WO2019192281A1 - 基于转录组学链霉菌次级代谢强启动子挖掘方法及应用 - Google Patents

基于转录组学链霉菌次级代谢强启动子挖掘方法及应用 Download PDF

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WO2019192281A1
WO2019192281A1 PCT/CN2019/077130 CN2019077130W WO2019192281A1 WO 2019192281 A1 WO2019192281 A1 WO 2019192281A1 CN 2019077130 W CN2019077130 W CN 2019077130W WO 2019192281 A1 WO2019192281 A1 WO 2019192281A1
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promoter
streptomyces
gene
secondary metabolism
metabolism
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毛旭明
李永泉
王凯
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浙江大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1051—Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
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  • the invention belongs to the field of biochemistry and molecular biology, and relates to a method and application for excavating a strong promoter based on transcriptomics Streptomyces secondary metabolism.
  • Streptomyces is a Gram-positive bacterium that has great industrial value due to its ability to produce abundant biologically active antibiotics, anticancer drugs and other natural products in secondary metabolism.
  • various metabolic engineering and synthetic biology tools for the precise control of biosynthetic gene clusters and related regulatory networks are needed.
  • Some efficient constitutive promoters have been well characterized, such as ermE*p, kasO*p and SF14p.
  • some inducible promoters have been used in various studies.
  • secondary metabolites are produced by biosynthesis of precursors such as acetyl-CoA and amino acids. These precursors are mainly derived from primary metabolism and are essential constituent units of cell structure and biologically active molecules during cell growth. Premature initiation of biosynthesis of natural products under the control of a constitutive promoter will inevitably consume these primary metabolites. At the same time, some secondary metabolites such as vancomycin with specific biological activity often have physiological effects of inhibiting the growth of bacterial cells. All of these will have a toxic effect on the growth and reproduction of cells, especially in the process of industrial expansion. These negative effects may eventually offset the positive effects of efficient constitutive promoters.
  • inducible promoters also have some negative effects on the physiology of bacteria due to the use of chemical inducers.
  • the inducer is usually too expensive.
  • the use of inducers requires careful control and avoidance of contamination.
  • a strong promoter that is consistent with the expression pattern of the gene cluster will be more suitable for overexpression of the gene cluster.
  • Streptomyces lignin L10 can produce macrolide antibiotics, natamycin.
  • the biosynthesis of natamycin requires a large amount of precursor substances such as acetyl-CoA and trehalose, some of which are toxic to the cells.
  • a promoter was screened with a relative expression level (36 hours/12 hour value) greater than 50 and verified to be active in secondary metabolism.
  • ermE*p is 7 times higher.
  • the high expression of natamycin positive regulators using this promoter showed a 30% higher natamycin production than ermE*p. This is a new approach to screening promoter elements that are automatically induced in secondary metabolism. The promoters screened by this method will be useful for the discovery and high yield transformation of natural products in Streptomyces.
  • the object of the present invention is to provide a transcriptome-based Streptomyces secondary metabolism strong promoter excavation method, aiming at excavating a strong promoter for the secondary metabolism of Streptomyces, and providing a transcriptomics-based method.
  • This is a new technique based on the Streptomyces transcriptome gene chip data to quickly analyze and efficiently track strong promoters of Streptomyces secondary metabolism.
  • the method of the invention is specifically implemented by the following steps:
  • the genes with relatively improved expression levels in the secondary metabolism of Streptomyces are initially screened; (2) according to the genes identified in step (1), the gene spacers are too short or Several genes are obtained after the gene of the gene spacer, and the promoter region of these genes is taken as an alternative promoter;
  • step (3) Amplifying the alternative promoter in step (2), and inserting the clone into the pIJ8660 vector, so that the candidate promoter is located upstream of the egfp gene, and after sequencing the correctness of the sequence, several sequences are sequentially obtained.
