KR20060040494A - Method for screening a substance capable of inhibiting either or both of the mevalonate pathway and nonmevalonate pathway using a host cell transformed with a dna encoding all enzymes associated with a foreign mevalonate pathway and having an inactivated indogenous nonmevalonate pathway - Google Patents

Abstract

In the present invention, a mevalonic acid pathway of a cell having a mevalonic acid pathway in a host cell having only a non-mevalonic acid pathway as a biosynthetic pathway of isopentenyl diphosphate (IPP) and one or more genes of the non-mevalonic acid pathway is inactivated Transformed host cells which have foreign mevalonic acid pathways as biosynthetic pathways of isopentenyl diphosphate (IPP), which are obtained by introducing DNA encoding all enzymes involved in Mevalonic acid comprising the step of culturing host cells in the presence of a test substance having only the non-mevalonic acid pathway as a biosynthetic pathway of isopentenyl diphosphate (IPP) and the gene of the non-mevalonic acid pathway is inactivated How to search for substances that inhibit either or both pathways and nonmevalonic acid pathways Provided.

Mevalonic acid, mevalonic acid pathway, nonmevalonic acid pathway

Description

The intrinsic nonmevalonic acid pathway is inactivated and inhibits either or both of the mevalonic acid pathway and the nonmevalonic acid pathway using a host cell transformed with DNA that encodes all enzymes involved in the foreign mevalonic acid pathway. {Method for screening a substance capable of inhibiting either or both of the mevalonate pathway and nonmevalonate pathway using a host cell transformed with a DNA encoding all enzymes associated with a foreign mevalonate pathway and having an inactivated indogenous nonmevalonate pathway}

1 is a diagram showing a mevalonic acid pathway and a non-mevalonic acid pathway as a biosynthetic pathway of IPP.

Figure 2 is a view showing the result of culturing the melononic acid E. coli ΔispC strain of the present invention in the presence of fosmidomycin or simvastatin.

In the present invention, either or both of the mevalonic acid pathway and the non-mevalonic acid pathway using a transformed host cell into which the intrinsic non-mevalonic acid pathway is inactivated and DNA encoding all enzymes related to the foreign mevalonic acid pathway are introduced. It relates to a method of searching for a substance that inhibits the.

Isoprenoids are a group of a wide variety of natural products having a common biosynthetic origin, i.e., isopentenyl diphosphate (IPP), which is a single precursor. The group of isoprenoid natural products includes all substances derived biosynthetically from the five carbon compound IPP. Isoprenoid is also called terpene or terpenoid.

As the biosynthetic pathway of isoprenoids, the mevalonic acid pathway and the nonmevalonic acid pathway are known. The mevalonic acid pathway is most eukaryotic (e.g. Saccharomyces cerevisiae ), the cytoplasm of plant cells, some bacteria (e.g. Streptococcus pneumoniae ) and paracoccal zeaxanthinipaci Paracoccus zeaxanthinifaciens ) and malaria cells. The nonmevalonic acid pathway is present in most bacteria (eg, E. coli), the plastids of plant cells. Plant cells and actinomycetes are known to have both of these pathways.

Several steps related to the mevalonic acid pathway have been known. The known mevalonic acid pathway consists of a reaction step by acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVA kinase, MVAP kinase, MVAPP decarboxylase and IPP isomerase ( See FIG. 1A). In Gram-positive bacteria Streptococcus pneumoniae, the enzyme is known to be encoded by the atoB , mvaS , mvaA , mvaK1 , mvaK2 and mvaD genes, respectively. In addition, mvaK1 , mvaK2 and mvaD genes (Genbank accession No. AF290098) and mvaS and mvaA genes (Genbank accession No. AF290099) are each present on the chromosome with one operon. In addition, the Gram-negative bacterium Paracacus zeaxanthinipanciens In Paracoccus zeaxanthinifaciens , enzymes involved in IPP synthesis from acetoacetyl-CoA are encoded by the mvaS , mvaA , mvaK1 , mvaK2 and mvaD genes, all of which form one operon (Genbank accession No. AJ431696). Known. Further, in the eukaryotic cell is Saccharomyces process as MY three Levy jiae, the enzyme according to the IPP is synthesized from acetoacetyl -CoA is HMGS (Genbank accession No. X96617), HMG1 (Genbank accession No. M22002), ERG12 (Genbank accession No X55875), ERG8 (Genbank accession No. M63648) and ERG19 (Genbank accession No. X97557) genes, each known to exist in separate operons.

Several steps related to the conventional nonmevalonic acid pathway have also been known. The known nonmevalonic acid pathway consists of a reaction step by DXP synthase, DXP reductoisomerase, CDP-ME synthase, CDP-ME kinase, MECP synthase, HMBPP synthase and HMBPP reductase (FIG. 1B). In E. coli, a gram-negative bacterium, the enzymes are dxs (Genbank accession No. AF035440), ispC (Genbank accession No. AB013300), ispD (Genbank accession No. AF230736), ispE (Genbank accession No. AF216300), and ispF (Genbank accession). No. AE000219), ispG (Genbank accession No. AE000338) and ispH (Genbank accession No. AE000113) genes are known to be encoded.

