WO2017000870A1 - Humanized lactate dehydrogenase (ldh) saccharomyces cerevisiae and construction method thereof - Google Patents

Humanized lactate dehydrogenase (ldh) saccharomyces cerevisiae and construction method thereof Download PDF

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WO2017000870A1
WO2017000870A1 PCT/CN2016/087449 CN2016087449W WO2017000870A1 WO 2017000870 A1 WO2017000870 A1 WO 2017000870A1 CN 2016087449 W CN2016087449 W CN 2016087449W WO 2017000870 A1 WO2017000870 A1 WO 2017000870A1
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saccharomyces cerevisiae
lactate dehydrogenase
humanized
ldhc
ldha
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高晓冬
中西秀树
李子杰
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江南大学
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Definitions

  • the invention relates to a lactate dehydrogenase humanized Saccharomyces cerevisiae and a construction method thereof, and belongs to the technical field of microorganisms and the technical field of molecular biology.
  • Saccharomyces cerevisiae uses the glycolysis pathway to produce capacity under anaerobic conditions and uses ethanol fermentation to maintain the glycolysis pathway. Ethanol fermentation involves two steps: first, pyruvate is converted to acetaldehyde by pyruvate decarboxylase (PDC), and then acetaldehyde is reduced to produce ethanol and NAD + regeneration under the action of alcohol dehydrogenase (ADH). Under aerobic conditions, Saccharomyces cerevisiae utilizes the electron transport chain in the mitochondria to produce.
  • PDC pyruvate decarboxylase
  • ADH alcohol dehydrogenase
  • LDH Lactate dehydrogenase
  • LDHA mainly localized in the cytoplasm, reversibly catalyzing the conversion of pyruvate to lactic acid while NADH is oxidized to NAD + . Therefore, lactic acid fermentation is necessary to maintain the glycolytic pathway of tumor cells and the regeneration of NAD + .
  • the human-derived LDH has three subunits, LDHA, LDHB, and LDHC.
  • LDHA is mainly found in anaerobic tissues, such as skeletal muscle
  • LDHB is mainly expressed in aerobic tissues, such as myocardium
  • LDHC is specifically expressed in testis.
  • LDH is a key enzyme in cancer cell proliferation and metastasis.
  • Human lactate dehydrogenase which is heterologously expressed in Saccharomyces cerevisiae, can be used as a potential screening tool for anticancer drugs.
  • researchers can use drugs to express heterologously.
  • the role of lactate dehydrogenase a preliminary evaluation of drug performance.
  • introduction of genes from human gene libraries into the LDH yeast humanization system can screen for genes involved in the regulation of LDH activity.
  • the present invention knocks out the genes PDC1, PDC5 and PDC6 of Saccharomyces cerevisiae encoding PDC, expresses human LDH in Saccharomyces cerevisiae and blocks electron chain transfer with antimycin A, so that humanized lactic acid fermentation replaces ethanol. Fermentation to maintain the progress of glycolysis.
  • a first object of the present invention is to provide a lactate dehydrogenase humanized Saccharomyces cerevisiae which lacks a pyruvate decarboxylase gene and which expresses human lactate dehydrogenase.
  • the deleted pyruvate decarboxylase genes are PDC1, PDC5 and PDC6.
  • the PDC1, PDC5, and PDC6, in one embodiment of the present invention are Genes with Gene IDs of 850733, 850825, and 852978, respectively, on NCBI.
  • the human lactate dehydrogenase is LDHA, LDHB or LDHC.
  • the human lactate dehydrogenase LDHA, LDHB or LDHC in one embodiment of the invention, is NCBI Genes with Gene ID 3939, 3945, 3948.
  • a second object of the present invention is a method for constructing the lactate dehydrogenase humanized Saccharomyces cerevisiae.
  • the constructing method in one embodiment of the present invention, is: (1) constructing a PDC1, PDC5, and PDC6 triple gene deletion strain, Saccharomyces cerevisiae pdc1 ⁇ pdc5 ⁇ pdc6 ⁇ ; (2) human lactate dehydrogenase LDHA, LDHB or LDHC It was ligated to the yeast expression plasmid and then transformed into S. cerevisiae pdc1 ⁇ pdc5 ⁇ pdc6 ⁇ to select the correct transformant, which is lactate dehydrogenase humanized Saccharomyces cerevisiae.
  • the three gene deletion bacteria which is an embodiment of the present invention, is constructed by using Saccharomyces cerevisiae W303-1A as a starting strain.
  • the construction method is, in an embodiment of the present invention, specifically: (1) using Saccharomyces cerevisiae W303-1A as a starting strain, and knocking out three genes of PDC1, PDC5 and PDC6 by homologous recombination method.
  • a third object of the present invention is to provide a method for expressing human lactate dehydrogenase using the lactate dehydrogenase humanized Saccharomyces cerevisiae.
  • the method is to culture lactate dehydrogenase humanized Saccharomyces cerevisiae in SD-Trp medium, and use antimycin A to block electron chain transfer, so that humanized lactic acid fermentation instead of ethanol fermentation to maintain glycolysis Going on.
  • the present invention also claims lactate dehydrogenase produced by humanized Saccharomyces cerevisiae lactate dehydrogenase, and its use in screening anticancer drugs, screening genes capable of regulating lactate dehydrogenase activity, and the like.
