WO2021259383A1 - Saccharomyces cerevisiae regulatory element and use thereof in carotenoid synthesis - Google Patents

Saccharomyces cerevisiae regulatory element and use thereof in carotenoid synthesis Download PDF

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WO2021259383A1
WO2021259383A1 PCT/CN2021/108448 CN2021108448W WO2021259383A1 WO 2021259383 A1 WO2021259383 A1 WO 2021259383A1 CN 2021108448 W CN2021108448 W CN 2021108448W WO 2021259383 A1 WO2021259383 A1 WO 2021259383A1
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saccharomyces cerevisiae
crte
gene
crtb
crti
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朱红惠
苏卜利
王东东
李安章
邓名荣
冯广达
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广东省科学院微生物研究所(广东省微生物分析检测中心)
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  • the present invention relates to the technical field of microorganisms, in particular to a regulatory element that antagonizes the positional effect of the Saccharomyces cerevisiae genome and its application.
  • Saccharomyces cerevisiae the position of the gene in the genome plays an important role in the expression of the gene, and its position often affects the transcription of the gene.
  • regulating the expression of endogenous or exogenous genes is the main way to construct microbial cell factories.
  • integrating foreign genes or synthetic pathways into the genome is the most commonly used method.
  • gene replication and transcription in microbial cells are controlled by the structure of chromosomes. This phenomenon is called positional effect, and it is usually manifested as genes exhibiting different levels of transcription at different sites. This phenomenon is found in many microbial species, including Saccharomyces cerevisiae, which is commonly used in industrial production.
  • the first objective of the present invention is to provide a regulatory element that can antagonize the positional effect of the Saccharomyces cerevisiae genome.
  • the present invention first selects 12 genomic sites and uses the lycopene synthesis pathway as the reporter gene to investigate the positional effect; and finds that the gene sequence in the synthesis pathway also has a significant impact on transcription; finally, it proposes to use insulators to antagonize low expression The position effect of the locus.
  • the nucleotide sequence of the regulatory element is shown in SEQ ID NO.1.
  • the second object of the present invention is to provide the application of the above-mentioned regulatory elements in antagonizing the positional effect of the gal80 locus of the Saccharomyces cerevisiae genome.
  • the above-mentioned regulatory elements are added to both ends of the gene transcription expression element inserted into the gal80 locus of the Saccharomyces cerevisiae genome.
  • regulatory elements are added to both ends of the gene transcription and expression element whose gene sequence is crtE-crtB-crtI.
  • the third object of the present invention is to provide a method for regulating the position effect of the Saccharomyces cerevisiae genome, which is to insert regulatory elements into the gal80 site or both ends of the Saccharomyces cerevisiae genome.
  • regulatory elements are added to both ends of the gene transcription and expression elements, and then inserted into the gal80 locus of the Saccharomyces cerevisiae genome.
  • regulatory elements are added to both ends of the gene transcription and expression element with the gene sequence crtE-crtB-crtI, and then inserted into the gal80 site of the Saccharomyces cerevisiae genome.
  • the fourth objective of the present invention is to provide a Saccharomyces cerevisiae with high lycopene production, which is to place crtI, crtE, and crtB under a promoter, and then connect and fuse in the order of crtI, crtE, and crtB, and insert them into Saccharomyces cerevisiae At the gal80 locus of the genome.
  • the present invention found that the chromosome near gal80 site of Saccharomyces cerevisiae has a special structure, and there may be other types of positional effects other than HM site (mating type allocation site) silencing effect.
  • the transcription activation region of TEF2 is added at both ends, and the results show that the transcription activation region of TEF2 can indeed antagonize the unknown position effect of gal80 and can significantly increase the content of lycopene.
  • its antagonistic effect is mainly to prevent the position effect from affecting gene transcription in the synthetic pathway.
  • Figure 1 shows the influence of different genomic sites on the pathway of exogenous synthesis.
  • A Schematic diagram of the integration of exogenous synthesis pathways;
  • B the influence of genomic sites on the content of lycopene, where ARS416d, 106a, etc. represent different genomic sites;
  • C the transcription of each gene in the exogenous pathway at different sites, where ARS416d, 106a, etc. represent different genomic loci.
  • Figure 2 shows the influence of different gene sequences on the pathway of exogenous synthesis.
  • A Schematic diagram of gene sequence integration of different exogenous synthesis pathways;
  • B The influence of different sequences on lycopene content;
  • EBI strain PE13, gene sequence is crtE-crtB-crtI;
  • IEB strain PE14 gene sequence is crtI-crtE -crtB;
  • BIE strain PE01, the gene sequence is crtB-crtI-crtE.
  • C The transcription of each gene in different order.
  • Figure 3 shows the influence of the insulator on the external source path.
  • A Schematic diagram of the use of insulators
  • B The effect of insulators on the content of lycopene, in which EBI is not inserted into the insulator, and EBI-ins is the inserted insulator
  • C the insulator affects the transcription of genes in the foreign synthesis pathway.
