KR20190079575A - Recombinant yeast with artificial cellular organelles and producing method for isoprenoids with same - Google Patents

Abstract

The present invention relates to a recombinant yeast in which an organelle is mutated by gene manipulation and a method for production of various isopreonoids by using the same. When a metabolic pathway of the useful product isoprenoid is introduced thereinto, a mutated yeast that is increased in the number or size of peroxisome through genetic recombination, can remarkably increase an isoprenoid production yield, compared to yeasts in which the organelle is not mutated, allowing the mass production of isoprenoid and finding useful applications in various industrial fields.

Description

[0001] The present invention relates to a recombinant yeast transformed with cell organelles and a method for producing isoprenoids using the recombinant yeast,

The present invention relates to a recombinant yeast in which cell organelles are mutated through gene manipulation, and a method for producing various isoprenoids using the recombinant yeast.

Control of functional metabolic pathways leads to various problems such as ineffective regulatory reactions, unoptimized physicochemical environment, loss of metabolic intermediates to competitive metabolism, metabolic toxicity. Yeast is the preferred industrial organism due to its low pH growth, simple nutritional requirements, low probability for viral infections and high resistance to substrate and metabolite toxicity, ease of gene manipulation, and ability to express complex heterologous enzymes . Yeast metabolism has been concentrated mainly on the metabolic pathway in the cytoplasm. Metabolic pathway optimization strategies, such as protein fusion and synthetic protein scaffolds, can effectively improve productivity due to some metabolic regulation, but the number of enzymes that can be built into the scaffold is limited by available binding domains, Has a negative effect on the enzyme activity. Therefore, various strategies to overcome this are being studied.

Isoprenoid refers to an organic compound containing isoprene as a constituent unit, which is also called "der-penoid" or "terpene". In addition to carbohydrates such as natural gums, compounds in which the terminal functional groups are alcohol, aldehyde, carboxylic acid or pyrophosphoric acid are known to appear in plant cells or animal cells. Depending on the number of constituent isoprene units (C5), monoterpene (C10), sesquiterpene (C15), diterpene (C20), triterpene (C30) and tetra terpene (C40) are also classified.

Isoprenoid compounds are a very chemically diverse substance with more than 23,000 known. Representative materials of these compounds are cholesterol, quinone, carotenoid, polyprenyl phosphate and rubber, which show diversity in function as well as structure. Polyprenyl phosphate, which is composed of 11 isoprene, is directly involved in the synthesis of prokaryotic cell walls.

Isoprenoid is a commercially important form of lipid and has excellent lubricity, oxidative stability, low pour point, low freezing point, and easy biodegradability. In addition, animals can not synthesize carotenoids, one of the isoprenoids, and therefore must be ingested through food. Due to the importance and necessity of such isoprenoids, a method of artificially synthesizing or extracting from some algae has been studied in order to provide isoprenoids more easily. Recently, with the development of genomics and bioinformatics, metabolism using microbial cells Technological mass production methods are being tried.

However, microorganisms whose metabolic processes have been altered so as to produce isoprenoids have not been widely reported, and there is a need for further studies.

Therefore, the inventors of the present invention have studied how to effectively produce isoprenoids. When the yeast cell organelles are mutated and mutations are caused in the isoprenoid production-related metabolism process, various isoprenoid production is more effectively achieved And the present invention has been completed.

Accordingly, an object of the present invention is to provide a genetically engineered recombinant yeast and a method for producing isoprenoid using the genetically modified yeast so that the productivity of isoprenoid can be improved through regulation of cell organelles.

In order to solve the problems as described above, the present invention PEX34 (Peroxisomal membrane protein 34) overexpression, PEX11 (Peroxisomal membrane protein 11) gene deletion and ATG36 And a deletion of a gene encoding a peroxisome autophagy-related protein (36) gene in a cell organelle-transformed recombinant yeast.

In another aspect, the present invention i) tHMG1 (truncated hydroxy-methyl glutaryl-CoA reductase), CrtE (Geranylgeranyl pyrophosphate synthase in the recombinant yeast is the organelle mutations) CrtYB (Phytoene synthase-lycopene cyclase), CrtI ( Phytoene desaturase) Gene set, and ii) tHMG1, CrtE , And a set of genes selected from the group consisting of a CDPS / KS (ent-copalyl diphosphate / kaurene synthase) gene set is introduced into a recombinant yeast strain having increased isoprenoid productivity.

The present invention also provides a method for producing a recombinant yeast, comprising culturing the recombinant yeast having increased isoprenoid productivity; ≪ RTI ID = 0.0 > isoprenoid < / RTI >

The introduction of the isoprenoid metabolic pathway, a useful product in mutant yeasts with an increased number or size of peroxidase, the organelle of the cell through genetic recombination, significantly increased isoprenoid production compared to yeast without cell organelle mutation And can mass-produce isoprenoids. Thus, it is useful for various industrial fields to which isoprenoid is applied.

FIG. 1 is a schematic diagram of a recombinant yeast having an increased number and size of peroxisome by the regulation of an artificial cellular organelle regulation.
FIG. 2 shows the results of confirming the number and size of peroxisome in a recombinant yeast having an artificial cellular organelles regulating ability by using a green fluorescent protein (GFP). FIG.
FIG. 3 is a diagram showing the result of confirming the structural change of a recombinant yeast having an artificial cellular organelles regulating ability through an energy filtration transmission electron microscope.
Figure 4 is a schematic diagram of the beta carotene biosynthetic pathway, one of the carotenoids.
FIG. 5 is a graph showing the growth curves (B) and beta-carotene yields (C) of the β-carotene-expressing cells (A), PX1-βCN transformed with the control yeast CEN-βCN and recombinant yeast Fig.
Figure 6 is a schematic diagram of the kaurin biosynthetic pathway, one of the gibberellin metabolites.
FIG. 7 is a graph showing the results of the strain growth (A) and the kaolin production (B) of PX1-KRN transformed with a control yeast CEN-KRN and a recombinant yeast having an artificial cellular organelles regulating ability.

The present invention relates to PEX34 (Peroxisomal membrane protein 34) gene, deletion of PEX11 (Peroxisomal membrane protein 11) gene and ATG36 And a deletion of a gene encoding a peroxisome autophagy-related protein (36) gene in a cell organelle-transformed recombinant yeast.

In the present invention, 'organellular transformation' refers to a variation in the number or size of peroxisome compared to the wild type yeast by introduction of the gene mutation as described above. Thus, the variation of the organelle of the present invention may be an increase in the number or size of peroxisome. Peroxisome is one of the intracellular organelles, which contains oxidase and catalase in the inside, and produces hydrogen peroxide through oxidation and converts hydrogen peroxide into water.

In the present invention, the recombinant yeast having an artificial cellular organotactic control ability having cell organelle mutation is PEX34 (Peroxisomal membrane protein 34) gene, deletion of PEX11 (Peroxisomal membrane protein 11) gene and ATG36 And a deletion of a gene encoding a peroxisome autophagy-related protein (36) gene, and the PEX34 (NCBI number: 850302) , PEX11 (NCBI number: 854018) And ATG36 (NCBI number: 853254) The gene was introduced into Saccharomyces cerevisiae cerevisiae . < / RTI >

In a preferred embodiment of the present invention, the cell organelles are mutated in a recombinant yeast, i) a recombinant yeast in which the ATG36 gene is deleted, ii) PEX34 Recombinant yeast overexpressing the gene, iii) PEX11 Gene deletion and PEX34 Recombinant yeast overexpressing the gene and iv) PEX11 Gene deletion, ATG36 gene deletion and PEX34 And a recombinant yeast in which the gene is over-expressed.