  • the beneficial effects of the invention knocking out the gene encoding the Saccharomyces cerevisiae pyruvate decarboxylase by gene homologous recombination, transforming the recombinant plasmid expressing human lactate dehydrogenase into the Saccharomyces cerevisiae pyruvate decarboxylase-deficient strain, 28 ° C
  • the single colony of Saccharomyces cerevisiae was grown at ⁇ 32 °C to achieve heterologous expression of human lactic acid bacteria dehydrogenase.
  • LDHA or LDHC was expressed in Saccharomyces cerevisiae pdc1 ⁇ pdc5 ⁇ pdc6 ⁇ three-deficient strain.
  • Lactate dehydrogenase humanized Saccharomyces cerevisiae will serve as a potential screening tool for anticancer drugs and genes that regulate lactate dehydrogenase activity.
  • Figure 1 Growth of humanized S. cerevisiae lactate dehydrogenase on plates containing antimycin A;
  • Figure 2 Determination of lactate production by humanized Saccharomyces cerevisiae lactate dehydrogenase
  • Figure 3 Western blot of the expression product
  • FIG. 4 Expression of human lactate dehydrogenase in S. cerevisiae.
  • lactated dehydrogenase humanized Saccharomyces cerevisiae includes the following steps:
  • the primers for the S. cerevisiae PDC6 gene sequence design are as follows:
  • Upstream primer HXO556 (sequence shown in SEQ ID NO. 1):
  • Downstream primer HXO557 (sequence as shown in SEQ ID NO. 2):
  • the PDC6 gene was amplified by the upstream primer and the downstream primer, and the His fragment derived from the plasmid pFA6a-HIS3MX6 was inserted into the PDC6 gene fragment to obtain a knock-out fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae PDC6 gene, and the method was transformed by lithium acetate/PEG.
  • the His-fragment amplified Hisis fragment was introduced into the Saccharomyces cerevisiae W303-1A haploid cells and screened; the transformed strain was applied to the SD-His-deficient plate, and a certain number of The colonies were extracted from the genome and verified by PCR, and compared with the genome of the wild type strain to obtain a pdc6 ⁇ deficient strain.
  • Upstream primer HXO552 (sequence shown in SEQ ID NO. 3):
  • Downstream primer HXO553 (sequence shown in SEQ ID NO. 4):
  • the PDC5 gene was amplified by the upstream primer and the downstream primer, and the Leu fragment derived from the plasmid pRS305 was inserted into the PDC5 gene fragment to obtain a knock-out fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae PDC5 gene, and the PCR was carried out by a lithium acetate/PEG transformation method.
  • the amplified Leu fragments were introduced into the Saccharomyces cerevisiae W303-1Apdc6 ⁇ deficient strain, respectively.
  • the recombinant plasmid pRS316TEF-PDC5 obtained by cloning the PDC5 gene into pRS316TEF was transformed into pdc6 ⁇ pdc5 ⁇ double-deficient strain, and the corresponding Saccharomyces cerevisiae recombinant strain was obtained. And knock out the PDC1 gene based on this recombinant strain.
  • Upstream primer HXO548 (sequence shown in SEQ ID NO. 5):
  • the downstream primer HXO549 (sequence as shown in SEQ ID NO. 6):
  • the PDC1 gene was amplified by the upstream primer and the downstream primer, and the Kan fragment derived from the plasmid pFA6a-kanMX6 was inserted into the PDC1 gene to obtain a knock-out fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae PDC1 gene, and the method of converting lithium acetate/PEG was carried out.
  • the Kan fragment amplified by PCR was introduced into the Saccharomyces cerevisiae pdc6 ⁇ pdc5 ⁇ /pRS316TEF-PDC5-deficient strain and screened; the transformed strain was applied to the SD-Leu-His-deficient plate containing G418 antibiotic, and the growth was delayed.
  • the LDHA, LDHB and LDHC genes amplified by PCR were inserted into the pRS424TEF plasmid, respectively, to obtain recombinant plasmids pRS424TEF-LDHA, pRS424TEF-LDHB and pRS424TEF-LDHC.
  • the recombinant plasmids pRS424TEF-LDHA, pRS424TEF-LDHB and pRS424TEF-LDHC were transferred to the Saccharomyces cerevisiae pdc1 ⁇ pdc5 ⁇ pdc6 ⁇ three-defective strain by the method of lithium acetate/PEG, and the transformed strain was coated in SD-Trp medium and grown at 30 °C. Single colonies, verified by sequencing, obtained lactated dehydrogenase humanized Saccharomyces cerevisiae expressing LDHA, LDHB and LDHC, respectively.
  • Example 3 Expression of human lactate dehydrogenase in pdc1 ⁇ pdc5 ⁇ pdc6 ⁇ triple-deficient strain
  • HPLC detection conditions Aminex HPX-87H analytical column (300 mm ⁇ 7.8 mm, Bio-Rad), mobile phase 5 mM H 2 SO 4 , flow rate 0.6 mL / min, column temperature 50 ° C, diode array detector, detection wavelength It is 210 nm.
  • the results of HPLC detection are shown in Fig. 2.
  • the results showed that LDHA or LDHC was expressed in the S. cerevisiae pdc1 ⁇ pdc5 ⁇ pdc6 ⁇ three-defective strain, and the production of lactic acid was detected after the addition of antimycin A.
  • western blot analysis was performed on the expression product of humanized Saccharomyces cerevisiae lactate dehydrogenase.
  • the results showed that LDHA, LDHB and LDHC could be expressed normally (Fig. 3).

