
<110> 辽宁斯韦尔生物科技有限公司<110> Liaoning Swell Biotechnology Co., Ltd.
上海交通大学Shanghai Jiaotong University
<120> 增强转录水平的高产安丝菌素菌株及其制备方法<120> Ansamitocin high-producing strain with enhanced transcription level and preparation method thereof
<130> 17-0837<130> 17-0837
<141> 2017-09-25<141> 2017-09-25
<160> 8<160> 8
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 885<211> 885
<212> DNA<212>DNA
<213> 珍贵束丝放线菌asm10(Actinosynnema pretiosum)<213> Actinosynnema pretiosum asm10 (Actinosynnema pretiosum)
<400> 1<400> 1
atgagcctcc cagcggactc accaccgccg gggccggtgg agatgcccga cgagagctgg 60atgagcctcc cagcggactc accaccgccg gggccggtgg agatgcccga cgagagctgg 60
acgtcgctgg gcaacgcggg ggcgcgggcg caggagtccg cgcgggccga ccggttgttc 120acgtcgctgg gcaacgcggg ggcgcgggcg caggagtccg cgcgggccga ccggttgttc 120
gacgacccgc tggcgcgggc gttcctggac gcggcgggga cggggaaccc gctgctgccc 180gacgacccgc tggcgcgggc gttcctggac gcggcgggga cggggaaccc gctgctgccc 180
gaccggggtg gcggtgatcc ggggctgctc gggcagatca cggacgtcat cgtggtcaag 240gaccggggtg gcggtgatcc ggggctgctc gggcagatca cggacgtcat cgtggtcaag 240
acggtgttct tcgacgcggt gctggcgcgg gcggcggcgg ccggggtgcg gcaggtggtg 300acggtgttct tcgacgcggt gctggcgcgg gcggcggcgg ccggggtgcg gcaggtggtg 300
ctgctggccg ccgggttgga cgcccgcgcg ttccggctgc cctggcccga gggggtggtg 360ctgctggccg ccgggttgga cgcccgcgcg ttccggctgc cctggcccga gggggtggtg 360
gtgttcgagg tggacctgcc ggacgtgctg gggttcaagg agcgcgtggt gcgggaggtc 420gtgttcgagg tggacctgcc ggacgtgctg gggttcaagg agcgcgtggt gcgggaggtc 420
ggggcggagc cctcgtgcga ccggcgggtc gtcgcggcgg acctgcgctc ggactgggtg 480ggggcggagc cctcgtgcga ccggcgggtc gtcgcggcgg acctgcgctc ggactgggtg 480
gccgcgctgg tcgccgccgg gctggaccgg gaggccccgg tggcctggct ggcggagggc 540gccgcgctgg tcgccgccgg gctggaccgg gaggccccgg tggcctggct ggcggagggc 540
gcgctggggc tgctggacga ggccgggtgc gaggagctga tggcggcggt gctcggggcc 600gcgctggggc tgctggacga ggccgggtgc gaggagctga tggcggcggt gctcggggcc 600
tccgcggcgg ggagccggtt cgcgctcgac cacacccacg acgggtggaa ggccggggag 660tccgcggcgg ggagccggtt cgcgctcgac cacacccacg acgggtggaa ggccggggag 660
gcgctgggcg ggtacctgga ggggaccggg gtctgcttgg ccgacctggt gaaggggggt 720gcgctgggcg ggtacctgga ggggaccggg gtctgcttgg ccgacctggt gaaggggggt 720
ccgcgggagc cgggcggggc gtggttggcg cggcacgggt ggcgggtggc ggagtacgac 780ccgcgggagc cgggcggggc gtggttggcg cggcacgggt ggcgggtggc ggagtacgac 780
gtcgtggcgg aggcggcacg gcacgggcgg cccgcgccga ccctgttccg ggtgccggag 840gtcgtggcgg aggcggcacg gcacgggcgg cccgcgccga ccctgttccg ggtgccggag 840
cggcgggcga acgcgacgat cctgttcgag gccgagctgg ggtag 885cggcgggcga acgcgacgat cctgttcgag gccgagctgg ggtag 885
<210> 8<210> 8
<211> 23<211> 23
<212> DNA<212>DNA
<213> 位点(RBS)<213> site (RBS)
<400> 8<400> 8
atctgagttg aagaggtgac gtc 23atctgagttg aagaggtgac gtc 23
<210> 8<210> 8
<211> 28<211> 28
<212> DNA<212>DNA
<213> 引物(asm10-R)<213> Primer (asm10-R)
<400> 8<400> 8
atagaattcc taccccagct cggcctcg 28atagaattcc taccccagct cggcctcg 28
<210> 6<210> 6
<211> 47<211> 47
<212> DNA<212>DNA
<213> 引物(asm10-F)<213> Primer (asm10-F)
<400> 6<400> 6
ataactagta tctgagttga agaggtgacg tcatgagcct cccagcg 47ataactagta tctgagttga agaggtgacg tcatgagcct cccagcg 47
<210> 2<210> 2
<211> 21<211> 21
<212> DNA<212>DNA
<213> 引物(asm10-RT-F)<213> Primer (asm10-RT-F)
<400> 2<400> 2
tctgcccgag cagccccgga t 21tctgcccgag cagccccgga t 21
<210> 3<210> 3
<211> 20<211> 20
<212> DNA<212>DNA
<213> 引物(asm10-RT-R)<213> Primer (asm10-RT-R)
<400> 3<400> 3
acgagagctg gacgtcgctg 20acgagagctg gacgtcgctg 20
<210> 4<210> 4
