KR100801633B1 - Process for preparing isoprenoid through mevalonate pathway - Google Patents

Abstract

The present invention relates to a microorganism transformed by a genetic combination of enzymes associated with mevalonate pathways derived from various organisms and a method for producing isoprenoids through the mevalonate pathway using the same. The microorganisms can be used to efficiently mass produce isoprenoids from microorganisms.

Description

PROCESS FOR PREPARING ISOPRENOID THROUGH MEVALONATE PATHWAY

1 is a schematic diagram of the mevalonate pathway,

2 is a schematic of the non-mevalonate pathway.

The present invention relates to a microorganism transformed by a combination of genes encoding enzymes involved in the mevalonate pathway and a method for producing isoprenoids using the same.

Isoprenoid is a generic term for compounds composed of isoprene having 5 carbon atoms as a basic unit. It is known that there are about 30,000 kinds of isoprenoid compounds in the natural world, and the single natural product class is the most common, and these isoprenoids are electron transport systems (quinoids), cell membranes, and cell walls (sterols). ), Signaling (prenylated protein) and photosynthesis (chlorophyl) play an essential role in the maintenance of life. Many isoprenoids are also anticancer and antioxidant (carotenoids such as lycopene, beta-carotene and astaxanthin), immunopotentiation, anti-inflammatory, analgesic, and various Its function is attracting attention in relation to human physiological activities such as prevention of adult diseases (coenzyme Q10), and it is also used as a fragrance, insecticide, antibiotics (Barkovich, R. et al . , Metab. Eng ., 3, 27- 39, 2001).

Recently, as interest in mass production of useful isoprenoids has increased, studies on efficient synthetic methods have been conducted. In particular, since the isoprene unit of the isoprenoid is biosynthesized from a precursor called isopentenyl pyrophosphate (IPP) of the formula (1) in an organism, studies on how to effectively produce isopentenylpyrophosphate as a precursor It's going on.

The biosynthesis pathway of such isopentenylpyrophosphate in organisms is largely divided into the mevalonate pathway as shown in FIG. 1 and the nonnmevalonate pathway as shown in FIG. 2 (MEP (2C-methyl-D-erythritol-). 4-phosphate) pathway is known (Boucher, Y. et al . , Mol. Microbiol ., 37, 703-716, 2003), and the mevalonate pathway is found in most eukaryotes, actinomycetes, and some bacteria, including humans. The nonmevalonate pathway is found in most prokaryotes, most bacteria including Escherichia coli.

Looking specifically at the mevalonate pathway, as shown in Figure 2, isopentenylpyrophosphate is biosynthesized by acetyl-CoA as a starting material by a six step enzyme reaction:

Step 1) acetyl-CoA acetyltransferase mediated, two molecules of acetyl-CoA is bound to biosynthesize acetoacetyl-CoA (acetoacetyl-CoA) molecules;

Step 2) One molecule of acetyl-CoA and one molecule of acetoacetyl-CoA, which is mediated by hydroxymethylglutaryl-CoA (HMG-CoA) synthase, binds to hydroxymethylgluta Biosynthesis of the reel-CoA molecule;

Step 3) reducing hydroxymethylglutaryl-CoA mediated by hydroxymethylglutaryl-CoA reductase to biosynthesize the mevalonate molecule;

Step 4) phosphorylation of mevalonate, mediated by mevalonate kinase;

Step 5) phosphorylation of phosphomevalonate, mediated by phosphomevalonate kinase; And

Step 6) biosynthesis of isopentenylpyrophosphate by decarboxylation of diphosphomevaloante, mediated by diphosphomevalonate decarboxylase.

Recently, methods for enhancing the biosynthesis of isoprenoids through manipulation of the non-mevalonate pathway have been reported (Kim, SW et al. , Biotechnol. Bioeng. , 72, 408-415, 2001; Harker, M. et al., FEBS Lett ., 448, 115-119, 1999; and Farmer, WR et al . , Biotechnol. Prog., 17, 57-61, 2001), these methods are insignificant and biosynthetic by host-specific metabolic pathway regulation mechanisms used. There is a problem that the enhancement effect is limited (Martin, V. JJ et al . , Nat. Biotechnol ., 21, 796-802, 2003). To overcome this problem, studies have been reported to increase the production of isoprenoids by introducing enzymes associated with mevalonate pathways derived from Saccharomyces cerevisiae into Escherichia coli with non-mevalonate pathways. (US Patent Publication Nos. 2003/0148479; and 2004/0005678), but only one type of mevalonate pathway derived from Saccharomyces cerevisiae was selected and introduced into the host E. coli. There was a limit to the biosynthetic efficiency of tenylpyrophosphate.

Therefore, the present inventors earnestly studied a method for producing isoprenoids using a mevalonate pathway in a microorganism having a non-mevalonate pathway, and as a result, a gene encoding six enzymes involved in the mevalonate pathway was identified. The present invention was completed by finding that the isoprenoid production ability was significantly improved when introduced into microorganisms having a non-mevalonate pathway in combination with those derived from various organisms.

It is an object of the present invention to provide a process for the preparation of isoprenoids via the mevalonate pathway in microorganisms.

Another object of the present invention is to provide a transformed microorganism capable of mass production of isoprenoids.

In accordance with the above object, in the present invention (a) Staphylococcus aureus ( Metalonate kinase derived from Staphylococcus aureus ), and Mevalonate Phosphate and Phosphate diphosphate derived from Streptococcus pneumoniae Nate decarboxylase; And (b-1) acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase and hydroxymethylglutaryl-CoA synthetase derived from Enterococcus faecalis , or (b-2) Acetyl-CoA acetyltransferase from Ralstonia eutropha , and hydroxymethylglutaryl-CoA synthetase and hydroxymethylglutaryl-CoA reductase from Streptococcus pneunomiae The present invention provides a method for producing an isoprenoid, which comprises introducing a gene encoding the gene into a host microorganism, transforming the same, and culturing the transformed host microorganism to biosynthesize the isoprenoid.