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Abstract

Provided are a humanized lactate dehydrogenase (LDH) Saccharomyces cerevisiae and the construction method thereof. In particular, the method of gene homologous recombination is used to knockout the genes PDC1, PDC5 and PDC6 which encode the PDC of Saccharomyces cerevisiae, LDH from humans is expressed in Saccharomyces cerevisiae, and antimycin A is used to block electronic chain transfers, thus making humanized lactic acid fermentation replace ethanol fermentation for maintaining glycolysis. Humanized lactate dehydrogenase (LDH) Saccharomyces cerevisiae obtained from the present method can express the lactate dehydrogenase (LDH) of humans. LDHA or LDHC is expressed in strains of Saccharomyces cerevisiae suffering from three deficiencies in pdc1Δ pdc6Δ pdc5Δ. After antimycin A is added, the strains can grow, and the production of lactic acid can be detected.

Description

一种乳酸脱氢酶人源化酿酒酵母及其构建方法Lactate dehydrogenase humanized Saccharomyces cerevisiae and construction method thereof 技术领域Technical field
本发明涉及一种乳酸脱氢酶人源化酿酒酵母及其构建方法，属于微生物技术领域和分子生物学技术领域。The invention relates to a lactate dehydrogenase humanized Saccharomyces cerevisiae and a construction method thereof, and belongs to the technical field of microorganisms and the technical field of molecular biology.
背景技术Background technique
酿酒酵母在无氧条件下利用糖酵解途径来产能并采用乙醇发酵来维持糖酵解途径的进行。乙醇发酵包含两个步骤：首先丙酮酸在丙酮酸脱羧酶(PDC)的作用下转化为乙醛，然后在乙醇脱氢酶(ADH)的作用下，还原乙醛生成乙醇同时NAD+再生。在有氧条件下，酿酒酵母利用线粒体中的电子传递链来产能。Saccharomyces cerevisiae uses the glycolysis pathway to produce capacity under anaerobic conditions and uses ethanol fermentation to maintain the glycolysis pathway. Ethanol fermentation involves two steps: first, pyruvate is converted to acetaldehyde by pyruvate decarboxylase (PDC), and then acetaldehyde is reduced to produce ethanol and NAD + regeneration under the action of alcohol dehydrogenase (ADH). Under aerobic conditions, Saccharomyces cerevisiae utilizes the electron transport chain in the mitochondria to produce.
在有氧条件下，癌细胞能够利用葡萄糖作为碳源经糖酵解代谢途径产生大量的乳酸。糖酵解途径对于肿瘤细胞的增殖、侵袭和转移是必需的。乳酸脱氢酶(LDH)主要是定位于细胞质中，可逆地催化丙酮酸转化为乳酸同时NADH被氧化为NAD+。因此，乳酸发酵对于维持肿瘤细胞糖酵解途径的进行以及NAD+的再生是必需的。来源于人的LDH有三个亚基组成，分别为LDHA、LDHB和LDHC。LDHA主要存在于厌氧组织，比如骨骼肌；LDHB主要是在有氧组织中表达，比如心肌；LDHC则是专门在睾丸中表达。Under aerobic conditions, cancer cells can use glucose as a carbon source to produce large amounts of lactic acid via the glycolysis pathway. The glycolytic pathway is essential for the proliferation, invasion and metastasis of tumor cells. Lactate dehydrogenase (LDH) is mainly localized in the cytoplasm, reversibly catalyzing the conversion of pyruvate to lactic acid while NADH is oxidized to NAD + . Therefore, lactic acid fermentation is necessary to maintain the glycolytic pathway of tumor cells and the regeneration of NAD + . The human-derived LDH has three subunits, LDHA, LDHB, and LDHC. LDHA is mainly found in anaerobic tissues, such as skeletal muscle; LDHB is mainly expressed in aerobic tissues, such as myocardium; LDHC is specifically expressed in testis.
LDH是癌细胞增殖和转移的关键酶，在酿酒酵母中异源表达的人的乳酸脱氢酶将可以作为一种潜在的抗癌药物的筛选工具，研究人员可以根据药物对异源表达的人的乳酸脱氢酶的作用情况，对药物性能进行初步评价。此外将人基因文库中的基因导入到LDH酵母人源化系统可以筛选能够调节LDH活性的相关基因。LDH is a key enzyme in cancer cell proliferation and metastasis. Human lactate dehydrogenase, which is heterologously expressed in Saccharomyces cerevisiae, can be used as a potential screening tool for anticancer drugs. Researchers can use drugs to express heterologously. The role of lactate dehydrogenase, a preliminary evaluation of drug performance. In addition, introduction of genes from human gene libraries into the LDH yeast humanization system can screen for genes involved in the regulation of LDH activity.
本发明将酿酒酵母编码PDC的基因PDC1，PDC5和PDC6敲除掉，在酿酒酵母中表达来源于人的LDH并用抗霉素A来阻断电子链传递，使得人源化的乳酸发酵来代替乙醇发酵来维持糖酵解的进行。The present invention knocks out the genes PDC1, PDC5 and PDC6 of Saccharomyces cerevisiae encoding PDC, expresses human LDH in Saccharomyces cerevisiae and blocks electron chain transfer with antimycin A, so that humanized lactic acid fermentation replaces ethanol. Fermentation to maintain the progress of glycolysis.
发明内容Summary of the invention
本发明的第一个目的是提供一种乳酸脱氢酶人源化酿酒酵母，所述酿酒酵母缺失了丙酮酸脱羧酶基因，且表达人的乳酸脱氢酶。A first object of the present invention is to provide a lactate dehydrogenase humanized Saccharomyces cerevisiae which lacks a pyruvate decarboxylase gene and which expresses human lactate dehydrogenase.
