KR102160215B1 - Yarrowia liplytica strain having improved xylose utilizing ability and the method for preparing the same - Google Patents

Abstract

The present specification describes a new Yarrowia lipolytica recombinant strain with improved xylose utilization ability, a preparing method thereof, and a bio-oil production method using the same. The present invention enables the production of high-yield bio-oil by transforming the wild-type Yarrowia lipolytica strain, which cannot use xylose as a carbon source, thereby improving the economics and sustainability of the production process of materials such as biofuels or cosmetics, health functional foods, oleochemical products and the like.

Description

Yarrowia liplytica strain having improved xylose utilizing ability and the method for preparing the same}

The present specification describes a novel microorganism with improved xylose utilization, a method for manufacturing the same, and a method for producing bio oil using the same.

As a new biodiesel production technology that can overcome the limitations of existing vegetable oil or microalgae-based biodiesel production in order to cope with the increasing biodiesel demand, it is a raw material for biodiesel from biomass using a lipid-producing yeast strain. Technologies that can improve the economic feasibility and sustainability of biodiesel by producing phosphorus lipids in high concentration are being developed. These lipids can be applied to the production of biodiesel as well as bio aviation oil, which is a technology that can greatly contribute to the production and supply of biofuels to reduce greenhouse gases.

The lipid production process using yeast strains is an economical process that can easily convert biomass into biofuel, but when non-edible biomass such as lignocellulosic biomass is used as a raw material, hexose derived from biomass (typically glucose, biofuel There is a problem in that conversion efficiency is deteriorated because only 50% of the mass is contained) and pentose (typically, xylose, containing 25% of the biomass) is not used. Therefore, it is necessary to develop a lipid-producing yeast strain capable of metabolizing xylose for more economical biodiesel production.

International Patent Publication WO2013192520A1

Rodrigo Ledesma Amaro et al., Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose, Metabolic Engineering, 2016, 38, 115-124 Gabriel M. Rodriguez et al., Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway, Biotechnology for Biofuels, 2016, 9:149 Haibo Li et al., Enabling xylose utilization in Yarrowia lipolytica for lipid production, Biotechnology Journal, 2016, 11, 1230-1240

In one aspect, the problem to be solved by the present invention is to provide a Yarrowia lipolytica transformed yeast strain with improved xylose availability.

In one aspect, the problem to be solved by the present invention is to provide a method for producing a Yarrowia lipolytica transformed yeast strain with improved xylose availability.

In one aspect, the problem to be solved by the present invention is to provide a bio-oil production method using a Yarrowia lipolytica transformed yeast strain with improved xylose availability.

In order to solve the above problem, an embodiment of the present invention is an adaptive evolution strain that is passaged in a minimal medium using xylose as the sole carbon source, and a gene encoding a xylose isomerase and a xylulose kinase comprising a gene encoding a (Xylulokinase), and the adaptive evolution strain provides 495_500delCAACTT comprising a mutation at a gene YALI0_D07634g, Yarrow subtotal Li poly urticae (Yarrowia lipolytica) transgenic yeast strains.

In order to achieve the above object, one embodiment of the present invention are passaged in a minimal medium for the above transformation Yeast production process, to the wild-type Yarrow subtotal Li poly urticae (Yarrowia lipolytica) as a parent strain, xylose as the only carbon source Culturing and adaptive evolution; And inserting a gene encoding a xylose isomerase and a gene encoding a xylulose kinase into the adapted strain.

In order to solve the above problems, an embodiment of the present invention is a bio-oil comprising the step of culturing the transformed yeast strain in a medium containing xylose as a sole carbon source, or a medium containing glucose and xylose as a carbon source. Provide production methods.

According to the present invention, a wild-type Yarrowia lipolytica strain that cannot use xylose as a carbon source is adaptively evolved through evolutionary engineering, and the metabolic pathway of xylose, a major sugar derived from lignocellulosic biomass, is based on xylose isomerase. Introduced to provide a transformed yeast strain with improved xylose metabolic efficiency. The yeast strain according to the present invention does not introduce an oxidoreductase-based xylose metabolic pathway, so there is no problem of cofactor imbalance. In addition, since xylose metabolism efficiency is improved, both glucose and xylose are used as carbon sources, so that the conversion rate from biomass to lipids can be about 50% to 75%. Therefore, the use of the yeast strain according to the present invention can greatly improve the economical efficiency and sustainability of the production process of bio-oil or oil-fat chemical material.

1 is a Yarrow subtotal Li poly urticae adapted evolved strain (YX1), wherein the YX1 strain xylose isomerase strain (YX1-A) introducing a gene coding for, xylene rule to the YX1 strain according to an embodiment of the present invention strains to introduce a gene coding for Ross kinases (YX1-K), wherein the YX1 strain xylose isomerase and xylene rule Los strain incorporating a gene encoding the kinase (YX1-AK), wild-type Yarrow subtotal Li poly It is a diagram showing the strain growth curve in the minimal medium using xylose as a carbon source of the strain (WT-AK) in which the genes encoding xylose isomerase and xylulose kinase were introduced into the tica strain.
Figure 2 is a Yarrow subtotal Li poly urticae adapted evolved strain (YX1), wherein the YX1 strain xylose isomerase strain (YX1-A) introducing a gene coding for, xylene rule to the YX1 strain according to an embodiment of the present invention A strain into which a gene encoding a Ross kinase enzyme was introduced (YX1-K), a strain in which a gene encoding a xylose isomerase and a xylulose kinase was introduced into the YX1 strain (YX1-AK), and a wild-type Yarrowia li A diagram showing the xylose utilization curve for 240 hours of culture in a minimal medium using xylose as a carbon source of a strain (WT-AK) in which a gene encoding xylose isomerase and xylulose kinase was introduced into a polytica strain. to be.
3 is compared with the one embodiment Yarrow subtotal Li poly urticae adapted evolved strain (YX1), a strain (YX1-AK) introducing a gene coding for xylose isomerase and xylene rule Los kinases in the YX1 strain of the present invention by way of example the strain growth curve in minimal medium of the Yarrow subtotal Li poly urticae strain (YSX) on xylose xylose of isomerase and xylene rule Los kinases strain (YSX-AK) introducing a gene coding for a carbon source It is a diagram shown.
4 is compared with an embodiment Yarrow subtotal Li poly urticae adapted evolved strain (YX1), a strain (YX1-AK) introducing a gene coding for xylose isomerase and xylene rule Los kinases in the YX1 strain of the present invention by way of example Yarrow subtotal Li poly urticae strain (YSX) to xylose isomerase and xylene rule for culturing 300 hours in a minimal medium for xylose of Los kinases strain (YSX-AK) introducing a gene coding for a carbon source It is a diagram showing the use curve of xylose.
5 is a transformed strain in which a wild-type Yarrowia lipolytica strain, which is an embodiment of the present invention, is adaptively evolved, and a gene encoding xylose isomerase and xylulose kinase is introduced, and diacylglycerol acyl transferase is overexpressed ( YX2-AK) is a diagram showing the use curves of glucose and xylose in the biomass saccharified solution in the bio-oil production process.