  • the promoter and the pIJ8660 recombinant plasmid of the egfp gene were selected, and the ermE*p promoter was amplified, and the same site was inserted into the pIJ8660 vector to obtain the pIJ8660 recombinant plasmid carrying the ermE*p promoter and the egfp gene in turn;
  • step (3) (4) combining all the recombinant plasmids in step (3) into Streptomyces, respectively, to obtain a plurality of mutant strains;
  • step (5) taking 24, 48, 72 and 96 hours of the fermentation broth of each mutant in step (5), collecting the cells by centrifugation, and ultrasonically breaking the cells to obtain total bacterial proteins;
  • step (6) Take step (6) a moderate amount of each sample for total protein Western blot analysis, detect the expression level of eGFP at 24, 48, 72 and 96 hours while using the same amount of each sample for protein gel electrophoresis after Coomassie bright Blue staining, to determine the total protein loading of each sample is consistent;
  • the method of the present invention is based on the Streptomyces transcriptome gene chip data to analyze a promoter having a significantly higher activity relative to primary metabolism in secondary metabolism of Streptomyces.
  • the method of the present invention inserts an alternative promoter into the upstream of the egfp gene, and uses the expression level of the eGFP protein to characterize the candidate promoter activity, thereby facilitating subsequent detection.
  • the promoter selected by the method of the present invention is a high-efficiency regulatory strong promoter of Streptomyces secondary metabolism confirmed by transcriptome gene chip screening and reporter gene egfp screening, which is characterized by low activity in primary metabolism and secondary metabolism.
  • the medium-high activity ensures that when the promoter is used, the primary metabolism of Streptomyces is unaffected and the secondary metabolites are efficiently produced in secondary metabolism.
  • the present invention has the distinct advantages of: 1) The present invention enables rapid screening of a strong promoter of Streptomyces secondary metabolism with a general procedure for efficient regulation. 2) The present invention provides a methodological tool for finding strong promoter elements for the biosynthesis of various natural products of Streptomyces and activation of recessive gene clusters. 3) The present invention is widely used in the biosynthesis of natural products, and all hosts capable of genetic manipulation can use the present invention for promoter excavation.
  • Figure 1 shows partial data of the Streptomyces chinensis L10 gene chip, and the relative expression intensity of 15 candidate genes increased significantly in secondary metabolism (36 hours / 12 hours value is greater than 50).
  • Figure 2 is a Western blot experiment to determine the expression level of the reporter gene egfp under the control of th1M4p in Streptomyces garcinia L10.
  • the expression level of egfp on day 3 was controlled by ermE*p as a control.
  • the expression levels of eGFP on days 1, 2, 3 and 4 were detected while using the same amount of protein for protein gel electrophoresis and Coomassie blue staining to determine the consistent sample loading.
  • Figure 3 is a graph showing the yield of natamycin of Streptomyces elegans L10 after high expression of scnRII by thlM4p and ermE*p.
  • Figure 4 is a dry weight curve of Streptomyces elegans L10 after high expression of scnRII with thlM4p and ermE*p.
  • step 2) According to the 57 genes identified in step 1), 15 candidate genes were obtained after excluding genes with too short or no intergenic spacers, and the promoter regions of these genes were used as alternative promoters. (See Table 1 and Figure 1)
  • step 2 3) by primer pair 1+2; 3+4; 5+6; 7+8; 9+10; 11+12; 13+14; 15+16; 17+18; 19+20; 21+22; +24;25+26;27+28;29+30; (see Table 2) Amplify the 15 alternative promoters in step 2), clone the BamHI/NdeI site inserted into the pIJ8660 vector, making 15 preparations
  • the selected promoters were located upstream of the egfp gene, and after sequencing the correctness of the sequence, 15 recombinant plasmids pIJ8660, which were followed by a candidate promoter and the egfp gene, were named pIJ8660-P1 to P15.
  • the ermE*p promoter was amplified from the primer pair 31+32 (see Table 2), and the BamHI/NdeI site of the pIJ8660 vector was cloned, and the pIJ8660 recombinant plasmid carrying the ermE*p promoter and the egfp gene was obtained and named.
