CN116144563A - Method for enhancing expression of glycoside hydrolase coding gene APASM_6114 transcription level to increase ansamitocin production - Google Patents
Method for enhancing expression of glycoside hydrolase coding gene APASM_6114 transcription level to increase ansamitocin productionDownload PDFInfo
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Abstract
Description
Translated fromChinese技术领域Technical Field
本发明属于生物医药技术领域，涉及一种通过增强水解蔗糖的胞外糖苷水解酶编码基因APASM_6114转录水平以提高安丝菌素产量的方法；尤其涉及一种通过增强水解蔗糖的胞外糖苷水解酶编码基因的转录水平、提高菌体蔗糖消耗量、从而提高碳源利用率，最终提高安丝菌素发酵水平的方法。The present invention belongs to the field of biomedicine technology, and relates to a method for increasing the production of ansamitocin by enhancing the transcription level of an extracellular glycoside hydrolase encoding gene APASM_6114 for hydrolyzing sucrose; in particular, it relates to a method for increasing the sucrose consumption of bacterial cells by enhancing the transcription level of an extracellular glycoside hydrolase encoding gene for hydrolyzing sucrose, thereby improving the carbon source utilization rate and ultimately improving the fermentation level of ansamitocin.
背景技术Background Art
安丝菌素(Ansamitocin)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位酯基上连接二硫键形成DM1分子，经DTT还原后可与不同的抗体连接形成抗体-药物偶联物(ADC)。目前，衍生于安丝菌素的多种ADC药物已经进入不同的临床检定阶段，其中由罗氏公司开发的用于治疗人类乳腺癌的Trastuzumab Emtansine(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菌、酵母、昆虫等其它真核生物的生长与繁殖。Ansamitocin is a macrolide antibiotic produced by Actinosynnema pretiosum. It can bind to the β subunit of tubulin, hinder microtubule assembly, and thus inhibit tumor cell division. Chari et al. from ImmunoGen, Inc. formed DM1 molecules by connecting disulfide bonds at the C-3 ester group, which can be connected with different antibodies to form antibody-drug conjugates (ADCs) after DTT reduction. At present, a variety of ADC drugs derived from ansamitocin have entered different clinical testing stages, among which Trastuzumab Emtansine (T-DM1) developed by Roche for the treatment of human breast cancer has been marketed. In addition to its anti-tumor activity, ansamitocin can also inhibit the growth and reproduction of other eukaryotic organisms such as fungi, yeast, and insects.
在安丝菌素的产生菌ATCC 31280的发酵过程中，蔗糖能够作为碳源被菌体有效利用，且一定浓度的蔗糖可为菌体生长提供碳骨架、能为初级代谢及次级代谢途径提供还原力、能量及提升次级代谢产物合成前体物的生成。本发明通过蛋白分级分离与活性追踪相结合的方式，找到了一个水解利用蔗糖的胞外糖苷水解酶及其编码基因：APASM_6114。通过增强水解蔗糖的胞外糖苷水解酶编码基因的转录水平可以提升菌体蔗糖消耗量，从而提高碳源利用率，最终可明显提高安丝菌素产量。In the fermentation process of ansamitocin-producing bacteria ATCC 31280, sucrose can be effectively utilized by the bacteria as a carbon source, and a certain concentration of sucrose can provide a carbon skeleton for bacterial growth, can provide reducing power and energy for primary and secondary metabolic pathways, and enhance the generation of precursors for the synthesis of secondary metabolites. The present invention combines protein fractionation and activity tracking to find an extracellular glycoside hydrolase and its encoding gene: APASM_6114 that hydrolyzes and utilizes sucrose. By enhancing the transcription level of the gene encoding the extracellular glycoside hydrolase that hydrolyzes sucrose, the sucrose consumption of the bacteria can be increased, thereby improving the utilization rate of the carbon source, and ultimately significantly increasing the yield of ansamitocin.
