WO2019214347A1 - 一种链霉菌生物合成基因簇负调控因子的遗传筛选方法 - Google Patents

一种链霉菌生物合成基因簇负调控因子的遗传筛选方法 Download PDF

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WO2019214347A1
WO2019214347A1 PCT/CN2019/078798 CN2019078798W WO2019214347A1 WO 2019214347 A1 WO2019214347 A1 WO 2019214347A1 CN 2019078798 W CN2019078798 W CN 2019078798W WO 2019214347 A1 WO2019214347 A1 WO 2019214347A1
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streptomyces
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李永泉
毛旭明
罗帅
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Zhejiang University
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    • 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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  • the invention relates to the fields of biochemistry and molecular biology, in particular to a novel genetic screening method for negative regulators of streptavidin biosynthesis gene clusters.
  • transposons are widely used in the genetic modification of eukaryotic cells.
  • the transposon can be randomly or specifically inserted into a position in the genome, thereby affecting the subsequent transcriptional translation process of the positional gene, resulting in loss of function of the target gene in the organism.
  • Streptomyces is an actinomycete and is a Gram-positive bacterium. Streptomyces has a complex life cycle, including morphological differentiation from matrix hyphae, aerial hyphae to spores. At different stages of the Streptomyces life cycle, regulatory proteins in the body regulate these life cycles in a smooth and orderly manner by activating or inhibiting the transcriptional levels of various genes. Most of the antibiotics currently used in the world are produced by secondary metabolism of Streptomyces. Therefore, the excavation research of the secondary metabolite synthesis gene cluster pathway-specific regulatory factors in Streptomyces is extremely important both in basic research and industrial production.
  • the regulation of the target gene cluster regulated by the regulatory protein is carried out by regulating the promoter activity of the gene cluster of interest.
  • the object of the present invention is to provide a genetic screening method for the negative regulatory factors of Streptomyces biosynthesis gene clusters, and to provide a novel method for regulating protein in vivo screening.
  • the present invention constructs a reporter system mediated by a promoter of the gene of interest in a Streptomyces cell, and then randomly mutates the Streptomyces carrying the reporter system using a random mutation system constructed based on the transposon Himar1.
  • the randomly mutated Streptomyces strain is subjected to enhanced screening to obtain a Streptomyces strain having high expression of the target gene.
  • the genome of the Streptomyces strain with high expression of the target gene is packaged by phage packaging and the cosmid with random insert is screened.
  • the position of the random insert insertion in the genome of the Streptomyces strain with high expression of the target gene was determined by DNA sequencing of cosmid. Specific steps are as follows:
  • step (3) integrating the promoter sequence in step (1) into the upstream of the reporter gene in the reporter plasmid system in step (2);
  • the reporter plasmid obtained in the step (3) is introduced into wild-type Streptomyces by conjugation, and verified;
  • step (5) using plasmid pKC1139 as a backbone, the three fragments amplified in step (5) were inserted into plasmid pKC1139, respectively, to obtain plasmid pLRM04;
  • the plasmid pLRM04 obtained in the step (6) is ligated into the Streptomyces containing the reporter plasmid obtained in the step (3), and verified;
  • the Streptomyces strain obtained in the step (8) is cultured, and a certain concentration of hygromycin is added during the culture to activate the expression of the tipAp-Himar1 gene, initiate transposon activity, and ITR-aac (3) After the IV-ITR fragment is randomly inserted into the Streptomyces genome, a large number of random mutant strains are collected;
  • the Streptomyces strain obtained in the step (10) is subjected to liquid culture, and a mass-enhancing genome is extracted. And evenly make a fragment of a certain size;
  • the fragment obtained in the step (11) was all blunt-ended and dephosphorylated using T4 DNA polymerase. After dephosphorylation of the linearized cosmid, the genomic fragments obtained in this step are ligated to each other by blunt-end enzyme digestion;
  • step (14) According to the sequencing result of step (14), using the DNA sequence alignment technique, the alignment of the random insertion fragment ITR-aac(3)IV-ITR in the Streptomyces genome is accurately located in the Streptomyces genome database. And the disrupted gene in the Streptomyces genome;
  • the Streptomyces used in the present invention is Streptomyces which can perform stable genetic manipulation under laboratory conditions.