  • pIJ8660-P16 For pIJ8660-P16.
  • step 3 The 16 recombinant plasmids in step 3) were separately ligated into Streptomyces garcinia L10, and 16 mutant strains were obtained.
  • step 6) Take 20 ⁇ g of each sample in step 6) for Western blot analysis of total bacterial proteins, and detect the expression levels of eGFP at 24, 48, 72, and 96 hours, and use the same amount of each sample for protein gel electrophoresis after Coomassie blue staining. The total protein loading of each sample was determined to be consistent.
  • pIJ8660-thlM4p-scnRII Named pIJ8660-thlM4p-scnRII.
  • the ermE*p promoter was amplified by primer pair 37+38 (see Table 3), and the scnRII gene was amplified by primer pair 39+40 (see Table 3), and the ermE*p promoter and scnRII gene were seamless.
  • the cloning method was inserted into the BamHI/NotI site of the pIJ8660 vector to obtain the pIJ8660 recombinant plasmid carrying the ermE*p promoter and the scnRII gene in sequence, and named pIJ8660-ermE*p-scnRII.
  • step 1) The two recombinant plasmids in step 1) were separately ligated into Streptomyces garcinia L10, and the corresponding two strains with high expression of scnRII were obtained.
  • step 3 Take the fermentation broth of each mutant in 24, 48, 72, 96 and 120 hours in step 3), and measure the yield of natamycin by HPLC after methanol extraction.
  • the yield of natamycin of the St. chinensis L10 mutant strain with high expression of scnRII by thlM4p was 30% higher than that of ercnE*p scnRII, and the dry weight of the strain was not found. obvious change. (See Figure 3 and Figure 4).
  • the thlM4p sequence is the same as in Example 1.

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Abstract

一种基于转录组学链霉菌次级代谢强启动子挖掘方法及应用，是通过比较分析链霉菌转录组基因芯片数据，筛选次级代谢中表达水平相对提高明显的基因，并扩增这些基因的启动子区域，作备选启动子。以强启动子ermE*p作参照，利用egfp报告基因表征备选启动子活性，确证启动子在次级代谢中相对高活性。此外，与ermE*p相比，应用筛选得到的启动子高表达恰塔努加链霉菌纳他霉素途径特异性正调控因子，使纳他霉素产量提高30％。该方法可应用于链霉菌次级代谢基因表达调控。