发明内容Summary of the invention
本发明的目的在于提供一种增强表达糖苷水解酶编码基因APASM_6114转录水平以提高安丝菌素产量的方法；具体来说，是一种通过增强水解蔗糖的胞外糖苷水解酶编码基因APASM_6114转录水平以提高安丝菌素发酵水平的方法；通过在珍贵束丝放线菌ATCC31280中增强表达内源性水解蔗糖的胞外糖苷水解酶编码基因APASM_6114，获得高产安丝菌素的突变菌株(ARE-11)，通过提升菌体碳源利用效率，最终提高安丝菌素的产量。The object of the present invention is to provide a method for enhancing the transcription level of glycoside hydrolase encoding gene APASM_6114 to increase the yield of ansamitocin; specifically, a method for improving the fermentation level of ansamitocin by enhancing the transcription level of extracellular glycoside hydrolase encoding gene APASM_6114 that hydrolyzes sucrose; by enhancing the expression of endogenous extracellular glycoside hydrolase encoding gene APASM_6114 that hydrolyzes sucrose in Actinomyces preciousis ATCC31280, a mutant strain (ARE-11) with high ansamitocin production is obtained, and the carbon source utilization efficiency of the bacteria is improved, thereby ultimately increasing the yield of ansamitocin.
为了实现上述目的，本发明采用了以下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
第一方面，本发明涉及安丝菌素高产菌株，在珍贵束丝放线菌中增强表达水解蔗糖的胞外糖苷水解酶编码基因。In a first aspect, the present invention relates to ansamitocin high-producing strain, wherein the expression of an extracellular glycoside hydrolase encoding gene for hydrolyzing sucrose is enhanced in Actinomyces preciousi.
作为本发明的一个实施方案，所述胞外糖苷水解酶编码基因为APASM_6114，其序列如SEQ ID NO.1所示。该编码基因是负责蔗糖水解的珍贵束丝放线菌内源性胞外糖苷水解酶编码基因。As an embodiment of the present invention, the exoglycoside hydrolase encoding gene is APASM_6114, and its sequence is shown in SEQ ID NO. 1. The encoding gene is an endogenous exoglycoside hydrolase encoding gene of Actinomyces preciousis responsible for sucrose hydrolysis.
作为本发明的一个实施方案，所述珍贵束丝放线菌为Actinosynnema pretiosumsubsp.pretiosum ATCC 31280。此时，该安丝菌素高产菌株由珍贵束丝放线菌ATCC 31280中增强表达内源性水解蔗糖的胞外糖苷水解酶编码基因APASM_6114后获得，获得的突变株基于增强表达内源性负责蔗糖水解的糖苷水解酶编码基因APASM_6114，通过促进蔗糖消耗量、提升菌体碳源利用效率，从而提高安丝菌素产量。As an embodiment of the present invention, the Actinosynnema pretiosum subsp. pretiosum ATCC 31280. At this time, the ansamitocin high-yield strain is obtained by enhancing the expression of the endogenous extracellular glycoside hydrolase encoding gene APASM_6114 for hydrolyzing sucrose in Actinosynnema pretiosum
第二方面，本发明涉及一种用于增强表达水解蔗糖的糖苷水解酶编码基因的整合型载体，所述载体包含(源于珍贵束丝放线菌的)胞外糖苷水解酶编码基因APASM_6114，其序列如SEQ ID NO.1。In a second aspect, the present invention relates to an integrative vector for enhancing the expression of a gene encoding a glycoside hydrolase for hydrolyzing sucrose, wherein the vector comprises an extracellular glycoside hydrolase encoding gene APASM_6114 (derived from Actinomyces preciousi), and the sequence thereof is shown in SEQ ID NO.1.
作为本发明的一个实施方案，所述胞外糖苷水解酶编码基因来源于珍贵束丝放线菌ATCC 31280。As an embodiment of the present invention, the gene encoding the extracellular glycoside hydrolase is derived from Actinomyces pretica ATCC 31280.