Hereinafter, embodiments of the present invention will be described in detail.

In the present specification, “lipid” refers to an organic material or organic compound composed of a fatty acid and glycerol. The lipid may include one or more of acylglycerol, glyceride and free fatty acids. The acyl glycerol may be at least one selected from the group consisting of triacylglycerol (TAG), diacylglycerol (DAG), and monoacylglycerol (MAG). The glyceride may be at least one selected from the group consisting of monoglyceride, diglyceride, and triglyceride. Alternatively, the lipid is butyric acid (butanoic acid, C4:0), caproic acid (hexanoic acid, C6:0), caprylic acid (octanoic acid, C8:0), capric acid (decanoic acid, C10:0), Lauric acid (dodecanoic acid, C12:0), myristic acid (tetradecanoic acid, C14:0), myristoleic acid (ω-5, C14:1), pentadecylic acid (C15:0), Palmitic acid (hexadecanoic acid, C16:0), palmitoleic acid (ω-7, C16:1), hexadecadienoic acid (C16:2), hexadecatrienoic acid (C16: 3), Margaric acid (C17:0), heptadenoic acid (C17:1), stearic acid (octadecanoic acid, C18:0), oleic acid (ω-9, C18:1), Linoleic acid (LA, ω-6, C18:2), alpha-linolenic acid (ALA, ω-3, C18:3), octadecatetraenoic acid (C18:4), nonadecylic acid, C19:0), nonadecylic acid (C19:1), arachidic acid (eiconic acid, C20:0), arachidonic acid (AA, ω-6, C20:4), eicosapentaenoic acid (EPA , ω-3, C20:5), behenic acid (docoic acid, C22:0), erucic acid (ω-9, C22:1), docosapentaenoic acid (DPA, ω-3, 22:5) , And docosahexaenoic acid (DHA, ω-3, C22: 6) may be one or more selected from the group consisting of, specifically C16:0, C16:1, C16:2, C16:3, C18:0, It may be one or more fatty acids selected from the group consisting of C18:1, C18:2, C18:3, and C18:4.

In the present specification, "wood-based biomass" includes herbaceous and woody biomass as a raw material that provides a carbon source necessary for the strain producing lipids according to an embodiment of the present invention. Lignocellulosic biomass is the most abundant carbon source, accounting for more than 90% of the total biomass produced on the planet, and is an eco-friendly and renewable resource. In addition, when biofuels are produced using woody biomass, the carbon reduction effect is greater than fossil fuels and first-generation biofuels. Such woody biomass includes waste wood, agricultural and forestry by-products, and energy crops. It is known that the content of glucose among the available carbon sources in the woody biomass is about 50% or less, and that the xylose content is about 25% or less.

An embodiment of the present invention Provides a Yarrowia lipolytica transformed yeast strain with improved xylose utilization.

Yarrow subtotal Li poly urticae (Yarrowia lipolytica) have been reported that can produce a lipid for typical yeast lipid produced mainly recombinant strains in glucose-based medium to about 90% of the cell dry weight. However, when the wild-type Yarrowia lipolytica strain produces lipids from lignocellulosic biomass rather than refined sugar, the use of carbon sources is limited because pentose (xylose), a representative hydrolyzate of lignocellulosic biomass, cannot be used. In addition, the wild-type Yarrowia lipolytica strain has an oxidoreductase-based xylose metabolic pathway that can endogenously use xylose, but its metabolism is impossible due to the low level of expression of related genes. International Patent Publication WO2013192520A1, Rodrigo Ledesma Amaro et al., Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose, Metabolic Engineering, 2016, 38, 115-124, Gabriel M. Rodriguez et al., Engineering xylose utilization in Yarrowia lipolytica by understanding its cryptic xylose pathway, Biotechnology for Biofuels, 2016, 9:149 and Haibo Li et al., Enabling xylose utilization in Yarrowia lipolytica for lipid production, Biotechnology Journal, 2016, 11, 1230-1240 . poly urticae and the xylose metabolic pathway of the oxidation-reduction enzyme-based development of a recombinant strain is reported introduction bar for imparting the ability to use xylose strain, the above document are incorporated herein by reference in its entirety. However, the recombinant strains reported in the above documents did not reach a satisfactory level of xylose utilization performance, and in particular, the oxidoreductase-based xylose metabolic pathway is not only a cofactor imbalance problem, but also NADPH, a cofactor that is highly consumed in the lipid production process. There is a problem that lipid production performance may be degraded because of consumption.

Accordingly, the present inventors applied the evolution method to Yarrowia lipolytica strains as a technology capable of producing high-yield bio-oils and materials, and improved the use of xylose by introducing a xylose pathway based on xylose isomerase. A new strain was developed.

Specifically, the present invention is an adaptive evolution strain passaged in a minimal medium using xylose as the sole carbon source, as an embodiment, and a gene encoding a xylose isomerase and a xylulokinase. It provides a Yarrowia lipolytica transformed yeast strain comprising the encoding gene.

As an example, the adaptive evolutionary strain may include a 495_500delCAACTT mutation in the YALI0_D07634g gene. In one embodiment the adaptive evolution strain YALI0_A00374g, YALI0_A00935g, YALI0_A02497g, YALI0_A02937g, YALI0_A15642g, YALI0_A15796g, YALI0_A15796g, YALI0_A17578g, YALI0_A17776g, YALI0_A17776g, YALI0_A17853g, YALI0_A17853g, YALI0_A18744g, YALI0_A19646g, YALI0_B00748g, YALI0_B05676g, YALI0_B18194g, YALI0_C07722g, YALI0_C07722g, YALI0_C08327g, YALI0_C08349g , YALI0_C08349g, YALI0_C08473g, YALI0_C09031g, YALI0_C09614g, YALI0_C13002g, YALI0_C13728g, YALI0_C13728g, YALI0_C13970g, YALI0_C15532g, YALI0_C15741g, YALI0_C16148g, YALI0_C16247g, YALI0_C16247g, YALI0_C16247g, YALI0_C16247g, YALI0_C16247g, YALI0_C16247g, YALI0_C16247g, YALI0_D00627g, YALI0_D09647g, YALI0_D14542g, YALI0_D15752g, YALI0_D15752g, YALI0_D17820g, YALI0_D17820g , YALI0_D18678g, YALI0_D19360g, YALI0_D20064g, YALI0_D20526g, YALI0_D20790g, YALI0_D24849g, YALI0_D25058g, YALI0_D26972g, YALI0_E07832g, YALI0_E08008g, YALI0_E13948g, YALI0_E18766g, YALI0_E19767g, YALI0_E20053g, YALI0_E20449g, YALI0_E23969g, YALI0_E23969g, YALI0_E29623g, YALI0_E31999g, YALI0 _E33891g, YALI0_E33891g, YALI0_E34089g, YALI0_E35046g, YALI0_F12573g, YALI0_F12771g, YALI0_F12793g, YALI0_F12793g, YALI0_F18568g, YALI0_F19030g, YALI0_F19030g, YALI0_F19030g, YALI0_F19030g, YALI0_F19030g, YALI0_F19030g, YALI0_F19030g, YALI0_F19030g