所述缺失的丙酮酸脱羧酶基因是PDC1、PDC5和PDC6。The deleted pyruvate decarboxylase genes are PDC1, PDC5 and PDC6.
所述PDC1、PDC5和PDC6，在本发明的一种实施方式中，分别是NCBI上Gene ID为850733、850825、852978的基因。The PDC1, PDC5, and PDC6, in one embodiment of the present invention, are Genes with Gene IDs of 850733, 850825, and 852978, respectively, on NCBI.
所述人的乳酸脱氢酶是LDHA、LDHB或LDHC。The human lactate dehydrogenase is LDHA, LDHB or LDHC.
所述人的乳酸脱氢酶LDHA、LDHB或LDHC，在本发明的一种实施方式中，分别是NCBI 上Gene ID为3939、3945、3948的基因。The human lactate dehydrogenase LDHA, LDHB or LDHC, in one embodiment of the invention, is NCBI Genes with Gene ID 3939, 3945, 3948.
本发明的第二个目的是一种所述乳酸脱氢酶人源化酿酒酵母的构建方法。A second object of the present invention is a method for constructing the lactate dehydrogenase humanized Saccharomyces cerevisiae.
所述构建方法，在本发明的一种实施方式中，是：(1)构建PDC1、PDC5和PDC6三基因缺失菌—酿酒酵母pdc1Δpdc5Δpdc6Δ；(2)将人的乳酸脱氢酶LDHA、LDHB或LDHC连接到酵母表达质粒上，然后转化到酿酒酵母pdc1Δpdc5Δpdc6Δ中，筛选正确的转化子，即为乳酸脱氢酶人源化酿酒酵母。The constructing method, in one embodiment of the present invention, is: (1) constructing a PDC1, PDC5, and PDC6 triple gene deletion strain, Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ; (2) human lactate dehydrogenase LDHA, LDHB or LDHC It was ligated to the yeast expression plasmid and then transformed into S. cerevisiae pdc1Δpdc5Δpdc6Δ to select the correct transformant, which is lactate dehydrogenase humanized Saccharomyces cerevisiae.
所述三基因缺失菌，在本发明的一种实施方式，是以酿酒酵母W303-1A为出发菌株构建得到的。The three gene deletion bacteria, which is an embodiment of the present invention, is constructed by using Saccharomyces cerevisiae W303-1A as a starting strain.
所述构建方法，在本发明的一种实施方式，具体是：(1)以酿酒酵母W303-1A为出发菌株，通过同源重组的方法，依次敲出其PDC1、PDC5、PDC6三个基因得到酿酒酵母pdc1Δpdc5Δpdc6Δ；(2)将LDHA、LDHB或LDHC连接到pRS424TEF质粒上，得到重组质粒pRS424TEF-LDHA，pRS424TEF-LDHB和pRS424TEF-LDHC，然后通过醋酸锂/PEG的转化方法将重组质粒导入酿酒酵母pdc1Δpdc5Δpdc6Δ中，筛选，测序验证。The construction method is, in an embodiment of the present invention, specifically: (1) using Saccharomyces cerevisiae W303-1A as a starting strain, and knocking out three genes of PDC1, PDC5 and PDC6 by homologous recombination method. Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ; (2) LDHA, LDHB or LDHC was ligated into pRS424TEF plasmid to obtain recombinant plasmids pRS424TEF-LDHA, pRS424TEF-LDHB and pRS424TEF-LDHC, and then the recombinant plasmid was introduced into Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ by lithium acetate/PEG transformation method. , screening, sequencing validation.
本发明的第三个目的是提供一种利用所述乳酸脱氢酶人源化酿酒酵母表达人的乳酸脱氢酶的方法。A third object of the present invention is to provide a method for expressing human lactate dehydrogenase using the lactate dehydrogenase humanized Saccharomyces cerevisiae.
所述方法是在SD-Trp培养基中培养乳酸脱氢酶人源化酿酒酵母，并用抗霉素A来阻断电子链传递，使得人源化的乳酸发酵来代替乙醇发酵来维持糖酵解的进行。The method is to culture lactate dehydrogenase humanized Saccharomyces cerevisiae in SD-Trp medium, and use antimycin A to block electron chain transfer, so that humanized lactic acid fermentation instead of ethanol fermentation to maintain glycolysis Going on.
所述方法，在本发明的一种实施方式是：挑取酵母单菌落培养至OD660＝0.8-1.0后，洗涤细胞并重悬于新鲜培养基中，细胞生长至1.5×107-1.7×107个/mL时加入抗霉素A，继续培养22-26h。The method, in one embodiment of the present invention, is: picking yeast single colony culture to OD 660 = 0.8-1.0, washing the cells and resuspending in fresh medium, and growing the cells to 1.5×10 7 -1.7×10 Antimycin A was added at 7 mL/mL and incubation was continued for 22-26 h.
所述方法，在本发明的一种实施方式，具体是：挑取酵母单菌落在SD-Trp培养基中生长至OD660＝1.0后，细胞用无菌水洗涤后，重新悬浮新鲜SD-Trp培养基并使细胞数为1.6×107个/mL并加入终浓度为10μg/mL的抗霉素A，继续培养24h。The method, in one embodiment of the present invention, specifically: picking a yeast single colony in SD-Trp medium and growing to OD 660 = 1.0, the cells are washed with sterile water, and then resuspending fresh SD-Trp The medium was adjusted to have a cell count of 1.6 × 10 7 /mL and antibiotic A was added to a final concentration of 10 μg/mL, and culture was continued for 24 hours.