第三方面，本发明涉及一种用于增强表达水解蔗糖的糖苷水解酶编码基因的整合型载体的构建方法，所述整合型质粒的具体构建方法是，通过PCR扩增的方式从ATCC31280基因组中获取(4281bp的)APASM_6114基因片段，通过酶切连接的方法连入整合型质粒pLQ648中人工强启动子KasOp*下游的NdeI/EcoRI位点。获得整合型载体记为pLQ2013。In the third aspect, the present invention relates to a method for constructing an integrative vector for enhancing the expression of a gene encoding a glycoside hydrolase for hydrolyzing sucrose, wherein the specific construction method of the integrative plasmid is to obtain a (4281 bp) APASM_6114 gene fragment from the ATCC31280 genome by PCR amplification, and connect it to the NdeI/EcoRI site downstream of the artificial strong promoter KasOp* in the integrative plasmid pLQ648 by enzyme cutting and ligation. The obtained integrative vector is recorded as pLQ2013.
作为本发明的一个实施方案，使用引物APASM_6114-F/R，通过PCR扩增得到APASM_6114基因。As one embodiment of the present invention, the APASM_6114 gene was obtained by PCR amplification using primers APASM_6114-F/R.
第四个方面，本发明涉及一株安丝菌素高产菌株，将前述的整合型质粒载体，或前述的方法构建得到的整合型质粒载体接合转移导入珍贵束丝放线菌中进行重组获得所述菌株。In a fourth aspect, the present invention relates to an ansamitocin high-producing strain, which is obtained by introducing the aforementioned integrative plasmid vector, or the integrative plasmid vector constructed by the aforementioned method, into Actinomyces pretica for recombination.
作为本发明的一个实施方案，本发明提供了一种高产安丝菌素的珍贵束丝放线菌突变株ARE-11，将前述的整合型质粒载体，或前述的方法构建得到的整合型质粒载体经接合转移导入受体菌珍贵束丝放线菌ATCC 31280中获得所述突变株。As one embodiment of the present invention, the present invention provides a mutant strain ARE-11 of Actinomyces pretica having high ansamitocin production, wherein the aforementioned integrative plasmid vector or the integrative plasmid vector constructed by the aforementioned method is introduced into a recipient bacterium Actinomyces pretica ATCC 31280 through conjugation transfer to obtain the mutant strain.
第五个方面，本发明涉及一种安丝菌素高产菌株的构建方法，将所述的整合型质粒载体构建方法得到的整合型质粒载体接合转移导入珍贵束丝放线菌中进行重组获得所述基因增强表达突变株。In a fifth aspect, the present invention relates to a method for constructing an ansamitocin high-yield strain, wherein the integrative plasmid vector obtained by the integrative plasmid vector construction method is introduced into Actinomyces pretti by conjugation transfer for recombination to obtain the gene-enhanced expression mutant strain.
作为本发明的一个实施方案，所述构建方法包括如下步骤：As an embodiment of the present invention, the construction method comprises the following steps:
S1，设计并构建用于增强表达胞外糖苷水解酶编码基因APASM_6114的整合型质粒；S1, design and construction of an integrative plasmid for enhanced expression of the exoglycoside hydrolase encoding gene APASM_6114;
S2，通过接合转移将所述整合型质粒导入受体菌株中，然后对突变株进行安普霉素抗性验证，并挑取菌丝体通过PCR产物片段大小的差异，筛选得到基因增强表达突变株。S2, introducing the integrative plasmid into the recipient strain by conjugation transfer, then verifying the mutant strain for apramycin resistance, and picking mycelium to screen for gene-enhanced expression mutant strains by the difference in PCR product fragment size.
作为本发明的一个实施方案，获得的突变株ARE-11基于增强表达内源性水解蔗糖的糖苷水解酶编码基因APASM_6114,促进蔗糖消耗量、提升菌体碳源利用效率，从而提高安丝菌素产量。As an embodiment of the present invention, the obtained mutant strain ARE-11 is based on enhancing the expression of the endogenous sucrose hydrolase encoding gene APASM_6114 that hydrolyzes sucrose, thereby promoting sucrose consumption and improving the carbon source utilization efficiency of the bacteria, thereby increasing the production of ansamitocin.
第六方面，本发明涉及一种提高安丝菌素产量(发酵水平)的方法，在珍贵束丝放线菌ATCC 31280中增强表达水解蔗糖的胞外糖苷水解酶编码基因，获得安丝菌素高产菌株；发酵，获得安丝菌素。In a sixth aspect, the present invention relates to a method for increasing the yield (fermentation level) of ansamitocin, wherein the expression of an extracellular glycoside hydrolase encoding gene for hydrolyzing sucrose is enhanced in Actinomycetes preciousis ATCC 31280 to obtain ansamitocin high-yield strain; and ansamitocin is obtained by fermentation.