According to another object, the present invention provides a transformed microorganism transformed with the enzyme genes to mass produce isoprenoids.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have described a combination of genes derived from various organisms into microorganisms having non-mevalonate pathways as genes of six enzymes associated with the mevalonate pathway, which is an intermediate of isoprenoid biosynthesis. Optimal gene combinations have been found that have been introduced to enhance the biosynthesis of isoprenoids, and this attempt was first performed in the present invention that has not been reported to date.

In the present invention, steps 1, 2 and 3 are divided into upper paths, and steps 4, 5 and 6 are divided into lower paths among the six steps of the entire mevalonate path shown in FIG. 1 for convenience. In this case, the upper path is a path for biosynthesis of mevalonate starting from acetyl-CoA, and the lower path is a path for biosynthesis of isopentenylpyrophosphate from mevalonate. Therefore, if only a combination of genes of the upper or lower mevalonate pathway related enzymes, but not the entire mevalonate pathway, is introduced into the host microorganism having the non-mevalonate pathway, the isoprenoid cannot be biosynthesized, In case of adding mevalonate to the culture, the isoprenoid can be biosynthesized by introducing only a combination of genes of lower mevalonate pathway related enzymes (Campos, N. et al. , Biochem. J., 353, 59-67, 2003). And Martin, V. JJ. Et al., Nat. Biotechnol ., 21, 797-802, 2003).

Each of the enzymes used in the present invention mediated in the mevalonate pathway, and the exemplary amino acid sequence of each enzyme and the nucleotide sequence of the gene encoding it are shown in Table 1 below.

In a preferred embodiment of the present invention, in order to identify an efficient combination of mevalonate pathway-associated enzymes, a combination of various organism-derived genes for the lower mevalonate pathway-associated enzymes is first introduced into a host microorganism, respectively. By comparing and evaluating the productivity of lycopene, one gene combination of enzymes related to the lower mevalonate pathway according to the present invention was selected (Staphylococcus aureus-derived mevalonate kinase, and Streptococcus pneumoniae-derived phosphate Nate kinase and diphosphate mevalonate decarboxylase), followed by a combination of various organism-derived genes for the enzymes associated with the upper mevalonate pathway, respectively, into the host microorganisms, resulting in the metabolo, the final product of the upper mevalonate pathway. By evaluating the productivity of Nate Two gene combinations of the top mevalonate pathway related enzymes according to the invention were selected. Therefore, the gene combination of the total mevalonate pathway related enzymes according to the present invention is a combination of the genes of the two upper mevalonate pathway related enzymes selected from the gene combination of the selected one lower mevalonate pathway related enzymes, respectively. It is obtained by combining.

Genes encoding the enzymes associated with the mevalonate pathway may be introduced into a host microorganism by inserting into an appropriate expression vector. At this time, any expression vector that is commonly used in the host microorganism selected as the expression vector may be used, and is commercialized in the art such as pUC19, pSTV28, pBBR1MCS, pBluscriptII, pBAD, pTrc99A, pET, pACYC184 and pBR322 series. Vector can be used.

Gene combinations of the enzymes associated with the mevalonate pathway according to the present invention can be introduced into a microorganism host by inserting each gene constituting the combination into one vector or by inserting the genes into two or more vectors. When two or more genes are inserted into a vector, the genes may be inserted in the form of an operon.

In addition, genes encoding the mevalonate pathway related enzymes according to the present invention can be introduced directly on the chromosome of the host microorganism, and some of the genes constituting the gene combination are introduced directly on the chromosome of the host microorganism and the rest Can also be introduced by inserting into a vector. As a method of introducing onto such a chromosome, known methods commonly used may be used, and for example, homologous recombination methods (Link, AJ et al., J. Bacteriol ., 179, 6228-6237, 1997) may be used. have.

As the host microorganism used in the present invention, any microorganism capable of biosynthesizing isoprenoids by introducing mevalonate pathway genes may be used. For example, a biosynthetic pathway of isopentenylpyrophosphate, a precursor of isoprenoids, may be used. E. coli , Bacillus sp., Pseudomonas sp., Agrobacterium sp., Rhodobacter sp. Bacteria, such as Erwinia sp., Preferably E. coli can be used.

If the host microorganism does not have the ability to convert isopentenylpyrophosphate to isoprenoids, the excess of isopentenylpyrophosphate produced in the microorganism may be toxic, causing the host to stop growing or die (Hamano, Y). Et al. , Biosci. Biotechnol. Biochem., 65, 1627-1635, 2001; and Martin, V. JJ. Et al. , Nat. Biotechnol ., 21, 797-802, 2003). Accordingly, in the present invention, a gene encoding an enzyme that biosynthesizes isoprenoids from isopentenylpyrophosphate may be further introduced into the host microorganism and transformed in accordance with the type of the final isoprenoid desired. You can choose the type of enzyme gene. Representative isoprenoids include lycopene; Carotenoids such as beta-carotene, zeaxanthin, astaxanthin and lutein; Quinones such as coenzyme qtene; Turfoids such as monoterpenoids, sesquiterpenoids, diterpenoids, and sesterterpenoids; Various steroids; And natural rubbers. In addition, the introduction of such additional genes can be performed simultaneously or sequentially with the introduction of genes encoding mevalonate pathway related enzymes.

The present invention also provides a host microorganism transformed with a combination of genes encoding the enzymes associated with the mevalonate pathway. At this time, the transformed host microorganism is transformed with plasmids pT-LYCm4 and pEFSASN, for example, E. coli, which is prepared by a preferred embodiment of the present invention and deposited with the Korea Biotechnology Research Institute Gene Bank on April 7, 2006. Converted E. coli MG1655 (Accession Number: KCTC 10932BP).