  • the reporter gene system selected in the step (2) is a reporter gene system which is available for the Streptomyces such as the resistance gene reporting system, the fluorescent protein reporter system, and the substrate color rendering system.
  • the threshold value selected in the step (5) corresponds to the corresponding reporting system
  • the resistance gene reporting system corresponds to the upper limit of the antibiotic concentration
  • the fluorescence protein reporting system corresponds to the fluorescence display intensity
  • the substrate color development reporting system corresponds to the color rendering intensity.
  • the cosmid used in step (12) is a cosmid that can be used for phage packaging.
  • the Escherichia coli selected in the step (13) is Escherichia coli which can be infected by phage.
  • the gene knockout system used in the step (16) is a homologous recombination knockout system, a cosmid knockout system, a cristr/cas9-mediated Streptomyces knockout system, and the like, which can stably knock out the knockout system of the target gene.
  • the target gene promoter is screened for negative regulators in Streptomyces, and the screening environment is stable, and the false positive factors of the negative regulatory factors are low, which can greatly reduce the verification of subsequent regulatory factors, and is efficient, accurate, and convenient to operate. .
  • the target gene promoter regulatory factor is globally screened on the Streptomyces genome, and the screening flux is high, and theoretically, all the negative regulatory factors of the target gene can be screened.
  • the present invention is widely used in Streptomyces, and all of the Streptomyces which can be genetically manipulated can use the present invention to screen for negative regulatory factors of the target gene.
  • the invention can be widely used in the field of high-yield screening and transformation of industrial streptomyces metabolites, and the transformed Streptomyces metabolites have high yield and good genetic stability, and are suitable for industrial production applications.
  • Figure 1 The phaR gene was knocked out using the crispr/cas9 system in Example 1: 1, 2, 3, 4, 5 are PCR amplified fragments of the knockout strain;
  • Figure 2 Results of EMSA experiments of the PhaR protein and the dptEp promoter in Example 1;
  • Figure 3 Results of qRT experiments of the phaR knockout strain ( ⁇ phaR) and the Streptomyces roseosporus L30 (WT) dptE gene in Example 1;
  • Figure 4 Comparison of the yield of the phaR knockout strain ( ⁇ phaR) and the Streptomyces roseophylla L30 (WT) daptomycin shake flask fermentation in Example 1;
  • a negative regulatory protein of the dptE gene in the daptomycin producing strain Streptomyces roseosporus L30 was screened by the method.
  • Streptomyces roseosporus L30 is a strain of Streptomyces that industrially produces daptomycin. Its genomic sequence was also determined and the daptomycin synthesis gene cluster was located. In the entire daptomycin synthesis gene cluster, the datomycin direct synthesis protein is encoded by five genes including dptE, dptF, dptA, dptBC, and dptD.
  • the reporter gene available in the selected Streptomyces roseosporus L30 is the kanamycin resistance gene neo, and the plasmid system in which the reporter gene system is genetically manipulated in the Streptomyces is constructed.
  • the Apra resistance gene was converted into the spectinomycin resistance gene Spec at the SacI digestion micro-point, and the kanamycin resistance gene neo was inserted between the NdeI and NotI restriction sites;
  • step (3) Integrating the promoter sequence dptEp in step (1) into step (2) between the BamHI and BglII upstream of the reporter gene in the reporter plasmid system;
  • the reporter plasmid obtained in the step (4) is introduced into the wild-type Streptomyces roseosporus L30 by conjugation, and verified;
  • step (5) using plasmid pKC1139 as a backbone, the three fragments amplified in step (5) were inserted into plasmid pKC1139, respectively, to obtain plasmid pLRM04;
  • the plasmid pLRM04 obtained in the step (6) is ligated into the Streptomyces roseosporus containing the reporter plasmid obtained in the step (3), and verified;
  • the random mutant strain obtained in the step (9) is screened by the reporter gene threshold obtained in the step (5), and the kanamycin screening concentration is 900 ⁇ g/mL, and the kanamycin resistant strain is selected. ;
  • the high kanamycin resistant strain obtained in the step (10) is subjected to TSB liquid culture, and a high-quality genome is extracted, and uniformly fragmented into about 40 Kb;
  • the fragment obtained in the step (11) was all blunt-ended and dephosphorylated using T4 DNA polymerase.