All of the recombinant yeasts of i to iv have various types of modified organelles, and preferably, the size and / or number of peroxisome is increased compared to the wild type yeast. In addition, the recombinant yeasts i to iv all exhibit improved isoprenoid production ability as compared with the wild-type yeast in which cell organelles are not mutated when introducing isoprenoid-related biosynthetic metabolism into cell organelles. Therefore, the organelle-transformed recombinant yeast of the present invention can be used for production of isoprenoid.

In the present invention, the recombinant yeast is not limited to yeast capable of achieving mutation of cell organelles by the mutation of the gene, and preferably includes S. cerevisiae, S. boulardii, S. bulderi, S. cariocanus, Scharomyces cerevisiae, S. aureus, and the like, S. cariocus, S. chevalieri, S. dairenensis, S. ellipsoideus, S. cerevisiae, S. eubayanus, S. exiguus, S. florentinus, S. kluyveri, Sakae, and the like. S. martiniae, S. monacensis, < RTI ID = 0.0 > SACARAMI < S. norbensis, S. paradoxus, S. pastorianus, S. spencerorum, and Sarkaromyces spp. In the case of S. turicensis, S. unisporus, S. uvarum, and S. zonatus, And one kind selected from the group consisting of

The present invention also provides a recombinant yeast with increased isoprenoid productivity by introducing an isoprenoid-related biosynthetic pathway into the recombinant yeast transformed with the cell organelles.

The isofrenoid productivity of the recombinant yeast is increased by: i) truncated hydroxy-methyl glutaryl-CoA reductase ( tHMG1 ) CrtE ( Geranylgeranyl pyrophosphate synthase), CrtYB (Phytoene synthase-lycopene cyclase) and CrtI ( Phytoene desaturase) Gene set, and ii) tHMG1, CrtE and And a set of genes selected from the group consisting of CDPS / KS (ent-copalyl diphosphate / kaurene synthase) gene set is introduced into a recombinant yeast having increased isoprenoid productivity.

The use of the recombinant yeast of the present invention can increase the ability of kaurin, one of the carotenoids, beta-carotene and gibberellin metabolites, to increase the ability to produce isoprenoid according to the introduction of the gene set and the variation of cell organelles Can be confirmed.

In the present invention, isoprenoids may include a variety of isoprenoids known in the art that can be increased in production upon introduction of the gene set of the present invention, but preferably beta-carotene, kaurin, carotinoid or gibberellin . ≪ / RTI >

The tHMG1 gene contained in the gene set of the present invention (NCBI number: 854900) is derived from Saccharomyces cerevisiae, and the CrtE (NCBI number: DQ016502) , CrtYB (NCBI number: AY177204) And CrtI (NCBI number: KR779665) is derived from xanthophyllomyces dendrorhous , and the CDPS / KS (NCBI number: AB013295) is derived from Fusarium fujikuroi . < / RTI >

In the present invention, the recombinant yeast is preferably selected from the group consisting of i) tHMG1 , CrtE , CrtYB And CrtI gene set, and the yeast into which the gene set is introduced increases the productivity of beta carotene, which is one of the carotenoids. Therefore, when the yeast into which the gene set is introduced is used, the productivity of carotenoids in isoprenoid Can be increased.

In the present invention, the recombinant yeast is selected from the group consisting of ii) tHMG1 , CrtE and The yeast in which the CDPS / KS gene set has been introduced can be used, and the yeast into which the gene set is introduced increases the productivity of kaurin, a metabolite of gibberellin. Therefore, when the yeast in which the gene set is introduced, The productivity of gibberellin can be increased.

In order to produce the recombinant yeast of the present invention, a recombinant method can be used by a conventional method known in the art. For example, a method of introducing the relevant genes into an appropriate expression vector and introducing the recombinant yeast can be used. As the expression vector, any expression vector conventionally used for the selected host microorganism can be used. For example, one or more vectors selected from the group consisting of plasmid, cosmid, phage particle and viral vector can be used, More preferably, pRS series, YEplac series, and pCEV series can be used. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.

In the present invention, genes encoding isoprenoid biosynthesis-related enzymes can be introduced directly onto the genome, and some of the genes constituting the corresponding gene combination are directly introduced into the chromosome of the cell organelle-regulating yeast, . Methods for introducing the cDNA on such chromosomes include, but are not limited to, conventional methods such as homologous recombination or CRISPR Cas9.

The present invention also relates to a method for producing a recombinant yeast, comprising: culturing the recombinant yeast; The present invention will now be described in detail with reference to the accompanying drawings, in which like reference characters designate the same or similar parts throughout the lifespan.

In the present invention, "cultivation" means that microorganisms are grown under moderately artificially controlled environmental conditions.

In the method of the present invention, the recombinant yeast is a yeast in which the intracellular organelles are mutated, and the efficiency of the introduced isoprenoid biosynthesis pathway is increased, so that the desired isoprenoid can be produced with higher efficiency .

The yeast can be grown in a conventional medium, for example, in a nutrient broth medium. The culture medium may contain nutrients required for culturing, that is, a microorganism to be cultured in order to cultivate a specific microorganism, and may be a mixture in which a substance for a special purpose is further added and mixed. The medium is also referred to as an incubator or a culture medium, and is a concept including both natural medium, synthetic medium and selective medium.

The medium used for cultivation should meet the requirements of a specific strain in a suitable manner while controlling temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used. The preferred culture medium may be, for example, LB, YT or M9 medium, which is a medium for the growth of transformants.

Isoprenoid, which is a target substance in the present invention, can be obtained by separating the compound through HPLC or the like or by separating the compound by a conventional method known in the art.

Hereinafter, the present invention will be described in detail by way of examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention in any way to the scope of the invention as defined by the appended claims. It will be obvious.

Example  1. Anthropogenic organelle Controllability  Production of recombinant yeast

1.1. Preparation of recombinant vectors containing gene combinations for enzymes involved in cellular organelle regulation

Cells oxy buffer, an enzyme involved in the metabolism of one of the peroxisome (peroxisome) of the scavenging some membrane protein PEX34 coding of the coding genes, PEX34, peroxisome membrane protein PEX11 (Peroxisomal membrane protein 11) of (Peroxisomal membrane protein 34) PEX11 gene, and peroxidase had little autolysis related protein ATG36 (peroxisome autophagy-related protein 36 ) to remove the gene encoding ATG36 using the polymerase chain reaction amplification. In order to confirm the changes in the organelles in the yeast cells, the Saccharomyces cerevisiae cerevisiae) were separated, amplified using the polymerase chain reaction of the 3-keto acyl -CoA thiol raised (3-ketoacyl-CoA thiolase, POT1) any of one of plasma membrane protein of some of the peroxide from the organelle. The forward and reverse primers used in the polymerase chain reaction can be obtained by sequencing the nucleotide sequence information of a gene encoding peroxisome and an endoplasmic reticulum-related enzyme to Saccharomyces cerevisiae using the NCBI (National Center for Biotechnology Information, http: /www.ncbi.nlm.nih.gov/), and the sequence of the forward and reverse primers used for the amplification of each gene and the type of the restriction enzyme contained in the primer Table 1 shows the results.