In order to search for substances that specifically inhibit the mevalonic acid pathway or the non-mevalonic acid pathway, the enzymes involved in each pathway are isolated and the enzyme activity is inhibited by reacting the isolated enzyme with the test substance in vitro . By determining whether or not. In addition, the method of searching for a substance that specifically inhibits the mevalonic acid pathway or the non-mevalonic acid pathway may be made by using a variant strain having a mutation in a gene encoding an enzyme related to the mevalonic acid pathway or the non-mevalonic acid pathway. have. For example, WO 02/31120 discloses transduced bacterial host cells that can be used to search for novel antibiotics and herbicides and methods for searching for antibiotics and herbicides using the same. The transduced bacterial host cell comprises a disrupted first intrinsic gene of the nonmevalonic acid pathway and a transgene that functionally replaces the disrupted first gene. The transduced bacterial host cell may also be introduced with a mini operon comprising an essential gene of the mevalonic acid (MVA) pathway. The method of searching for antibiotics and herbicides using the transduced bacterial host cells includes culturing the strain in a medium comprising a chemical supplement to rescue the disrupted first gene. According to this method, antibiotics and herbicides which specifically inhibit the non-mevalonic acid pathway can be searched by culturing the transduced bacterial host cells in the presence of a test substance. However, only substances that specifically inhibit the non-mevalonic acid pathway can be searched for, and the medium always contains a chemical supplement capable of rescue of the destroyed first gene and mevalonic acid for the action of the mini-operon. Should be U.S. Patent Publication No. 2003/0170662 discloses a method for searching for substances that specifically inhibit the non-mevalonate pathway by using individuals that can use both the mevalonate and non-mevalonate pathways as biosynthetic pathways of IPP and DMAPP. It is starting. Individuals that can use both the mevalonate and non-mevalonate pathways are those who can use the mevalonate route only in the presence of mevalonic acid or those who can use the mevalonate route in the absence. Specifically, the search method is to culture the individual that can use the mevalonate route only in the presence of mevalonic acid in the medium containing the test substance in the presence or absence of mevalonic acid, grow in the presence of mevalonic acid, but in the absence of mevalonic acid Substances that do not grow are selected as substances that specifically inhibit the non-mevalonic acid pathway. In addition, the search method is cultured in the medium containing the test substance in the absence of mevalonic acid and transformed individuals that can use the mevalonate pathway in the absence of mevalonic acid and wild strain having only the non-mevalonic acid pathway, mevalonic acid Substances that inhibit only the growth of wild strains without pathways are selected as substances that specifically inhibit the non-mevalonic acid pathway. According to this method, when searching for a substance that specifically inhibits the non-mevalonic acid pathway using one cell, mevalonic acid should be added to the medium, and mevalonic acid should be added when using two types of cells. There is no. However, the present method can only search for substances that specifically inhibit the non-mevalonic acid pathway in both cases.

Therefore, there is still a need for a method of searching for a substance that specifically inhibits the mevalonic acid pathway, while at the same time searching for a substance that specifically inhibits the non-mevalonic acid pathway.

It is an object of the present invention to employ either or both of the mevalonic acid pathway and the non-mevalonic acid pathway using strains in which the intrinsic non-mevalonic acid pathway is inactivated and DNA encoding all enzymes involved in the foreign mevalonic acid pathway is introduced. It is to provide a method for searching for inhibitors.

One aspect of the present invention is a biosynthetic pathway of isopentenyl diphosphate (IPP) of a cell having a mevalonic acid pathway in a host cell having only the non-mevalonic acid pathway and at least one gene of the non-mevalonic acid pathway is inactivated. The biosynthetic pathway of isopentenyl diphosphate (IPP), obtained by introducing DNA encoding all enzymes involved in the mevalonic acid pathway, has a foreign mevalonic acid pathway, and the intrinsic nonmevalonic acid pathway is inactivated and transformed. Culturing host cells in the presence of a test substance having only the non-mevalonic acid pathway as a biosynthetic pathway of the host cell and isopentenyl diphosphate (IPP) and the gene of the non-mevalonic acid pathway is inactivated. Search for substances that inhibit either or both of the mevalonic acid pathway and the non-mevalonic acid pathway Provide a way to.

The host cell used in the method of the present invention contains only the non-mevalonic acid pathway as the biosynthetic pathway of isopentenyl diphosphate (IPP), and any one of the non-mevalonic acid pathways is inactivated. . For example, E. coli, Salmonella typimurium , and Plasmodium falsiparum are included, but are not limited to these examples.