  • the reagents involved in the embodiments of the present invention are all commercially available products, and all can be purchased through commercial channels.
  • the Saccharomyces cerevisiae medium in the examples is YPM medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate heptahydrate 5g/L, potassium sulfate 3.5g/ L, sodium phosphate 2.5g/, TMS solution 1 ml/L (magnesium chloride hexahydrate 250mg/L, calcium chloride dihydrate 104.5mg/L, mg/L, copper sulfate pentahydrate 0.4mg/L, sodium iodide 0.08mg /L, manganese chloride tetrahydrate 0.1mg/L, sodium molybdate dihydrate 0.5mg/L, boric acid 1mg/L, cobalt chloride hexahydrate 0.3mg/L, zinc sulfate heptahydrate 6.25mg/L, sulfuric acid heptahydrate Ferrous iron 3.5mg/
  • Example 1 The influence of genomic locus on the pathway of exogenous synthesis
  • the present invention uses the lycopene synthesis pathway as a transcription module, and selects 12 genomic sites for genomic integration of the exogenous synthesis pathway.
  • the invention name is a recombinant yeast strain and the pHCas9M-gRNA plasmid constructed in Example 1 in its application as a template
  • the primers in the primer list 2 were used to construct YDR448W, YGR240C, YGR038W, YPRC ⁇ 15, YPRCt3, YORW ⁇ 22, ARS308a, 911b, 720a, gal80, 106a, ARS416d genomic integration plasmids (Table 1);
  • crtE, crtB, crtI modules use the modules constructed in the invention patent 201910267866.1 as templates, and use the relevant primers in the primer list to amplify different positions Point of function module.
  • the modules at different positions are integrated with the corresponding genome integration plasmids,
  • the lycopene content of each strain was determined according to the standard lycopene determination method. As shown in Figure 1B, the lycopene content of each strain is significantly related to the genomic site where it is located, and there is a 3.8-fold difference between different sites, and the content is in the range of 0.3 to 1.2 mg/g dry cell weight. There is a 58-fold difference in the transcription level of each gene in the synthetic pathway ( Figure 1C). At the same time, we found that among the strains with the highest lycopene content, the crtE gene also showed the highest transcription.
  • fluorescence quantitative PCR measures the expression levels of crtI, crtB and crtE genes.
  • the specific steps are as follows.
  • the PCR reaction system is: Cham QUniversal SYBR qPCR Master Mix 10 ⁇ L, forward primer and reverse primer each 0.4 ⁇ L, Template cDNA 1 ⁇ L, water supplement Add to 20 ⁇ L; the two-step reaction procedure is: 95°C pre-denaturation 30s; 95°C denaturation 10s, 60°C annealing, extension 30s, the above cycle 40 times, the dissolution curve: 95°C 15s; 95°C 60s, 60°C 15s.
  • the fluorescent quantitative PCR primers for crtI, crtB and crtE genes are:
  • Example 2 The effect of gene sequence in the exogenous synthesis pathway on carotenoid synthesis
  • Example 1 the lycopene synthesis pathway gene sequence is crtB-crtI-crtE, and the pathway integrated into the gal80 site of Saccharomyces cerevisiae is BIE (PE00).
  • the corresponding primers in the primer list were used to amplify the functional modules of crtE, crtB, and crtI respectively, and the genomic integration plasmid of gal80 site of Saccharomyces cerevisiae was used to integrate the genomic sequence of the crtE-crtB-crtI synthetic pathway to construct the strain EBI (PE13).
  • the content of synthetic lycopene of the strain EBI, strain BIE, and strain IEB constructed above was determined.
  • the crtI, crtE, and crtB genes in each strain were used to determine the transcription of the three genes by fluorescence quantitative PCR.
  • Fig. 2B The lycopene yield of each strain is shown in Fig. 2B. It can be seen from Fig. 2B that the lycopene content of the strain IEB is the highest, which is better than that of the strain BIE and the strain EBI.
  • Figure 2C shows the transcription levels of the crtI, crtB and crtE genes of the constructed strain EBI, strain BIE, and strain IEB.
  • the transcription amount of each gene in each strain is different.
  • the crtE transcription amount is the highest in the strain IEB, followed by the strain BIE, and the lowest is the strain EBI, which is consistent with the content of lycopene.
  • Example 3 The influence of insulators on external synthesis pathways
  • positional effects has brought unpredictable results to the construction of exogenous synthetic pathways, so how to eliminate or weaken positional effects is particularly important, because in the previous results, we can also see that they are different at the same site. There are also different transcriptional characteristics of the genes, which means that the position effect may also have specificity.