Strain gene vector direction Base sequence Restriction enzyme SAKAROMAISE Serebijie
( Saccharomyces cerevisiae ) PEX34 pRS424- PGK1 Forward SEQ ID NO: 1 BamHI Reverse SEQ ID NO: 2 SalI ATG36 Del cassette pUC57_URA Blast Forward SEQ ID NO: 3 - Reverse SEQ ID NO: 4 - PEX11 Del - PEX34 O / E cassette pUC57_URA Blast- PGK1-PEX34 Forward SEQ ID NO: 5 - Reverse SEQ ID NO: 6 - PEX34 O / E cassette Forward SEQ ID NO: 7 - Reverse SEQ ID NO: 8 - POT1 - EGFP cassette pUC57_URA Blast Forward SEQ ID NO: 9 - Reverse SEQ ID NO: 10 - * Del: deletion, O / E: over-expression

The chromosomal DNA template of Saccharomyces cerevisiae and the primers having the nucleotide sequences of SEQ ID NOS: 1 and 2 were used to amplify the PEX34 gene. The amplified gene fragments were treated with BamHI and SalI, and then pRS426 - PGK1 vector to prepare plasmid pRS426- PGK1 - PEX34 .

Thereafter, the pRS426- PGK1 - PEX34 plasmid gene, PGK1 Promoter and terminator was treated with Sac I and Kpn I and inserted into the plasmid pUC57_URA Blast cut with the same enzyme to prepare plasmid pUC57_URA Blast- PGK1 - PEX34 .

Since, pUC57_URA Blast and pUC57_URA Blast- PGK1 - PEX34 nutritional needs factors selected indicators (Auxotroph selection marker) gene or the URA3 gene promoter and each module and select indicators, including nutritional factors required terminator of the plasmid (Auxotroph selection marker) gene is URA3 Were amplified using a primer having the nucleotide sequence of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9 and 10 to obtain ATG36 Del- URA3, PEX11 Del- PGK1-PEX34- CYC1-URA3, and PGK1 - PEX34 - CYC1 - URA3, POT1 for green fluorescent protein, an indicator of cell organelles produced transgenic - EGFP - CYC1 - a URA3 cassette was prepared.

1.2 Preparation of Recombinant Yeast and Verification of Cell Organelle Changes

The homologous recombinant transformation cassettes PEX11 Del- URA3 , ATG36 Del- URA3 , and PGK1 - PEX34 - CYC1 - URA3 prepared in the above 1.1 were introduced into wild-type yeast CEN PK2.1D, respectively, and transformed into the following recombinant Yeast was prepared.

designation Gene combination CEN CEN-PK2.1D wild type PX1 CEN -? ATG36 PX2 CEN - PEX34 PX3 CEN - ΔPEX11 - PEX34 PX4 CEN - ΔATG36 - ΔPEX11 - PEX34

The homologous recombinant transformation cassette POT1 - EGFP - CYC1 - URA3 prepared in 1.1 above was transformed into wild-type yeast Saccharomyces cerevisiae CEN PK2.1D, recombinant yeast PX1, PX2, PX3, and by removing the stop codon to the C- terminus of the POT1 gene and introduction of PX4 and transgenic CEN- POT1-EGFP, PX1- POT1 - EGFP, PX2- POT1 - EGFP , PX3- POT1 - EGFP , and PX4- POT1 - EGFP recombinant yeast were prepared.

A single colony of each transformed recombinant yeast was inoculated into SD medium (6.7 g / L yeast nitrogen base without amino acids) containing 0.77 g / L of DO supplement-URA (Clontech) and 20 g / L glucose) and incubated at 30 ° C for 24 hours with shaking. 5 ml of the obtained culture was inoculated into 100 ml SD medium containing 100 μg / ml DO supplement-URA, Time shake culture. Then, 1 ml of each culture was taken at 24 hour intervals, and the presence or absence of changes in the organelle was observed by fluorescence microscopy through the expression of green fluorescent protein. The presence or absence of changes in the organelle of the recombinant yeasts PX1, PX2, PX3 and PX4 identified by fluorescence microscopy is shown in Fig.

As shown in FIG. 2, the expression of green fluorescent protein was changed in the recombinant yeast in which recombinant yeast PX1, PX2, PX3 and PX4 corresponding to pex34 were overexpressed and atg36 and / or pex11 were deleted, All of the organelles were changed. In particular, no change was observed in the organelles of the pex30 or pex34 deletion group, whereas only the fluorescence of the wild type was observed in the pex34 deletion group. Therefore, it was confirmed that the combination of overexpression of pex34 and deletion of atg36 and / or pex11 is the optimal combination to change the expression of peroxisome, a cell organelle.

A single colony of each transformed recombinant yeast was inoculated into SD medium (6.7 g / L yeast nitrogen base without amino acids) containing 0.77 g / L of DO supplement-UA (Clontech) 20 g / L glucose) and shake-cultured at 30 ° C for 24 hours. 5 ml of the resulting culture was inoculated into 100 ml of SD medium containing 100 μg / ml of DO supplement-URA, Lt; / RTI > for 72 hours. Then, 10 ml of each culture was centrifuged to obtain cells, which were then dissolved in 5 ml of phosphate buffered saline (PBS) to prepare samples for transmission electron microscopy (TEM) analysis. Sample preparation and analysis for TEM analysis were conducted by NICEM (National Institute of Agronomy Science, Seoul National University).

For the TEM analysis, the cells were separated by centrifugation in phosphate buffered saline, and then fixed with a modified Karnovsky's fixation solution (2% paraformaldehyde and 2% glutaraldehyde in 0.05M sodium cacodylate buffer (pH 7.2)). , Immobilized for 4 hours, and washed three times with 0.05M sodium cacodylate buffer (pH 7.2). After washing, the supernatant was removed by centrifugation. Secondary immobilization was performed by adding 1 ml of 2% Osmium tetroxide and 1 ml of 0.1 M Cacodylate buffer, followed by washing twice with sterilized water And the cells were loosened by the addition of 0.5% Uranyl acetate, followed by en-bloc staining for 24 hours. Dehydration was carried out three times with various concentrations of ethanol (30, 50, 70, 80, 90, and 100%) sequentially and propylene oxide was added for 10 minutes After the reaction was completed, propylene oxide and spurr's resin (4.1 g ERL 4221, 1.43 g DER 736, 5.9 g NSA, and 0.1 g DMAE) were charged and stirred for 1 day. After centrifugation, the cells were separated and re-packed with spurr's resin (4.1 g ERL 4221, 1.43 g DER 736, 5.9 g NSA, and 0.1 g DMAE) and dried at 70 ° C to obtain an EM UC7 ultra- Ultramicrotome was used to prepare cut slices and the differences in cell organelles of wild-type yeast and recombinant yeast with an artificial cell organellar regulating ability were analyzed using LIBRA 120 energy-filtering transmission electron microscopy (TEM). The results are shown in Fig.

As shown in FIG. 3, the gene PEX34 encoding the peroxisome membrane protein PEX34, the peroxisome membrane protein PEX 11, and the peroxisome autolysis-related protein ATG36, enzymes involved in peroxisome metabolism, one of the cell organelles, , PEX11 , and ATG36 regulated recombinant yeast PX1 to PX4 increased the number and the size of peroxisome.