597_598insGGCTCCGCTGCCGCCTCC mutation in the YALI0_A00374g gene;

202_203insGCTC mutation in the YALI0_A00935g gene;

427_429dupAAC mutation in the YALI0_A02497g gene;

20_21insGGAGACCGTGGGGGAGACCGTGGA mutation in the YALI0_A02937g gene;

147G>A mutation in the YALI0_A15642g gene;

576T>G mutation in the YALI0_A15796g gene;

567_568insACA mutation in the YALI0_A15796g gene;

434_435insGGACCCGCC mutation in the YALI0_A17578g gene;

1810_1811insACAACAACACCC mutation in the YALI0_A17776g gene;

959_960insACAGCAGAT mutation in the YALI0_A17776g gene;

1940_1942delAGG mutation in the YALI0_A17853g gene;

1990_1991insAGGAGGAGGCTAAGAAGA mutation in the YALI0_A17853g gene;

2512_2513insCTGGAGGGGGTGGAAGCG mutation in the YALI0_A18744g gene;

1467delA mutation in the YALI0_A19646g gene;

522_523insAACTCC mutation in the YALI0_B00748g gene;

1369_1370insCAGCAGCGT mutation in the YALI0_B05676g gene;

1695_1697delCGG mutation in the YALI0_B18194g gene;

1553C>T mutation in the YALI0_C07722g gene;

1552A>G mutation in the YALI0_C07722g gene;

1221_1250delTCCTCACGTGCAGCCTCCTCATGCGCAGCC mutation in the YALI0_C08327g gene;

2455_2456insCTCCCATCTCCTCCT mutation in the YALI0_C08349g gene;

2471_2472insTCCCATCTCCTCCGA mutation in the YALI0_C08349g gene;

1326_1327insGCACCTGCGACTTCTTCG mutation in the YALI0_C08473g gene;

1789_1790insCTCCCGAGTCCTCTGCTGAGCCTACCAGCGAAGAGACTTCTTCCG mutation in the YALI0_C09031g gene;

1617_1622delACAACA mutation in the YALI0_C09614g gene;

815_816insTCTAA mutation in the YALI0_C13002g gene;

99_100insAAAAAGTGGCCGAAAGAGTGGCCA mutation in the YALI0_C13728g gene;

129_130insTGGCCGAAAAAGTGGCCAAAA mutation in the YALI0_C13728g gene;

1587_1588insGCT mutation in the YALI0_C13970g gene;

1611_1616delCAGCTT mutation in the YALI0_C15532g gene;

198_209delCTATTCAAACGA mutation in the YALI0_C15741g gene;

957_968delCAGCAGCAGCAG mutation in the YALI0_C16148g gene;

1292A>G mutation in the YALI0_C16247g gene;

1412T>A mutation in the YALI0_C16247g gene;

1441G>A mutation in the YALI0_C16247g gene;

1513A>G mutation in the YALI0_C16247g gene;

1534A>C mutation in the YALI0_C16247g gene;

1544T>A mutation in the YALI0_C16247g gene;

1847C>T mutation in the YALI0_C16247g gene;

2603A>C mutation in the YALI0_D00627g gene;

491_492insAAAGCAGCAGCAGCA mutation in the YALI0_D09647g gene;

80_81insC mutation in the YALI0_D14542g gene;

1038_1039insCAG mutation in the YALI0_D15752g gene;

1038_1039insCGGCAG mutation in the YALI0_D15752g gene;

615_616insCCCCACCACCCGCAA mutation in the YALI0_D17820g gene;

641_642insGCAAACCTACCACCA mutation in the YALI0_D17820g gene;

1300_1323 delACCCAGAACCAGACCCAGAACCAG mutation in the YALI0_D18678g gene;

2132_2155delGGAGTGAGAGTGGGAGTGAGAGTG mutation in the YALI0_D19360g gene;

69_70delCT mutation in the YALI0_D20064g gene;

355_356insCCACCGGCG mutation in the YALI0_D20526g gene;

39_40insCGTGAAAGGAGCCGAACC mutation in the YALI0_D20790g gene;

103G>A mutation in the YALI0_D24849g gene;

592_594dupAAG mutation in the YALI0_D25058g gene;

36_41delACAGCA mutation in the YALI0_D26972g gene;

499_500insCCAAGCCCCCCGCTTCCAAGCCCACCGCTT mutation in the YALI0_E07832g gene;

794_795insCTCTTCCTCTTCCTCTTCCTCTTCCTCTTC mutation in the YALI0_E08008g gene;

825_830delCAGCAA mutation in the YALI0_E13948g gene;

1100_1101insTTCTGCCGCTTA mutation in the YALI0_E18766g gene;

2699A>G mutation in the YALI0_E19767g gene;

533_534insGGCCGAGGAGGC mutation in the YALI0_E20053g gene;

902_903insGGCCAA mutation in the YALI0_E20449g gene;

480_481insTCCGCTACCACCGAG mutation in the YALI0_E23969g gene;

497A>C mutation in the YALI0_E23969g gene;

10A>G mutation in the YALI0_E29623g gene;

269_270insTGTGTATATATGCAGAAAAAAAAAAA mutation in the YALI0_E31999g gene;

237C>A mutation in the YALI0_E33891g gene;

239G>C mutation in the YALI0_E33891g gene;

72_73insCG mutation in the YALI0_E34089g gene;

1926T>C mutation in the YALI0_E35046g gene;

75_76insTCCCCG mutation in the YALI0_F12573g gene;

1019_1021delAGT mutation in the YALI0_F12771g gene;

1855_1860delTCTTCT mutation in the YALI0_F12793g gene;

4102_4103insTAGCCG mutation in the YALI0_F12793g gene;

718_723delCAGCAG mutation in the YALI0_F18568g gene;

2099C>A mutation in the YALI0_F19030g gene;

2098A>G mutation in the YALI0_F19030g gene;

2065T>A mutation in the YALI0_F19030g gene;

1344_1345insCCTACTCCCTCCGAGGTT mutation in the YALI0_F19030g gene.