The host cell used in the method of the present invention is one or more genes of the intrinsic nonmevalonic acid pathway are inactivated. Methods of inactivating genes are well known in the art. For example, site specific mutagenesis and mutagenesis by homologous recombination may be used. According to the method of mutagenesis by homologous recombination, DNA containing the gene to be inactivated and homologous sequence but partly deleted is introduced into the host cell, homologous recombination occurs within the cell, and then the inactivated cell is removed. It can manufacture by selecting. The gene to be inactivated may be a gene encoding any enzyme involved in the intrinsic nonmevalonic acid pathway. For example, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, DXP reductoisomerase, CDP-methyl-D-erythritol-4-phosphate (CDP-ME) synthetase , CDP-ME kinase, MECDP synthase, HMB-PP synthase and HMB-PP reductase. These DNAs include, for example, E. coli dxs (Genbank accession No. AF035440), ispC (Genbank accession No. AB013300), ispD (Genbank accession No. AF230736), ispE (Genbank accession No. AF216300), ispF (Genbank). accession No. AE000219), ispG (Genbank accession No. AE000338) and ispH (Genbank accession No. AE000113) genes, preferably ispC (Genbank accession No. AB013300).

The transformed host cell used in the method of the present invention is a DNA encoding all the enzymes involved in the mevalonic acid pathway of a cell having a mevalonic acid pathway in a host cell in which one or more genes of the non-mevalonic acid pathway are inactivated. It is obtained by introduction. The cells having the mevalonic acid pathway are not particularly limited as long as they have the mevalonic acid pathway, and for example, Streptococcus pneumoniae, Paracoccus zeaxanthinipassiens and Saccharomyces cerevisiae may be included. In the present invention, "all enzymes involved in the mevalonic acid pathway" refers to all enzymes involved in synthesizing isopentenyl diphosphate (IPP) from acetoacetyl-CoA as essential enzymes involved in the mevalonic acid pathway. do. Thus, the DNA encoding all of the enzymes involved in the mevalonic acid pathway is (a) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5), (b) HMG-CoA reductase (EC 1.1.1.34), (c) mevalonate kinase (EC 2.7.1.36), (d) phosphomevalonate kinase (EC 2.7.4.2) and (e) mevalonate diphosphate dica DNA encoding the reboxylase (EC 4.1.1.33). The DNA may further include DNA encoding acetoacetyl CoA thiolase (EC 2.3.1.9) and isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2). DNA encoding all the enzymes involved in the mevalonic acid pathway is, for example, phbA gene from Alcaligenes eutrophus H16 (Genbank accession No. J04987), mvaS from Streptococcus pneumoniae , mvaA , mvaK1, mvaK2 and mvaD idi gene and the gene of Escherichia coli-derived DNA containing (Genbank accession No. AF119715), Paracoccus claim aksan Tiny Pacific Enschede of mvaS, mvaA, mvaK1, mvaK2, DNA containing the idi gene and mvaD, Or DNA including HMGS , HMG1 , ERG12 , ERG8 , ERG19 and E. coli-derived idi gene (Genbank accession No. AF119715) from Saccharomyces cerevisiae.

Methods of introducing DNA into host cells are well known in the art. For example, transformation, transfection, transduction, electroporation and particle collision may be used, but are not limited to these examples. The DNA to be introduced includes not only the coding sequence but also regulatory sequences located upstream (5′-noncoding sequence), inside and downstream (3′-noncoding sequence) of the coding sequence. The regulatory sequence affects transcription, RNA processing or stability, or translation of the associated coding sequence. Such regulation may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The DNA to be introduced may be introduced into the host cell while being inserted into the DNA itself or the carrier. Such carriers may include “plasmids”, “vectors” and “cassettes,” which refer to extrachromosomal elements that often have genes that are not part of the cell's central metabolism and are usually double-stranded circular DNA fragments. As used herein, "plasmid", "vector" and "cassette" are used interchangeably. The element is a unique construct from which a large number of nucleotide sequences are linked or recombined and is an independently replicating sequence derived from any source capable of introducing into the cell DNA encoding a selected gene product with a promoter and a suitable 3 ′ untranslated sequence. , Genomic insertion sequences, phage or nucleotide sequences, linear or circular, single or double stranded DNA or RNA. Preferably, the vector is pBAD24, pSTV28, or pBBR1MCS.

The method of the present invention inhibits the growth of the transformed host cell but does not inhibit the growth of the host cell in which the gene of the non-mevalonic acid pathway is inactivated is a candidate that specifically inhibits the mevalonic acid pathway. The method may further include selecting a material.

In addition, the test method of the present invention does not inhibit the growth of the transformed host cell, but the test substance that inhibits the growth of the host cell in which the gene of the non-mevalonic acid pathway is not inactivated, specifically inhibits the non-mevalonic acid pathway. The method may further include selecting a candidate substance to inhibit.

In addition, the test method of the present invention inhibits both the growth of the transformed host cell and the host cell in which the gene of the non-mevalonic acid pathway is not inactivated inhibits both the mevalonic acid pathway and the non-mevalonic acid pathway. The method may further include selecting a candidate substance.

In the method of the present invention, the host cell in which the gene of the non-mevalonic acid pathway is not inactivated has only the non-mevalonic acid pathway as a biosynthetic pathway of isopentenyl diphosphate (IPP), and one of the non-mevalonic acid pathways. The empty vector containing no DNA may be introduced into a vector used to introduce a DNA encoding all enzymes involved in the mevalonic acid pathway of a cell having a mevalonic acid pathway to a host cell in which the above gene is inactivated. have. In the present invention, a host cell in which the gene of the non-mevalonic acid pathway is not inactivated is used as a control culture and is an experimental group for searching for non-mevalonic acid specific inhibitors.