Thus, it was confirmed that the recombinant yeast having an artificial cellular organelles regulating ability that the number and size of the peroxidase, a cell organelle, was remarkably increased could be produced through the regulation of PEX34 , PEX11 and ATG36 .

Example  2. Carotenoid Production capacity  Preparation of improved yeast

To determine whether carotenoids in isoprenoid, which is a useful substance, could be produced by using recombinant yeast having an artificial cellular organelles regulating ability prepared in Example 1, yeasts that do not have a carotenoid biosynthetic pathway A new gene cassette was introduced to produce beta carotene, one of the representative C ₄₀ carotenoids, and to compare the productivity through regulation of cell organelles.

2.1. Production of recombinant vector containing carotenoid biosynthesis-related enzyme gene combinations

Mai Seth Serenity busy as Saccharomyces (Saccharomyces cerevisiae) gene encoding the carotene cannabinoid inhibition portion of the HMG1 gene of the enzyme hydroxymethyl glutaryl -CoA reductase involved in the biosynthesis of precursor removed from the part truncated HMG1 (tHMG1), xanthophylls Roman Isis dendeuro house (xanthophyllomyces CrtE , a gene encoding a geranyl geranyl pyrophosphate synthase, an enzyme involved in carotenoid biosynthesis from dendrorhous , CrtYB , a gene encoding phytoene synthase and lycopene cyclic enzyme, and a gene encoding phytoene- unsaturated enzyme Were isolated and amplified by polymerase chain reaction. A schematic diagram of the beta carotene biosynthetic pathway including MVA and MEP pathway for synthesis of beta carotene, one of the carotenoids, is shown in FIG.

The forward and reverse primers used for the polymerase chain reaction were obtained by sequencing the nucleotide sequence information of the gene encoding Saccharomyces cerevisiae, the carotenoid of Zanthophylloma isolis dendrobians, and the enzyme involved in the precursor biosynthesis pathway with the NCBI for Biotechnology Information, http://www.ncbi.nlm.nih.gov/), and included in the base sequence and primer of the forward and reverse primers used for the amplification of each gene The types of restriction enzymes are shown in Table 3 below.

Strain gene vector direction Base sequence Restriction enzyme Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) tHMG1 pRS424-GPD Forward SEQ ID NO: 11 Note I Reverse SEQ ID NO: 12 Spe I Zontopil Roma Isis Dendro House
( xanthophyllomyces dendrorhous ) CrtE pRS426- TEF1 Forward SEQ ID NO: 17 Spe I Reverse SEQ ID NO: 18 Xma I CrtYB pRS424- PGK1 Forward SEQ ID NO: 19 Bam HI Reverse SEQ ID NO: 20 Sal I CrtI pRS423- PGK1 Forward SEQ ID NO: 21 Bam HI Reverse SEQ ID NO: 22 Sal I SAKAROMAISE Serebijie
( Saccharomyces cerevisiae ) ypl062w pSNR52-sg TEF1 Forward SEQ ID NO: 23 Eco RI Reverse SEQ ID NO: 24 - Forward SEQ ID NO: 25 - Reverse SEQ ID NO: 26 Kpn I ROX1 Forward SEQ ID NO: 23 Eco RI Reverse SEQ ID NO: 27 - Forward SEQ ID NO: 28 - Reverse SEQ ID NO: 26 Kpn I yjl064w Forward SEQ ID NO: 23 Eco RI Reverse SEQ ID NO: 29 - Forward SEQ ID NO: 30 - Reverse SEQ ID NO: 26 Kpn I GPD - tHMG1 YIplac128 Forward SEQ ID NO: 13 Pst I Reverse SEQ ID NO: 14 Sal I GPD - tHMG1 - LEU2 YIplac128-GPD
-tHMG1 Forward SEQ ID NO: 15 - Reverse SEQ ID NO: 16 - Zontopil Roma Isis Dendro House
( xanthophyllomyces dendrorhous ) TEF1 - CrtE - CYC1 pRS426- TEF1 - CrtE Forward SEQ ID NO: 31 - Reverse SEQ ID NO: 32 - PGK1 - CrtYB - CYC1 pRS424- PGK1 - CrtYB Forward SEQ ID NO: 33 - Reverse SEQ ID NO: 34 - PGK1 - CrtI - ADH2 pRS423- PGK1 - CrtI Forward SEQ ID NO: 35 - Reverse SEQ ID NO: 36 -

First, the tHMG1 gene was amplified using the DNA template of the gene synthesized based on the gene information on Saccharomyces cerevisiae and a primer having the nucleotide sequence of SEQ ID NOS: 11 and 12. The amplified gene fragment was amplified by Not I After treatment with Spe I, pRS424- GPD digested with the same enzyme Vector to prepare plasmid pRS424- GPD - tHMG1 .

Thereafter, the gene of pRS424- GPD - tHMG1 plasmid, GPD Promoter, and terminator was amplified using primers having the nucleotide sequences of SEQ ID NOS: 13 and 14, and then treated with Pst I and Sal I and inserted into plasmid YIplac128 digested with the same enzyme to obtain plasmid YIplac128- GPD - tHMG1 .

Thereafter, a DNA template containing each gene module including a promoter and terminator of YIplac126- GPD - tHMG1 plasmid and LEU2 gene as an auxotroph selection marker gene was amplified using primers having the nucleotide sequences of SEQ ID NOs: 15 and 16 To produce GPD - tHMG1 -CYC1-LEU2 cassettes for yeast transformation using homologous recombination.

CDNA was synthesized by reverse transcriptase using as a template the messenger RNA of Zanthorpylomyces dendrovus as a template. The cDNA template and the primers having the nucleotide sequences of SEQ ID NOs: 17, 18, 19, 20, 21 and 22 Using crtE , crtYB , crtI Each gene was amplified and the amplified gene fragment was treated with restriction enzymes Spe I and Xma I, Bam HI and Sal I, or Bam HI and Sal I. After inserted into the pRS426- TEF1, PGK1 pRS423-, and pRS424- PGK1 vector digested with the same restriction enzyme plasmid pRS426- TEF1 - was prepared crtI - crtE, pRS423- PGK1-crtYB , and pRS424- PGK1.

In order to introduce the gene on a chromosome-Cas9 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats-Cas9) using the method CrtE, CrtYB, CrtI and of the yeast ypl062w, ROX1, and on yjl064w Respectively. Ypl062w , ROX1 , and & lt ; RTI ID = 0.0 & gt ; For the construction of the sgRNA expression vector recognizing the base sequence of yjl064w, the pSRNR52-sg TEF1 vector was used as a template strand, and a primer having the nucleotide sequence of SEQ ID NO: 23 containing the Eco RI enzyme recognition site and a primer having the PAM sequence , 27, and 29 were used to amplify the first fragment, and primers having the nucleotide sequences of SEQ ID NOS: 25, 28, and 30 containing the PAM sequence containing 5'-NGG-3 ' The second fragment was amplified using a primer having the nucleotide sequence of SEQ ID NO: 26 containing the recognition site of the Kpn I enzyme. Using these two fragments as a template strand, a primer having the nucleotide sequence of SEQ ID NO: 23 containing the Eco RI enzyme recognition site and an extension extension PCR using the primer having the nucleotide sequence of SEQ ID NO: 26 containing the recognition site of the Kpn I enzyme To obtain a DNA fragment containing the sgRNA sequence. The DNA fragment of Kpn I and Eco RI and then treated with the enzyme, and then inserted into the treated with the same restriction enzyme pSNR52-sg TEF1 vector to introduce CrtE, CrtYB, and CrtI ypl062w, ROX1, and pSNR52 -sg ypl062w , SNR52-sg ROX1 and SNR52-sg yjl064w vectors recognizing the yjl064w site were prepared.