In one embodiment the Yarrow subtotal Li poly urticae adaptive evolution strain may be a strain deposited as accession number of KCTC 13911BP.

Yarrow parent strain of a subtotal Li poly Mathematica to transform the one embodiment may be a wild-type strain Yarrow subtotal Li poly Mathematica. As an example, it may be confirmed that the wild-type Yarrowia lipolytica strain is commercially available, or is preserved in a reliable preservation institution and can be freely distributed by a catalog issued by a preservation institution. . In one embodiment, the wild-type Yarrowia lipolytica strain may be a strain having accession number ATCC MYA-2613, but the wild-type Yarrowia lipolytica strain is not limited thereto and may be included.

In one embodiment, the gene encoding the xylose isomerase is a gene encoding an enzyme that converts D-xylose and D-xylulose to each other, and isomerization of xylose. If possible, it is not limited thereto and may be included. As an embodiment, the gene encoding the xylose isomerase may include xylA3* represented by the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In one embodiment, the gene encoding the xylulose kinase is an enzyme that produces D-xylulose-5-phosphate from D-xylulose. As a coding gene, If phosphorylation of xylose is possible, it is not limited thereto, and all may be included. As an embodiment, the gene encoding the xylulose kinase may include ylXK (xylulokinase) represented by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

In the present specification, the mutations can be introduced by treating microorganisms with any chemical means and/or physical means known in the art as capable of causing mutations. The chemical means may include chemicals such as NTG (nitrosoguanidine), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), benzopyrene, etc., which are effective guanidine derivatives as substances (mutations) that cause mutations, and the physical means is ultraviolet , X-rays, γ-rays, and the like, but are not limited thereto. In addition, the mutant gene may be introduced using a molecular biological method, and specifically, gene scissors, for example, CRISPR-Cas9 may be used, but the present invention is not limited thereto.

As an embodiment, the transforming yeast strain may be a first vector including a gene encoding a xylose isomerase and a second vector including a gene encoding a xylulose kinase. Alternatively, as another embodiment, the transforming yeast strain may be introduced with a third vector including both a gene encoding a xylose isomerase and a gene encoding a xylulose kinase.

As an example, the first to third vectors may be genomic DNA or plasmid, but are not limited thereto. In addition, the expression "first, second, or third" is only for distinguishing the type of vector, and does not limit the order or method of transformation. As an example, the genes introduced into the first vector, the second vector, and the third vector may be inserted into genomic DNA or expressed in a plasmid form.

As an example, the first vector may include a UAS1B enhancer, a translational elongation factor (TEF) promoter, and a gene encoding a xylose isomerase. For example, the first vector is SEQ ID NO: It may consist of the nucleotide sequence of 3 (pMCS-UAS1B16-TEF-XylA3*-CYC1t). As an example, the second vector may include a UAS1B enhancer, a translational elongation factor (TEF) promoter, and a gene encoding a xylulose kinase, for example, the second vector has a sequence It may consist of the nucleotide sequence number 4 (pMCSu-UAS1B12-TEF-XK-CYC1t). As an embodiment, the third vector may include a UAS1B enhancer, a translational elongation factor (TEF) promoter, a xylose isomerase-encoding gene, and a xylulose kinase-encoding gene. For example, the third vector may be composed of the nucleotide sequence of SEQ ID NO: 5 (pMCSu-UAS1B12-TEF-ylXK-CYC1t-TEFint-XylA3*-Syn7t).

In addition, as an embodiment of the present invention, the transformed yeast strain may be one overexpressing diacylglycerol acyltransferase. Specifically, the strain deletes peroxisomal biogenesis factor 10 represented by SEQ ID NO: 6 nucleotide sequence using the CRISPR/Cas9 system, and UAS1B enhancer, TEF (Translational elongation factor) Diacylglycerol acyl transferase (SEQ ID NO: 8, pMCS-16TEF-DGA1-CYC1t) containing a fourth vector (SEQ ID NO: 8; Diacylglycerol acyltransferase) may be overexpressed.

As an example may be a strain deposited with the accession number of KCTC 13912BP to diacylglycerol acyl transferase (Diacylglycerol acyltransferase) the Yarrow subtotal Li poly Mathematica transformed yeast overexpressing.

Since xylose accounts for up to about 25% and glucose in about 50% in lignocellulosic biomass, the transgenic yeast strain according to an embodiment of the present invention uses both glucose and xylose as carbon sources, Up to 75% of the available resources in the mass can be used. Therefore, it is possible to significantly improve the yield of lipid production for a unit biomass, thereby greatly improving the economics and sustainability of the production of biooils and biomaterials using lignocellulosic biomass.

As an embodiment, the present invention may include a culture of the Yarrowia lipolytica transformed yeast strain described above. The culture may contain lipids.

In the present specification, "culture" is applicable without limitation as long as it is by a culture method known in the art. As an embodiment, the culture of the yeast strain may be culture by any one selected from the group consisting of shaking culture, stationary culture, batch culture, fed-batch culture, and continuous culture. The shaking culture means a method of culturing while shaking a culture inoculated with a microorganism, and the stationary culture means a method of culturing a liquid culture inoculated with a microorganism without shaking. The batch culture refers to a method of fixing the volume of the culture and culturing without adding a new culture from the outside, and an apparatus capable of realizing such a culture method is called a batch reactor. The fed-batch culture refers to a method of culturing by first adding a small amount of elements and adding the raw materials to this as a contrast to a single culture (batch) in which all raw materials are first put into a culture tank and then cultured. A device that can be realized is called a fed-batch reactor. The continuous culture refers to a culture method in which a new nutrient medium is continuously supplied and cultures including cells and products are continuously removed, and an apparatus capable of realizing such a culture method is called a continuous reactor. Specifically, the culture may be carried out in a batch reactor, a continuous reactor, or a fed-batch reactor.

As an embodiment, the cultivation is performed by adjusting the temperature and pH in a conventional medium containing at least one selected from the group consisting of an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., while satisfying the cultivation requirements of the yeast strain in an appropriate manner. I can. For example, carbon sources that can be used include sugars and carbohydrates such as glucose, xylose, sucrose, lactose, fructose, maltose, starch, cellulose, oils such as soybean oil, sunflower oil, castor oil, coconut oil, etc. Fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, volatile fatty acids such as acetic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, isovaleric acid, caproic acid, etc. acid, VFA), etc. may be included. These materials may be included individually or in combination. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and corresponding sodium-containing salt as personnel. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used as the inorganic compound. Finally, substances such as amino acids and vitamins can be used in addition to the above substances.