One embodiment of the method of the present invention is as follows. In this embodiment, the intrinsic non-mevalonic acid pathway is inactivated by the inactivation of the ispC gene, and the mevalonic acid pathway derived from Streptococcus pneumoniae, Paracoccus zanthic acid panifaciens , or Saccharomyces cerevisiae E. coli (hereinafter referred to as "mevalonic Escherichia coli ΔispC strain") into which DNA encoding all the enzymes involved in E. coli is introduced, and E. coli (hereinafter referred to as "control E. coli") into which only an empty vector except the DNA is introduced is used. First, 20 μl of approximately 200 μl of LB medium and test substance were dispensed into 2 wells in a 96-well or 384-well plate using a separator.Then, cultures of Mevalonic Escherichia coli ΔispC strain and Control Escherichia coli 20, respectively. Inoculate μl. At this time, the initial cell concentration is adjusted to a concentration corresponding to an OD ₆₀₀ value of 0.1, and the cell concentration is measured at regular time intervals (eg, 6 hours and 12 hours after incubation) using a microplate reader. As a negative control, 20 μl of distilled water is added in place of the test substance, and as a positive control, simvastatin and fosmidomycin are added to the E. coli ΔispC strain and the control strain, respectively. As a result of the culture, the degree to which the test substance inhibits the growth of each strain is determined in comparison with the control group, respectively. As a result of the culture, the substance which inhibited growth only in the microwell plate inoculated with the E. coli ΔispC strain was determined as the mevalonic acid pathway specific inhibitor, and the substance which inhibited the growth only in the microwell plate inoculated with the control E. coli strain The lonic acid pathway is specifically identified as an inhibitor, and the substances that inhibit growth in both microwell plates are identified as candidates that inhibit both the mevalonic acid pathway and the non-mevalonic acid pathway.

The present invention also provides a cell of a cell having a mevalonic acid pathway in a host cell having only the non-mevalonic acid pathway as a biosynthetic pathway of isopentenyl diphosphate (IPP) and in which at least one gene of the non-mevalonic acid pathway is inactivated. A transgenic host that has a foreign mevalonic acid pathway as the biosynthetic pathway of isopentenyl diphosphate (IPP) obtained by introducing DNA encoding all enzymes involved in the lonic acid pathway, and the intrinsic nonmevalonic acid pathway is inactivated. As a cell,

MvaS and mvaA (Genbank accession No. AF290099), mvaK1 , mvaK2 and mvaD (Genbank accession No. AF290098) of Streptococcus pneumoniae in Escherichia coli in which ispC (Genbank accession No. AB013300) gene of the non-mevalonic acid pathway is inactivated , alkali presence oil Trojan crispus H16 phbA gene derived from the origin (Genbank accession No. J04987), and the idi gene derived from E. coli (Genbank accession No. AF119715) with the DNA, Paracoccus claim aksan Tiny Pacific Enschede of mvaS, mvaA, mvaK1 , mvaK2, mvaD and idi genes (Genbank accession No. AJ431696) and a DNA having a saccharide in the My process three Levy jiae HMGS (Genbank accession No. X96617), HMG1 (Genbank accession No. M22002), ERG12 (Genbank accession No X55875), ERG8 (Genbank accession No. M63648), ERG19 (Genbank accession No. X97557) gene and E. coli-derived idi gene (Genbank accession No. AF119715) obtained by introducing a DNA selected from the group consisting of Provides host cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

In the following embodiment of the present invention, DNA encoding the enzymes associated with the mevalonic acid pathway from Streptococcus pneumoniae, Paracoccus t. Antifaciens, or Saccharomyces cerevisiae, This was introduced into Escherichia coli in which the intrinsic non-mevalonic acid pathway was inactivated by the deletion of the ispC gene, thereby preparing E. coli with the intrinsic non-mevalonic acid pathway inactivated and the foreign mevalonic acid pathway. Next, the transformed Escherichia coli and Escherichia coli having an intrinsic non-mevalonic acid pathway transformed with an empty vector except for the DNA are cultured in the presence of a known mevalonic acid pathway or a inhibitor of the vimevalonic acid pathway. It was checked for usefulness. Hereinafter, the molecular biological method used to prepare the recombinant strain of the present invention in this Example used a known method (J. Sambrook & DW Russel, (Ed.) Molecular Cloning 3rd ed., Cold Spring Harbor Laboratory Press).

Example 1: Preparation of a vector comprising DNA encoding all enzymes involved in Streptococcus pneumoniae-derived mevalonic acid pathway

In this example, Gram-positive bacteria Streptococcus pneumoniae The gene coding for the enzyme involved in the mevalonic acid-derived path of mvaS, mvaA, mvaK1, mvaK2 and mvaD and alkaline presence oil Trojan crispus H16 origin of the phbA gene (Genbank accession No. J04987), and the E. coli origin of the idi gene (Genbank accession No. AF119715) was used to prepare a plurality of expression vectors.