本发明还要求保护乳酸脱氢酶人源化酿酒酵母生产的乳酸脱氢酶，以及其在筛选抗癌药物、筛选能够调节乳酸脱氢酶活性的基因等方面的应用。The present invention also claims lactate dehydrogenase produced by humanized Saccharomyces cerevisiae lactate dehydrogenase, and its use in screening anticancer drugs, screening genes capable of regulating lactate dehydrogenase activity, and the like.
本发明的有益效果：采用基因同源重组的方法敲除负责编码酿酒酵母丙酮酸脱羧酶基因，将表达人的乳酸脱氢酶的重组质粒转化到酿酒酵母丙酮酸脱羧酶缺陷型菌株，28℃～32℃培养长出酿酒酵母单菌落，实现了人的乳酸菌脱氢酶的异源表达，在酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株中表达LDHA或LDHC，加入抗霉素A后，能够生长并检测到乳酸的产生。乳酸脱氢酶人源化酿酒酵母将可以作为一种潜在的筛选抗癌药物以及能够调节乳酸脱氢酶活性相关基因的工具。 The beneficial effects of the invention: knocking out the gene encoding the Saccharomyces cerevisiae pyruvate decarboxylase by gene homologous recombination, transforming the recombinant plasmid expressing human lactate dehydrogenase into the Saccharomyces cerevisiae pyruvate decarboxylase-deficient strain, 28 ° C The single colony of Saccharomyces cerevisiae was grown at ~32 °C to achieve heterologous expression of human lactic acid bacteria dehydrogenase. LDHA or LDHC was expressed in Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ three-deficient strain. After addition of antimycin A, lactic acid could be grown and detected. The production. Lactate dehydrogenase humanized Saccharomyces cerevisiae will serve as a potential screening tool for anticancer drugs and genes that regulate lactate dehydrogenase activity.
附图说明DRAWINGS
图1：乳酸脱氢酶人源化酿酒酵母在含有抗霉素A的平板上的生长情况；Figure 1: Growth of humanized S. cerevisiae lactate dehydrogenase on plates containing antimycin A;
图2：乳酸脱氢酶人源化酿酒酵母产乳酸情况的测定；Figure 2: Determination of lactate production by humanized Saccharomyces cerevisiae lactate dehydrogenase;
图3：表达产物的western blot；Figure 3: Western blot of the expression product;
图4：人的乳酸脱氢酶在酿酒酵母中的表达定位。Figure 4: Expression of human lactate dehydrogenase in S. cerevisiae.
具体实施方式detailed description
实施例1：pdc1Δpdc5Δpdc6Δ三基因缺陷菌株的构建Example 1: Construction of a pdc1Δpdc5Δpdc6ΔTrigenic Deficient Strain
乳酸脱氢酶人源化酿酒酵母的构建，包括以下步骤：The construction of lactated dehydrogenase humanized Saccharomyces cerevisiae includes the following steps:
(1)pdc6Δ缺陷型菌株的构建(1) Construction of pdc6Δ deficient strain
酿酒酵母PDC6基因序列设计引物如下：The primers for the S. cerevisiae PDC6 gene sequence design are as follows:
上游引物HXO556(序列如SEQ ID NO.1所示)：Upstream primer HXO556 (sequence shown in SEQ ID NO. 1):
Figure PCTCN2016087449-appb-000001
Figure PCTCN2016087449-appb-000001
下游引物HXO557(序列如SEQ ID NO.2所示)：Downstream primer HXO557 (sequence as shown in SEQ ID NO. 2):
Figure PCTCN2016087449-appb-000002
Figure PCTCN2016087449-appb-000002
利用上游引物和下游引物扩增得到PDC6基因，将来源于质粒pFA6a-HIS3MX6的His片段插入PDC6基因片段内部，获得含酿酒酵母PDC6基因上下游序列的敲除片段，通过醋酸锂/PEG的转化方法将PCR扩增得到的His片段导入到酿酒酵母W303-1A单倍体细胞中，并进行筛选；将转化菌株涂布到SD-His缺陷型平板上，待长出菌落后，挑取一定数目的菌落提取基因组，并进行PCR验证，并以野生型菌株的基因组进行对比，获得pdc6Δ缺陷型菌株。The PDC6 gene was amplified by the upstream primer and the downstream primer, and the His fragment derived from the plasmid pFA6a-HIS3MX6 was inserted into the PDC6 gene fragment to obtain a knock-out fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae PDC6 gene, and the method was transformed by lithium acetate/PEG. The His-fragment amplified Hisis fragment was introduced into the Saccharomyces cerevisiae W303-1A haploid cells and screened; the transformed strain was applied to the SD-His-deficient plate, and a certain number of The colonies were extracted from the genome and verified by PCR, and compared with the genome of the wild type strain to obtain a pdc6Δ deficient strain.