  • TEF2 transcription activation region of TEF2 (insulator, nucleotide sequence is ccctgccggctgtgagggcgccataaccaaggtatctatagaccgccaatcagcaaactacctccgtacattcatgttgcacccacacatttat acacccagaccgcacccacacatttat acacccagcagcagcaaactacctccgtacattcatgttgcacccacacatttat acacccagaccgcgacaaa) at both ends of the synthetic pathway of gene sequence crtE-crtB-crtI.
  • the corresponding primers in the list amplify the functional modules of crtE, crtB, and crtI containing insulators, and use the similar integration method in Example 1 for genome integration.
  • the schematic diagram of the specific gene sequence integration is shown in Figure 3A, and the Insulator in Figure 3A represents the transcription of TEF2. Activate the area.
  • the specific results are shown in Figure 3.
  • the results show that the transcription activation region of TEF2 can indeed antagonize the unknown position effect of the gal80 locus, and can significantly improve lycopene.
  • it also shows that its antagonistic effect is mainly to prevent the influence of positional effect on gene transcription in the synthetic pathway (a 10-fold increase, Figure 3C).
  • Table 1 Strains and plasmids used in the present invention

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Abstract

Provided are a regulatory element having a nucleotide sequence as shown in SEQ ID NO:1 that antagonizes the genomic position effect of Saccharomyces cerevisiae, and the use thereof. Chromosomes near the gal80 site of Saccharomyces cerevisiae have special structures, and may have other types of position effects that are non-HM site (mating type allocation site) silencing effects. The transcription activation region of TEF2 is added to two ends of a synthetic pathway with a gene sequence of crtE-crtB-crtI, which can antagonize the unknown position effect of the gal80 site and significantly increase the content of lycopene. The antagonistic effect mainly prevents the influence of the position effect on gene transcription in the synthetic pathway.

Description

一种酿酒酵母调控元件及其在类胡萝卜素合成中的应用Saccharomyces cerevisiae regulatory element and its application in carotenoid synthesis 技术领域Technical field
本发明涉及微生物技术领域，特别涉及一种拮抗酿酒酵母基因组位置效应的调控元件及其应用。The present invention relates to the technical field of microorganisms, in particular to a regulatory element that antagonizes the positional effect of the Saccharomyces cerevisiae genome and its application.
背景技术Background technique
在酿酒酵母中，基因在基因组中的位置对基因的表达有着重要的作用，其位点往往会影响基因的转录。通常情况下，通过调控内源或者外源基因的表达是构建微生物细胞工厂的主要方式。在改造过程中，为了进行稳定的大规模生产，将外源基因或合成途径整合进基因组是最常采用的方法。然而，在微生物细胞中基因的复制和转录会受到染色体结构的控制。这种现象被称作位置效应，通常表现为基因在不同位点呈现出不同的转录水平。这种现象在众多微生物种都有发现，包括工业生产中常用的酿酒酵母。In Saccharomyces cerevisiae, the position of the gene in the genome plays an important role in the expression of the gene, and its position often affects the transcription of the gene. Normally, regulating the expression of endogenous or exogenous genes is the main way to construct microbial cell factories. In the process of transformation, in order to carry out stable large-scale production, integrating foreign genes or synthetic pathways into the genome is the most commonly used method. However, gene replication and transcription in microbial cells are controlled by the structure of chromosomes. This phenomenon is called positional effect, and it is usually manifested as genes exhibiting different levels of transcription at different sites. This phenomenon is found in many microbial species, including Saccharomyces cerevisiae, which is commonly used in industrial production.
由于在工业生产中的广泛应用，酿酒酵母位置效应已有众多研究。例如，以LacZ为报告基因，20个位点可以呈现8.7倍的差异；同样以LacZ为报告基因，在18个位点中可以呈现14倍的差异。为了获得基因组组学水平的位置效应，分别以GFP和RFP为报告基因，考察了482个和1044个位点的位置效应。这些研究都是以单个基因为报告基因，以一条合成途径为报告基因的还未见报道。并且，这些研究只是呈现不同位点的差异，并没有提出如何解决低表达位点的办法。Due to its wide application in industrial production, there have been many studies on the positional effect of Saccharomyces cerevisiae. For example, with LacZ as the reporter gene, 20 loci can present an 8.7-fold difference; also with LacZ as the reporter gene, a 14-fold difference can be present at 18 loci. In order to obtain the position effect at the genomics level, using GFP and RFP as reporter genes, respectively, the position effect of 482 and 1044 loci were investigated. These studies all use a single gene as the reporter gene, and there is no report that uses a synthetic pathway as the reporter gene. Moreover, these studies only show the differences between different sites, and do not propose how to solve the low expression sites.
发明内容Summary of the invention
本发明的第一个目的是提供一种可以拮抗酿酒酵母基因组位置效应的调控元件。The first objective of the present invention is to provide a regulatory element that can antagonize the positional effect of the Saccharomyces cerevisiae genome.