First, Streptococcus pneumoniae PCR reactions were performed using oligonucleotides of SEQ ID NOs: 1 and 2, in which chromosomes were used as templates and KpnI and XbaI restriction enzyme cleavage sequences were introduced, respectively. As a result, Streptococcus pneumoniae The 2.8 kb size of operon DNA including the derived mvaK1, mvaK2 and mvaD genes were amplified. Next, the amplified DNA product was digested with KpnI / XbaI restriction enzyme and inserted into pBAD24 expression vector (Genbank accession No. X81837) digested with KpnI / XbaI to prepare a pDSN12D vector.

Next, PCR reactions were performed using oligonucleotides of SEQ ID NOs: 3 and 4, in which E. coli chromosome DNA was used as a template and SmaI and SphI restriction enzyme cleavage sequences were introduced, respectively. As a result, idi gene DNA (Genbank accession No. AF119715) of 0.55 kb size was amplified. Next, the amplified DNA product was digested with SmaI / SphI restriction enzyme and inserted into the pDSN12D expression vector digested with SmaI / SphI to prepare a pDSN12Didi vector.

Next, Streptococcus pneumoniae PCR reactions were performed using oligonucleotides of SEQ ID NOs: 5 and 6, in which the chromosomes were used as templates and EcoRI and KpnI restriction enzyme cleavage sequences were introduced, respectively. As a result, Streptococcus pneumoniae 2.5 kb of operon DNA (Genbank accession No. AF290099) containing the derived mvaS and mvaA genes were amplified. Next, the amplified DNA product was digested with EcoRI / KpnI restriction enzyme and inserted into the pDSN12Didi expression vector digested with EcoRI / KpnI to prepare a pDSNSA12DI vector.

Next, PCR reactions were performed using oligonucleotides of SEQ ID NOs: 7 and 8, in which the chromosomal DNA of alkaligen eutropus H16 was used as a template, and introduced with NheI and EcoRI restriction enzyme cleavage sequences, respectively. As a result, a 1.2 kb phbA gene DNA (Genbank accession No. J04987) was amplified. Next, the amplified DNA product was digested with NheI / EcoRI restriction enzyme and inserted into the pDSNSA12DI expression vector digested with NheI / EcoRI to finally prepare a pDASNSA12DI expression vector. The obtained pDASNSA12DI was Streptococcus pneumoniae DNAs encoding the mvaS, mvaA , mvaK1, mvaK2 and mvaD genes derived from the phbA derived from the alkaline gene Eutropus H16 and the idi gene derived from E. coli are included.

Streptococcus pneumoniae to pSTV28 vector (Takara Shuzo Co., Ltd.) and pBBR1MCS vector (Genbank accession No. U02374) through a process similar to the manufacturing process of the pDASNSA12DI expression vector. By introducing the DNA encoding the mvaS, mvaA, mvaK1, mvaK2 idi gene and the gene and the E. coli origin of mvaD origin was prepared pSSNSA12DI and pBSNSA12DI expression vector. The pSTV28 vector used in the preparation of the expression vector is a low copy number vector and the pBBR1MCS vector is a vector with a wide host range.

Example 2 Preparation of a Vector Containing DNA Encoding All Enzymes Associated with Paracacus zanthicipanciens Derived Mevalonic Acid Pathway

In the present embodiment, the Gram-negative bacterium Paracacus zeaxanthinipineciens A plurality of expression vectors including mvaS, mvaA , mvaK1, mvaK2, mvaD and idi , which are genes encoding enzymes involved in the derived mevalonic acid pathway, and atoB genes derived from E. coli were prepared.

First, Paracoccus zeaksan tiffany faciens PCR reactions were carried out using oligonucleotides of SEQ ID NOs: 9 and 10, in which chromosomes were used as templates and NheI and XbaI restriction enzyme cleavage sequences were introduced, respectively. As a result, Paracacus zeaksan tiffany faciens The operon DNA of 6.3 kb size including the derived mvaS, mvaA , mvaK1, mvaK2, mvaD and idi genes was amplified. Next, the amplified DNA product was digested with NheI / XbaI restriction enzyme and inserted into pBAD24 expression vector digested with NheI / XbaI to prepare a pDPZSA12DI vector.

Through a process similar to the preparation of the pDPZSA12DI expression vector, paracoccus zeaxanthin thyfaciens is also applied to the pSTV28 vector (Takara Shuzo Co., Ltd.) and the pBBR1MCS vector (Genbank accession No. U02374). Derived mvaS, mvaA , mvaK1, mvaK2, mvaD and idi gene DNA were introduced to prepare pSPZSA12DI and pBPZSA12DI expression vectors. The pSTV28 vector used in the preparation of the expression vector is a low copy number vector and the pBBR1MCS vector is a vector with a wide host range.

Example 3 Preparation of a Vector Containing DNA Encoding All Enzymes Associated with Saccharomyces cerevisiae Derived Mevalonic Acid Pathway

In this example, Saccharomyces cerevisiae, a eukaryotic cell. To prepare a plurality of types including the idi gene in the gene coding for the enzyme involved in the mevalonic acid-derived path of HMGS, HMG1, ERG12, ERG8, and ERG19, and E. coli-derived expression vector.