As an example, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the culture process, but are not particularly limited thereto. An appropriate concentration of a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid can be used in an appropriate amount and manner to adjust the pH of the culture.

An embodiment according to the present invention may provide a method for producing the Yarrowia lipolytica transformed yeast strain.

The production method according to an embodiment, the wild-type Yarrow subtotal Li poly urticae (Yarrowia lipolytica) to a parent strain by subculture to adaptive evolution in a minimal medium for the xylose as the only carbon source comprising: (adaptive evolution), and the adaptive evolution It may include the step of inserting a gene encoding a xylose isomerase and a gene encoding a xylulose kinase into the strain. In an embodiment, the gene can be inserted using CRISPR/Cas9 or by introducing a plasmid.

In one embodiment the method comprising the step of said adaptive evolution, insert the wild-type gene encoding a gene and poly Yarrow subtotal Lee Giles rule Los kinases encoding the xylose isomerase in the Mathematica (Yarrowia lipolytica); Subculturing the inserted strain in a minimal medium containing xylose as the sole carbon source; And removing a gene encoding a xylose isomerase and a gene encoding a xylulose kinase from the subcultured strain.

In addition, as an embodiment, the method may further include overexpressing diacylglycerol acyltransferase. Specifically, the above step is to delete the peroxisomal biogenesis factor 10 using the CRISPR / Cas9 system as described above, UAS1B enhancer (enhancer), TEF (Translational elongation factor) promoter, diacil It may include overexpressing diacylglycerol acyltransferase using a fourth vector (pMCS-16TEF-DGA1-CYC1t) containing a gene encoding a glycerol acyl transferase.

As an embodiment, the insertion or deletion step of the genes may include all of the sequences of the genes to be inserted or deleted are not limited to the order described herein.

As an example, the adaptive evolution is a method for obtaining a strain with good performance by inducing a process of evolution occurring in nature to occur quickly and effectively in a laboratory. The method may include a method of subculturing the strain in a medium containing xylose as a carbon source, and then screening a strain having good xylose availability to select a strain having improved xylose utilization. The minimal medium may be a medium containing xylose as a carbon source and to which nutrients necessary for growth of other strains are added. For example, the minimum medium is YSC medium (Yeast Synthetic Complete medium). As described above, by introducing a xylose metabolic pathway based on a xylose isomerase into a yeast strain adaptively evolved through metabolic engineering and evolutionary engineering, a transformed yeast strain having improved xylose utilization can be prepared. As an embodiment, the adaptive evolution may include adaptive evolution to have xylose usability by subculturing 2 times, 3 times, 4 times, 5 times or more in a medium using xylose as the sole carbon source, It is not limited thereto.

As an embodiment, the gene insertion or deletion may be performed using CRISPR/Cas9, but any method capable of inserting or deleting the gene is not limited thereto and may be included. As an example, when the strain is transformed using the CRISPR/Cas9, it is easy to further improve the transformed yeast strain, such as introducing a metabolic pathway for producing other materials in addition to bio-oil.

An embodiment according to the present invention may provide a method for producing bio-oil comprising the step of culturing the Yarrowia lipolytica transformed yeast strain in a medium containing xylose as a sole carbon source. The description of the transformed yeast strain and the cultivation of the strain are as described above.

In one embodiment, the step of culturing the Yarrow subtotal Li poly urticae transform the yeast strains for xylose minimal medium as a sole carbon source: In was inoculated to (YSC medium Yeast Synthetic Complete medium) 24-32 ℃ 24-300 Incubating for a period of time may be included.

Another embodiment may provide a bio-oil production method comprising culturing the Yarrowia lipolytica transformed yeast strain in a medium containing glucose and xylose as a carbon source. Specifically, the step of culturing the Yarrow subtotal Li poly Mathematica the transformed yeast strains and xylose minimal medium for glucose as a carbon source: 24-300 hours at 24-32 ℃ was inoculated to (YSC medium Yeast Synthetic Complete medium) It may include the step of culturing during.

As an embodiment, the medium may contain lignocellulosic biomass, and the bio-oil production method decomposes the lignocellulosic biomass before culturing the yeast strain in the medium to reduce one or more of glucose and xylose. It may further include the step of producing. At this time, the step of decomposing the lignocellulosic biomass to produce one or more of glucose and xylose may include, for example, strong acid saccharification, weak acid saccharification, and pretreatment with an organic solvent, but glucose and xylose from the lignocellulosic biomass Any method that can produce one or more of them is not limited thereto, but includes all.

As an embodiment, the method may further include producing biodiesel as bio-oil using the culture of the strain. Specifically, the step may further include transesterifying the lipid obtained in the culturing step to obtain biodiesel. Examples of the produced culture include biodiesel or raw materials capable of producing biomaterials that can be used in cosmetics or health functional foods. As an example, the culture of the strain may contain lipids, but is not limited thereto.

In the present specification, “biodiesel” is one of biofuels, and a fatty acid methyl ester made through a transesterification method of producing a fatty acid ester after separating glycerol from triglycerides in which glycerol is bound to a trivalent fatty acid using methanol ( fatty acid methyl ester, FAME). The step of transesterifying the produced lipid to obtain biodiesel is not limited as long as it is a method of obtaining biodiesel from the lipid.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples and drawings. However, the following examples and drawings are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] Preparation of transformed yeast strain

As an example of the present invention, a Yarrowia lipolytica transformed yeast strain having xylose utilization was prepared by the following method.

First, the wild-type Yarrowia lipolytica strain (accession number: ATCC MYA-2613) was introduced with xylose isomerase and xylulose kinase, and 20g/L of xylose was present as a single carbon source (YSC medium: Yeast Synthetic Complete medium), followed by adaptive evolution, and then removing the xylose isomerase and xylulose kinase again to prepare an adaptive evolutionary strain.

Specifically, the wild-type Yarrow subtotal Li poly urticae strain (Accession No: ATCC MYA-2613) amplified the chair (Enhancer, UAS1B) is added TEF (Translational elongation factor) using a promoter xylose isomerase gene is SEQ ID NO: coding for the enzyme the ylXK represented by xylA3 * and xylene rule base sequence of SEQ ID NO: Los kinase gene encoding a second represented by the nucleotide sequence of one was expressed by each introduced into plasmid forms. At this time, the base sequence of the plasmid containing the gene encoding the xylose isomerase is represented by SEQ ID NO: 3, and the base sequence of the plasmid containing the gene encoding the xylose kinase gene is represented by SEQ ID NO: 4. will be. Then, the minimum medium (CSM (Complete Supplement Mixture)-Leu-Ura medium (6.7g/L yeast nitrogen base, 20g) using xylose as the only carbon source for the strains into which the xylose isomerase and xylulose kinase were introduced) /L xylose, CSM-Leu-Ura (MP biomedicals, Solon, USA), inoculated with about 0.5% (v/v) strains of the exponential phase in new xylose medium and cultured, then exponential When the growth phase was reached, the plasmids of SEQ ID NO: 3 and SEQ ID NO: 4, which were previously introduced, were removed again by subculture 3 times by inoculating a new medium at about 0.5%.