本发明首先选择了12个基因组位点，以番茄红素合成途径为报告基因，考察位置效应； 并发现了合成途径中的基因顺序对转录也存在显著影响；最后，提出用绝缘子来拮抗低表达位点的位置效应。The present invention first selects 12 genomic sites and uses the lycopene synthesis pathway as the reporter gene to investigate the positional effect; and finds that the gene sequence in the synthesis pathway also has a significant impact on transcription; finally, it proposes to use insulators to antagonize low expression The position effect of the locus.
所述的调控元件，其核苷酸序列如SEQ ID NO.1所示。The nucleotide sequence of the regulatory element is shown in SEQ ID NO.1.
本发明的第二个目的是提供上述调控元件在拮抗酿酒酵母基因组gal80位点的位置效应中的应用。The second object of the present invention is to provide the application of the above-mentioned regulatory elements in antagonizing the positional effect of the gal80 locus of the Saccharomyces cerevisiae genome.
优选，是在插入酿酒酵母基因组gal80位点位置的基因转录表达元件两端加上上述调控元件。Preferably, the above-mentioned regulatory elements are added to both ends of the gene transcription expression element inserted into the gal80 locus of the Saccharomyces cerevisiae genome.
优选，是在基因顺序为crtE-crtB-crtI的基因转录表达元件两端加上调控元件。Preferably, regulatory elements are added to both ends of the gene transcription and expression element whose gene sequence is crtE-crtB-crtI.
本发明的第三个目是提供一种调控酿酒酵母基因组位置效应的方法，其是在酿酒酵母基因组gal80位点的插入或两端各插入调控元件。The third object of the present invention is to provide a method for regulating the position effect of the Saccharomyces cerevisiae genome, which is to insert regulatory elements into the gal80 site or both ends of the Saccharomyces cerevisiae genome.
优选，是在基因转录表达元件两端加上调控元件，然后再插入酿酒酵母基因组gal80位点。Preferably, regulatory elements are added to both ends of the gene transcription and expression elements, and then inserted into the gal80 locus of the Saccharomyces cerevisiae genome.
优选，是在基因顺序为crtE-crtB-crtI的基因转录表达元件两端加上调控元件，然后再插入酿酒酵母基因组gal80位点。Preferably, regulatory elements are added to both ends of the gene transcription and expression element with the gene sequence crtE-crtB-crtI, and then inserted into the gal80 site of the Saccharomyces cerevisiae genome.
本发明的第四个目的是提供一种高产番茄红素的酿酒酵母菌，其是将crtI、crtE、crtB置于启动子下，然后按照crtI、crtE、crtB的顺序连接融合，插入到酿酒酵母基因组gal80位点处。The fourth objective of the present invention is to provide a Saccharomyces cerevisiae with high lycopene production, which is to place crtI, crtE, and crtB under a promoter, and then connect and fuse in the order of crtI, crtE, and crtB, and insert them into Saccharomyces cerevisiae At the gal80 locus of the genome.
本发明发现酿酒酵母gal80位点附近染色体存在特殊结构，可能存在非HM位点(交配型分配位点)沉默效应的其它类型的位置效应，本发明尝试在基因顺序为crtE-crtB-crtI合成途径两端加上TEF2的转录激活区域，结果表明，TEF2的转录激活区域确实可以拮抗gal80 位点的未知位置效应，可以显著提高番茄红素的含量。同时，也表明其拮抗作用主要是阻止了位置效应对合成途径中基因转录的影响。The present invention found that the chromosome near gal80 site of Saccharomyces cerevisiae has a special structure, and there may be other types of positional effects other than HM site (mating type allocation site) silencing effect. The transcription activation region of TEF2 is added at both ends, and the results show that the transcription activation region of TEF2 can indeed antagonize the unknown position effect of gal80 and can significantly increase the content of lycopene. At the same time, it also shows that its antagonistic effect is mainly to prevent the position effect from affecting gene transcription in the synthetic pathway.
附图说明Description of the drawings
图1是不同基因组位点对外源合成途径的影响。(A)外源合成途径整合示意图；(B)基因组位点对番茄红素含量的影响，其中ARS416d，106a等代表不同基因组位点；(C)不同位点外源途径各基因转录情况，其中ARS416d，106a等代表不同基因组位点。Figure 1 shows the influence of different genomic sites on the pathway of exogenous synthesis. (A) Schematic diagram of the integration of exogenous synthesis pathways; (B) the influence of genomic sites on the content of lycopene, where ARS416d, 106a, etc. represent different genomic sites; (C) the transcription of each gene in the exogenous pathway at different sites, where ARS416d, 106a, etc. represent different genomic loci.