First, Saccharomyces cerevisiae SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and PSE and HindIII, SalI and PstI, SacI and SmaI, NcoI and EcoRI and EcoRI and SalI restriction enzyme cleavage sequences introduced, respectively 16, SEQ ID NO: 17 and 18 and SEQ ID NO: 19 and 20 oligonucleotides were used as primers, respectively, PCR reaction was performed. As a result, Saccharomyces cerevisiae Derived HMGS ( corresponds to mvaS ), HGM1 ( corresponds to mvaA ), ERG12 ( corresponds to mvaK1 ), ERG8 ( corresponds to mvaK2 ) and ERG19 ( corresponds to mvaD ) DNAs of 1.5, 1.5, 1.3, 1.3 and 1.2 kb size, including genes, were amplified, respectively.

Next, PCR reactions were performed using the oligonucleotides of SEQ ID NOs: 19 and 20, in which E. coli chromosomal DNA was used as a template and introduced with SmaI and SalI restriction enzyme cleavage sequences, respectively. As a result, idi gene DNA of 0.55 kb size was amplified.

Next, pTSCSA12DI was prepared by digesting the amplified DNA products with the corresponding restriction enzymes and inserting them into the pTrc99A vector (Genbank accession No. M22744) in the order of idi, HMGS, ERG8, ERG19, ERG12, and HGM1 .

Through the process similar to the preparation of the pTSCSA12DI expression vector, pSTV28, pBBR1MCS and pBAD24 are also derived from E. coli idi gene and Saccharomyces cerevisiae HMGS, ERG8, ERG19, ERG12 and HGM1 gene DNA derived were introduced to prepare pSSCSA12DI, pBSCSA12DI and pDSCSA12DI expression vectors.

Example 4: Inactivation of the non-mevalonic acid pathway of E. coli and introduction of enzyme genes involved in the foreign mevalonic acid pathway

In this embodiment, the expression vector having the foreign mevalonic acid pathway prepared in Examples 1 to 3 was introduced into a strain obtained by inactivating the ispC gene encoding the DXP reductoisomerase involved in the non-mevalonic acid pathway of E. coli. E. coli strains with foreign mevalonic acid pathways were prepared.

The pDSN12Didi vector of Example 1 contains mvaK1, mvaK2, mvaD and idi genes encoding enzymes of the mevalolic acid pathway involved in IPP and DMAPP synthesis from mevalonic acid. Cleavage of pDSN12Didi with BamHI and SalI restriction enzymes yields a DNA fragment containing these genes. This DNA fragment was inserted into pSTV28 vector digested with BamHI and SalI restriction enzyme to prepare pSSN12Didi vector. The obtained pSSN12Didi vector was introduced into Escherichia coli FS1576 to prepare transformed Escherichia coli. E. coli can express enzymes involved in the synthesis of IPP and DMAPP from mevalonic acid in the mevalonic acid pathway, and can be grown under a medium in which mevalonic acid is present.

Next, a mutant strain in which the kanamycin resistance gene ( aphII ) was inserted into the E. coli transformed with the pSSN12Didi vector by homologous recombination, inactivating the endogenous non-mevalonic acid pathway. PCR reaction was performed using the chromosome of E. coli as a template and oligonucleotides of SEQ ID NOs: 22 and 23 as primers. As a result, 1.2 kb of ispC gene DNA (Genbank accession No. AB013300) was amplified. Next, the amplified DNA was inserted into pBluescript SK (+) (Stratagen) expression vector digested with EcoRV to prepare a pBispC vector. SalI was digested with pUK4K (Pharmacia) to obtain kanamycin gene resistant fragments and blunt-terminated with Klenow. The kanamycin gene resistant fragment was cut into an ispC gene region with NcoI and inserted into pBispC vector blunt-terminated with Klenau to prepare pBCK. To increase the efficiency of homologous recombination, pBCK was digested with BspH1 restriction enzyme, linearized, and electroporated into E. coli FS1576 transformed with the pSSN12Didi vector. To find a transformant lacking the gene isp C, it was replicated in LB solid medium containing 50 μg / ml kanamycin and 1 mM mevalonic acid and LB solid medium containing 50 μg / ml kanamycin by replica method. Isp C deficiency strains were prepared by selecting colonies that grew only in LB solid medium containing 50 μg / ml kanamycin and 1 mM mevalonic acid, but not in LB solid medium containing only μg / ml kanamycin.

Escherichia coli obtained as a result of homologous recombination was cultured in a medium to which mevalonic acid was added and a medium to which no mevalonic acid was added. .