In the host microorganism of the present invention, genes encoding six enzymes involved in the mevalonate pathway are composed of various organism-derived genes capable of maximizing the production of isoprenoids. Can be produced by

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Gene Combination Selection for Lower Mevalonate Pathway Related Enzymes

Step 1) Preparation of a Recombinant Vector Comprising Gene Combinations for Lower Mevalonate Pathway Related Enzymes

MvaK1 , the gene of the mevalonate kinase mediating step 4 of the lower mevalonate pathway, mvaK2 , the gene of the mevalonate phosphatase mediating step 5, and divalbartaphate decarboxylate mediating the step 6 To amplify mvaD , a gene of the enzyme, from various organisms, Streptococcus pneumoniae ( ATCC6314), Enterococcus faecalis ( ATCC700802), Staphylococcus aureus ( ATCC35556), and Chromosome DNA of Streptococcus pyogenes ( ATCC12344) was obtained from the American Type Culture Collection (ATCC). Meanwhile, in order to amplify the genes of HMGS, PMK (ERG8), MDD (ERG19), MVK (ERG12) and HMGR, enzymes related to the entire mevalonate pathway of Saccharomyces cerevisiae , Saccharomyces cerevisiae Seth Cerebizia Chromosomal DNA was extracted from the L32Q strain (ATCC 204508) using a DNeasy Tissue Kit from Qiagen. In addition, in order to amplify idi , which is an isopentenylpyrophosphate isomerase gene for increasing productivity of isopentenylpyrophosphate, using a DNiae kit (DNeasy Kit) of Qiagen from E. coli MG1655 (ATCC 700926) Chromosomal DNA was extracted.

The amplification of each gene was performed by polymerase chain reaction (PCR) using pfu polymerase (Promega) using the chromosomal DNA as a template, and the amplified gene was purified by DNA purification kit (Qiagen) and then Eco. The nucleotide sequence was confirmed by inserting into a pBluescriptII KS vector (Stratagene) cut with RV.

Forward and reverse primers used in the polymerase chain reaction, the base sequence information of the gene encoding the enzyme related to the methalonate sub-path of each organism is NCBI (National Center for Biotechnology Information, http: //www.ncbi. nlm.nih.gov/) was prepared based on this, and the base sequence of the forward and reverse primers used for amplification of each gene and the types of restriction enzymes contained in the primers are shown in Table 2 below.

First, a Streptococcus pneumoniae chromosome DNA template and SEQ ID NO: 9, and using the 10 primers having the nucleotide sequence of were amplified mvaK1, mvaD and mvaK2 operon, an amplified gene fragment was treated with Kpn I and Xba I The plasmid pDSN12D was then prepared by inserting the pBAD24 vector (Guzman, LM et al . , J. Bacteriol. , 177, 4121-4130, 1995) digested with the same enzyme. In addition, the idi gene fragment was amplified using an E. coli chromosome DNA template and primers having the nucleotide sequences of SEQ ID NOs: 27 and 28, and then treated with Sma I and Sph I and inserted into the plasmid pDSN12D digested with the same enzyme. pDSN12Didi was prepared. Thereafter, the plasmid pDSN12Didi was digested with Bam HI and Sal I to obtain a gene fragment containing the four genes, which were then inserted into a pSTV28 vector (Takara) treated with the same enzyme to prepare a plasmid pSSN12Didi.

The mvaK1 , mvaD and mvaK2 operons were amplified using the chromosomal DNA template of Enterococcus faecalis and the nucleotide sequences of SEQ ID NOs: 11 and 12, and the amplified gene fragments were treated with Nco I and Sal I and then the same enzyme. Plasmid pDEF12D was prepared by inserting into a pBAD24 vector treated with. Plasmid pDEF12Didi was prepared by inserting the idi gene into the prepared plasmid pDEF12D in the same manner as described above. The plasmid pDEF12D was digested with Bam HI and Hind III to obtain a gene fragment containing the four genes, which was then treated with the same enzyme. Inserted into to prepare plasmid pSEF12Didi.

The mvaK1 , mvaD and mvaK2 operons were amplified using the chromosomal DNA template of Staphylococcus aureus and the nucleotide sequences of SEQ ID NOs: 13 and 14, and the amplified gene fragments were treated with Nco I and Xba I. Plasmid pDSA12D was prepared by inserting into the pBAD24 vector treated with the same enzyme. The plasmid pDSA12Didi was prepared by inserting the idi gene into the prepared plasmid pDSA12D in the same manner as above. The plasmid pDSA12Didi was digested with Eco RI and Pst I to obtain a gene fragment containing the four genes, which were then treated with the same enzyme. Inserted into to prepare plasmid pSSA12Didi.

Streptococcus Pio chromosomal DNA template and SEQ ID NO: of jeneseu: 15 and was using primers having the nucleotide sequence of 16 to amplify the mvaK1, mvaD and mvaK2 operon, it processes the amplified gene fragment with Kpn I and Xba I and then the same enzyme Plasmid pDSY12D was prepared by inserting into a pBAD24 vector treated with. By inserting the idi gene with a cut made pDSY12D plasmid with Xba I and then made blunt ends by Hinckley Now (Klenow) polymerase (New England Biolabs, Inc.) treated with Sal I, and then the same procedure as above was prepared pDSY12Didi . This was digested with Eco RI and Sal I to obtain a gene fragment containing the four genes, which were then inserted into a pSTV28 vector treated with the same enzyme to prepare plasmid pSSY12Didi.

In order to replace the mvaK1 gene derived from Streptococcus pneumoniae of the plasmid pSSN12Didi prepared above with the mvaK1 gene derived from Staphylococcus aureus, the pSSA12Didi recombinant plasmid was digested with Eco RI and Nde I and mvaK1 derived from Staphylococcus aureus. After the gene fragment was isolated, it was treated with the same enzyme and inserted into the pSSN12Didi recombinant plasmid from which the mvaK1 gene was removed to prepare a pSSA1SN2Didi recombinant plasmid.