图2是不同基因顺序对外源合成途径的影响。(A)不同外源合成途径基因顺序整合示意图；(B)不同顺序对番茄红素含量的影响；EBI,菌株PE13，基因顺序为crtE-crtB-crtI；IEB,菌株PE14基因顺序为crtI-crtE-crtB；BIE,菌株PE01，基因顺序为crtB-crtI-crtE。(C)不同顺序各基因转录情况。Figure 2 shows the influence of different gene sequences on the pathway of exogenous synthesis. (A) Schematic diagram of gene sequence integration of different exogenous synthesis pathways; (B) The influence of different sequences on lycopene content; EBI, strain PE13, gene sequence is crtE-crtB-crtI; IEB, strain PE14 gene sequence is crtI-crtE -crtB; BIE, strain PE01, the gene sequence is crtB-crtI-crtE. (C) The transcription of each gene in different order.
图3是绝缘子对外源途径的影响。(A)绝缘子使用示意图(B)绝缘子对番茄红素含量的影响，其中EBI是未插入绝缘子Insulator的，EBI-ins是插入绝缘子Insulator；(C)绝缘子对外源合成途径各基因转录影响。Figure 3 shows the influence of the insulator on the external source path. (A) Schematic diagram of the use of insulators (B) The effect of insulators on the content of lycopene, in which EBI is not inserted into the insulator, and EBI-ins is the inserted insulator; (C) the insulator affects the transcription of genes in the foreign synthesis pathway.
具体实施方式detailed description
为了使本技术领域的人员更好地理解本发明方案，下面结合附图和具体实施方式对本发明作进一步的详细说明。In order to enable those skilled in the art to better understand the solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products, and all can be purchased through commercial channels.
实施例中酿酒酵母培养基为YPM培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，磷酸二氢钾10g/L，七水硫酸镁5g/L，硫酸钾3.5g/L，磷酸钠2.5g/，TMS溶液1 ml/L(六水氯化镁250mg/L，二水氯化钙104.5mg/L，mg/L，五水合硫酸铜0.4mg/L，碘化钠0.08mg/L，四水合氯化锰0.1mg/L，二水合钼酸钠0.5mg/L，硼酸1mg/L，六水合氯化钴0.3mg/L，七水硫酸锌6.25mg/L，七水合硫酸亚铁3.5mg/L，余量为水)，余量为水。其配制方法是将各个成分混合均匀，灭菌制得。The Saccharomyces cerevisiae medium in the examples is YPM medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate heptahydrate 5g/L, potassium sulfate 3.5g/ L, sodium phosphate 2.5g/, TMS solution 1 ml/L (magnesium chloride hexahydrate 250mg/L, calcium chloride dihydrate 104.5mg/L, mg/L, copper sulfate pentahydrate 0.4mg/L, sodium iodide 0.08mg /L, manganese chloride tetrahydrate 0.1mg/L, sodium molybdate dihydrate 0.5mg/L, boric acid 1mg/L, cobalt chloride hexahydrate 0.3mg/L, zinc sulfate heptahydrate 6.25mg/L, sulfuric acid heptahydrate Ferrous iron 3.5mg/L, the balance is water), the balance is water. The preparation method is to mix the ingredients uniformly and sterilize.
实施例1 基因组位点对外源合成途径的影响Example 1 The influence of genomic locus on the pathway of exogenous synthesis
为了考察基因组位置效应，本发明以番茄红素合成途径为一个转录模块，选择12个基因组位点进行外源合成途径的基因组整合。以申请号201910267866.1，发明名称为一种重组酵母菌株及其应用中实施例1构建的pHCas9M-gRNA质粒为模板，分别使用引物列表2中的引物构建YDR448W，YGR240C，YGR038W，YPRCΔ15，YPRCt3，YORWΔ22，ARS308a，911b，720a，gal80，106a，ARS416d位点的基因组整合质粒(表1)；crtE、crtB、crtI模块以发明专利201910267866.1中构建的模块为模板，使用引物列表中相关引物扩增针对不同位点的功能模块。将不同位点的模块与对应的基因组整合质粒，进行不同位点的基因组整合，整合的基因顺序为crtB-crtI-crtE(图1A)。In order to investigate the effect of genomic position, the present invention uses the lycopene synthesis pathway as a transcription module, and selects 12 genomic sites for genomic integration of the exogenous synthesis pathway. Using the application number 201910267866.1, the invention name is a recombinant yeast strain and the pHCas9M-gRNA plasmid constructed in Example 1 in its application as a template, the primers in the primer list 2 were used to construct YDR448W, YGR240C, YGR038W, YPRCΔ15, YPRCt3, YORWΔ22, ARS308a, 911b, 720a, gal80, 106a, ARS416d genomic integration plasmids (Table 1); crtE, crtB, crtI modules use the modules constructed in the invention patent 201910267866.1 as templates, and use the relevant primers in the primer list to amplify different positions Point of function module. The modules at different positions are integrated with the corresponding genome integration plasmids, and the genome integration at different positions is performed. The integrated gene sequence is crtB-crtI-crtE (Figure 1A).