作为本发明的一个实施方案，通过增强水解蔗糖的胞外糖苷水解酶编码基因APASM_6114转录水平，获得增强表达突变株，发酵，获得安丝菌素。作为一个具体示例，在珍贵束丝放线菌ATCC 31280中利用人工强启动子KasOp*增强表达内源性水解蔗糖的胞外糖苷水解酶编码基因APASM_6114，促进蔗糖消耗量、提升菌体碳源利用效率，从而提高安丝菌素产量。As one embodiment of the present invention, the transcription level of the gene APASM_6114 encoding the extracellular glycoside hydrolase that hydrolyzes sucrose is enhanced to obtain ansamitocin by obtaining an enhanced expression mutant strain, fermenting, and obtaining ansamitocin. As a specific example, the artificial strong promoter KasOp* is used in Actinomyces preciousis ATCC 31280 to enhance the expression of the endogenous gene APASM_6114 encoding the extracellular glycoside hydrolase that hydrolyzes sucrose, promote sucrose consumption, improve the carbon source utilization efficiency of the bacteria, and thus increase the yield of ansamitocin.
作为本发明的一个实施方案，所述发酵包含下列步骤：将水解蔗糖的胞外糖苷水解酶的编码基因增强表达突变株(ARE-11)在固体培养基上活化，然后将活化后的菌丝体在一级种子培养基中，30℃、220rpm条件下培养24小时；按3.3％-6.6％接种量转接至二级种子培养基中，30℃、220rpm的转速下培养24小时；按10％的接种量转接至发酵培养基中，25℃、220rpm的转速下发酵7天后收集发酵液并进行萃取。As an embodiment of the present invention, the fermentation comprises the following steps: activating an enhanced expression mutant strain (ARE-11) encoding a gene encoding an extracellular glycoside hydrolase that hydrolyzes sucrose on a solid culture medium, and then culturing the activated mycelium in a primary seed culture medium at 30°C and 220rpm for 24 hours; transferring the inoculation amount to a secondary seed culture medium at 3.3%-6.6%, and culturing at 30°C and 220rpm for 24 hours; transferring the inoculation amount to a fermentation culture medium at 10%, and collecting the fermentation broth after fermentation at 25°C and 220rpm for 7 days for extraction.
作为一个具体实施例，将安丝菌素高产菌株在固体培养基上活化，然后将活化后的菌丝体在一级种子培养基中，30℃、220rpm条件下培养24小时；按6.6％接种量转接至二级种子培养基中，30℃、220rpm的转速下培养24小时；按10％的接种量转接至发酵培养基中，25℃、220rpm的转速下发酵7天后收集发酵液并进行萃取。作为具体对比示例，将野生型ATCC 31280与基因增强表达的突变株在固体培养基上活化，然后将活化后的菌丝体在一级种子培养基中，30℃、220rpm条件下培养24小时；按6.6％接种量转接至二级种子培养基中，30℃、220rpm的转速下培养24小时；按10％的接种量转接至发酵培养基中，25℃、220rpm的转速下发酵7天后收集发酵液并进行萃取。As a specific example, the high-yield strain of ansamitocin was activated on a solid culture medium, and then the activated mycelium was cultured in a primary seed culture medium at 30°C and 220rpm for 24 hours; it was transferred to a secondary seed culture medium at a 6.6% inoculation rate, and cultured at 30°C and 220rpm for 24 hours; it was transferred to a fermentation medium at a 10% inoculation rate, and fermented at 25°C and 220rpm for 7 days, and then the fermentation liquid was collected and extracted. As a specific comparative example, the wild-
作为本发明的一个实施方案，所述固体培养基包括0.4w/v％的酵母提取物、1w/v％的麦芽提取物、0.4w/v％的葡萄糖、1.6 -2％的琼脂粉。As an embodiment of the present invention, the solid culture medium comprises 0.4 w/v% yeast extract, 1 w/v% malt extract, 0.4 w/v% glucose, and 1.6-2% agar powder.