Next, the pDSNSA12DI vector prepared in Example 1 was introduced into the E. coli in which the endogenous non-mevalonic acid pathway was inactivated, and then subcultured in LB liquid medium to which 100 μg / ml ampicillin was added to lose the pSSN12Didi plasmid from the E. coli. It was made. The subculture was carried out five times at 37 ° C. for 12 hours. The passaged E. coli was plated in LB solid medium containing 100 μg / ml ampicillin and incubated at 37 ° C. for 24 hours. Colonies formed in the solid medium were transferred to the LB solid medium containing 100 μg / ml ampicillin and the LB solid medium containing 50 μg / ml chloramphenicol by Replica method and not grown in the LB solid medium containing 50 μg / ml chloramphenicol. Colonies growing only in LB solid medium containing 100 μg / ml ampicillin were selected. That is, E. coli (hereinafter referred to as "Mevalonic acid Escherichia coli ΔispC strain" or "MEC strain") in which the intrinsic non-mevalonic acid pathway was inactivated and the foreign mevalonic acid pathway was introduced, in which only pDSNSA12Didi was introduced, was prepared.

Example 5 Screening of Inhibitor Substances That Specifically Inhibit Any or All of the Mevalonic Acid and Non-Mevalonic Acid Pathways Using the E. coli ΔispC Strain

In this example, the mevalonic acid Escherichia coli ΔispC strain obtained in Example 4 was cultured in the presence of simvastatin and phosmidomycin, which are known as specific inhibitors of the mevalonic acid pathway and the non-mevalonic acid pathway, respectively. It was confirmed whether a substance that specifically inhibits any or all can be searched.

First, two kinds of LB agar plates (diameter 9 cm plates) to which simvastatin and fosmidomycin were added were prepared. Next, each of the LB agar plates was plated with the E. coli ΔispC strain obtained in Example 4 and E. coli introduced only with the empty vector, and incubated at 37 ° C. for 20 hours. The growth of Escherichia coli was compared from the plate obtained as a result of culture. Fig. 2 is a diagram showing the results of culturing the E. coli ΔispC strain of the present invention in the presence of fosmidomycin (A) or simvastatin (B). As shown in FIG. 2, only the mevalonic acid Escherichia coli ΔispC strain was grown on the plate added with phosmidomycin, and the growth of the mevalonic acid Escherichia coli ΔispC strain was inhibited on the plate to which simvastatin was added, and only the empty vector was introduced. Only grew.

As a result of this embodiment, it was confirmed that the inhibitor material can be specifically searched for either or both of the mevalonic acid and non-mevalonic acid pathways using the E. coli ΔispC strain and the E. coli strain introduced only with the empty vector. .

According to the method for screening a substance that inhibits any or all of the mevalonic acid and non-mevalonic acid pathways of the present invention, media of mevalonic acid or non-mevalonic acid pathways such as mevalonic acid or 2-methylerythritol-4-phosphate Without administration in the middle, it is possible to simultaneously search for inhibitors of the non-mevalonic acid pathway as well as substances that inhibit the mevalonic acid pathway or both. Therefore, according to the method of the present invention, the cost is low and efficient.

<110> Industry-Academic cooperation foundation Gyeongsang National University <120> Method for screening a substance capable of inhibiting either or          both of the mevalonate pathway and nonmevalonate pathway using a          host cell transformed with a DNA encoding all enzymes associated          with a foreign mevalonate pathway and having an inactivated          indogenous nonmevalonate pathway <130> PN056154 <160> 23 <170> KopatentIn 1.71 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cggtaccaat gacaaaaaaa gttggtgtcg g 31 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ttctagatta cgatttgtcg tcatgtcc 28 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccccgggagg agagaaatta tgcaaacgga ac 32 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgcatgctta tttaagctgg gtaaatg 27 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gggtaccgat tcctctgagt ttttatgat 29 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cggctagcag gactacacaa tgactga 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cggctagcag gactacacaa tgactga 27 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cggaattctc ctgcagttat ttgcgctc 28 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggctagcagg agagaccaga tgatttccca tac 33 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gctctagagt ttaacgcccc tcgaacgg 28 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gctgcagagg aggtgcagca tgaaactctc 30 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 caagcttatg caatcgatgg gggaa 25 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cgtcgacagg aggttatact atggaccaat tggtg 35 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctgcagtta ggatttaatg caggtgacgg 30 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgagctcagg agctgtcaat atgttattac cgtt 34 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gcccgggtta tgaagtccat ggtaaattcg 30 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cccatggaag agttgagagc cttcagtg 28 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cgaattctta tcaagataag tttccggat 29 <210> 19 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggaattcagg agccagcaca atgaacgttt acac 34 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ggagctctta ttcctttggt agaccagt 28 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ggtcgactta tttaagctgg gtaaatg 27 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 atgaagcaac tcaccattct 20 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tcagcttgcg agacgcatca cc 22  

Claims (14)