Description

基于转录组学链霉菌次级代谢强启动子挖掘方法及应用 技术领域
本发明属于生物化学与分子生物学领域，涉及基于转录组学链霉菌次级代谢强启动子挖掘方法及应用。
背景技术
链霉菌是一种革兰氏阳性菌，由于其在次级代谢过程中能产生丰富的具有重要生物活性的抗生素，抗癌药物等天然产物而具有巨大工业价值。为了大量地生产重要的天然产物，需要各种用于生物合成基因簇和相关调控网络精确控制的代谢工程和合成生物学工具。一些高效的组成型启动子已经被很好地表征，例如ermE*p,kasO*p以及SF14p。另外，还有一些诱导型启动子被用于各类研究。
众所周知，次级代谢产物是由乙酰辅酶A，氨基酸等前体生物合成产生的。这些前体主要来自初级代谢，并且在菌体生长过程中是细胞结构和生物活性分子的必要组成单位。由组成型启动子控制下的天然产物生物合成过早开始将不可避免地消耗这些初级代谢产物。同时一些有着特定生物活性的万古霉素等次级代谢产物，经常有抑制菌体细胞生长的生理学效应。上述这些都会对细胞的生长繁殖有毒害作用，尤其是在工业化扩大生产的过程中。这些负面作用可能最终将会抵消高效的组成型启动子的积极作用。
另一方面，由于化学诱导剂的使用，诱导性启动子在菌体生理上也有一些负面的作用。同时在工业生产中，诱导剂通常过于昂贵。此外，为了最佳的诱导效果，诱导剂的使用需要精心控制并避免污染。
所以，与基因簇表达模式相符合的强启动子将会更适用于基因簇的过表达。为了这一目标，就要设法寻找在初级代谢阶段沉默，而在细胞进入次级代谢后高效表达的启动子。在这样的情况下，由于启动子的活性依赖于生理开关，所以不需要诱导剂。
工业生产菌，恰塔努加链霉菌L10能产生大环内酯类抗生素，纳他霉素。纳他霉素的生物合成需要大量的乙酰辅酶A和海藻糖等前体物质，其中一些代谢产物对于菌体有毒害作用。基于恰塔努加链霉菌L10的转录组基因芯片数据，经筛选得到了一个启动子，其相对表达水平(36小时/12小时值)大于50，并且经验证，在次级代谢中其活性比ermE*p高7倍。除此之外，使用这个启动子高表达纳他霉素正调控因子显示出比ermE*p高30％的纳他霉素产量。这是一种筛选在次级代谢中自动诱导的启动子元件的新方法。这种方法筛选的启动子将可以用于链霉菌中天然产物的发现与高产改造。
基于以上考虑，我们发明了基于转录组学链霉菌次级代谢强启动子挖掘方法。该方法高效，准确，操作方便，为寻找内源的链霉菌次级代谢高效调控强启动子提供了一种新的方法。
发明内容
本发明的目的是提供基于转录组学链霉菌次级代谢强启动子挖掘方法，针对的是为链霉菌次级代谢高效调控强启动子的挖掘，提供一种基于转录组学的方法。这是一种基于链霉菌转录组基因芯片数据，快速分析挖掘链霉菌次级代谢高效调控强启动子的新技术。本发明方法具体用以下步骤实现：
(1)根据链霉菌转录组基因芯片数据，初步筛选出链霉菌次级代谢中表达水平相对提高明显的基因；(2)根据步骤(1)中确定的基因，剔除其中基因间隔区过短或者没有基因间隔区的基因后得到若干个基因，取这些基因的启动子区域作为备选启动子；
(3)扩增出步骤(2)中的备选启动子，克隆插入pIJ8660载体中，使得备选启动子位于egfp基因的上游，测序检验序列正确性后，即得到若干分别依次带有一段备选启动子和egfp基因的pIJ8660重组质粒，同时扩增出ermE*p启动子，克隆插入pIJ8660载体的相同位点，得到依次带有ermE*p启动子和egfp基因的pIJ8660重组质粒；
(4)将步骤(3)中的所有重组质粒分别接合转导进入链霉菌中，得到突变株若干；
(5)将步骤(4)中的突变株进行发酵；
(6)取步骤(5)中24、48、72以及96小时的各突变株发酵液，离心收取菌体，超声破碎细胞获得菌体总蛋白；
(7)取步骤(6)中等量的各样品进行菌体总蛋白Western blot实验，检测eGFP在24、48、72以及96小时的表达水平同时利用等量的各样品进行蛋白胶电泳后考马斯亮蓝染色，确定各样品总蛋白上样量一致；
(8)根据步骤(7)中Western blot实验相对定量检测eGFP的结果，进一步确认在链霉菌中次级代谢高效调控强启动子，其特点为在初级代谢中低活性并且在次级代谢中高活性。
本发明的目的是提供所述方法在链霉菌次级代谢基因表达调控中的应用。
1.本发明方法是根据链霉菌转录组基因芯片数据分析出在链霉菌次级代谢中相对于初级代谢具有明显较高活性的启动子。