按标准的番茄红素测定方法进行各菌株番茄红素含量测定。如图1B所示，各菌株的番茄红素含量与其所在的基因组位点显著相关，不同位点之间呈现出3.8倍差异，含量在0.3到1.2mg/g细胞干重范围内。而合成途径中各基因转录水平有58倍的差异(图1C)。同时我们发现番茄红素含量最高的菌株中，crtE基因也呈现出最高的转录量。The lycopene content of each strain was determined according to the standard lycopene determination method. As shown in Figure 1B, the lycopene content of each strain is significantly related to the genomic site where it is located, and there is a 3.8-fold difference between different sites, and the content is in the range of 0.3 to 1.2 mg/g dry cell weight. There is a 58-fold difference in the transcription level of each gene in the synthetic pathway (Figure 1C). At the same time, we found that among the strains with the highest lycopene content, the crtE gene also showed the highest transcription.
其中荧光定量PCR测定crtI、crtB和crtE基因的表达量，具体步骤如下，PCR的反应体系为：Cham QUniversal SYBR qPCR Master Mix 10μL，正向引物和反向引物各0.4μL，Template cDNA 1μL，水补加至20μL；两步法反应程序为：95℃预变性30s；95℃变性10s、 60℃退火、延伸30s，以上循环40次，溶解曲线：95℃15s；95℃60s、60℃15s。Among them, fluorescence quantitative PCR measures the expression levels of crtI, crtB and crtE genes. The specific steps are as follows. The PCR reaction system is: Cham QUniversal SYBR qPCR Master Mix 10μL, forward primer and reverse primer each 0.4μL, Template cDNA 1μL, water supplement Add to 20μL; the two-step reaction procedure is: 95°C pre-denaturation 30s; 95°C denaturation 10s, 60°C annealing, extension 30s, the above cycle 40 times, the dissolution curve: 95°C 15s; 95°C 60s, 60°C 15s.
crtI、crtB和crtE基因荧光定量PCR引物为：The fluorescent quantitative PCR primers for crtI, crtB and crtE genes are:
Figure PCTCN2021108448-appb-000001
Figure PCTCN2021108448-appb-000001
实施例2 外源合成途径中基因顺序对类胡萝卜素合成的影响Example 2 The effect of gene sequence in the exogenous synthesis pathway on carotenoid synthesis
实施例1中番茄红素合成途径基因顺序为crtB-crtI-crtE的途径整合于酿酒酵母的gal80位点菌株为BIE(PE00)。以引物列表中对应引物分别扩增crtE，crtB，crtI功能模块，与酿酒酵母的gal80位点基因组整合质粒，进行基因顺序为crtE-crtB-crtI合成途径的基因组整合，构建菌株EBI(PE13)。以引物列表中对应引物分别扩增crtI，crtE，crtB功能模块，与酿酒酵母的gal80位点基因组整合质粒，进行基因顺序为crtI-crtE-crtB合成途径的基因组整合，构建菌株IEB(PE14)。In Example 1, the lycopene synthesis pathway gene sequence is crtB-crtI-crtE, and the pathway integrated into the gal80 site of Saccharomyces cerevisiae is BIE (PE00). The corresponding primers in the primer list were used to amplify the functional modules of crtE, crtB, and crtI respectively, and the genomic integration plasmid of gal80 site of Saccharomyces cerevisiae was used to integrate the genomic sequence of the crtE-crtB-crtI synthetic pathway to construct the strain EBI (PE13). Use the corresponding primers in the primer list to amplify the functional modules of crtI, crtE, and crtB respectively, and integrate the plasmid with the gal80 locus genome of Saccharomyces cerevisiae, and carry out the genomic integration of the crtI-crtE-crtB synthetic pathway to construct the strain IEB (PE14).
菌株EBI、菌株BIE、菌株IEB的外源合成途径基因顺序整合示意图如图2A所示。The schematic diagram of the sequence integration of the exogenous synthetic pathway genes of strain EBI, strain BIE, and strain IEB is shown in Figure 2A.
按标准的番茄红素测定方法(在YPM培养基摇瓶发酵条件下)，对上述构建的菌株EBI、菌株BIE、菌株IEB合成番茄红素的含量进行测定。另外对各菌株中的crtI、crtE、crtB基因通过荧光定量PCR测定三个基因的转录情况。According to the standard lycopene determination method (under YPM medium shake flask fermentation conditions), the content of synthetic lycopene of the strain EBI, strain BIE, and strain IEB constructed above was determined. In addition, the crtI, crtE, and crtB genes in each strain were used to determine the transcription of the three genes by fluorescence quantitative PCR.
各菌株番茄红素的产量如图2B，由图2B可知，菌株IEB的番茄红素的含量最高，优于菌株BIE和菌株EBI。The lycopene yield of each strain is shown in Fig. 2B. It can be seen from Fig. 2B that the lycopene content of the strain IEB is the highest, which is better than that of the strain BIE and the strain EBI.