The adaptive evolutionary strain was named YX1, and deposited under the accession number KCTC 13911BP. It was confirmed that the adaptive evolutionary strain was mutated as follows.

No. Gene name Genetic variation Amino acid mutation One YALI0_A00374g 597_598insGGCTCCGCTGCCGCCTCC Ser199_Gly200insGlySerAlaAlaAlaSer 2 YALI0_A00935g 202_203insGCTC Tyr68fs 3 YALI0_A02497g 427_429dupAAC Asn143dup 4 YALI0_A02937g 20_21insGGAGACCGTGGGGGAGACCGTGGA Val7_Glu8insGluThrValGlyGluThrValGlu 5 YALI0_A15642g 147G>A Trp49* 6 YALI0_A15796g 576T>G Asp192Glu 7 YALI0_A15796g 567_568insACA Thr189dup 8 YALI0_A17578g 434_435insGGACCCGCC Ser145_Glu146insAspProPro 9 YALI0_A17776g 1810_1811insACAACAACACCC Asn604_Asn605insAsnAsnThrHis 10 YALI0_A17776g 959_960insACAGCAGAT Pro320_Gln321insGlnGlnIle 11 YALI0_A17853g 1940_1942delAGG Glu648del 12 YALI0_A17853g 1990_1991insAGGAGGAGGCTAAGAAGA Glu664_Glu665insGluGluAlaLysLysLys 13 YALI0_A18744g 2512_2513insCTGGAGGGGGTGGAAGCG Pro838_Gly839insGlyGlyGlyGlySerAla 14 YALI0_A19646g 1467delA Gly490fs 15 YALI0_B00748g 522_523insAACTCC Ser174_Asp175insAsnSer 16 YALI0_B05676g 1369_1370insCAGCAGCGT Ala457_Ala458insAlaAlaSer 17 YALI0_B18194g 1695_1697delCGG Gly566del 18 YALI0_C07722g 1553C>T Thr518Ile 19 YALI0_C07722g 1552A>G Thr518Ala 20 YALI0_C08327g 1221_1250delTCCTCACGTGCAGCCTCCTCATGCGCAGCC Pro408_Pro417del 21 YALI0_C08349g 2455_2456insCTCCCATCTCCTCCT Ala819_Pro820insProIleSerSerSe 22 YALI0_C08349g 2471_2472insTCCCATCTCCTCCGA Ala824_Pro825insProIleSerSerAsp 23 YALI0_C08473g 1326_1327insGCACCTGCGACTTCTTCG Ser442_Thr443insAlaProAlaThrSerSer 24 YALI0_C09031g 1789_1790insCTCCCGAGTCCTCTGCTGAGCCTACCAGCGAAGAGACTTCTTCCG Ser596_Val597insAlaProGluSerSerAlaGluProThrSerGluGluThrSerSer 25 YALI0_C09614g 1617_1622delACAACA Gln540_Gln541del 26 YALI0_C13002g 815_816insTCTAA Glu272fs 27 YALI0_C13728g 99_100insAAAAAGTGGCCGAAAGAGTGGCCA Pro33_Lys34insLysLysTrpProLysGluTrpPro 28 YALI0_C13728g 129_130insTGGCCGAAAAAGTGGCCAAAA Lys43_Lys44insTrpProLysLysTrpProLys 29 YALI0_C13970g 1587_1588insGCT Gln529dup 30 YALI0_C15532g 1611_1616delCAGCTT Ser538_Phe539del 31 YALI0_C15741g 198_209delCTATTCAAACGA Asp66_Asn69del 32 YALI0_C16148g 957_968delCAGCAGCAGCAG Ser320_Ser323del 33 YALI0_C16247g 1292A>G Asp431Gly 34 YALI0_C16247g 1412T>A Val471Asp 35 YALI0_C16247g 1441G>A Asp481Asn 36 YALI0_C16247g 1513A>G Asn505Asp 37 YALI0_C16247g 1534A>C Ser512Arg 38 YALI0_C16247g 1544T>A Val515Glu 39 YALI0_C16247g 1847C>T Ala616Val 40 YALI0_D00627g 2603A>C Asn868Thr 41 YALI0_D07634g 495_500delCAACTT Asn166_Phe167del 42 YALI0_D09647g 491_492insAAAGCAGCAGCAGCA Gln164_Gln165insLysGlnGlnGlnGln 43 YALI0_D14542g 80_81insC Ala30fs 44 YALI0_D15752g 1038_1039insCAG Pro346_Gln347insGln 45 YALI0_D15752g 1038_1039insCGGCAG Pro346_Gln347insArgGln 46 YALI0_D17820g 615_616insCCCCACCACCCGCAA Gln205_Thr206insProHisHisProGln 47 YALI0_D17820g 641_642insGCAAACCTACCACCA Gln214_Gln215insGlnThrTyrHisGln 48 YALI0_D18678g 1300_1323delACCCAGAACCAGACCCAGAACCAG Thr434_Gln441del 49 YALI0_D19360g 2132_2155delGGAGTGAGAGTGGGAGTGAGAGTG Gly711_Ser718del 50 YALI0_D20064g 69_70delCT Tyr23fs 51 YALI0_D20526g 355_356insCCACCGGCG Ser119_Thr120insThrGlyAla 52 YALI0_D20790g 39_40insCGTGAAAGGAGCCGAACC Ser13_Arg14insArgGluArgSerArgThr 53 YALI0_D24849g 103G>A Ala35Thr 54 YALI0_D25058g 592_594dupAAG Lys198dup 55 YALI0_D26972g 36_41delACAGCA Gln13_Gln14del 56 YALI0_E07832g 499_500insCCAAGCCCCCCGCTTCCAAGCCCACCGCTT Pro167_Lys168insLysProProAlaSerLysProThrAlaSer 57 YALI0_E08008g 794_795insCTCTTCCTCTTCCTCTTCCTCTTCCTCTTC Gly265_Ser266insSerSerSerSerSerSerSerSerSerSer 58 YALI0_E13948g 825_830delCAGCAA Ser276_Asn277del 59 YALI0_E18766g 1100_1101insTTCTGCCGCTTA Ser367_Ser368insSerAlaAlaTyr 60 YALI0_E19767g 2699A>G Asp900Gly 61 YALI0_E20053g 533_534insGGCCGAGGAGGC Asp178delinsGluAlaGluGluAla 62 YALI0_E20449g 902_903insGGCCAA Ala300_Asn301insLysAla 63 YALI0_E23969g 480_481insTCCGCTACCACCGAG Ser160_Ser161insSerAlaThrThrGlu 64 YALI0_E23969g 497A>C Tyr166Ser 65 YALI0_E29623g 10A>G Thr4Ala 66 YALI0_E31999g 269_270insTGTGTATATATGCAGAAAAAAAAAAA Glu91fs 67 YALI0_E33891g 237C>A Gly79Gly 68 YALI0_E33891g 239G>C Gly80Ala 69 YALI0_E34089g 72_73insCG Val25fs 70 YALI0_E35046g 1926T>C Val642Val 71 YALI0_F12573g 75_76insTCCCCG Thr25_Leu26insSerPro 72 YALI0_F12771g 1019_1021delAGT Glu340_Ser341delinsAla 73 YALI0_F12793g 1855_1860delTCTTCT Ser619_Ser620de 74 YALI0_F12793g 4102_4103insTAGCCG Ala1367_Gly1368insValAla 75 YALI0_F18568g 718_723delCAGCAG Gln240_Gln241del 76 YALI0_F19030g 2099C>A Thr700Asn 77 YALI0_F19030g 2098A>G Thr700Ala 78 YALI0_F19030g 2065T>A Ser689Thr 79 YALI0_F19030g 1344_1345insCCTACTCCCTCCGAGGTT Val448_Pro449insProThrProSerGluVal