The DNA template containing each gene module including the promoter and terminator of pRS426- TEF1 - CrtE , pRS423- PGK1 - CrtYB , and pRS424- PGK1 - CrtI plasmid was designated SEQ ID NOS: 31, 32, 33, 34, 35, and 36 PCR primers were used to prepare TEF1 - CrtE - CYC1 , PGK1 - CrtYB - CYC1 , and PGK1 - CrtI - ADH1 cassettes for yeast transformation using the CRISPR - Cas9 method .

2.2 Anthropogenic organelles Controllability  Comparison of Carotenoid Productivity Improvement Using Recombinant Yeast

The transformant cassette GPD - tHMG1 - CYC1 prepared in the above 2.1 was transformed into wild-type yeast and recombinant yeast PX 1 having an artificial cell organotactic control ability prepared in Example 1, and then transformed cassette TEF1 - CrtE -CYC1, PGK1 - CrtYB - CYC1, and PGK1 - CrtI - ADH2, Cas9 expression vector Cas9-NAT (Addgene, Plasmid # 64329) vector, and ypl062w, ROX1, sgRNA expression vector pSNR52-sg that recognizes each site yjl064w The vector was introduced into the yeast PX1 from which wild type yeast and the peroxisome autolysis related protein ATG36 ( ATG36 ) were removed from the recombinant yeast having an artificial cell organotypic regulating ability prepared in Example 1. Each transformed recombinant yeast was named CEN-βCN (control) and PX1-βCN.

A single colony of each transformed recombinant yeast was inoculated in 5 ml of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) Each 5 ml of the obtained culture was inoculated into 50 ml of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) Time shake culture. After that, 1 ml of each culture was taken and the absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and the amount of the cells was determined by multiplying by the dilution factor. The cultured 50-ml medium was centrifuged to obtain cells, which were then extracted with 5 ml of 100% acetone to extract the expressed carotenoids. Carotenoids were extracted with 10 ml of hexane and water (H ₂ O) The nodal layer was split to separate the carotenoids. The separated carotenoids were transferred to a glass test tube and dried in a high pressure dryer for 30 minutes. The dried carotenoid extract was dissolved in 1 ml of ethyl acetate, and 20 μl of the extract was subjected to high pressure liquid chromatography (HPLC) photodiode array detector (DAD ) Was used to measure beta-carotene. The measured value was substituted into the equation obtained through the calibration curve, and the amount of beta carotene was calculated by calculating the dilution factor. For the calibration curve, standard beta-carotene (Sigma aldrich) was purchased and dissolved in ethyl acetate. The concentration of beta-carotene was measured using a high-pressure liquid chromatography photodiode array detector and diluted with ethyl acetate at different concentrations. A calibration curve was prepared. The conditions of the high pressure liquid chromatography performed are as follows.

- High Pressure Liquid Chromatography: Agilent Technologies 1200 series model with auto-sampler

- Photodiode array detector: Agilent Technologies 1200 series model

Column: Agilent Zorbax XDB-C18 (4.6 x 150 mm, 5-micron)

- Mobile phase: Acetonitrile: 80 / Methanol: 15 / Isoprepanol: 5 (v / v%)

- Rate: 1.0 ml / min

- Column temperature: 35 ℃

- Measuring device: DAD (Diode array detector)

- Sample injection amount: 20 μl

- Detection: spectrometry method (190nm ~ 900nm)

The amount of the recombinant yeast having the ability to produce beta carotene and the amount of each beta carotene produced through the artificial cell organellar regulation, confirmed by the above method, And 5.

Maximal device
(OD600) Beta-carotene
Absolute quantitative value (mg / L) CEN-βCN (control group) 6.38 ± 0.24 4.85 ± 0.29 PX1-βCN 5.06 ± 0.12 6.31 ± 0.01

As shown in Table 4 and FIG. 5, the recombinant yeast having an artificial cellular organotypic regulation ability of Example 1, an improved hydroxymethylglutaryl-CoA reductase, an enzyme that produces beta carotene, which is one of the carotenoids, geranyl geranyl pyrophosphate synthase, payito yen synthetic lycopene and cyclic enzymes, and payito yen transgenic cassettes that this DNA, coding for the enzyme expression unsaturated GPD - tHMG1 - CYC1, TEF1 - CrtE - CYC1, PGK1 -CrtYB- CYC1, and PGK1 - CrtI - When introducing ADH2, as compared with the introduction of the same transformation cassette wild-type yeast, although slightly reduced value up to an absolute growth production of beta-carotene was confirmed to increase. As shown in FIG. 5A, even when the cell growth value was decreased, it was not significantly different from the wild type at the time of recovery of the cells, and showed a deep orange color due to the increase of beta-carotene compared to the wild type.

As shown in FIG. 5B, when the carotenoid is over-expressed in the microorganism, the carotenoid is accumulated in the cell membrane and the cell growth is inhibited. Therefore, the maximum growth value of the recombinant yeast having the ability to produce beta carotene improved through artificial cellular organelle regulation But the production of beta-carotene was rather increased as shown in Fig. 5C. Specifically, the production of beta-carotene was further increased up to 130% based on the culture volume and up to 165% based on the unit cell. Thus, it was confirmed that the recombinant yeast having an artificial cellular organelle control ability exhibited high carotenoid productivity.

Example  4. Gibberellin  Metabolite Production capacity  Preparation of improved yeast

To confirm whether kaurene, a major metabolite of gibberellin in isoprenoid, which is a useful substance, can be produced using recombinant yeast having an artificial cellular organelle control ability prepared in Example 1, A novel gene cassette was introduced into yeast that did not have a pathway for the production of cholines, and productivity was regulated through regulation of cell organelles.

4.1 Gibberellin  Preparation of a recombinant vector containing a combination of enzyme genes related to metabolic biosynthesis

Mai Seth Serenity busy as Saccharomyces (Saccharomyces cerevisiae) gene encoding the active portion of the suppressing gene HMG1 the enzyme hydroxymethyl glutaryl -CoA reductase enzymes involved in gibberellin biosynthesis precursor removed from the part truncated HMG1 (tHMG1), xanthophylls Roman Isis dendeuro house (xanthophyllomyces dendrorhous ), a gene encoding a geranyl geranyl pyrophosphate synthase, which is an enzyme involved in biosynthesis of gibberellin metabolite, CrtE , Fusarium fujium fujikuroi ) was isolated and amplified from CDPS / KS , which is a gene encoding coarylpyrophosphate and kaurin synthase, among enzymes involved in the biosynthesis of gibberellin metabolites. A schematic diagram of the metabolic pathway including MVA and MEP pathway for kaurin biosynthesis, one of the gibberellin metabolites, is shown in FIG.

The forward and reverse primers used for the polymerase chain reaction were prepared by sequencing the nucleotide sequences of the genes encoding Saccharomyces cerevisiae, Zonthorphemaeisstendendorhaus, and the gibberellin and the precursor biosynthetic pathway-related enzyme of Fusarium fujicullo The information was compiled from the results of comparative analysis with the information in the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/), and the forward and reverse primers The nucleotide sequences and types of restriction enzymes contained in the primers are shown in Table 5 below.