On the other hand, using the chromosomal DNA of the yeast Saccharomyces cerevisiae as a template, the primer having the nucleotide sequence of SEQ ID NO: 17 and 18 for the HMGS gene, SEQ ID NO: 19 and 20 for the PMK (ERG8) gene A primer having a nucleotide sequence of SEQ ID NO: 21, a primer having a nucleotide sequence of SEQ ID NOs: 21 and 22 for MDD (ERG19) gene, a primer having a nucleotide sequence of SEQ ID NOs: 23 and 24 for MVK (ERG12) gene, In the case of the HMGR gene, fragments of each gene were amplified using primers having the nucleotide sequences of SEQ ID NOs: 25 and 26. The amplified HMGS gene fragment was treated with Pst I and Hind III and then inserted into a pTrc99A vector (Amann, E. et al., Gene , 69, 301-305, 1998) treated with the same enzyme to prepare plasmid pTMV1. The plasmid pTMV2 was prepared by inserting the PMK (ERG8) gene fragment into plasmid pTMV1 treated with Nco I and Eco RI and then treated with the same enzyme. In addition, amplified MDD (ERG19) gene fragments were treated with Eco RI and Sac I and then inserted into plasmid pTMV2 treated with the same enzyme to prepare plasmid pTMV3, and the amplified MVK (ERG12) gene fragments were prepared as Sac I and Sma I. The plasmid pTMV4 was prepared by digestion with plasmid pTMV3 treated with the same enzyme. Finally, the amplified HMGR gene was treated with Sal I and Pst I, and then inserted into plasmid pTMV4 treated with the same enzyme. A plasmid pTMV5 was prepared containing a genetic combination of Levi's entire mevalonate pathway related enzymes. Meanwhile, using the chromosomal DNA template of Ralstonia eutropha ( KCTC1006) and the primers of SEQ ID NOs: 35 and 36, the gene of acetyl-CoA acetyltransferase, which mediates step 1 of the mevalobiite pathway, PhbA was amplified and treated with Pst I and inserted into plasmid pTMV5 treated with the same enzyme to prepare plasmid pTMV7.

The prepared plasmid pTMV7 was treated with Eco RI and Sac I to cut out the MDD (ERG19) gene, and then inserted into the pSTV28 vector treated with the same enzyme to prepare plasmid pSSCD, and pTMV7 was treated with Sac I and Sma I to treat MVK ( EFG12) was cut out and inserted into pSSCD treated with the same enzyme to prepare plasmid pSSC1D. Plasmid pSSC1Didi was prepared by inserting the idi gene in the same manner as above. Finally, pTMV7 was treated with Nco I and Eco RI to cut the PMK (ERG8) gene, which was then treated with Klenow polymerase treated with the same enzyme to make a blunt end, and then inserted into Sma I-treated plasmid pSSC1Didi. A plasmid pSSC12Didi containing a genetic combination of enzymes related to the lower mevalonate pathway of Mrs. cerevisiae was prepared.

Step 2) Preparation of Plasmids Containing Lycopene Biosynthetic Enzyme Genes

To prepare a recombinant vector containing a lycopene biosynthetic enzyme gene, the crtE , crtB and crtI genes of Erwinia herbicola and the IPP isomerase of Hematococcus pluvialis were used. A plasmid pT-LYCO4 (Kim, SW et al . , Metab . Eng ., 72, 408-415, 2001), which includes the ipiHP1 gene encoding and capable of expressing red lycopene in E. coli, was used.

A primer having a 31 and a base sequence of 32, and: The pT-LYCO4 using the vector as a template, in the case of the crtE gene SEQ ID NO: For the primer having the nucleotide sequence of 29 and 30, crtB and crtI gene SEQ ID NO: For the ipiHP1 gene, the respective fragments were amplified using primers having the nucleotide sequences of SEQ ID NOs: 33 and 34. The amplified gene fragments by restriction enzyme treatment according to the restriction sites included in the primers were each inserted into a vector, particularly crtE Were prepared for gene Eco RI and Xho I and then treated with the pACYC184 vector treated with the same restriction enzyme plasmid pAC-crtE was inserted into the (New England Biolabs Inc.), crtB And In the case of the crtI gene, the plasmid pAC-crtEBI was prepared by cleaving with Xho I and Xba I and inserting the same enzyme-treated plasmid pAC-crtE. The ipiHP1 gene was treated with Xba I and Sal I and then treated with the same enzyme. Plasmid pAC-LYCm4 was prepared by insertion into the plasmid pAC-crtEBI. The prepared plasmid pAC-LYCm4 was digested with Eco RI and Sal I to separate gene fragments containing the four genes, and inserted into the pTrc99A vector treated with the same enzyme to prepare plasmid pT-LYCm4.

Step 3) Comparing Lycopene Productivity of Gene Combinations for Lower Mevalonate Pathway Related Enzymes

The plasmids pSSN12Didi, pSSC12Didi, pSEF12Didi, pSSA12Didi, pSSY12Didi and pSSA1SN2Didi prepared in step 1) were transformed with the plasmid pT-LYCm4 prepared in step 2) and introduced into E. coli MG1655 obtained from ATCC. A single colony of each transformed Escherichia coli was collected in 2YT medium (16 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCl) containing 100 μg / ml of ampicillin and 50 μg / ml of chloramphenicol. Inoculated with ㎖ and incubated shaking at 37 ℃, 600 μl of the obtained culture was inoculated in 300 ml of 2YT medium containing 1% glycerol, 100 μg / ml ampicillin, 50 μg / ml chloramphenicol and 10 mM mevalonate, respectively, at 29 ° C. Incubated for 48 hours at.