Then, using a TEF (Translational elongation factor) promoter to which an amplifier (UAS1B) is added to the adaptive evolutionary strain YX1, xylA3* and xylA3* expressed by the nucleotide sequence of SEQ ID NO: 1, a gene encoding a xylose isomerase, and xyl The ylXK represented by the nucleotide sequence of SEQ ID NO: 2, which is a gene encoding cellulose kinase, was introduced and expressed in the form of a plasmid, respectively. At this time, the base sequence of the plasmid containing the gene encoding the xylose isomerase is represented by SEQ ID NO: 3, and the base sequence of the plasmid containing the gene encoding the xylose kinase gene is represented by SEQ ID NO: 4. will be.

The strain was named YX1-AK.

[Test Example 1]

The following experiment was conducted to confirm the use of xylose of the Yarrowia lipolytica transformed yeast strain of Example 1 provided as an example of the present invention.

As a comparative example of the present invention, both the gene encoding xylose isomerase and the gene encoding xylulose kinase were introduced into the wild-type Yarrowia lipolytica strain, which is the parent strain of Example 1, in the same manner as in Example 1. a WT-AK (Comparative example 1), the Yarrow subtotal Li prepared in the same manner as in example 1, poly urticae adaptive evolution strain YX1 (Comparative example 2), the Yarrow subtotal Li poly urticae prepared in the same manner as in example 1, introduced by adaptive evolution strains only genes encoding the xylose isomerase in the same manner YX1-a (Comparative example 3) and a Yarrow subtotal Li poly xylene rule Los phosphorylation in Mathematica adaptive evolution strain produced in the same manner as in example 1, YX1-K (Comparative Example 4) in which only the gene encoding the enzyme was introduced by the same method was prepared. Yarrow subtotal Li poly urticae adaptive evolution YX1 strain of Comparative Example 2 was introduced into the xylA3 * empty vector without inserted gene and both genes are ylXK the adaptive evolution strain.

Then, the strains of Example 1 and Comparative Examples 1 to 4 were inoculated in a minimum medium (YSC medium: Yeast Synthetic Complete medium) containing about 20 g/L of xylose as a carbon source, respectively, and stirred at 28° C. at 200 rpm. While incubating for 240 hours, the growth of the strain using xylose was confirmed according to the culture time, and the xylose utilization ability of each strain was evaluated based on this. Growth of the strain was confirmed by measuring the OD ₆₀₀ of the medium using a spectrophotometer (spectrophotometer). In the present specification, “OD ₆₀₀ ”means absorbance or optical density in light of a wavelength of 600 nm, and the density or concentration of microorganisms or cells in a solution or sample can be confirmed through the measured OD ₆₀₀ .

As a result, in the case of WT-AK (Comparative Example 1), as shown in FIG. 1, xylose was used even when a xylose metabolic pathway including both a gene encoding a xylose isomerase and a gene encoding a xylulose kinase was introduced. Thus, the strain did not grow. Yarrow subtotal Li poly urticae adaptive evolution strain YX1 (Comparative Example 2) and the adaptive evolution strains in xylose isomerase (for comparison 3) YX1-A respectively introduced a gene encoding a gene or xylene rule Los kinases encoding the And in the case of YX1-K (Comparative Example 4), the strain did not grow using xylose. On the other hand, YX1-AK (Example 1) according to an embodiment of the present invention was confirmed that the strain was grown using xylose.

In addition, as shown in FIG. 2, in any of Comparative Examples 1 to 4, xylose consumption was not observed when the strain was grown. On the other hand, since YX1-AK (Example 1) according to an embodiment of the present invention uses xylose for strain growth, it can be seen that about 22.8 g/L of xylose was consumed for 240 hours of culturing the strain.

[Example 2]

As another embodiment of the present invention, a plasmid having a gene encoding a xylose isomerase and a gene encoding a xylose kinase for ease of further engineering, although the genetic composition is substantially the same as in Example 1 above. Yarrowia lipolytica transformed yeast strain introduced by being included in was prepared.

Specifically, the adaptive evolution of Yarrow subtotal Li poly urticae strain sequence (YX1, accession No. KCTC 13911BP) amplification chair (Enhancer, UAS1B) is added the gene coding for xylose isomerase using (Translational elongation factor) TEF promoter in It was expressed by introducing a xylA3 ylXK * and represented by the base sequence of the gene of SEQ ID NO: 2 coding for Ross Giles rule kinases represented by the base sequence of No. 1 in the form of a plasmid containing both. At this time, the base sequence of the plasmid containing both the xylose isomerase-encoding gene and the xylose kinase gene-encoding gene is represented by SEQ ID NO: 5.

[Test Example 2]

As an embodiment of the present invention, the following experiment was conducted to confirm the degree of improvement in the use of xylose of the Yarrowia lipolytica transformed yeast strain.

As a comparative example of the present invention further Yarrow subtotal Li poly urticae adaptive evolution strain YSX (Accession number KCTC13615BP, Comparative Example 5) and YSX Example 2 by the same method by introducing a xylose path isomerase based on the strain YSX-AK (Comparative Example 6) was prepared.