Strain gene vector direction Base sequence Restriction enzyme Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) tHMG1 pRS424-GPD Forward SEQ ID NO: 11 Note I Reverse SEQ ID NO: 12 Spe I Xanthophyllomyces dendrorhous CrtE pRS426- TEF1 Forward SEQ ID NO: 17 Spe I Reverse SEQ ID NO: 18 Xma I Fujarium Fujikuroi
( Fusarium fujikuroi ) CDPS / KS pRS426- PGK1 Forward SEQ ID NO: 37 Spe I Reverse SEQ ID NO: 38 Xho I SAKAROMAISE Serebijie
( Saccharomyces cerevisiae ) ypl062w pSNR52-sg TEF1 Forward SEQ ID NO: 23 Eco RI Reverse SEQ ID NO: 24 - Forward SEQ ID NO: 25 - Reverse SEQ ID NO: 26 Kpn I ROX1 Forward SEQ ID NO: 23 Eco RI Reverse SEQ ID NO: 27 - Forward SEQ ID NO: 28 - Reverse SEQ ID NO: 26 Kpn I GPD - tHMG1 YIplac128 Forward SEQ ID NO: 13 Pst I Reverse SEQ ID NO: 14 Sal I GPD - tHMG1 - LEU2 YIplac128-GPD
-tHMG1 Forward SEQ ID NO: 15 - Reverse SEQ ID NO: 16 - Zontopil Roma Isis Dendro House
( xanthophyllomyces dendrorhous ) TEF1 - CrtE - CYC1 pRS426- TEF1 - CrtE Forward SEQ ID NO: 31 - Reverse SEQ ID NO: 32 - Fujarium Fujikuroi
( Fusarium fujikuroi ) PGK1 - CDPS / KS-TEF2 pRS426- PGK1 - CDPS / KS Forward SEQ ID NO: 33 - Reverse SEQ ID NO: 34 -

First, the tHMG1 gene was amplified using a DNA template of the gene synthesized based on the genomic information of Saccharomyces cerevisiae and a primer having a nucleotide sequence of SEQ ID NOs: 11 and 12. The amplified gene fragment was amplified by Not I And Spe I, and then digested with the same enzyme, pRS424- GPD Vector to prepare plasmid pRS424- GPD - tHMG1 .

Thereafter, the gene of pRS424- GPD - tHMG1 plasmid, GPD Promoter, and terminator was amplified using primers having the nucleotide sequences of SEQ ID NOS: 13 and 14, and then treated with Pst I and Sal I and inserted into plasmid YIplac128 digested with the same enzyme to obtain plasmid YIplac128- GPD - tHMG1 .

Thereafter, a DNA template containing each gene module including a promoter and terminator of YIplac126- GPD - tHMG1 plasmid and LEU2 gene as an auxotroph selection marker gene was amplified using primers having the nucleotide sequences of SEQ ID NOs: 15 and 16 To produce GPD - tHMG1 -CYC1-LEU2 cassettes for yeast transformation using homologous recombination.

In addition, cDNA was synthesized through reverse transcriptase reaction using messenger RNA of Zanthorpylomyces staine dendrohaeus and Fusarium pujicullo as a template, and the cDNA template and the nucleotide sequence of SEQ ID NOs: 17, 18, 37 and 38 RTI ID = 0.0 > rtE < / RTI > And CDPS / KS genes were amplified. The amplified gene fragments were treated with restriction enzymes Spe I, Xma I, Spe I and Xho I and inserted into the pRS426- TEF1 and pRS426- PGK1 vectors digested with the same enzymes to obtain plasmids pRS426- TEF1 - crtE and pRS426- PGK1 - CDPS / KS .

Then, CrtE and CDPS / KS were incubated with yeast's yepl062w And ROX1 YPl062W for transfection. And To construct an sgRNA expression vector recognizing the ROX1 nucleotide sequence, a pSRNR52-sg TEF1 vector was used as a template strand, and a primer having the nucleotide sequence of SEQ ID NO: 23 containing the recognition region of the Eco RI enzyme and a primer having the nucleotide sequence of SEQ ID NO: And 27 were used to amplify the first fragment. A primer having the nucleotide sequence of SEQ ID NOs: 25 and 28 containing the PAM sequence containing 5'-NGG-3 'and a primer having the nucleotide sequence of SEQ ID NO: 26 including the recognition site of the Kpn I enzyme were used for the second fragment Lt; / RTI > Using these two fragments as a template strand, a primer having the nucleotide sequence of SEQ ID NO: 23 containing the Eco RI enzyme recognition site and an extension extension PCR using the primer having the nucleotide sequence of SEQ ID NO: 26 containing the recognition site of the Kpn I enzyme To obtain a DNA fragment containing the sgRNA sequence. After treating this DNA fragment with Kpn I and Eco RI enzymes, and then inserted into the treated with the same restriction enzyme pSNR52-sg TEF1 ypl062w vector to introduce and CrtE CDPS / KS And PSNR52 -sg ypl062w and SNR52-sg ROX1 vectors recognizing the ROX1 region were prepared.

The DNA template containing each gene module including the promoter and terminator of pRS426 - TEF1 - CrtE and pRS426- TEF1 - CDPS / KS plasmid was amplified using primers having the nucleotide sequences of SEQ ID NOS: 31, 32, 33 and 34 TEF1 - CrtE - CYC1 for yeast transformation using the CRISPR-Cas9 method And PGK1 - CDPS / KS- TEF2 cassette.

4.2 Anthropogenic organelles Controllability  With recombinant yeast Kaurin  Productivity improvement comparison

The transformant cassette GPD - tHMG1 - CYC1 prepared in the above 3.1 was transformed into wild-type yeast and recombinant yeast PX 1 having an artificial cell organotactic control ability prepared in Example 1, and then transformed cassette TEF1 - CrtE -CYC1 and PGK1 - CDPS / KS- TEF2 , Cas9 -NAT (Addgene, Plasmid # 64329) vector, Cas9 expression vector, and ypl062w And The sgRNA expression vectors pSNR52-sg ypl062w and SNR52-sg ROX1 , which recognize the ROX1 region, The vector was transformed into wild-type yeast and PX1, a yeast strain from which the peroxisome auto-digestion-related protein ATG36 ( ATG36 ) was removed from recombinant yeast having an artificial cell organotypic regulating ability prepared in Example 1, and transformed into the control CEN- KRN and transformed PX1-KRN.

A single colony of each transformed recombinant yeast was inoculated in 5 ml of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) Each 5 ml of the obtained culture was inoculated into 50 ml of YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) Time shake culture. After that, 1 ml of each culture was taken and the absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and the amount of the cells was determined by multiplying by the dilution factor. In addition, each 50 ml culture medium was centrifuged to separate the cells and the culture medium. The cells were then extracted with 5 ml of 100% acetone to extract the expressed kaurin, and the cells were extracted with 10 ml of hexane and water (H ₂ O) to separate kaolin. The separated kaurin was transferred to a glass test tube and dried for 30 minutes using a high-pressure dryer. The dried kaurin extract was dissolved in 1 ml of hexane, and 1 μl of the extract was analyzed by gas chromatography (GC) mass spectrometry (MS) The kaurin content of the cells was measured. For the medium, 10 ml of hexane was added to a 10 ml medium to separate the kaolin layer to separate kaolin. The separated kaurin was transferred to a glass test tube and dried for 30 minutes using a high-pressure dryer. The dried kaurin extract was dissolved in 1 ml of hexane, and 1 μl of the extract was analyzed by gas chromatography (GC) mass spectrometry (MS) The kaolin of the medium was measured. The measured value was substituted into the equation obtained from the calibration curve, and the amount of kaolin was calculated by calculating the dilution factor. In order to prepare the calibration curve, the own kaurin was purified and its weight was measured, and it was dissolved in hexane, diluted with hexane at different concentrations, and the amount of kaolin was measured using a gas chromatography mass spectrometry system. Respectively. The gas chromatographic conditions used in the experiment are as follows.