作为本发明的一个实施方案，所述一级种子培养基包括TSB 3w/v％、酵母提取物0.5w/v％、蔗糖7.0w/v％。二级种子培养基包括TSB 3w/v％、酵母提取物0.6-0.8w/v％、蔗糖7.0w/v％、异丁醇0.05v/v％、异丙醇0.05v/v％。As one embodiment of the present invention, the primary seed culture medium comprises TSB 3w/v%, yeast extract 0.5w/v%, and sucrose 7.0w/v%. The secondary seed culture medium comprises TSB 3w/v%, yeast extract 0.6-0.8w/v%, sucrose 7.0w/v%, isobutanol 0.05v/v%, and isopropanol 0.05v/v%.
作为本发明的一个实施方案，所述发酵培养基包括酵母提取物3.3-6.6w/v％、麦芽提取物1w/v％、蔗糖7.0w/v％、缬氨酸40mmol/L、异丁醇0.5v/v％、异丙醇1.2v/v％、MgCl2 2mmol/L。As an embodiment of the present invention, the fermentation medium comprises 3.3-6.6 w/v% yeast extract, 1 w/v% malt extract, 7.0 w/v% sucrose, 40 mmol/L valine, 0.5 v/v% isobutanol, 1.2 v/v% isopropanol, and2 mmol/L MgCl 2 .
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1)通过在珍贵束丝放线菌中增强水解蔗糖的胞外糖苷水解酶编码基因APASM_6114，促进蔗糖消耗量、提升菌体碳源利用效率，从而提高安丝菌素产量。1) By enhancing the extracellular glycoside hydrolase encoding gene APASM_6114 that hydrolyzes sucrose in Actinomycetes precious, the sucrose consumption is promoted and the carbon source utilization efficiency of the fungus is improved, thereby increasing the production of ansamitocin.
2)本发明中通过在珍贵束丝放线菌中增强水解蔗糖的胞外糖苷水解酶编码基因APASM_6114，得到高产菌株；本发明所得到的高产菌株ARE-11安丝菌素发酵产量在实验室摇瓶发酵水平下达到63.7mg/L，较对照菌株提高36.9％。2) In the present invention, the extracellular glycoside hydrolase encoding gene APASM_6114 for hydrolyzing sucrose is enhanced in Actinomycetes precious, thereby obtaining a high-yield strain; the ansamitocin fermentation yield of the high-yield strain ARE-11 obtained in the present invention reaches 63.7 mg/L at the laboratory shake flask fermentation level, which is 36.9% higher than that of the control strain.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:
图1为增强表达基因APASM_6114的质粒构建示意图；FIG1 is a schematic diagram of the plasmid construction for enhancing the expression of gene APASM_6114;
图2为水解蔗糖的胞外糖苷水解酶编码基因增强表达突变株ARE-11与对照菌株ATCC 31280::pSET152的安丝菌素发酵产量示意图。FIG. 2 is a schematic diagram of ansamitocin fermentation yields of the mutant strain ARE-11 with enhanced expression of the gene encoding the extracellular glycoside hydrolase that hydrolyzes sucrose and the control strain ATCC 31280::pSET152.
具体实施方式DETAILED DESCRIPTION
下面通过实施例对本发明作进一步说明。本实施例在以本发明技术方案为前提下进行实施，并给出了详细的实施方式和过程，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，按照常规条件或制造厂商的建议条件。The present invention is further described below by way of examples. This example is implemented on the premise of the technical solution of the present invention, and provides detailed implementation methods and processes, but the protection scope of the present invention is not limited to the following examples. The experimental methods in the following examples that do not specify specific conditions are based on conventional conditions or the conditions recommended by the manufacturer.
本发明所涉及的质粒pLQ648已经在SCI数据库文献《Liu,X.；Wu,Y.；Zhang,X.；Kang,Q.；Yan,Y.；Bai,L.Comparative Transcriptome-Based Mining of Genes Involvedin the Export of Polyether Antibiotics for Titer Improvement.Antibiotics2022,11,600.》中记载。The plasmid pLQ648 involved in the present invention has been recorded in the SCI database document "Liu, X.; Wu, Y.; Zhang, X.; Kang, Q.; Yan, Y.; Bai, L. Comparative Transcriptome-Based Mining of Genes Involved in the Export of Polyether Antibiotics for Titer Improvement.