  • the cosmid pHAQ31 was linearized with restriction endonuclease NheI and then dephosphorylated, and then digested with StuI into two segments to separate the two cos sites.
  • the genomic fragments obtained in this step are ligated to each other using a T4 ligase;
  • step (12) using the phage protein to encapsulate the ligation product obtained in step (12), and infecting E. coli DH10B, and then coating on LB plate with ampicillin and apramycin;
  • step (14) According to the sequencing result of step (14), using the DNA sequence alignment technique, the alignment of the random insertion fragment ITR-aac(3)IV-ITR in the Streptomyces genome is accurately determined in the S. roseosporus SW0702 genome database. The location of the insertion. The gene to be inserted into the mutation is determined to be phaR (SEQ ID No: 3);
  • the phaR knockout strain obtained in the step (16) is fermented with Streptomyces roseosporus L30 (see Table 1 for fermentation conditions);
  • the Streptomyces roseosporus L30 is classified as: Streptomyces roseosporus L30, and the depository is: General Microbiology Center of China Microbial Culture Collection Management Committee, deposit number: CGMCC No.15745, and the deposit date: 2018 On the 9th, the deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

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Abstract

提供了一种链霉菌生物合成基因簇负调控因子的筛选方法，该方法包括：在链霉菌细胞内构建一个以自身目的基因的启动子介导的报告系统，再使用基于转座子Himar1构建的随机突变体系对带报告系统的链霉菌作随机突变，将突变后的链霉菌菌株进行强化筛选，得到目的基因高表达的链霉菌菌株，对目的基因高表达的链霉菌菌株的基因组进行噬菌体包装并筛选出带有随机插入片段的粘粒，对粘粒的DNA测序确定目的基因高表达的链霉菌菌株基因组中随机插入片段插入的位置。

Description

一种链霉菌生物合成基因簇负调控因子的遗传筛选方法 技术领域
本发明涉及生物化学与分子生物学领域，特别涉及一种新的链霉菌生物合成基因簇负调控因子的遗传筛选方法。
背景技术