- Gas chromatography: Agilent Technologies 7890A (Agilent Technologies 7890C) model with auto-sampler

- Mass spectrometry system: Agilent Technologies 5975C (Agilent Technologies 5975C) model

Column: Agilent HP5-MS (0.25 mm x 30 m, 0.25-micron)

- Electron ionization mode: 70ev

- Rate: 1.0 ml / min

- Sample injection temperature: 80 ℃

- oven temperature: maintained at 80 캜 for 1 minute, then heated to 245 캜 at a rate of 15 캜 / minute, then heated to 300 캜 at a rate of 5 캜 / minute

- Sample injection amount: 1μ (Splitless mode)

- Detection: MS results (90 m / z to 600 m / z)

The amount of the recombinant yeast having the ability to produce kaolin improved through artificial cell organellar control and the amount of each kaurin produced, confirmed by the above method, are shown in Table 6 and FIG.

Maximal device
(OD600) Kaurin
Absolute quantitative value (mg / L) CEN-KRN (control group) 12.28 + 0.07 9.08 ± 0.29 PX1-KRN 13.32 ± 0.24 10.07 ± 0.19

As shown in Table 6 and FIG. 7, in the recombinant yeast having an artificial cellular organelles regulating ability of Example 1, an improved hydroxymethylglutaryl-CoA reductase enzyme, which is an enzyme that produces kaurin, one of the gibberellin metabolites , Transgenic cassettes GPD - tHMG1 - CYC1 , TEF1 - CrtE - CYC1 , and PGK1 - CDPS / KS- TEF2 expressing a gene encoding a geranyl geranyl pyrophosphate synthase and a gene encoding a coaryl pyrophosphate and a kaurin synthase (Fig. 7A), kaurin production was further increased up to 120% (Fig. 7B), compared with the wild-type yeast in which the same transfection cassette was introduced, When the recombinant yeast having the regulatory ability was used, it was confirmed that the productivity of the high gibberellin metabolite was exhibited.

                         SEQUENCE LISTING <110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION   <120> Recombinant yeast with artificial cellular organelles and        producing method for isoprenoids with same <130> AJOU1-85P-1 <150> KR10-2017-0181134 <151> 2017-12-27 <160> 38 <170> PatentIn version 3.2 <210> 1 <211> 33 <212> DNA <213> Artificial <220> <223> PEX34 forward primer <400> 1 agcggatcca aaatggtttc gaagaaaaat acg 33 <210> 2 <211> 32 <212> DNA <213> Artificial <220> <223> PEX34 reverse primer <400> 2 tacgtcgact tatacaatta ttctacaaag tg 32 <210> 3 <211> 66 <212> DNA <213> Artificial <220> <223> ATG36 Del cassette forward primer <400> 3 tgtattcagg gcttaaaata ctaaaatttg gtggtcagta cagttcatta gacgttgtaa 60 aacgac 66 <210> 4 <211> 68 <212> DNA <213> Artificial <220> <223> ATG36 Del cassette reverse primer <400> 4 aactgatggt gttcggacaa cgttttagaa tgagggtatc taactttctt caggaaacag 60 ctatgacc 68 <210> 5 <211> 70 <212> DNA <213> Artificial <220> <223> PEX11 Del - PEX34 O / E cassette forward primer <400> 5 tgtctacatc tactacttca aagacttcat caagtaatag tataatcaat gacgttgtaa 60 aacgacggcc 70 <210> 6 <211> 72 <212> DNA <213> Artificial <220> <223> PEX11 Del - PEX34 O / E cassette reverse primer <400> 6 atcaaacata agcggagaat agccaaataa aaaaaaagat gaaaagaaag cacacaggaa 60 acagctatga cc 72 <210> 7 <211> 70 <212> DNA <213> Artificial <220> <223> PEX34 O / E cassette forward primer <400> 7 ccaaaatttg tttactaaaa acacatgtgg atatcttgac tgatttttcc gacgttgtaa 60 aacgacggcc 70 <210> 8 <211> 72 <212> DNA <213> Artificial <220> <223> PEX34 O / E cassette reverse primer <400> 8 gctctaattt gtgagtttag tatacatgca tttacttata atacagtttt cacacaggaa 60 acagctatga cc 72 <210> 9 <211> 70 <212> DNA <213> Artificial <220> <223> POT1-EGFP cassette forward primer <400> 9 gtatgtgtat cggtactggt atgggtgccg ccgccatctt tattaaagaa cggatccccg 60 ggttaattaa 70 <210> 10 <211> 70 <212> DNA <213> Artificial <220> <223> POT1-EGFP cassette reverse primer <400> 10 ttttaatact tgataatagt taatattctc cctttttatt atgctcatat gaattcgagc 60 tcgtttaaac 70 <210> 11 <211> 32 <212> DNA <213> Artificial <220> <223> tHMG1 forward primer <400> 11 cggcggccgc aaaatgtcta ttccagaaac tc 32 <210> 12 <211> 28 <212> DNA <213> Artificial <220> <223> tHMG1 reverse primer <400> 12 gcactagttt atttagaagt gtcaacaa 28 <210> 13 <211> 29 <212> DNA <213> Artificial <220> <223> GPD-tHMG1 forward primer <400> 13 gcctgcagag tttatcatta tcaatactc 29 <210> 14 <211> 25 <212> DNA <213> Artificial <220> <223> GPD-tHMG1 reverse primer <400> 14 cgcgtcgacg gccgcaaatt aaagc 25 <210> 15 <211> 71 <212> DNA <213> Artificial <220> GPD-tHMG1-LEU2 forward primer <400> 15 acgttggtca agaaatcaca gccgaagcca ttaaggttct taaagctatt agtttatcat 60 tatcaatact c 71 <210> 16 <211> 71 <212> DNA <213> Artificial <220> GPD-tHMG1-LEU2 reverse primer <400> 16 atggccttac cttcttcagg caagttcaat gacaatttca acatcattgc attatcatga 60 cattaaccta t 71 <210> 17 <211> 29 <212> DNA <213> Artificial <220> <223> CrtE forward primer <400> 17 gactagtaaa aatggattac gcgaacatc 29 <210> 18 <211> 26 <212> DNA <213> Artificial <220> <223> CrtE reverse primer <400> 18 ccccccgggt cacagaggga tatcgg 26 <210> 19 <211> 31 <212> DNA <213> Artificial <220> <223> CrtYB forward primer <400> 19 cgggatccaa aatgacggct ctcgcatatt a 31 <210> 20 <211> 28 <212> DNA <213> Artificial <220> <223> CrtYB reverse primer <400> 20 acgcgtcgac ttactgccct tcccatcc 28 <210> 21 <211> 33 <212> DNA <213> Artificial <220> <223> CrtI forward primer <400> 21 cgggatccaa aatgggaaaa gaacaagatc agg 33 <210> 22 <211> 31 <212> DNA <213> Artificial <220> <223> CrtI reverse primer <400> 22 acgcgtcgac tcagaaagca agaacaccaa c 31 <210> 23 <211> 17 <212> DNA <213> Artificial <220> <223> ypl062w forward primer or ROX1 forward primer or yjl064w forward primer <400> 23 gaattggagc tcactcg 17 <210> 24 <211> 40 <212> DNA <213> Artificial <220> <223> ypl062w reverse primer <400> 24 cggcgcgccc accctctttt gatcatttat ctttcactgc 40 <210> 25 <211> 41 <212> DNA <213> Artificial <220> <223> ypl062w forward primer <400> 25 gt; <210> 26 <211> 18 <212> DNA <213> Artificial <220> <223> ypl062w reverse primer or ROX1 reverse primer or yjl064w reverse primer <400> 26 gaacaaaagc tggtaccg 18 <210> 27 <211> 40 <212> DNA <213> Artificial <220> <223> ROX1 reverse primer <400> 27 ccccctctat taccttctct gatcatttat ctttcactgc 40 <210> 28 <211> 41 <212> DNA <213> Artificial <220> <223> ROX1 forward primer <400> 28 agagaaggta atagaggggg gttttagagc tagaaatagc a 41 <210> 29 <211> 40 <212> DNA <213> Artificial <220> <223> yjl064w forward primer <400> 29 ggtgctgaca catgagtccc gatcatttat ctttcactgc 40 <210> 30 <211> 41 <212> DNA <213> Artificial <220> <223> yjl064w forward primer <400> 30 gggactcatg tgtcagcacc gttttagagc tagaaatagc a 41 <210> 31 <211> 65 <212> DNA <213> Artificial <220> <223> TEF1-CrtE-CYC1 forward primer <400> 31 caggtcagga actgccgtca catacgacac tgcccctcac gtaagggcaa gcgcgcaatt 60 aaccc 65 <210> 32 <211> 68 <212> DNA <213> Artificial <220> <223> TEF1-CrtE-CYC1 reverse primer <400> 32 aatccccctc accccgaatt tattacgaat ttgcccacat ggtcggtgct atagggcgaa 60 ttgggtac 68 <210> 33 <211> 67 <212> DNA <213> Artificial <220> PGK1-CrtYB-CYC1 forward primer or PGK1-CDPS / KS-TEF2 forward primer <400> 33 ccagaaaata ctaatacttc ttcacacaaa agaacgcagt tagacaatcc aagcgcgcaa 60 ttaaccc 67 <210> 34 <211> 66 <212> DNA <213> Artificial <220> &Lt; 223 > PGK1-CDPS / KS-TEF2 reverse primer or PGK1-CrtYB-CYC1 reverse        primer <400> 34 gttaaaggga atatagtata atataatata acggaaagaa gaaatgggca gattgtactg 60 agagtg 66 <210> 35 <211> 67 <212> DNA <213> Artificial <220> <223> PGK1-CrtI-ADH2 forward primer <400> 35 cggagccgta tcgttcacca cataggcgga gtaaacttca ttagggggcg cgcgcaatta 60 accctca 67 <210> 36 <211> 66 <212> DNA <213> Artificial <220> &Lt; 223 > PGK1-CrtI-ADH2 reverse primer <400> 36 cagaagaaac aagagagaat agcgtcagga tagctcgctc gatgtgacac tatagggcga 60 attggg 66 <210> 37 <211> 29 <212> DNA <213> Artificial <220> <223> CDPS / KS forward primer <400> 37 ggactagtca aaatgcctgg caaaatcga 29 <210> 38 <211> 30 <212> DNA <213> Artificial <220> <223> CDPS / KS reverse primer <400> 38 ccgctcgagt cacttcatgc tgcttgaaag 30