1 mL of each obtained culture solution was taken, and the absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and the number of cells was multiplied by the dilution factor. In addition, centrifuged 20 μl from each culture medium to obtain the cells, suspended with 400 μl of acetone and left at 55 ° C. for 15 minutes, and then 600 μl of acetone was added thereto and left at 55 ° C. for 15 minutes to extract lycopene. It was. The obtained extract was centrifuged at 14,000 rpm for 10 minutes, and then the absorbance of the supernatant was measured at a wavelength of 474.5 nm using a spectrophotometer. The measured values were substituted into the equation obtained through the calibration curve, and the amount of lycopene was calculated by calculating the dilution factor. At this time, for preparing a calibration curve, a standard lycopene (Sigma) was purchased, dissolved in acetone, and diluted in acetone at different concentrations to measure absorbance using a 474.5 nm wavelength in a spectrophotometer. The cell mass and the lycopene content per cell of E. coli transformed with each plasmid confirmed from the above method are shown in Table 3 below.

As a result, according to the present invention, a combination of genes encoding mevalonate kinase derived from Staphylococcus aureus, and mevalonate phosphatase derived from Streptococcus pneumoniae and mevalonate decarboxylase from diphosphate When introducing the plasmid pSSA1SN2Didi containing the transformed E. coli showed the highest lycopene productivity.

Example 2: Gene Combination Selection for Upper Mevalonate Pathway Related Enzymes

Step 1) Preparation of a Recombinant Vector Comprising Gene Combinations for Upper Mevalonate Pathway Related Enzymes

PhbA , a gene of acetyl-CoA acetyltransferase mediating step 1 of the upper mevalonate pathway, mvaS , a gene of hydroxymethylglutaryl-CoA synthase mediating step 2, hydroxymethyl mediating step 3 Mediated to mvaA , the gene of glutaryl- CoA reductase, and steps 1 and 3 Streptococcus pneumoniae (ATCC6314), Enterococcus faecalis (ATCC14508) and Staphylo to amplify mvaE , a gene of acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase, from a variety of organisms Chromosome DNA of Caucus aureus (ATCC35566) was obtained from ATCC, and Ralstonia The chromosomal DNA of eutropha , KCTC1006), Streptomyces griseolosporeus (KCCM12200), and Paracoccus zeaxanthinifaciens ( ATCC21588) was extracted according to a conventional method. Gene combinations encoding mevalonate upstream related enzymes from Saccharomyces cerevisiae were obtained from plasmid pTMV7 prepared in Example 1 above.

The amplification of each gene, the preparation of the forward and reverse primers used for each gene amplification and the nucleotide sequence confirmation of the amplified gene fragments were performed in the same manner as in Example 1, the forward and reverse primers used for amplification of each gene The base sequence and the type of restriction enzyme contained in the primers are shown in Table 4 below.

First, the phbA gene was amplified using a chromosomal DNA template of Ralstonia eutropa and a primer having nucleotide sequences of SEQ ID NOs: 35 and 36. The amplified gene was treated with Pst I and Mungbean nuclease. ) To make a smooth end, and then inserted into a pTrc99A vector treated with Sma I to prepare a plasmid pTphbA.

The mvaA and mva S genes were amplified using the chromosomal DNA of Paracoccus ziazynifaciens as a template and primers of SEQ ID NOs: 37 and 38, and SEQ ID NOs: 39 and 40, respectively. The amplified mvaA gene was treated with Nco I and Sma I and then inserted into the pTrc99A vector treated with the same enzyme to prepare plasmid pTPZmvaA. The amplified mvaS gene was treated with Sma I and Xba I and then treated with the same enzyme. Plasmid pTPZmvaAS was prepared by inserting into pTPZmvaA. After that, processes the phbA gene was separated by treating the plasmid pTphbA prepared in the Kpn I zero meongbin nuclease and process the Hind III and then, it was treated with Xba I and process the Cleveland Now polymerase Hind III The plasmid pTPZmvaASP was prepared by inserting into the treated plasmid pTPZmvaAS.

The mvaA and mva S genes were amplified using the chromosomal DNA of Streptomyces gryolospororeus as a template and primers of SEQ ID NOs: 41 and 42, and SEQ ID NOs: 43 and 44, respectively. The amplified mvaA gene was treated with Bsp HI and Eco RI, and then inserted into a pTrc99A vector treated with Nco I and Eco RI to prepare plasmid pTSGmvaA. The amplified mvaS gene was treated with Eco RI and Sac I and then treated with the same enzyme. Plasmid pTSGmvaAS was prepared by insertion into the treated plasmid pTSGmvaA. Subsequently, the plasmid pTMV7 prepared in Example 1 was treated with Pst I to prepare a blunt end by treatment with an empty nuclease, followed by insertion into a plasmid pTSGmvaAS treated with Sma I to prepare plasmid pTSGmvaASP. It was.

The plasmid pTMV7 prepared in Example 1 was treated with Nco I to remove gene combinations of the lower mevalonate pathway related enzymes, and then relinked to combine the genes of the upper mevalonate pathway related enzymes of Saccharomyces cerevisiae. A plasmid pTMV7-1 containing was prepared.

The mvaA and mva S genes were amplified using chromosomal DNA of Staphylococcus aureus as a template and primers having the nucleotide sequences of SEQ ID NOs: 45 and 46, and SEQ ID NOs: 47 and 48, respectively. The amplified mvaA gene was to produce plasmid pTSAmvaA was inserted Pci I and pTrc99A vector was treated with Sac I treatment the Nco I and Eco RI, the amplified mvaS gene with the same restriction enzyme and then treated with Sac I and Kpn I Plasmid pTSAmvaAS was prepared by insertion into the treated plasmid pTSAmvaA. Then, the plasmid pTSAmvaASP was prepared by inserting the phbA gene into the plasmid pTSAmvaAS in the same manner as the preparation method of the plasmid pTSGmvaASP.

Using the chromosomal DNA of Enterococcus faecalis as a template and amplifying the mvaE and mva S genes using primers having the nucleotide sequences of SEQ ID NOs: 49 and 50, and SEQ ID NOs: 51 and 52, respectively, The amplified mvaE gene was treated with Sac I and Sma I and inserted into the pTrc99A vector treated with the same enzyme to prepare plasmid pTEFAmvaE. The amplified mvaS gene was treated with Sma I and Bam HI and then treated with the same enzyme. Plasmid pTEFAmvaES was prepared by insertion into pTEFAmvaE.