(2)pdc6Δpdc5Δ双缺陷菌株的构建(2) Construction of pdc6Δpdc5Δ double-deficient strain
根据酿酒酵母PDC5基因序列设计引物如下：Primers were designed based on the S. cerevisiae PDC5 gene sequence as follows:
上游引物HXO552(序列如SEQ ID NO.3所示)：Upstream primer HXO552 (sequence shown in SEQ ID NO. 3):
Figure PCTCN2016087449-appb-000003
Figure PCTCN2016087449-appb-000003
下游引物HXO553(序列如SEQ ID NO.4所示)：Downstream primer HXO553 (sequence shown in SEQ ID NO. 4):
Figure PCTCN2016087449-appb-000004
Figure PCTCN2016087449-appb-000004
利用上游引物和下游引物扩增得到PDC5基因，将来源于质粒pRS305的Leu片段插入PDC5基因片段内部，获得含酿酒酵母PDC5基因上下游序列的敲除片段，通过醋酸锂/PEG的转化方法将PCR扩增得到的Leu片段分别导入到酿酒酵母W303-1Apdc6Δ缺陷型菌株，并进 行筛选；将转化菌株涂布到SD-Leu-His缺陷型平板上，待长出菌落后，挑取一定数目的菌落提取基因组，并进行PCR验证，并以野生型菌株的基因组进行对比，从而得到pdc6Δpdc5Δ双缺陷菌株。The PDC5 gene was amplified by the upstream primer and the downstream primer, and the Leu fragment derived from the plasmid pRS305 was inserted into the PDC5 gene fragment to obtain a knock-out fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae PDC5 gene, and the PCR was carried out by a lithium acetate/PEG transformation method. The amplified Leu fragments were introduced into the Saccharomyces cerevisiae W303-1Apdc6Δ deficient strain, respectively. Screening; transforming the strains onto SD-Leu-His-deficient plates, picking up a certain number of colonies to extract the genome, and performing PCR verification, and comparing them with the genome of the wild-type strain, A pdc6Δpdc5Δ double-deficient strain was obtained.
(3)pdc1Δpdc5Δpdc6Δ三缺陷菌株的构建(3) Construction of pdc1Δpdc5Δpdc6Δ three-deficient strain
由于酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株生长比较慢，将把PDC5基因克隆到pRS316TEF(带有强启动子TEF的pRS316质粒)得到的重组质粒pRS316TEF-PDC5转化pdc6Δpdc5Δ双缺陷型菌株，得到相应的酿酒酵母重组菌株，并在此重组菌株的基础上敲除PDC1基因。Due to the slow growth of the S. cerevisiae pdc1Δpdc5Δpdc6Δ three-deficient strain, the recombinant plasmid pRS316TEF-PDC5 obtained by cloning the PDC5 gene into pRS316TEF (pRS316 plasmid with strong promoter TEF) was transformed into pdc6Δpdc5Δ double-deficient strain, and the corresponding Saccharomyces cerevisiae recombinant strain was obtained. And knock out the PDC1 gene based on this recombinant strain.
根据酿酒酵母PDC1基因序列设计引物如下：Primers were designed based on the S. cerevisiae PDC1 gene sequence as follows:
上游引物HXO548(序列如SEQ ID NO.5所示)：Upstream primer HXO548 (sequence shown in SEQ ID NO. 5):
Figure PCTCN2016087449-appb-000005
Figure PCTCN2016087449-appb-000005
下游引物HXO549(序列如SEQ ID NO.6所示)：The downstream primer HXO549 (sequence as shown in SEQ ID NO. 6):
Figure PCTCN2016087449-appb-000006
Figure PCTCN2016087449-appb-000006
利用上游引物和下游引物扩增得到PDC1基因，将来源于质粒pFA6a-kanMX6的Kan片段插入到PDC1基因内部，获得含酿酒酵母PDC1基因上下游序列的敲除片段，通过醋酸锂/PEG的转化方法将PCR扩增得到的Kan片段导入到酿酒酵母pdc6Δpdc5Δ/pRS316TEF-PDC5缺陷型菌株，并进行筛选；将转化菌株涂布到含有G418抗生素的SD-Leu-His缺陷型平板上，待长出菌落后，挑取一定数目的菌落提取基因组，并进行PCR验证，并以野生型菌株的基因组进行对比，从而得到pdc1Δpdc6Δpdc5Δ/pRS316TEF-PDC5三缺陷菌株。最后在含有500μg/ml 5-氟乳清酸的SD培养基消除掉pRS316TEF-PDC5质粒，得到pdc1Δpdc5Δpdc6Δ三缺陷菌株。The PDC1 gene was amplified by the upstream primer and the downstream primer, and the Kan fragment derived from the plasmid pFA6a-kanMX6 was inserted into the PDC1 gene to obtain a knock-out fragment containing the upstream and downstream sequences of the Saccharomyces cerevisiae PDC1 gene, and the method of converting lithium acetate/PEG was carried out. The Kan fragment amplified by PCR was introduced into the Saccharomyces cerevisiae pdc6Δpdc5Δ/pRS316TEF-PDC5-deficient strain and screened; the transformed strain was applied to the SD-Leu-His-deficient plate containing G418 antibiotic, and the growth was delayed. A certain number of colony-extracted genomes were picked and verified by PCR and compared with the genome of the wild-type strain to obtain a pdc1Δpdc6Δpdc5Δ/pRS316TEF-PDC5 trideficient strain. Finally, the pRS316TEF-PDC5 plasmid was eliminated in SD medium containing 500 μg/ml of 5-fluoroorotic acid to obtain a pdc1Δpdc5Δpdc6Δ trideficient strain.