Claims (13)

PEX34 (Peroxisomal membrane protein 34) overexpression, PEX11 (Peroxisomal membrane protein 11) gene deletion and ATG36 And a deletion of a gene encoding a peroxisome autophagy-related protein (36) gene in a cell organelle-transformed recombinant yeast.
2. The method according to claim 1, wherein the recombinant yeast is
i) ATG36 Recombinant yeast lacking the gene
ii) PEX34 Recombinant yeast overexpressing the gene
iii) PEX11 Gene deletion and PEX34 Recombinant yeast overexpressing the gene and
iv) PEX11 Gene deletion, ATG36 gene deletion and PEX34 Wherein the recombinant yeast is a yeast selected from the group consisting of a recombinant yeast overexpressing the gene and a cell organellated mutant.
The method of claim 1, wherein the PEX34 Gene, PEX11 Wherein the gene and the ATG36 gene are derived from Saccharomyces cerevisiae.
The recombinant yeast according to claim 1, wherein the recombinant yeast is selected from the group consisting of S. cerevisiae, S. bayanus, S. boulardii, S. bulderi, S. cariocanus, S. cariocus, S. chevalieri, and S. cariocanus, S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, and S. cerevisiae, For example, S. florentinus, S. kluyveri, S. martiniae, S. monacensis, and S. cerevisiae, S. norbensis, S. paradoxus, S. pastorius, and the like, an S. cerevisiae, S. spencerorum, S. turicensis, S. unisporus, S saccharomyces utavorum (S wherein the recombinant yeast is one selected from the group consisting of yeast, uvarum, and S. zonatus.
5. The recombinant yeast according to any one of claims 1 to 4, wherein the mutation of the cell organelles is an increase in the number or size of peroxisome.
The recombinant yeast of claim 1,
i) tHMG1 (truncated hydroxy-methyl glutaryl-CoA reductase) , CrtE (Geranylgeranyl pyrophosphate synthase), CrtYB (Phytoene synthase-lycopene cyclase) and CrtI ( Phytoene desaturase) Gene set and
ii) tHMG1, CrtE and And a set of genes selected from the group consisting of CDPS / KS (ent-copalyl diphosphate / kaurene synthase) gene sets.
7. The recombinant yeast according to claim 6, wherein the isoprenoid is beta carotene, kaurin, carotinoid or gibberellin.
7. The method of claim 6, wherein the tHMGl is It is derived from Saccharomyces cerevisiae. The CrtE, CrtYB And CrtI Wherein the recombinant yeast is derived from xanthophyllomyces dendrorhous , and wherein the CDPS / KS is derived from Fusarium fujikuroi .
The method of claim 6, wherein the recombinant yeast is i) tHMG1 (truncated HMG1), CrtE, CrtYB and A recombinant yeast with increased isoprenoid productivity, wherein the isoformic acid is a carotenoid, wherein the CrtI gene set is introduced.
10. The recombinant yeast according to claim 9, wherein the recombinant yeast is increased in beta-carotene productivity.
7. The method according to claim 6, wherein the recombinant yeast is selected from the group consisting of tHMG1 , CrtE And A recombinant yeast with enhanced isoprenoid productivity, wherein the isoprenoid is gibberellin, wherein the CDPS / KS gene set is introduced.
12. The recombinant yeast according to claim 11, wherein the recombinant yeast is increased in kaolin productivity.
Culturing the recombinant yeast of any one of claims 6 to 12; &Lt; / RTI &gt;

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