The biosynthetic pathway of isopentenyl diphosphate (IPP), which has only the nonmevalonic acid pathway and is involved in the mevalonic acid pathway of a cell having the mevalonic acid pathway in a host cell in which one or more genes of the nonmevalonic acid pathway is inactivated. Transformed host cells and isopentenyl, which have foreign mevalonic acid pathways as biosynthetic pathways of isopentenyl diphosphate (IPP), obtained by introducing DNA encoding all enzymes, and the intrinsic nonmevalonic acid pathways are inactivated A mevalonic acid pathway, comprising culturing a host cell having only the non-mevalonic acid pathway as a biosynthetic pathway of diphosphate (IPP), and wherein the gene of the non-mevalonic acid pathway is inactivated in the presence of a test substance; and A method of searching for substances that inhibit either or both of the nonmevalonic acid pathways. The method of claim 1, wherein the host cell is Escherichia coli, Salmonella typimurium or Plasmodium falsiparum . The method of claim 1, wherein the cell with the mevalonic acid pathway is Streptococcus pneumoniae , Paracoccus zeaxanthinifaciens or Saccharomyces cerevisiae . The method of claim 1, wherein the gene of the inactivated non mevalonic acid pathway is one or more genes selected from genes consisting of dxs , ispC , ispD , ispE , ispF , ispG and ispH . The method of claim 1 wherein the DNA encoding all the enzymes involved in the mevalonic acid pathway is HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase and mevalonate diphosphate dicar Method of DNA encoding a carboxylase. The method of claim 5, wherein the DNA further comprises a DNA encoding acetoacetyl-CoA thiolase or isopentenyl diphosphate isomerase. The method of claim 1, wherein the DNA encoding all the enzymes involved in the mevalonic acid pathway is the mvaS and mvaA (Genbank accession No. AF290099), mvaK1 , mvaK2 and mvaD (Genbank accession No.) of Streptococcus pneumoniae AF290098), phbA gene from Alcaligenes eutrophus H16 (Genbank accession No. J04987) and idi gene from E. coli (Genbank accession No. AF119715). The method of claim 1, wherein the DNA encoding all the enzymes involved in the mevalonic acid pathway is the mvaS , mvaA , mvaK1 , mvaK2 , mvaD and idi genes of Paracoccus zeaxanthinifaciens (Genbank accession No. AJ431696). According to claim 1, HMGS (Genbank accession No. X96617), HMG1 (Genbank accession No. M22002) of DNA in Saccharomyces has three Levy MY process jiae (Saccharomyces cerevisiae) encoding all enzymes involved in the mevalonic acid route, ERG12 (Genbank accession No. X55875), ERG8 (Genbank accession No. M63648), ERG19 (Genbank accession No. X97557) gene and E. coli-derived idi gene (Genbank accession No. AF119715). The test substance according to claim 1, wherein the test substance that inhibits the growth of the transformed host cell but does not inhibit the growth of the host cell in which the gene of the non-mevalonic acid pathway is not inactivated specifically inhibits the mevalonic acid pathway. Selecting as a candidate substance. The test substance according to claim 1, wherein the test substance which does not inhibit the growth of the transformed host cell but inhibits the growth of the host cell in which the gene of the non-mevalonic acid pathway is not inactivated specifically inhibits the non-mevalonic acid pathway. Selecting as a candidate substance. The test substance for inhibiting the growth of the transformed host cell and the host cell in which the gene of the non-mevalonic acid pathway is not inactivated is a candidate substance that inhibits both the mevalonic acid pathway and the non-mevalonic acid pathway. The method comprising the step of selecting. The host cell of claim 1, wherein the host cell of which the gene of the non-mevalonic acid pathway is not inactivated has only the non-mevalonic acid pathway as a biosynthetic pathway of isopentenyl diphosphate (IPP), and one of the non-mevalonic acid pathways. An empty vector containing no DNA is introduced into a vector used to introduce a DNA encoding all enzymes involved in the mevalonic acid pathway of a cell having a mevalonic acid pathway to a host cell in which the above gene is inactivated. Way. The biosynthetic pathway of isopentenyl diphosphate (IPP), which has only the nonmevalonic acid pathway and is involved in the mevalonic acid pathway of a cell having the mevalonic acid pathway in a host cell in which one or more genes of the nonmevalonic acid pathway is inactivated. As a biosynthetic pathway of isopentenyl diphosphate (IPP) obtained by introducing DNA encoding all enzymes, the intrinsic bivalonic acid pathway is an inactivated transformed host cell, E. coli in which the ispC (Genbank accession No. AB013300) gene of the non-mevalonic acid pathway is inactivated, mvaS and mvaA (Genbank accession No. AF290099), mvaK1 , mvaK2 and mvaD (Genbank accession) of Streptococcus pneumoniae No. AF290098), DNA having a phbA gene from Alcaligenes eutrophus H16 (Genbank accession No. J04987) and an idi gene from E. coli (Genbank accession No. AF119715), Paracacus zeaxanthinipanciens mvaS, mvaA, mvaK1, mvaK2, HMGS (Genbank accession No. X96617) of mvaD and idi genes (Genbank accession No. AJ431696) the my process three Levy jiae (Saccharomyces cerevisiae) and to the DNA having the saccharide (Paracoccus zeaxanthinifaciens), HMG1 (Genbank accession No. M22002), ERG12 (Genbank accession No. X55875), ERG8 (Genbank accession No. M63648), ERG19 (Genbank accession No. X97557) idi gene and the gene derived from E. coli (Genbank accessi A host cell obtained by introducing a DNA selected from the group consisting of DNA having on No. AF119715).

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