过去几个世纪，科学家们从自然界中分离筛选出了数以万计的天然产物。然而近几十年来，科学家发现并分离新天然产物的难度与日俱增。自本世纪初开始蓬勃发展的基因组学，为我们发现新天然产物指明了新的方向。许多蛋白质在生物体内具有调控作用，其中有一类调控蛋白可以参与基因的表达的调控，它们可以激活或者抑制特定基因的转录水平。研究人员们企图通过寻找特异性的调控因子，激活并高表达生物体内新的次级代谢基因簇，从而发现并分离出更多有意思的化合物。然而，我们遏待简单、准确、高效的分子生物学手段，来寻找被沉默基因簇的途径特异性调控因子，以进一步激活并高表达生物体内被沉默或抑制表达的新化合物合成基因簇。
作为一个强而有力的基因组修饰工具，转座子广泛地被应用于真核细胞的基因改造中。转座子可以随机或特异性地插入基因组中的某个位置，从而影响该位置基因后续转录翻译进程，造成生物体内目的基因的功能缺失。
链霉菌属于放线菌，是革兰氏阳性菌。链霉菌具有复杂的生命周期，包括从基质菌丝、气生菌丝到孢子的形态分化。在链霉菌生命周期的不同阶段，其体内的调控蛋白通过激活或者抑制各种基因的转录水平，调控这些生命周期平稳有序地进行。世界上现在使用的大部分的抗生素都是由链霉菌次级代谢生产。因此链霉菌体内次级代谢产物合成基因簇途径特异性调控因子的发掘研究不论是从基础科研还是从工业生产上来说，都具有极其重要的意义。
调控蛋白对其调控的目的基因簇的调控作用是通过调节目的基因簇的启动子活性进行的。基于以上基础，我们发明了一种新的链霉菌生物合成基因簇未知负调控因子的体内遗传筛选方法。该方法高效，准确，操作方便，为链霉菌体内目的基因簇调控因子的筛选提供了一种新的研究方式。
发明内容
本发明的目的是针对链霉菌体内基因簇调控蛋白的遗传筛选研究，提供一种链霉菌生物合成基因簇负调控因子的遗传筛选方法，是一种调控蛋白体内筛选的新方法。
本发明在链霉菌细胞内构建一个以自身目的基因的启动子介导的报告系统，然后使用基于转座子Himar1构建的随机突变体系对带有报告系统的链霉菌进行随机突变。接着将随机突变后的链霉菌菌株进行强化筛选，得到目的基因高表达的链霉菌菌株。第四步通过噬菌 体包装的方法，对目的基因高表达的链霉菌菌株的基因组进行包装并筛选出带有随机插入片段的cosmid。最后通过对cosmid的DNA测序确定目的基因高表达的链霉菌菌株基因组中随机插入片段插入的位置。具体步骤如下：
(1)选定链霉菌基因组中需要筛选调控因子的目的基因，扩增出目的基因的上游启动子序列；
(2)选择链霉菌体内可用的报告基因系统，构建该报告基因系统在该链霉菌体内可遗传操作的质粒体系，并确定质粒体系中，报告基因上游无启动子；
(3)将步骤(1)中的启动子序列整合入步骤(2)中报告质粒系统内报告基因上游；
(4)将步骤(3)得到的报告质粒通过接合转导入野生型链霉菌中，并验证；
(5)根据选择的报告基因系统，对步骤(4)得到的链霉菌菌株进行报告基因表达水平的阈值筛选；
(6)扩增三个DNA片段：①潮霉素抗性基因hph；②潮霉素诱导启动子以及转座子tipAp-Himar1；③中间加入阿泊拉霉素抗性基因的随机插入片段ITR-aac(3)IV-ITR；
(7)以质粒pKC1139为骨架，将步骤(5)中扩增出来的三个片段分别插入质粒pKC1139中，得到质粒pLRM04；
(8)将步骤(6)得到的质粒pLRM04，接合转导入步骤(3)得到的含有报告质粒的链霉菌中，并验证；
(9)将步骤(8)得到的链霉菌菌株进行培养，并在培养过程中加入一定浓度的潮霉素，以激活tipAp-Himar1基因的表达，启动转座子活性，将ITR-aac(3)IV-ITR片段随机插入链霉菌基因组后，收集到大量的随机突变菌株；
(10)将步骤(9)得到的随机突变菌株，以步骤(5)得到的报告基因阈值进行筛选，筛选出表型高于步骤(5)阈值的菌株；
(11)将步骤(10)得到的链霉菌菌株进行液体培养后抽提高质量的基因组。并均匀打成一定大小的片段；
(12)使用T4DNA聚合酶将步骤(11)得到的片段全部补成平末端，并去磷酸化。将线性化后的cosmid去磷酸化后，使用平末端酶酶切，将与本步骤得到的基因组片段相互连接；
(13)使用噬菌体蛋白包裹步骤(12)得到的连接产物，并侵染大肠杆菌后，涂布于带有cosmid抗性相应抗生素和阿泊拉霉素的LB平板上；
(14)将步骤(13)中LB平板上生长出来的大肠杆菌单菌落扩大培养，抽提cosmid，并测序；
(15)根据步骤(14)的测序结果，使用DNA序列比对技术，在链霉菌基因组数据库 中比对，精确定位随机插入片段ITR-aac(3)IV-ITR在链霉菌基因组中插入的位置，以及链霉菌基因组中被破坏的基因；