Using the chromosomal DNA of Streptococcus pneumoniae as a template and amplifying mvaA and mva S genes using primers having the nucleotide sequences of SEQ ID NOs: 53 and 54, and SEQ ID NOs: 55 and 56, respectively, The amplified mvaA gene, Eco RI and Kpn after treatment with I was prepared plasmid pTSNmvaA inserted into the pTrc99A vector treated with the same restriction enzyme, the amplified mvaS is treated with the same enzymes and then treated with Kpn I and Xba I plasmid pTSNmvaA Inserted in to prepare plasmid pTSNmvaAS. Then, the plasmid pTSNmvaASP was prepared by incorporating the phbA gene amplified using the chromosomal DNA template of Ralstonia eutropa and the primers of SEQ ID NOs: 35 and 36 with Pst I, and then inserting the same enzyme-treated plasmid pTSNmvaAS. .

Step 2) Comparison of Mevalonate Productivity of Gene Combinations for Top Mevalonate Pathway Related Enzymes

The plasmids pTEFAmvaES, pTMV7-1, pTPZmvaASP, pTSAmvaASP, pTSGmvaASP and pTSNmvaASP prepared in Example 2 were transformed into Escherichia coli MG1655, respectively. A single colony of each transformed Escherichia coli was inoculated in 5 ml of 2YT medium containing 100 µg / ml ampicillin and shaken at 37 ° C. 200 µl of the obtained culture solution was diluted with 0.1 mM IPTG (isopropyl) containing 100 µg / ml ampicillin. TT medium with or without -β-D-thiogalactopyranoside (20 g glucose, 1 g distilled water, 16 g K ₂ HPO ₄ , 14 g KH ₂ PO ₄ , 5 g peptone, 1 g citric acid, 25 mg magnesium sulfate) MgSO ₄ · 7H ₂ O), 50 mg iron sulfate (FeSO ₄ · 7H ₂ O), and 8 mg thiamine (HCl) were added to 5 ml of medium adjusted to pH 7.2 using 4 N sodium hydroxide). Inoculation was incubated for 48 hours at 30 ° C.

200 μl each of the cultured medium was taken, and the absorbance was measured at a wavelength of 600 nm using a spectrophotometer to check the cell mass. In addition, in order to confirm the mevalonate productivity, 400 μl of the culture solution was taken from each culture medium, and centrifuged at 14,000 rpm for 10 minutes, and then 350 μl of the supernatant was added thereto. After adjusting to 2.0, it was left at 45 ℃ for 1 hour. To this, crystalline sodium sulfate was added with shaking until saturation, 1 ml of ethyl acetate was added thereto, followed by shaking for 5 minutes to extract mevalonate. Centrifuged for 10 minutes, the supernatant was taken twice to obtain a mevalonate extract. The extract was evaporated under vacuum to completely remove ethyl acetate, and then contained 25% buttaldehyde, an internal standard. The content of mevalonate was analyzed by dissolving in 400 μl of ethyl acetate and performing gas chromatography under the following conditions.

Gas Chromatography: Agilent Techonolies 6890N model with auto-sampler

Column: HP-5-433 (30.0 m × 0.25 mm × 0.25 mm noninal, 5% phenyl methyl siloxane, Hewlett-Packard)

Mobile phase: hydrogen

Rate: 1.5 ml / min

-Oven temperature: 180 ℃

Mode: Isocratic Mode

Sample injection volume: 1 μl

Detection: FID method

The cell weight and the lycopene content per cell of the E. coli transformed with each plasmid confirmed from the above method are shown in Table 5 below.

As a result, as shown in Table 5, the acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase and hydroxymethylglutaryl-CoA synthetase derived from Enterococcus faecalis according to the present invention. Plasmid pTEFAmvaES containing a combination of genes (0.15 to 4.16 g / L), acetyl-CoA acetyltransferase derived from Ralstonia eutropha, and hydroxymethylglutaryl-derived from Streptococcus pneumoniae Transgenic Escherichia coli exhibited excellent mevalonate productivity when plasmid pTSNmvaASP was introduced (0.86-1.32 g / L) containing a combination of genes encoding CoA synthase and hydroxymethylglutaryl-CoA reductase. It was.

Example  3: full Mevalonate  Genetic combinations for pathway related enzymes In E. coli Lycopene  Productivity Check

Step 1) Preparation of a Plasmid Containing Gene Combinations for All Mevalonate Pathway Related Enzymes

Two gene combinations encoding the lower mevalonate pathway related enzymes selected in Example 1 were combined with two gene combinations encoding the upper mevalonate pathway related enzymes selected in Example 2, respectively. Plasmids each containing a gene combination encoding the balonate pathway related enzymes were prepared as follows.

Two gene combination fragments encoding the upper mevalonate pathway related enzymes were amplified using the plasmids pTEFAmvaES and pTSNmvaASP prepared in Example 2, respectively, wherein the amplification method of each gene, the omnidirectional and Preparation of the reverse primer and confirming the nucleotide sequence of the amplified gene fragments in the same manner as in Example 1. The base sequence of the forward and reverse primers used for amplification of each gene and the restriction enzyme types included in the primers are shown in Table 6 below.

First, a plasmid pTSNmvaASP as a template and SEQ ID NO: using primers having the nucleotide sequences of 57 and 58 Lal Stony ah oil Trojan wave derived phbA, and was amplified gene fragment containing the Streptococcus pneumoniae derived mvaA and mvaS , After treatment with Sal I, treated with Klenow polymerase and inserted into the plasmid pSSA1SN2Didi, which made the blunt end, ralstonia utropa-derived acetyl-CoA acetyltransferase, Streptococcus pneumoniae-derived hydroxymethylgluta Reyl-CoA Synthetase and hydroxymethylglutaryl-CoA Reductase, Staphylococcus aureus-derived mevalonate kinase, and Streptococcus pneumoniae-phosphate mevalonate kinase and diphosphorate valate A plasmid pSNSASN was prepared comprising a combination of genes encoding carboxylase.