图2C为构建的菌株EBI、菌株BIE、菌株IEB菌株的crtI、crtB和crtE基因的转录量。各菌株中各基因的转录量不相同，其中番茄红素的含量最高菌株IEB中，其crtE转录量最高，其次是菌株BIE，最低是菌株EBI，与番茄红素的含量相一致。Figure 2C shows the transcription levels of the crtI, crtB and crtE genes of the constructed strain EBI, strain BIE, and strain IEB. The transcription amount of each gene in each strain is different. Among the strains with the highest lycopene content, the crtE transcription amount is the highest in the strain IEB, followed by the strain BIE, and the lowest is the strain EBI, which is consistent with the content of lycopene.
从上面可以看出，将合成途径中基因的顺序变成crtE-crtB-crtI进行基因组整合时，番茄红素的含量会显著降低，这预示着合成途径中基因的顺序可能会对代谢途径各基因的转录造成影响。这一点与理论不太相符，因为酿酒酵母中大部分基因是一个独立的转录本，彼此之间没有影响。我们构建的番茄红素合成途径也是这样，三个基因分别有各自的启动子和终止子，理论上变换顺序，彼此之间不会产生影响，也不会存在转录的差异。为了更清楚了解合成途径中基因顺序对基因转录的影响，我们又构建了crtI-crtE-crtB顺序的菌株，三种不同基因顺序的菌株番茄红素含量呈现出14倍的差异。经基因转录分析，不同基因顺序造成了30倍转录差异。这说明gal80位点附近染色体存在特殊结构，可能存在非HM位点(交配型分配位点)沉默效应的其它类型的位置效应。It can be seen from the above that when the sequence of genes in the synthetic pathway is changed to crtE-crtB-crtI for genome integration, the content of lycopene will be significantly reduced, which indicates that the sequence of genes in the synthetic pathway may affect the genes in the metabolic pathway. The transcription affects. This is not in line with theory, because most genes in Saccharomyces cerevisiae are independent transcripts and have no influence on each other. The lycopene synthesis pathway we constructed is the same. The three genes have their own promoters and terminators. Theoretically, the sequence of the three genes will be changed. There will be no influence on each other, and there will be no differences in transcription. In order to have a clearer understanding of the effect of gene sequence on gene transcription in the synthetic pathway, we constructed strains with the sequence crtI-crtE-crtB. The lycopene content of the three strains with different gene sequences showed a 14-fold difference. According to gene transcription analysis, different gene sequences caused a 30-fold transcription difference. This indicates that there is a special structure of the chromosome near the gal80 locus, and there may be other types of positional effects other than the silencing effect of the HM locus (mating-type allocation site).
实施例3 绝缘子对外源合成途径的影响Example 3 The influence of insulators on external synthesis pathways
位置效应的存在为外源合成途径的构建带来了不可预期的结果，那么怎么去消除或削弱位置效应就显得尤为重要，因为在前面的结果中，我们也可以看到，在同一位点不同的基因也会存在不同的转录特性，这就说明位置效应可能还存在特异性。我们尝试在基因顺序为crtE-crtB-crtI合成途径两端加上TEF2的转录激活区域(绝缘子，核苷酸序列为ccctgccggctgtgagggcgccataaccaaggtatctatagaccgccaatcagcaaactacctccgtacattcatgttgcacccacacatttat acacccagaccgcgacaaa，具体核苷酸序列如SEQ ID NO.1所示)，用引物列表中对应引物扩增含有绝缘子的crtE，crtB，crtI功能模块，利用实施例1中类似的整合方法进行基因组整合，具体基因顺序整合示意图如图3A所示，图3A中的Insulator代表TEF2的转录激活区域。参照实施例1进行番茄红素的产量测定和各基因转录量测定，具体结果如图3所示，结果表明，TEF2的转录激活区域确实可以拮抗gal80位点的未知位置效应，可以显著提高番茄红素的含量(7倍，图3B)。同时，也表明其拮抗作用主要是阻止了位置效应对合成途径中基因转录的影响(提高10倍，图3C)。The existence of positional effects has brought unpredictable results to the construction of exogenous synthetic pathways, so how to eliminate or weaken positional effects is particularly important, because in the previous results, we can also see that they are different at the same site. There are also different transcriptional characteristics of the genes, which means that the position effect may also have specificity. We tried to add the transcription activation region of TEF2 (insulator, nucleotide sequence is ccctgccggctgtgagggcgccataaccaaggtatctatagaccgccaatcagcaaactacctccgtacattcatgttgcacccacacatttat acacccagaccgcacccacacatttat acacccagcagcagcaaactacctccgtacattcatgttgcacccacacatttat acacccagaccgcgacaaa) at both ends of the synthetic pathway of gene sequence crtE-crtB-crtI. The corresponding primers in the list amplify the functional modules of crtE, crtB, and crtI containing insulators, and use the similar integration method in Example 1 for genome integration. The schematic diagram of the specific gene sequence integration is shown in Figure 3A, and the Insulator in Figure 3A represents the transcription of TEF2. Activate the area. Refer to Example 1 for the determination of the yield of lycopene and the determination of the transcription amount of each gene. The specific results are shown in Figure 3. The results show that the transcription activation region of TEF2 can indeed antagonize the unknown position effect of the gal80 locus, and can significantly improve lycopene. Element content (7 times, Figure 3B). At the same time, it also shows that its antagonistic effect is mainly to prevent the influence of positional effect on gene transcription in the synthetic pathway (a 10-fold increase, Figure 3C).