(16)设计基因敲除方案，对步骤(15)定位的基因进行敲除。并验证该基因对目的基因的调控机制。
本发明中所用到的链霉菌是在实验室条件下可以进行稳定遗传操作的链霉菌。
步骤(2)中选取的报告基因系统为抗性基因报告系统，荧光蛋白报告系统，底物显色报告系统等链霉菌可用的报告基因系统。
步骤(5)中所筛选的阈值对应的是相应的报告系统，抗性基因报告系统对应抗生素浓度上限，荧光蛋白报告系统对应荧光显示强度，底物显色报告系统对应显色强度等。
步骤(12)中使用的cosmid是可以用于噬菌体包装的cosmid。
步骤(13)中所选取的大肠杆菌是可以被噬菌体侵染的大肠杆菌。
步骤(16)中所使用的基因敲除体系是同源重组敲除体系，cosmid敲除体系，crispr/cas9介导的链霉菌敲除体系等可以稳定敲除目的基因的敲除体系。
本发明与现有技术相比具有的优势为：
1)本发明将目的基因启动子在链霉菌体内进行负调控因子的筛选，筛选环境稳定，出来的负调控因子假阳性低，可以极大减少后续调控因子的验证工作，高效，准确，操作方便。
2)本发明对目的基因启动子调控因子在链霉菌基因组上进行全局筛选，筛选通量高，理论上可以筛选出所有目的基因的负调控因子。
3)本发明在链霉菌中应用广泛，所有可以进行遗传操作的链霉菌都可以使用本发明进行目的基因的负调控因子筛选。
4)本发明可以广泛用于工业用链霉菌代谢产物高产筛选改造领域，筛选改造出来的链霉菌代谢产物产量高，遗传稳定性好，适用于工业生产应用。
附图说明
图1：实施例1中使用crispr/cas9体系敲除phaR基因：图中1，2，3，4，5为敲除菌株的PCR扩增片段；
图2：实施例1中PhaR蛋白与dptEp启动子的EMSA实验结果；
图3：实施例1中phaR敲除菌株(ΔphaR)和玫瑰孢链霉菌L30(WT)dptE基因qRT实验结果；
图4：实施例1中phaR基因敲除菌株(ΔphaR)与玫瑰孢链霉菌L30(WT)达托霉素摇瓶发酵产量对比；
具体实施方式
下面结合附图和具体实施例来进一步详细描述本发明。
实施例1
以使用本方法筛选达托霉素生产菌种玫瑰孢链霉菌L30中dptE基因的负调控蛋白为例。玫瑰孢链霉菌L30是工业生产达托霉素的链霉菌菌株。其基因组序列也被测定并且定位了达托霉素合成基因簇。整个达托霉素合成基因簇中，达托霉素直接合成蛋白由dptE，dptF,dptA，dptBC，dptD等5个基因编码。这五个基因组成一个顺反子，而dptE基因的启动子dptEp调控整个顺反子的转录翻译步骤。已有的研究表明，高表达dptE，dptF,dptA，dptBC，dptD等5个基因，可以有效地提高达托霉素的工业发酵产量。因此，我们通过本方法筛选玫瑰孢链霉菌L30基因组中启动子dptEp的负调控因子并敲除，以实现该菌株在工业发酵的过程中对达托霉素的高产。具体实施步骤如下：
(1)选定达托霉素生产菌株玫瑰孢链霉菌L30基因组中基因dptE(SEQ ID No:1)，扩增出dptE基因的上游启动子序列dptEp(SEQ ID No:2)；
(2)选定玫瑰孢链霉菌L30体内可用的报告基因为卡那霉素抗性基因neo，构建该报告基因系统在该链霉菌体内可遗传操作的质粒体系。以pIJ8660为骨架质粒，在SacI酶切微点将Apra抗性基因转换为壮观霉素抗性基因Spec，在NdeI和NotI酶切位点之间插入卡那霉素抗性基因neo；
(3)将步骤(1)中的启动子序列dptEp，整合入步骤(2)中报告质粒系统内报告基因上游BamHI和BglII酶切为点之间；
(4)将步骤(4)得到的报告质粒通过接合转导入野生型玫瑰孢链霉菌L30中，并验证；
(5)根据选择的neo卡那霉素抗性基因报告系统，对步骤(4)得到的玫瑰孢链霉菌菌株进行报告基因表达水平的阈值筛选。筛选结果为，在YMG平板上，步骤(4)得到的菌株卡那霉素最高抗性浓度为300μg/mL；
(6)扩增三个DNA片段：①潮霉素抗性基因hph；②潮霉素诱导启动子以及转座子tipAp-Himar1；③中间加入阿泊拉霉素抗性基因的随机插入片段ITR-aac(3)IV-ITR；
(7)以质粒pKC1139为骨架，将步骤(5)中扩增出来的三个片段分别插入质粒pKC1139中，得到质粒pLRM04；