In addition, using a primer having the plasmid pTEFAmvaES as a template and the nucleotide sequences of SEQ ID NOs: 59 and 60, amplified gene fragments containing mvaE and mvaS derived from Enterococcus faecalis were treated with Sal I after treatment with Xho I. Inserted into the treated plasmid pSSA1SN2Didi, enterococcus faecalis-derived acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase and hydroxymethylglutaryl-CoA synthetase, Staphylococcus aureus-derived mes. A plasmid pEFSASN was prepared comprising a combination of a balonate kinase, and a gene combination encoding Streptococcus pneumoniae phosphate mevalonate phosphatase and a divalphate valonate decarboxylase.

Step 2) Confirmation of Lycopene Productivity in Escherichia Coli with Gene Combinations for Total Mevalonate Pathway-Related Enzymes

The two plasmids pSNSASN and pEFSASN obtained in step 1) were introduced into E. coli MG1655 with plasmid pT-LYCm4 containing the gene for the lycopene biosynthesis enzyme obtained in Example 1, respectively, and transformed into plasmid pT- as a control. Only LYCm4 was transformed into Escherichia coli MG1655. Among them, Escherichia coli MG1655 transformed with plasmids pEFSASN and pT-LYCm4 was deposited on April 7, 2006 to the Korea Biotechnology Research Institute Gene Bank with accession number KCTC 10932BP. A single colony of each transformed E. coli was inoculated in 5 ml of 2YT medium containing 100 μg / ml ampicillin and 50 μg / ml chloramphenicol and shaken at 37 ° C., and 200 μl of the obtained culture was added to 1% glycerol, 100 μg. 5 ml of 2YT medium containing / ml of ampicillin and 50 g / ml of chloramphenicol were then incubated for 48 hours at 29 ° C.

Cell culture and lycopene content was confirmed in the same manner as in step 3) of Example 1 from each culture medium, and the cell weight and lycopene content per cell of E. coli transformed from each plasmid identified therefrom are shown in Table 7 below. It was.

As a result, as shown in Table 7, it was confirmed that the lycopene productivity of the transformed Escherichia coli increased by a factor of five or more compared to Escherichia coli without introducing the gene combination encoding the enzyme of the mevalonate pathway according to the present invention. It was.

Example 4: Confirmation of the productivity of beta-carotene and zeaxanthin in E. coli introduced gene combinations for the entire mevalonate pathway related enzymes

A fragment of the gene crtY (lycopene beta-cyclase gene), which encodes an enzyme for biosynthesis of beta-carotene from lycopene, was used as a template using chromosomal DNA of the Pantoea ananas (KCCM40419) strain, SEQ ID NO: 61 And amplified in the same manner as in Example 1 using the forward and reverse primers having the nucleotide sequence of 62, and the plasmid pT-LYCm4 treated with Sal I and Hind III and then treated with the same enzyme after the amplified crtY gene Insertion to prepare plasmid pT-CARO.

In addition, a fragment of the gene crtZ (beta-carotene hydroxylase gene) encoding an enzyme for biosynthesis of zeaxanthin from beta-carotene was used as a template using the chromosomal DNA of Pantoea ananas strain obtained from ATCC as a template. : Amplified in the same manner as in Example 1 using the forward and reverse primers having base sequences of 63 and 64, the amplified crtZ gene was treated with Hind III and inserted into the plasmid pT-CARO treated with the same enzyme To prepare plasmid pT-ZEA.

The plasmids pSNSASN and pEFSASN prepared in Example 3 were transformed with E. coli MG1655 with the plasmids pT-CARO or pT-ZEA obtained above, respectively, and the control group was introduced into E. coli MG1655 with only plasmids pT-CARO or pT-ZEA. And transformed. A single colony of each transformed Escherichia coli was inoculated and incubated in the same manner as in Example 3, and the cell mass and the content of beta-carotene or zeaxanthin were confirmed by the same method as in step 3) of Example 1 from each culture solution obtained. It was. At this time, extraction and quantification of beta-carotene and zeaxanthin were performed in the same manner as in step 3) of Example 1, and samples of standard beta-carotene and zeaxanthin were obtained from Sigma. The cell weight of the transformed Escherichia coli, and the content of beta-carotene or zeaxanthin per cell are shown in Tables 8 and 9 below.

As a result, as shown in Tables 8 and 9, the beta-carotene and zeaxanthin productivity of the transformed Escherichia coli was introduced into E. coli by introducing a gene combination encoding the enzyme of the mevalonate pathway according to the present invention. It was confirmed that the increase more than five times.

Example 5: Confirmation of the productivity of coenzyme qtene in E. coli with the introduction of gene combinations for the entire mevalonate pathway related enzymes

A fragment of the gene dds , which encodes the decaprenylpyrophosphate synthase, which biosynthesizes coenzyme cutten, is used as a template, and the chromosomal DNA of the strain Rhodobacter sphaeroides 2.4.1 , ATCC BAA-808 is used as a template. Using the forward and reverse primers having the nucleotide sequences of SEQ ID NOs: 65 and 66, and amplified in the same manner as in Example 1, and the amplified dds gene was treated with Eco RI and Sac I, followed by the same enzyme. Plasmid pT-dds was prepared by insertion into the treated pTrc99A vector.

The plasmids pSNSASN and pEFSASN prepared in Example 3 were transformed with E. coli MG1655 with the plasmid pT-dds obtained above, respectively, and the control group was transformed by introducing only plasmid pT-dds into E. coli MG1655. A single colony of each transformed E. coli was inoculated and cultured in the same manner as in Example 3, and the cell mass was confirmed in the same manner as in step 3) of Example 1 from each culture medium. Extraction and content determination of coenzyme cuene are described in reference [Park, YC, et al . , App. Microbiol. Biotechnol. , 67, 192-196, 2005.