本发明所涉及的菌株珍贵束丝放线菌ATCC 31280已在文献《Wenqin Pan,QianjinKang,Lei Wang,Linquan Bai*&Zixin Deng:Asm8,a specific LAL-type activator of3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production.Science ChinaLife Sciences 2013(7):601-608》中记载。The strain Actinomyces preciousi ATCC 31280 involved in the present invention has been recorded in the document "Wenqin Pan, Qianjin Kang, Lei Wang, Linquan Bai* & Zixin Deng: Asm8, a specific LAL-type activator of 3-amino-5-hydroxybenzoate biosynthesis in ansamitocin production. Science China Life Sciences 2013(7):601-608".
实施例Example
本实施例为获取负责蔗糖水解的胞外糖苷水解酶编码基因APASM_6114增强表达突变株ARE-11的具体过程。具体操作步骤如下：This example is a specific process for obtaining the mutant strain ARE-11 with enhanced expression of the gene APASM_6114 encoding the extracellular glycoside hydrolase responsible for sucrose hydrolysis. The specific steps are as follows:
步骤一：质粒pLQ2013的构建Step 1: Construction of plasmid pLQ2013
以珍贵束丝放线菌ATCC 31280基因组DNA(GenBank assembly accession:CP029607.1)作为模板，使用在两端引入NdeI/EcoRI酶切位点的引物APASM_6114-F/R，通过PCR扩增得到APASM_6114基因片段(4281bp)。在质粒pLQ-648中强启动子kasOp*下游的NdeI/EcoRI位点插入酶切后的扩增片段(NdeI/EcoRI)，得到质粒pLQ2013，如图1所示。Using the genomic DNA of Actinomycetes precious ATCC 31280 (GenBank assembly accession: CP029607.1) as a template, the primers APASM_6114-F/R with NdeI/EcoRI restriction sites introduced at both ends were used to amplify the APASM_6114 gene fragment (4281 bp) by PCR. The amplified fragment (NdeI/EcoRI) after restriction digestion was inserted into the NdeI/EcoRI site downstream of the strong promoter kasOp* in the plasmid pLQ-648 to obtain the plasmid pLQ2013, as shown in Figure 1.
步骤二：将第一步构建完成的基因增强表达的质粒pLQ2013通过接合转移导入受体菌ATCC 31280中，通过PCR和抗性验证的方法验证正确的基因增强表达接合子。具体包括以下步骤：Step 2: The gene-enhanced expression plasmid pLQ2013 constructed in the first step is introduced into the
将已构建完成的基因增强表达的质粒pLQ2013转化进入宿主E.coli ET12567(含有pUZ8002质粒)中。取E.coli ET12567(pUZ8002)于含有30μg/mL安普霉素、50μg/mL卡那霉素和25μg/mL氯霉素的LB中37℃过夜培养，用相同的培养基，将过夜培养物按1％的比例转接一次并培养4-5小时至OD600达到0.6-0.8，然后用新鲜的LB溶液漂洗菌体以除去培养物中的抗生素。与此同时制备ATCC 31280新鲜菌丝体(约16小时培养物)，用LB溶液漂洗2～3次之后，将其与之前制备的宿主菌ET12567(pUZ8002)混合(受体菌细胞和供体菌的比例约为1：10)均匀后涂布于含有10mM镁离子的固体培养基上，于37℃培养箱倒置培养。16小时后取出平板，分别将安普霉素(终浓度100μg/mL)和萘啶酮酸(终浓度100μg/mL)两种抗生素加入1.5mL无菌水中混匀后覆盖在YMG固体培养基上，将固体培养基晾干后转移至30℃培养箱中倒置培养。一般3～5天后可见平板上有接合子长出，将其通过转接于含有安普霉素(终浓度100μg/mL)和萘啶酮酸(终浓度100μg/mL)两种抗生素的固体培养基上扩大培养，挑取菌丝体，采用kasOp-F和APASM_6114-ver-R为引物，通过PCR和抗性验证的方法验证接合子，获得正确的基因增强表达突变株ARE-11。