实施例2：乳酸脱氢酶人源化酿酒酵母的构建Example 2: Construction of humanized Saccharomyces cerevisiae with lactate dehydrogenase
将通过PCR扩增的LDHA，LDHB和LDHC基因分别插入到pRS424TEF质粒上，得到重组质粒pRS424TEF-LDHA，pRS424TEF-LDHB和pRS424TEF-LDHC。通过醋酸锂/PEG的转化方法分别将重组质粒pRS424TEF-LDHA，pRS424TEF-LDHB和pRS424TEF-LDHC转到酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株，将转化菌株涂布在SD-Trp培养基中，30℃培养长出单菌落，经测序验证，即得到分别表达LDHA、LDHB、LDHC的乳酸脱氢酶人源化酿酒酵母。The LDHA, LDHB and LDHC genes amplified by PCR were inserted into the pRS424TEF plasmid, respectively, to obtain recombinant plasmids pRS424TEF-LDHA, pRS424TEF-LDHB and pRS424TEF-LDHC. The recombinant plasmids pRS424TEF-LDHA, pRS424TEF-LDHB and pRS424TEF-LDHC were transferred to the Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ three-defective strain by the method of lithium acetate/PEG, and the transformed strain was coated in SD-Trp medium and grown at 30 °C. Single colonies, verified by sequencing, obtained lactated dehydrogenase humanized Saccharomyces cerevisiae expressing LDHA, LDHB and LDHC, respectively.
实施例3：人的乳酸脱氢酶在pdc1Δpdc5Δpdc6Δ三缺陷菌株中的表达 Example 3: Expression of human lactate dehydrogenase in pdc1Δpdc5Δpdc6Δ triple-deficient strain
(1)将表达LDHA、LDHB、LDHC的乳酸脱氢酶人源化酿酒酵母，在含有和不含有抗霉素A的平板上划线后培养，以导入pRS424TEF的三缺陷菌株为对照，结果如图1所示。结果显示，加入抗霉素A后，LDHA或LDHC能够维持酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株的生长，LDHB不能弥补酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株的生长。(1) Humanized Saccharomyces cerevisiae expressing lactate dehydrogenase of LDHA, LDHB, and LDHC, streaked on a plate containing and without antimycin A, and then introduced into a three-defective strain of pRS424TEF as a control. Figure 1 shows. The results showed that LDHA or LDHC could maintain the growth of S. cerevisiae pdc1Δpdc5Δpdc6Δ trideficient strain after the addition of antimycin A, and LDHB could not compensate for the growth of S. cerevisiae pdc1Δpdc5Δpdc6Δ three-deficient strain.
(2)挑取乳酸脱氢酶人源化酿酒酵母单菌落在SD-Trp培养基中生长至OD660约为1.0后，细胞用无菌水洗涤后，重新悬浮在5mL新鲜配置的SD-Trp培养基并使细胞数为1.6×107/mL并加入终浓度为10μg/mL的抗霉素A。继续培养24h后，将培养液在转速2,100×g下离心5min，上清液经0.45μm滤膜过滤后，用HPLC进行检测乳酸的产生。HPLC检测条件：Aminex HPX-87H分析柱(300mm×7.8mm，Bio-Rad)，流动相为5mM H2SO4，流速为0.6mL/min，柱温为50℃，二极管阵列检测器，检测波长为210nm。HPLC检测结果如图2所示，结果表明，在酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株中表达LDHA或LDHC，加入抗霉素A后，能够检测到乳酸的产生。同时对乳酸脱氢酶人源化酿酒酵母表达产物进行了western blot检测，结果显示，LDHA、LDHB、LDHC都能得到正常表达(如图3)。(2) Picking lactate dehydrogenase Humanized Saccharomyces cerevisiae Single colonies were grown in SD-Trp medium until OD 660 was about 1.0. After washing with sterile water, the cells were resuspended in 5 mL of freshly prepared SD-Trp. The medium was adjusted to have a cell count of 1.6 × 10 7 /mL and antimycin A was added to a final concentration of 10 μg/mL. After continuing to culture for 24 hours, the culture solution was centrifuged at 2,100 × g for 5 min, and the supernatant was filtered through a 0.45 μm filter, and the production of lactic acid was detected by HPLC. HPLC detection conditions: Aminex HPX-87H analytical column (300 mm × 7.8 mm, Bio-Rad), mobile phase 5 mM H 2 SO 4 , flow rate 0.6 mL / min, column temperature 50 ° C, diode array detector, detection wavelength It is 210 nm. The results of HPLC detection are shown in Fig. 2. The results showed that LDHA or LDHC was expressed in the S. cerevisiae pdc1Δpdc5Δpdc6Δ three-defective strain, and the production of lactic acid was detected after the addition of antimycin A. At the same time, western blot analysis was performed on the expression product of humanized Saccharomyces cerevisiae lactate dehydrogenase. The results showed that LDHA, LDHB and LDHC could be expressed normally (Fig. 3).
为了研究人的乳酸脱氢酶(LDHA、LDHB、LDHC)在酿酒酵母细胞中的表达定位情况，将LDHA、LDHB、LDHC的C端分别与红色荧光蛋白RFP进行融合表达。结果显示：LDHA-RFP和LDHC-RFP融合蛋白均定位于细胞质中，然而LDHB-RFP融合蛋白在酵母中表达形成异常的泡状结构(如图4)。此外结果显示当LDHA和LDHC与RFP进行融合表达时，RFP不会影响LDHA和LDHC的活性，仍然能维持酿酒酵母pdc1Δpdc5Δpdc6Δ三缺陷菌株的生长。In order to study the expression and localization of human lactate dehydrogenase (LDHA, LDHB, LDHC) in Saccharomyces cerevisiae cells, the C-termini of LDHA, LDHB and LDHC were fused to the red fluorescent protein RFP, respectively. The results showed that both the LDHA-RFP and LDHC-RFP fusion proteins were localized in the cytoplasm, whereas the LDHB-RFP fusion protein expressed in yeast to form an abnormal vesicular structure (Fig. 4). In addition, the results showed that when LDHA and LDHC were expressed in fusion with RFP, RFP did not affect the activity of LDHA and LDHC, and still maintained the growth of S. cerevisiae pdc1Δpdc5Δpdc6Δ three-deficient strain.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。 Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.