2.本发明方法是将备选启动子插入egfp基因上游，利用eGFP蛋白的表达水平来表征备选启动子活性，从而便于后续的检测。
3.本发明方法筛选得到的启动子是经转录组基因芯片筛选和报告基因egfp筛选后确认的链霉菌次级代谢高效调控强启动子，其特征是在初级代谢中低活性并且在次级代谢中高活性，从而能够保证使用该启动子时，链霉菌初级代谢中不受影响而在次级代谢中保证相关次级代谢产物高效产出。
本发明具有的明显优点：1)本发明能够利用通用步骤快速筛选出链霉菌次级代谢高效调 控强启动子。2)本发明为链霉菌各种天然产物的生物合成以及隐性基因簇的激活提供了寻找强启动子元件的方法工具。3)本发明在天然产物的生物合成中应用广泛，所有可以进行遗传操作的宿主都可以使用本发明进行启动子挖掘。
附图说明
图1为恰塔努加链霉菌L10基因芯片部分数据，15个备选基因的在次级代谢中相对表达强度提高明显(36小时/12小时值大于50)。
图2为测定恰塔努加链霉菌L10中，thlM4p调控下的报告基因egfp的表达水平的Westernblot实验结果，实验以ermE*p调控下egfp在第3天时的表达水平为对照。检测eGFP在第1、2、3以及4天的表达水平同时利用等量的蛋白进行蛋白胶电泳后考马斯亮蓝染色，确定各样品上样量一致。
图3为以thlM4p和ermE*p高表达scnRII后，恰塔努加链霉菌L10的纳他霉素的产量曲线。
图4为以thlM4p和ermE*p高表达scnRII后，恰塔努加链霉菌L10的干重曲线。
具体实施方式
下面结合附图和具体实施例作进一步详细描述。
实施例1
以使用本方法挖掘恰塔努加链霉菌L10次级代谢高效调控强启动子为例，详细描述本发明。具体实施步骤如下：
1)根据恰塔努加链霉菌L10转录组基因芯片数据，初步筛选出恰塔努加链霉菌L10次级代谢中表达水平相对提高明显(36小时/12小时值大于50)的基因，得到57个基因。所述恰塔努加链霉菌L10，分类命名为：Streptomyces chattanoogensis L10，保藏于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 2644，保藏日：2008年8月27日，保藏单位地址：北京市朝阳区北辰西路1号院中国科学院微生物研究所，邮编：100101；
2)根据步骤1)中确定的57个基因，剔除其中基因间隔区过短或者没有基因间隔区的基因后得到15个备选基因，取这些基因的启动子区域作为备选启动子。(见表1与附图1)
表1 15个基因间隔区大小合适的备选基因的相对表达水平
  16h/12h 24h/12h 36h/12h
ORF 0749 2.46 129 71.3
ORF 2643 1.03 287 330
ORF 2644 0.92 319 425
ORF 2645 0.45 249 409
ORF 2889 0.62 30.1 147
ORF 4226 2.55 47.7 104
ORF 5195 3.25 175 234
ORF 5885 2.01 31.3 74.9
ORF 6166 4.27 16 179
ORF 6378 57.9 228 430
ORF 6409 25 195 57.8
ORF 6490 4.38 71 188
ORF 7592 23.3 277 201
ORF 7689 1.92 33.5 121
ORF 7906 1.83 17.2 62.6
3)由引物对1+2；3+4；5+6；7+8；9+10；11+12；13+14；15+16；17+18；19+20；21+22；23+24；25+26；27+28；29+30；(见表2)扩增出步骤2)中的15个备选启动子，克隆插入pIJ8660载体的BamHI/NdeI位点，使得15个备选启动子分别位于egfp基因的上游，测序检验序列正确性后，即得到15个分别依次带有一段备选启动子和egfp基因的pIJ8660重组质粒，命名为pIJ8660-P1～P15。同时由引物对31+32(见表2)扩增出ermE*p启动子，克隆插入pIJ8660载体的BamHI/NdeI位点，得到依次带有ermE*p启动子和egfp基因的pIJ8660重组质粒，命名为pIJ8660-P16。
表2构建报告质粒所用引物
引物 序列
1 cgagatctgatatcgaggggttcggtacgcagcc
2 ttgctcaccatggccattgatgctcccctcatg