The constructed gene-enhanced expression plasmid pLQ2013 was transformed into the host E. coli ET12567 (containing the pUZ8002 plasmid). E. coli ET12567 (pUZ8002) was cultured overnight at 37°C in LB containing 30 μg/mL apramycin, 50 μg/mL kanamycin and 25 μg/mL chloramphenicol. The overnight culture was transferred once at a ratio of 1% using the same culture medium and cultured for 4-5 hours until the OD600 reached 0.6-0.8, and then the cells were rinsed with fresh LB solution to remove the antibiotics in the culture. At the same time, prepare fresh mycelium of ATCC 31280 (about 16 hours of culture), rinse it with LB solution 2-3 times, mix it with the previously prepared host bacteria ET12567 (pUZ8002) (the ratio of recipient bacteria cells to donor bacteria is about 1:10), and evenly spread it on a solid culture medium containing 10mM magnesium ions, and invert it in a 37°C incubator. After 16 hours, take out the plate, add two antibiotics, apramycin (final concentration 100μg/mL) and nalidixic acid (final concentration 100μg/mL) to 1.5mL of sterile water, mix well, and cover it on the YMG solid culture medium. After the solid culture medium is dried, transfer it to a 30°C incubator for inverted culture. Generally, after 3 to 5 days, conjugates can be seen growing on the plate. They are expanded by transferring to a solid culture medium containing two antibiotics, apramycin (final concentration 100 μg/mL) and nalidixic acid (final concentration 100 μg/mL). Mycelium is picked, and kasOp-F and APASM_6114-ver-R are used as primers. The conjugates are verified by PCR and resistance verification methods to obtain the correct gene-enhanced expression mutant strain ARE-11.
上述步骤一中所涉及的内切酶识别位点(酶切位点)如下表1：The endonuclease recognition sites (enzyme cutting sites) involved in the above step 1 are shown in Table 1 below:
表1Table 1
上述步骤一、二中所用到的引物序列如表2所示：The primer sequences used in the above steps 1 and 2 are shown in Table 2:
表2Table 2
上述步骤一、二中基因片段制备所采用的PCR体系及条件：The PCR system and conditions used for the preparation of gene fragments in steps 1 and 2 above are:
PCR反应体系：DNA模板30ng，引物30pmol，50％ DMSO 3μL，25mM Mg2+2μL，缓冲液3μL，KOD聚合酶1个单位，加纯水补齐至30μL；PCR reaction system: 30 ng DNA template, 30 pmol primers, 3 μL 50% DMSO, 2 μL 25 mM Mg2+ , 3 μL buffer, 1 unit KOD polymerase, add pure water to make up to 30 μL;
PCR条件：95℃5min；95℃30s，64℃30s，72℃4min 20s；循环30次；72℃5min。PCR conditions: 95°C for 5 min; 95°C for 30 s, 64°C for 30 s, 72°C for 4 min and 20 s; 30 cycles; 72°C for 5 min.
引物序列为：The primer sequences are:
APASM_6114-F:CCCATATGATGGGGCACATGCGGGGCAGGCGGAG SEQ ID NO.2APASM_6114-F:CCCATATGATGGGGCACATGCGGGGCAGGCGGAG SEQ ID NO.2
APASM_6114-R:CGGAATTCCTACAACCCCGTGCACTGCCCCGAC SEQ ID NO.3上述步骤二中通过PCR验证筛选突变株时所采用的PCR体系及条件：APASM_6114-R: CGGAATTCCTACAACCCCGTGCACTGCCCCGAC SEQ ID NO.3 The PCR system and conditions used in the PCR verification and screening of mutants in the above step 2:
PCR反应体系：DNA模板10～100ng，引物10pmol，50％DMSO 2μL，2×Mix缓冲液10μL，加纯水补齐至20μL；PCR reaction system: DNA template 10-100 ng, primer 10 pmol, 50% DMSO 2 μL, 2×Mix buffer 10 μL, add pure water to make up to 20 μL;
PCR条件：95℃10min；95℃15s；64℃15s；72℃1min；循环30次；72℃5min。PCR conditions: 95°C for 10 min; 95°C for 15 s; 64°C for 15 s; 72°C for 1 min; 30 cycles; 72°C for 5 min.