Figure PCTCN2016087449-appb-000007
Figure PCTCN2016087449-appb-000007
Figure PCTCN2016087449-appb-000008
Figure PCTCN2016087449-appb-000008
Figure PCTCN2016087449-appb-000009
Figure PCTCN2016087449-appb-000009

Claims (9)

  1. 一种乳酸脱氢酶人源化酿酒酵母，其特征在于，所述酿酒酵母缺失了丙酮酸脱羧酶基因，且表达人的乳酸脱氢酶。A lactate dehydrogenase humanized Saccharomyces cerevisiae characterized in that the Saccharomyces cerevisiae lacks a pyruvate decarboxylase gene and expresses a human lactate dehydrogenase.
  2. 根据权利要求1所述的酿酒酵母，其特征在于，所述缺失的丙酮酸脱羧酶基因是PDC1、PDC5和PDC6；所述人的乳酸脱氢酶是LDHA、LDHB或LDHC。The Saccharomyces cerevisiae according to claim 1, wherein the deleted pyruvate decarboxylase gene is PDC1, PDC5 and PDC6; and the human lactate dehydrogenase is LDHA, LDHB or LDHC.
  3. 根据权利要求2所述的酿酒酵母，其特征在于，所述PDC1、PDC5和PDC6分别是NCBI上登录号为850733、850825、852978的基因；所述人的乳酸脱氢酶是LDHA、LDHB或LDHC分别是NCBI上登录号为3939、3945、3948的基因。The Saccharomyces cerevisiae according to claim 2, wherein the PDC1, PDC5 and PDC6 are genes of accession numbers 850733, 850825, 852978 on NCBI, respectively; the human lactate dehydrogenase is LDHA, LDHB or LDHC These are the genes for accession numbers 3939, 3945, and 3948 on NCBI.
  4. 一种权利要求1-3任一所述酿酒酵母的构建方法，其特征在于，所述方法是：(1)构建PDC1、PDC5和PDC6三基因缺失菌—酿酒酵母pdc1Δpdc5Δpdc6Δ；(2)将人的乳酸脱氢酶LDHA、LDHB或LDHC连接到酵母表达质粒上，然后转化到酿酒酵母pdc1Δpdc5Δpdc6Δ中，筛选正确的转化子，即为乳酸脱氢酶人源化酿酒酵母。A method for constructing a Saccharomyces cerevisiae according to any one of claims 1 to 3, which is characterized in that: (1) constructing a PDC1, PDC5 and PDC6 triple gene deletion bacteria - Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ; (2) Lactate dehydrogenase LDHA, LDHB or LDHC was ligated to the yeast expression plasmid and then transformed into S. cerevisiae pdc1Δpdc5Δpdc6Δ to screen for the correct transformant, namely lactate dehydrogenase humanized Saccharomyces cerevisiae.
  5. 根据权利要求4所述的方法，其特征在于，所述方法具体是：(1)以酿酒酵母W303-1A为出发菌株，通过同源重组的方法，敲除其PDC1、PDC5、PDC6三个基因得到酿酒酵母pdc1Δpdc5Δpdc6Δ；(2)将LDHA、LDHB或LDHC连接到pRS424TEF质粒上，得到重组质粒pRS424TEF-LDHA、pRS424TEF-LDHB或pRS424TEF-LDHC，然后通过醋酸锂/PEG的转化方法将重组质粒导入酿酒酵母pdc1Δpdc5Δpdc6Δ中，筛选，测序验证。The method according to claim 4, wherein the method is specifically: (1) using Saccharomyces cerevisiae W303-1A as a starting strain, knocking out three genes of PDC1, PDC5 and PDC6 by homologous recombination The Saccharomyces cerevisiae pdc1Δpdc5Δpdc6Δ was obtained; (2) LDHA, LDHB or LDHC was ligated to the pRS424TEF plasmid to obtain the recombinant plasmid pRS424TEF-LDHA, pRS424TEF-LDHB or pRS424TEF-LDHC, and then the recombinant plasmid was introduced into Saccharomyces cerevisiae by the conversion method of lithium acetate/PEG. In pdc1Δpdc5Δpdc6Δ, screening, sequencing verification.
  6. 一种利用权利要求1所述酿酒酵母表达人的乳酸脱氢酶的方法，其特征在于，所述方法是在SD-Trp培养基中培养乳酸脱氢酶人源化酿酒酵母，并用抗霉素A来阻断电子链传递，使得人源化的乳酸发酵来代替乙醇发酵来维持糖酵解的进行。A method for expressing human lactate dehydrogenase by using the Saccharomyces cerevisiae according to claim 1, wherein the method comprises culturing lactate dehydrogenase humanized Saccharomyces cerevisiae in SD-Trp medium, and using antimycin A to block the transmission of electron chains, so that humanized lactic acid fermentation instead of ethanol fermentation to maintain glycolysis.
  7. 权利要求1-3任一所述乳酸脱氢酶人源化酿酒酵母生产的乳酸脱氢酶。A lactate dehydrogenase produced by the human lactated yeast of lactate dehydrogenase according to any one of claims 1 to 3.
  8. 权利要求7所述乳酸脱氢酶在筛选抗癌药物方面的应用。Use of the lactate dehydrogenase of claim 7 for screening anticancer drugs.
  9. 权利要求7所述乳酸脱氢酶在筛选调节乳酸脱氢酶活性的基因的应用。 The use of the lactate dehydrogenase of claim 7 for screening for a gene regulating lactate dehydrogenase activity.
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