以上对本发明所提供的一种调控元件对基因组位置效应拮抗作用进行了详细介绍。本发明中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，比如在基因组其它低表达位点进行绝缘子调控，或利用其它强启动子的转录激活区域，这些改进和修饰也落入本发明权利要求的保护范围内。The antagonistic effect of a regulatory element provided by the present invention on the positional effect of the genome has been described in detail above. In the present invention, specific examples are used to describe the principle and implementation of the present invention, and the description of the above embodiments is only used to help understand the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, such as insulator regulation at other low expression sites in the genome, or the use of other strong These improvements and modifications of the transcriptional activation region of the promoter also fall within the protection scope of the claims of the present invention.
表1：本发明使用菌株和质粒Table 1: Strains and plasmids used in the present invention
Figure PCTCN2021108448-appb-000002
Figure PCTCN2021108448-appb-000002
Figure PCTCN2021108448-appb-000003
Figure PCTCN2021108448-appb-000003
表2：本发明所使用引物Table 2: Primers used in the present invention
Figure PCTCN2021108448-appb-000004
Figure PCTCN2021108448-appb-000004
Figure PCTCN2021108448-appb-000005
Figure PCTCN2021108448-appb-000005
Figure PCTCN2021108448-appb-000006
Figure PCTCN2021108448-appb-000006
Figure PCTCN2021108448-appb-000007
Figure PCTCN2021108448-appb-000007

Claims (8)

  1. 一种拮抗酿酒酵母基因组位置效应的调控元件，其特征在于，所述的调控元件，其核苷酸序列如SEQ ID NO.1所示。A regulatory element that antagonizes the positional effect of the Saccharomyces cerevisiae genome is characterized in that the nucleotide sequence of the regulatory element is shown in SEQ ID NO.1.
  2. 权利要求1所述的调控元件在拮抗酿酒酵母基因组gal80位点的位置效应中的应用。The use of the regulatory element of claim 1 in antagonizing the positional effect of the gal80 locus of the Saccharomyces cerevisiae genome.
  3. 根据权利要求2所述的应用，其特征在于，是在插入酿酒酵母基因组gal80位点位置的基因转录表达元件两端加上调控元件。The application according to claim 2, characterized in that the regulation element is added at both ends of the gene transcription expression element inserted into the gal80 locus of the Saccharomyces cerevisiae genome.
  4. 根据权利要求3所述的应用，其特征在于，是在基因顺序为crtE-crtB-crtI的基因转录表达元件两端加上调控元件。The application according to claim 3, characterized in that a regulatory element is added to both ends of the gene transcription and expression element whose gene sequence is crtE-crtB-crtI.
  5. 一种调控酿酒酵母基因组位置效应的方法，其特征在于，是在酿酒酵母基因组gal80位点的插入或两端各插入权利要求1所述的调控元件。A method for regulating the positional effect of the Saccharomyces cerevisiae genome, which is characterized in that the regulatory element according to claim 1 is inserted into the gal80 position or both ends of the Saccharomyces cerevisiae genome.
  6. 根据权利要求5所述的方法，其特征在于，是在基因转录表达元件两端加上调控元件，然后再插入酿酒酵母基因组gal80位点。The method according to claim 5, characterized in that the regulatory elements are added to both ends of the gene transcription and expression elements, and then inserted into the gal80 site of the Saccharomyces cerevisiae genome.
  7. 根据权利要求6所述的方法，其特征在于，是在基因顺序为crtE-crtB-crtI的基因转录表达元件两端加上调控元件，然后再插入酿酒酵母基因组gal80位点。The method according to claim 6, characterized in that regulatory elements are added to both ends of the gene transcription and expression element with the gene sequence crtE-crtB-crtI, and then inserted into the gal80 site of the Saccharomyces cerevisiae genome.
  8. 一种高产番茄红素的酿酒酵母菌，其特征在于，是分别将crtI、crtE、crtB置于启动子下，然后按照crtI、crtE、crtB的顺序连接融合，插入到酿酒酵母基因组gal80位点处。A Saccharomyces cerevisiae with high lycopene production, characterized in that crtI, crtE, and crtB are respectively placed under a promoter, and then connected and fused in the order of crtI, crtE, and crtB, and inserted into the gal80 site of the Saccharomyces cerevisiae genome .
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