引物序列为：The primer sequences are:
kasOp-F:GACAACATGCTGTGCGGTGTTGT SEQ ID NO.4kasOp-F:GACAACATGCTGTGCGGTGTTGT SEQ ID NO.4
APASM_6114-ver-R：GGTAGGTGCCGTCGGAGAACGC SEQ ID NO.5APASM_6114-ver-R：GGTAGGTGCCGTCGGAGAACGC SEQ ID NO.5
步骤三，利用HPLC检测安丝菌素的发酵产量Step 3: Detect the fermentation yield of ansamitocin using HPLC
采用安捷伦公司的Agilent 1200系列HPLC进行色谱分析，并采用DAD紫外吸收检测器测定254nm下的色谱吸收峰。Agilent 1200 series HPLC was used for chromatographic analysis, and a DAD ultraviolet absorption detector was used to measure the chromatographic absorption peak at 254 nm.
其中，HPLC参数如下：Among them, the HPLC parameters are as follows:
色谱柱：Agilent ZORBAX SB-C18,2.1×150mm,3.5μm；Chromatographic column: Agilent ZORBAX SB-C18, 2.1×150mm, 3.5μm;
流动相流速：1mL/min；Mobile phase flow rate: 1 mL/min;
流动相：纯乙腈溶液和1‰HPLC级甲醇梯度洗脱。Mobile phase: pure acetonitrile solution and 1‰ HPLC grade methanol gradient elution.
柱温：室温。Column temperature: room temperature.
图2为增强表达水解蔗糖的胞外糖苷水解酶编码基因APASM_6114的突变株ARE-11的安丝菌素发酵水平检测结果。结果表明增强表达以上基因之后，安丝菌素发酵水平有明显的提高，和出发菌株相比，本发明所得到的高产菌株ARE-11安丝菌素产量在实验室摇瓶发酵水平下达到63.7mg/L，较对照菌株提高36.9％。Figure 2 shows the results of the fermentation level test of ansamitocin of the mutant strain ARE-11 with enhanced expression of the gene APASM_6114 encoding the extracellular glycoside hydrolase that hydrolyzes sucrose. The results show that after the enhanced expression of the above genes, the fermentation level of ansamitocin is significantly improved. Compared with the starting strain, the ansamitocin yield of the high-yield strain ARE-11 obtained in the present invention reaches 63.7 mg/L at the laboratory shake flask fermentation level, which is 36.9% higher than that of the control strain.
综上所述，本发明通过在珍贵束丝放线菌ATCC 31280中利用强启动子kasOp*增强表达珍贵束丝放线菌内源性糖苷水解酶编码基因APASM_6114，得到高产安丝菌素的突变株ARE-11，促进珍贵束丝放线菌的蔗糖消耗、提高碳源利用率，最终可明显提高安丝菌素产量。通过本发明可显著提高安丝菌素的发酵产量，同时实现发酵成本的降低。In summary, the present invention uses the strong promoter kasOp* to enhance the expression of the endogenous glycoside hydrolase encoding gene APASM_6114 of
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005020883A2 (en)* | 2003-05-08 | 2005-03-10 | Immunogen, Inc. | Methods for the production of ansamitocins |
| CN107881137A (en)* | 2017-09-25 | 2018-04-06 | 辽宁斯韦尔生物科技有限公司 | Strengthen high yield ansamitocin bacterial strain of transcriptional level and preparation method thereof |
| 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 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005020883A2 (en)* | 2003-05-08 | 2005-03-10 | Immunogen, Inc. | Methods for the production of ansamitocins |
| CN107881137A (en)* | 2017-09-25 | 2018-04-06 | 辽宁斯韦尔生物科技有限公司 | Strengthen high yield ansamitocin bacterial strain of transcriptional level and preparation method thereof |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 李婷兰;韦柳静;范宇翔;马玉栋;白林泉;花强;: "奇迹束丝放线菌合成安丝菌素过程中支链氨基酸的添加效果分析", 华东理工大学学报(自然科学版), no. 06, 30 December 2013 (2013-12-30), pages 675 - 680* |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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