(8)将步骤(6)得到的质粒pLRM04，接合转导入步骤(3)得到的含有报告质粒的玫瑰孢链霉菌中，并验证；
(9)将步骤(8)得到的玫瑰孢链霉菌菌株进行培养，并在培养过程中加入4μg/mL的潮霉素，以激活tipAp-Himar1基因的表达，启动转座子活性。将ITR-aac(3)IV-ITR片段 随机插入链霉菌基因组序列后，收集到大量的随机突变玫瑰孢链霉菌菌株；
(10)将步骤(9)得到的随机突变菌株，以步骤(5)得到的报告基因阈值进行筛选，卡那霉素的筛选浓度为900μg/mL，筛选出高卡那霉素抗性的菌株；
(11)将步骤(10)得到的高卡那霉素抗性菌株进行TSB液体培养后抽提出高质量的基因组，并均匀打成40Kb左右的片段；
(12)使用T4DNA聚合酶将步骤(11)得到的片段全部补成平末端，去磷酸化。将cosmid pHAQ31使用限制性内切酶NheI线性化后去磷酸化，再用StuI酶切成两段，以分开两个cos位点。将本步骤得到的基因组片段使用T4连接酶相互连接；
(13)使用噬菌体蛋白包裹步骤(12)得到的连接产物，并侵染大肠杆菌DH10B后，涂布于带有氨苄青霉素和阿泊拉霉素的LB平板上；
(14)将步骤(13)中LB平板上生长出来的大肠杆菌单菌落液体扩大培养，抽提cosmid，并测序；
(15)根据步骤(14)的测序结果，使用DNA序列比对技术，在玫瑰孢链霉菌SW0702基因组数据库中比对，精确确定随机插入片段ITR-aac(3)IV-ITR在链霉菌基因组中插入的位置。确定被插入突变的基因为phaR(SEQ ID No:3)；
(16)设计crispr/cas9介导的基因敲除方案，对phaR基因进行敲除(图1)；
(17)体外表达纯化PhaR蛋白，使用EMSA实验验证PhaR蛋白与dptEp可以相互结合(图2)；
(18)将phaR基因敲除菌株和玫瑰孢链霉菌L30进行液体培养，再抽提RNA，进行荧光定量PCR分析。结果表明phaR基因敲除菌株中dptE基因的表达量是野生型玫瑰孢链霉菌中dptE基因的表达量的2-3倍(图3)；
(19)将步骤(16)得到的phaR基因敲除菌株与玫瑰孢链霉菌L30进行发酵(发酵条件见表1)；
(20)将phaR基因敲除菌株与玫瑰孢链霉菌L30发酵产物进行HPCL检测(检测结果见图4)。两个菌株的发酵结果表明，phaR基因敲除菌株的达托霉素产量明显上升，进一步证明phaR基因敲除菌株中达托霉素合成基因簇的高表达。
所述玫瑰孢链霉菌L30，分类命名为：玫瑰孢链霉菌(Streptomyces roseosporus)L30，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.15745，保藏日：2018年5月9日，保藏地址：北京市朝阳区北辰西路1号院3号。
以上实验结果证明，PhaR蛋白是dptE基因的负调控因子，其基因敲除菌株中dptE，及其下游基因得到了高表达。从而证明本发明的有效性。
表1：实施例1中玫瑰孢链霉菌的发酵工艺
种子培养基 2％TSB，5％PEG6000液体培养基
种子培养工艺 30ml培养基/250ml容器，30℃，22～26h，250rpm
发酵培养基 0.3％酵母提取物，0.3％麦芽提取物，0.5％胰蛋白胨，4％葡萄糖
发酵培养工艺 30ml/250ml，30℃，144～168h，250rpm
补料工艺 癸酸，发酵开始36h后，每次以体积比1/1000添加，2次/天。

Claims (8)

  1. 一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，在链霉菌细胞内构建一个以自身目的基因的启动子介导的报告系统，然后使用基于转座子Himar1构建的随机突变体系对带有报告系统的链霉菌进行随机突变，接着将随机突变后的链霉菌菌株进行强化筛选，得到目的基因高表达的链霉菌菌株，再通过噬菌体包装的方法，对目的基因高表达的链霉菌菌株的基因组进行包装并筛选出带有随机插入片段的cosmid，最后通过对cosmid的DNA测序确定目的基因高表达的链霉菌菌株基因组中随机插入片段插入的准确位置。
  2. 根据权利要求1所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，具体步骤如下：