3 cgagatctgatatcgccgtcgcccagggtgacctc
4 ttgctcaccatggccatgcttctcctgtcgggtgaag
5 cgagatctgatatcgcggggggtgcggcatcagtg
6 ttgctcaccatggccatggaattcctcgggtgtcttcg
7 cgagatctgatatcgcggttctggtggacatcggtg
8 ttgctcaccatggccatgtgggacctcccaaaaggtg
9 cgagatctgatatcgacgcctggacctatgagccgtc
10 ttgctcaccatggccattgctcctccggcagagtcgtac
11 cgagatctgatatcggccgcctcctcgtccttcgc
12 ttgctcaccatggccatccatgcctccccgcgtggatgaatg
13 cgagatctgatatcgcggtgatcagcgggctgg
14 ttgctcaccatggccattcgtcctccttgacgtttg
15 cgagatctgatatcgcgcaccggactccagcccca
16 ttgctcaccatggccatctcgcgccccccgtcgtag
17 cgagatctgatatcgcgccgagggcacggtggaca
18 ttgctcaccatggccatggagcggagccacc
19 cgagatctgatatcgtcgcccgttttgtcgtcccg
20 ttgctcaccatggccatggtgcgtctgggggagg
21 cgagatctgatatcgtgaactgctgcccaacctggtg
22 ttgctcaccatggccatggtggtggggatccccttcg
23 cgagatctgatatcggcggcgacttgagcggcag
24 ttgctcaccatggccatggcacttccttccggtcgtc
25 cgagatctgatatcgcgacaaccactccatcaagcac
26 ttgctcaccatggccatgatacggcggtaccgctgc
27 cgagatctgatatcgcgccacgatgccttgggagt
28 ttgctcaccatggccataggcccactcctccccatg
29 cgagatctgatatcgcgggctggtcatcgtggtct
30 ttgctcaccatggccattccccccgtctcatgcg
31 ttagatcctcgagatctgatatcggatccgatccggcggcttgcgcccgatg
32 tcgcccttgctcaccatggcca
4)将步骤3)中的16个重组质粒分别接合转导进入恰塔努加链霉菌L10中，得到相应16株突变株。
5)将步骤4)中的16株恰塔努加链霉菌L10突变株进行发酵。
6)取步骤5)中24、48、72以及96小时的各突变株发酵液，离心收取菌体，超声破碎细胞获得菌体总蛋白。
7)取步骤6)中各样品20μg进行菌体总蛋白Western blot实验，检测eGFP在24、48、72以及96小时的表达水平同时利用等量的各样品进行蛋白胶电泳后考马斯亮蓝染色，确定 各样品总蛋白上样量一致。
8)根据步骤7)中Western blot实验相对定量检测eGFP的结果，进一步确认了在恰塔努加链霉菌L10中次级代谢高效调控强启动子thlM4p(见附图2)，其特点为在初级代谢中低活性并且在次级代谢中高活性。
thlM4p序列:
Figure PCTCN2019077130-appb-000001
实施例2
1)选取恰塔努加链霉菌L10中纳他霉素合成途径特异性正调控基因scnRII作为靶基因，利用引物对33+34(见表3)扩增thlM4p启动子，同时利用引物对35+36(见表3)扩增scnRII基因，将thlM4p启动子和scnRII基因以无缝克隆的方法插入pIJ8660载体的BamHI/NotI位点，即得到依次带有thlM4p启动子和scnRII基因的pIJ8660重组质粒，命名为pIJ8660-thlM4p-scnRII。另外，利用引物对37+38(见表3)扩增ermE*p启动子，同时利用引物对39+40(见表3)扩增scnRII基因，将ermE*p启动子和scnRII基因以无缝克隆的方法插入pIJ8660载体的BamHI/NotI位点，即得到依次带有ermE*p启动子和scnRII基因的pIJ8660重组质粒，命名为pIJ8660-ermE*p-scnRII。