<211> 20<211> 20
<212> DNA<212>DNA
<213> 引物(hrdB-RT-F)<213> Primer (hrdB-RT-F)
<400> 4<400> 4
gttcccccaa ggcgaagaag 20gttcccccaa ggcgaagaag 20
<210> 5<210> 5
<211> 20<211> 20
<212> DNA<212>DNA
<213> 引物(hrdB-RT-R)<213> Primer (hrdB-RT-R)
<400> 5<400> 5
gcttggcgtt ctcctcctcg 20gcttggcgtt ctcctcctcg 20
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710870997.XACN107881137A (en) | 2017-09-25 | 2017-09-25 | Strengthen high yield ansamitocin bacterial strain of transcriptional level and preparation method thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710870997.XACN107881137A (en) | 2017-09-25 | 2017-09-25 | Strengthen high yield ansamitocin bacterial strain of transcriptional level and preparation method thereof |
| Publication Number | Publication Date |
|---|---|
| CN107881137Atrue CN107881137A (en) | 2018-04-06 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710870997.XAPendingCN107881137A (en) | 2017-09-25 | 2017-09-25 | Strengthen high yield ansamitocin bacterial strain of transcriptional level and preparation method thereof |
| Country | Link |
|---|---|
| CN (1) | CN107881137A (en) |
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|---|---|---|---|---|
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| CN116144563A (en)* | 2022-10-24 | 2023-05-23 | 上海交通大学 | Method for enhancing expression of glycoside hydrolase coding gene APASM_6114 transcription level to increase ansamitocin production |
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| US20090117127A1 (en)* | 2006-03-31 | 2009-05-07 | Sabine Gaisser | Novel Compounds and Methods for Their Production |
| CN104059863A (en)* | 2014-05-06 | 2014-09-24 | 江南大学 | Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine |
| CN104357506A (en)* | 2014-10-28 | 2015-02-18 | 上海交通大学 | Method for improving fermentation level of salinomycin by increasing supply of precursors |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060084141A1 (en)* | 2001-11-21 | 2006-04-20 | Floss Heinz G | Biosynthetic gene cluster for the maytansinoid antitumor agent ansamitocin |
| WO2005020883A2 (en)* | 2003-05-08 | 2005-03-10 | Immunogen, Inc. | Methods for the production of ansamitocins |
| US20090117127A1 (en)* | 2006-03-31 | 2009-05-07 | Sabine Gaisser | Novel Compounds and Methods for Their Production |
| CN104059863A (en)* | 2014-05-06 | 2014-09-24 | 江南大学 | Metabolic transformation method for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine |
| CN104357506A (en)* | 2014-10-28 | 2015-02-18 | 上海交通大学 | Method for improving fermentation level of salinomycin by increasing supply of precursors |
| Title |
|---|
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| CN113980982A (en)* | 2021-10-18 | 2022-01-28 | 上海交通大学 | A method for high-yielding ansothrin for enhancing the expression of target protein genes in vivo |
| CN113980982B (en)* | 2021-10-18 | 2024-02-06 | 上海交通大学 | A method for high-yield ansiothricin to enhance the gene expression of ansiothricin target protein in vivo |
| CN116144563A (en)* | 2022-10-24 | 2023-05-23 | 上海交通大学 | Method for enhancing expression of glycoside hydrolase coding gene APASM_6114 transcription level to increase ansamitocin production |
| CN116144563B (en)* | 2022-10-24 | 2025-06-06 | 上海交通大学 | Method for enhancing the transcription level of glycoside hydrolase encoding gene APASM_6114 to increase the production of ansamitocin |
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