    (1)选定链霉菌基因组中需要筛选调控因子的目的基因，扩增出目的基因的上游启动子序列；
    (2)选择链霉菌体内可用的报告基因系统，构建该报告基因系统在该链霉菌体内可遗传操作的质粒体系，并确定质粒体系中，报告基因上游无启动子；
    (3)将步骤(1)中的启动子序列整合入步骤(2)中报告质粒系统内报告基因上游；
    (4)将步骤(3)得到的报告质粒通过接合转导入野生型链霉菌中，并验证；
    (5)根据选择的报告基因系统，对步骤(4)得到的链霉菌菌株进行报告基因表达水平的阈值筛选；
    (6)扩增三个DNA片段：①潮霉素抗性基因hph；②潮霉素诱导启动子以及转座子tipAp-Himar1；③中间加入阿泊拉霉素抗性基因的随机插入片段ITR-aac(3)IV-ITR；
    (7)以质粒pKC1139为骨架，将步骤(5)中扩增出来的三个片段分别插入质粒pKC1139中，得到质粒pLRM04；
    (8)将步骤(6)得到的质粒pLRM04，接合转导入步骤(3)得到的含有报告质粒的链霉菌中，并验证；
    (9)将步骤(8)得到的链霉菌菌株进行培养，并在培养过程中加入一定浓度的潮霉素，以激活tipAp-Himar1基因的表达，启动转座子活性，将ITR-aac(3)IV-ITR片段随机插入链霉菌基因组后，收集到大量的随机突变菌株；
    (10)将步骤(9)得到的随机突变菌株，以步骤(5)得到的报告基因阈值进行筛选，筛选出表型高于步骤(5)阈值的菌株；
    (11)将步骤(10)得到的链霉菌菌株进行液体培养后抽提高质量的基因组。并均匀打成一定大小的片段；
    (12)使用T4DNA聚合酶将步骤(11)得到的片段全部补成平末端，并去磷酸化。将线性化后的cosmid去磷酸化后，使用平末端酶酶切，将与本步骤得到的基因组片段相互连接；
    (13)使用噬菌体蛋白包裹步骤(12)得到的连接产物，并侵染大肠杆菌后，涂布于带有cosmid抗性相应抗生素和阿泊拉霉素的LB平板上；
    (14)将步骤(13)中LB平板上生长出来的大肠杆菌单菌落扩大培养，抽提cosmid，并测序；
    (15)根据步骤(14)的测序结果，使用DNA序列比对技术，在链霉菌基因组数据库中比对，精确定位随机插入片段ITR-aac(3)IV-ITR在链霉菌基因组中插入的位置，以及链霉菌基因组中被破坏的基因；
    (16)设计基因敲除方案，对步骤(15)定位的基因进行敲除，并验证该基因对目的基因的调控机制。
  3. 根据权利要求2所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，所用到的链霉菌是在实验室条件下可以进行稳定遗传操作的链霉菌。
  4. 根据权利要求2所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，步骤(2)中选取的报告基因系统为抗性基因报告系统，荧光蛋白报告系统，底物显色报告系统链霉菌可用的报告基因系统。
  5. 根据权利要求2所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，步骤(5)中所筛选的阈值对应的是相应的报告系统，抗性基因报告系统对应抗生素浓度上限，荧光蛋白报告系统对应荧光显示强度，底物显色报告系统对应显色强度。
  6. 根据权利要求2所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，步骤(12)中使用的cosmid是用于噬菌体包装的cosmid。
  7. 根据权利要求2所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，步骤(13)中所选取的大肠杆菌是被噬菌体侵染的大肠杆菌。
  8. 根据权利要求2所述的一种链霉菌生物合成基因簇负调控因子的筛选方法，其特征在于，步骤(16)中所使用的基因敲除体系是同源重组敲除体系，cosmid敲除体系，crispr/cas9介导的链霉菌敲除体系，可以稳定敲除目的基因的敲除体系。
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