Each plasmid identified from the method described above shows the cell weight of E. coli and the content of coenzyme cuene per cell in Table 10.

As a result, as shown in Table 10, the coenzyme qten productivity of E. coli transformed by introducing a gene combination encoding the enzyme associated with the mevalonate pathway according to the present invention increased more than three times compared to E. coli without introducing it. Confirmed.

As described above, according to the present invention, a method of preparing isoprenoids by introducing gene combinations of enzymes related to mevalonate pathways derived from various organisms into microorganisms effectively increases the biosynthesis of isoprenoids compared to conventional methods. Therefore, it can be usefully used for mass production development of isoprenoids.

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Claims (18)

(a) Mevalonate kinase derived from Staphylococcus aureus , phosphate mevalonate kinase derived from Streptococcus pneumoniae , and diphosphate metal derived from Streptococcus pneumoniae Nate decarboxylase, acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase from Enterococcus faecalis and hydroxymethylglutaryl-CoA synthase from enterococcus faecalis A combination of genes each encoding a; or (b) methalonate kinase derived from Staphylococcus aureus, metholonate phosphatase derived from Streptococcus pneumoniae, mevalonate decarboxylase diphosphate derived from Streptococcus pneumoniae, ralstony Acetyl-CoA acetyltransferase from Ralstonia eutropha , hydroxymethylglutaryl-CoA synthetase from Streptococcus pneumoniae and hydroxymethylglutaryl-CoA from Streptococcus pneumoniae Combination of genes encoding reductase And introducing the host microorganism into the host microorganism, followed by culturing the transformed host microorganism, thereby producing an isoprenoid. The method of claim 1, A mevalonate kinase derived from Staphylococcus aureus is SEQ ID NO: 6, and a methalonate phosphatase derived from Streptococcus pneumoniae is SEQ ID NO: 7, biphosphate mevalonate from Streptococcus pneumoniae. Decarboxylase is SEQ ID NO: 8, acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase derived from Enterococcus faecalis is SEQ ID NO: 1, hydroxymethyl gluta derived from Enterococcus faecalis Reyl-CoA synthetase is SEQ ID NO: 2, acetyl-CoA acetyltransferase from Ralstropa eutropa is SEQ ID NO: 3, hydroxymethylglutaryl-CoA synthetase is derived from Streptococcus pneumoniae No. 4 and the hydroxymethylglutaryl-CoA reductase from Streptococcus pneumoniae has the amino acid sequence of SEQ ID NO: 5. 15. delete The method of claim 1, Method for introducing into the host microorganism in the form of a recombinant vector prepared by inserting the gene into the expression vector. delete delete The method of claim 1, The genes are introduced directly on the chromosome of the host microorganism. The method of claim 1, Wherein the genes are partially introduced directly on the chromosome of the host microorganism and the remainder are introduced in the form of a recombinant vector inserted into the vector. The method of claim 1, Wherein the host microorganism has a non-mevalonate pathway as a biosynthetic pathway of isopentenylpyrophosphate, a precursor of isoprenoids. The method of claim 9, Host microorganisms include E. coli , Bacillus sp., Pseudomonas sp., Agrobacterium sp., Rhodobacter sp. And Erwinia. sp.) A method characterized in that it is selected from among bacteria including strains. The method of claim 10, The host microorganism is characterized in that E. coli. The method of claim 1, Isoprenoids are lycopene; Carotenoids including beta-carotene, zeaxanthin, astaxanthin and lutein; Quinones including coenzyme qtene; Turfoids including monoterpenoids, sesquiterpenoids, diterpenoids, and sesterterpenoids; Steroids; And it is selected from the group consisting of natural rubber. delete (a) Mevalonate kinase derived from Staphylococcus aureus , phosphate mevalonate kinase derived from Streptococcus pneumoniae , and diphosphate metal derived from Streptococcus pneumoniae Nate decarboxylase, acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase from Enterococcus faecalis and hydroxymethylglutaryl-CoA synthase from enterococcus faecalis A combination of genes each encoding a; or (b) methalonate kinase derived from Staphylococcus aureus, metholonate phosphatase derived from Streptococcus pneumoniae, mevalonate decarboxylase diphosphate derived from Streptococcus pneumoniae, ralstony Acetyl-CoA acetyltransferase from Ralstonia eutropha , hydroxymethylglutaryl-CoA synthetase from Streptococcus pneumoniae and hydroxymethylglutaryl-CoA from Streptococcus pneumoniae Combination of genes encoding reductase Microorganisms transformed by introducing. The method of claim 14, A mevalonate kinase derived from Staphylococcus aureus is SEQ ID NO: 6, and a methalonate phosphatase derived from Streptococcus pneumoniae is SEQ ID NO: 7, biphosphate mevalonate from Streptococcus pneumoniae. Decarboxylase is SEQ ID NO: 8, acetyl-CoA acetyltransferase / hydroxymethylglutaryl-CoA reductase derived from Enterococcus faecalis is SEQ ID NO: 1, hydroxymethyl gluta derived from Enterococcus faecalis Reyl-CoA synthetase is SEQ ID NO: 2, acetyl-CoA acetyltransferase from Ralstropa eutropa is SEQ ID NO: 3, hydroxymethylglutaryl-CoA synthetase is derived from Streptococcus pneumoniae No. 4 and a hydroxymethylglutaryl-CoA reductase from Streptococcus pneumoniae have the amino acid sequence of SEQ ID NO: 5. delete The method of claim 14, E. coli , Bacillus sp.), Pseudomonas sp.), Agrobacterium sp., Rhodobacter sp.) and Erwinia sp. The method of claim 17, The microorganism characterized in that E. coli is Escherichia coli MG1655 deposited with accession number KCTC 10932BP.

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