表3构建高表达质粒所用引物
33 cgagatctgatatcgtcgcccgttttgtcgtcccg
34 ttatcaaggctcgccatggtgcgtctgggggagg
35 atggcgagccttgataaaacgttgac
36 cgggctgcagccgggcggccgctcacttcacgaagtcgtccacaac
37 ttagatcctcgagatctgatatcggatccgatccggcggcttgcgcccgatg
38 tatgtccgcctcctttggtcactcagtcag
39 gaccaaaggaggcggacatatggcgagccttgataaaacgttgac
40 cgggctgcagccgggcggccgctcacttcacgaagtcgtccacaac
2)将步骤1)中的2个重组质粒分别接合转导进入恰塔努加链霉菌L10中，得到相应2 株高表达scnRII的突变株。
3)将步骤2)中的2株恰塔努加链霉菌L10突变株进行发酵。
4)取步骤3)中24、48、72、96以及120小时的各突变株发酵液，甲醇提取后HPLC测定纳他霉素产量。经检测thlM4p高表达scnRII的恰塔努加链霉菌L10突变株纳他霉素的产量比ermE*p高表达scnRII的恰塔努加链霉菌L10突变株高30％，同时菌体干重未发现明显变化。(见附图3与附图4)。thlM4p序列同实施例1所示。

Claims (6)

  1. 基于转录组学链霉菌次级代谢强启动子挖掘方法及应用，其特征是，通过比较分析链霉菌转录组基因芯片数据，筛选出次级代谢中表达水平相对提高明显的基因，并扩增出这些基因的启动子区域，作为备选启动子，利用egfp报告基因次级代谢中表达水平进一步确认和表征备选启动子活性，同时以强启动子ermE*p作为参照，确证启动子在初级代谢中低活性但在次级代谢中高活性。
  2. 根据权利要求1所述的基于转录组学链霉菌次级代谢强启动子挖掘方法，其特征是，通过以下步骤实现：
    (1)根据链霉菌转录组基因芯片数据，初步筛选出链霉菌次级代谢中表达水平相对提高明显的基因；
    (2)根据步骤(1)中确定的基因，剔除其中基因间隔区过短或者没有基因间隔区的基因后得到若干个基因，取这些基因的启动子区域作为备选启动子；
    (3)扩增出步骤(2)中的备选启动子，克隆插入pIJ8660载体中，使得备选启动子位于egfp基因的上游，测序检验序列正确性后，即得到若干分别依次带有一段备选启动子和egfp基因的pIJ8660重组质粒，同时扩增出ermE*p启动子，克隆插入pIJ8660载体的相同位点，得到依次带有ermE*p启动子和egfp基因的pIJ8660重组质粒；
    (4)将步骤(3)中的所有重组质粒分别接合转导进入链霉菌中，得到突变株若干；
    (5)将步骤(4)中的突变株进行发酵；
    (6)取步骤(5)中24、48、72以及96小时的各突变株发酵液，离心收取菌体，超声破碎细胞获得菌体总蛋白；
    (7)取步骤(6)中等量的各样品进行菌体总蛋白Western blot实验，检测eGFP在24、48、72以及96小时的表达水平同时利用等量的各样品进行蛋白胶电泳后考马斯亮蓝染色，确定各样品总蛋白上样量一致；
    (8)根据步骤(7)中Western blot实验相对定量检测eGFP的结果，进一步确认在链霉菌次级代谢高效调控强启动子，其特点为在初级代谢中低活性并且在次级代谢中高活性。
  3. 权利要求1或2所述方法在链霉菌次级代谢基因表达调控中的应用
  4. 根据权利要求3所述的应用，其特征是，根据链霉菌转录组基因芯片数据筛选出链霉菌次级代谢中表达水平相对提高明显的基因，取这些基因的启动子区域作为备选启动子。
  5. 根据权利要求3所述的应用，其特征是，将备选启动子插入egfp基因上游，利用eGFP蛋白的表达水平来表征备选启动子活性，便于检测。
  6. 根据权利要求3所述的应用，其特征是，筛选得到的启动子在初级代谢中低活性并且在次级代谢中高活性，从而能够保证使用该启动子时，链霉菌初级代谢中不受影响而在次级 代谢中保证相关次级代谢产物高效产出。
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