Then, the strains of Example 2, Comparative Examples 2, 5, and 6 were inoculated in a minimum medium (YSC medium: Yeast Synthetic Complete medium) containing about 20 g/L of xylose as a carbon source, respectively, and then at 28° C. at 200 rpm. While incubating for 300 hours while stirring, the growth of the strain using xylose was confirmed according to the cultivation time, and the xylose availability of each strain was evaluated based on this. Growth of the strain was confirmed by measuring the OD ₆₀₀ of the medium using a spectrophotometer (spectrophotometer).

As a result, in the case of YSX-AK (Comparative Example 6), as shown in FIG. 3, the strain concentration increased to about 6.2 based on OD ₆₀₀ during a culture time of about 300 hours, but YX1-AK (Example 2 ), it can be seen that the OD ₆₀₀ is increased to about 15.33, and xylose-based growth capacity is improved by about 2.4 times or more compared to the conventional strain. In addition, as shown in FIG. 4, in the case of YSX-AK (Comparative Example 6), about 5.9 g/L of xylose was used for about 300 hours, whereas in the case of YX1-AK (Example 2), all xylose (18.2 g) in the medium was used. By consuming /L), it can be seen that the strain according to an embodiment of the present invention has improved performance by about 3 times or more compared to the conventional strain in xylose availability. This conventional Yarrow subtotal Li poly different genetic variation (mutation), obtained in the process of evolution adapted that the new strains in accordance with an embodiment of the Mathematica adaptive evolution strain YSX strains with the invention, particularly in accordance with one embodiment of the present invention It means that mutation of 495_500delCAACTT in YALI0_D07634g gene according to adaptive evolution plays an important role in increasing xylose utilization efficiency.

[Example 3]

As another embodiment of the present invention, peroxisome-forming factor, a lipid degradation-related gene, using the CRISPR/Cas9 system in the transformed yeast strain of Example 2 so that the strain of Example 2 can produce a higher concentration of lipids. 10 (peroxisomal biogenesis factor 10, SEQ ID NO: 6) is deleted, and a gene encoding a diacylic glycerol acyl transferase expressed by the nucleotide sequence of SEQ ID NO: 7 is a UAS1B enhancer (enhancer), TEF (Translational elongation factor) promoter. The fourth vector (SEQ ID NO: 8, pMCS-16TEF-DGA1-CYC1t) was used to overexpress diacylglycerol acyltransferase, a key gene in the lipid production metabolic pathway.

The diacylglycerol was a acyl transferase overexpression Yarrow subtotal Li poly Mathematica transformed yeast strains termed YX2-AK, were deposited as accession number of KCTC 13912BP.

[Test Example 3]

The following experiment was conducted to confirm whether the Yarrowia lipolytica transformed yeast strain according to an embodiment of the present invention can convert the integrated sugar (glucose/xylose) in the biomass saccharification solution to produce high-concentration lipids. .

Specifically, the YX2-AK (Example 3) strain was fed-batch cultivation in a reactor, and the utilization per consolidation and lipid production were confirmed. The reactor was operated at a different stirring speed (250-800rpm) to maintain the dissolved oxygen concentration above 50% at 28°C, and 2.5M NaOH solution was added if necessary to maintain the pH in the reactor at 3.5. The biomass saccharification solution was saccharified and neutralized using dilute sulfuric acid, and then concentrated to increase the sugar concentration. After that, the saccharified solution that had undergone a detoxification process using activated carbon was purchased from Sugar & Co., Ltd. and used. When operating the reactor for lipid production, the initial batch operation started with the concentration of glucose and xylose in the medium at 30.1 g/L and 32.7 g/L, respectively.After 117 hours of operation of the reactor, glucose xylose was added to the reactor for a total of 156 hours. Was driven.

As a result, as shown in FIG. 5, YX2-AK (Example 3) consumes sugar at a very slow rate at the initial stage of the reactor operation, and then glucose and xyl alcohol from about 40 hours after the concentration of the strain in the reactor reaches a certain level or higher. Ross was consumed rapidly. Thereafter, when additional saccharification solution was supplied, it was confirmed that all sugar was consumed within about 24 hours after additional saccharification solution was supplied. During the operation of the batch reactor, about 6.6 g/L of lipid was produced by consuming 30 g/L and 25 g/L of glucose and xylose derived from the saccharified solution of giant silver grass. At this time, the lipid content was 60.5%, the lipid conversion rate was 0.11, and the lipid production rate was 1.36 g/L. Thereafter, during the operation of the fed-batch reactor, 29.3 g/L of glucose and 35.7 g/L of xylose were additionally consumed, and about 15.4 g/L of lipid was produced during 156 hours of the entire reactor operation. The lipid content at this time was 81%, the lipid conversion rate was 0.12, and the lipid production rate was 2.37 g/L. In particular, looking at the lipid productivity performance of YX2-AKD after the first saccharification solution was added, 8.8 g/L of lipid was produced for about 39 hours, lipid conversion rate (0.13 g/g) and lipid production rate (5.39 g/L/d) ) Showed a very high lipid production performance.

Korea Research Institute of Bioscience and Biotechnology KCTC13911BP 20190809 Korea Research Institute of Bioscience and Biotechnology KCTC13912BP 20190809

<110> Korea Institute of Science of Technology <120> Yarrowia liplytica strain having improved xylose utilizing ability and the method for preparing the same <130> 19P300IND <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 1314 <212> DNA <213> Artificial Sequence <220> <223> XylA3* sequence <400> 1 atggctaagg agtatttccc tcagattcag aagatcaagt tcgacggcaa ggacagcaaa 60 aaccccctgg ctttccatta ttacgacgcc gagaaggaag tgatgggcaa gaagatgaag 120 gactggctgc ggttcgcaat ggcatggtgg cacaccctgt gcgcagaggg agcagatcag 180 ttcggaggag gcaccaagtc ttttccatgg aacgagggca cagacgccat cgagatcgcc 240 aagcagaagg tggatgccgg cttcgagatc atgcagaagc tgggcatccc ctactattgt 300 tttcacgacg tggatctggt gtccgagggc aactctatcg gcgagtacga gtccaatctg 360 aaggccgtgg tggcctatct gaaggacaag cagaaggaga ccggcatcaa gctgctgtgg 420 agctccgcca acgtgttcgg ccacaagagg tacatgaacg gcgcctctac caatcccgac 480 tttgatgtgg tggcccgcgc catcgtgcag atcaagaacg ccatcgatac aggcatcgag 540 ctgggcgccg agaattacgt gttttggggc ggccgggagg gctatatgtc tctgctgaat 600 acagaccaga agagagagaa ggagcacatg gccaccatgc tgacaatggc cagggattac 660 gcccgcagca agggcttcaa gggcaccttt ctgatcgagc ccaagcctat ggagcctaca 720 aagcaccagt atgacgtgga taccgagaca gccatcggct tcctgaaggc ccacaatctg 780 gacaaggact tcaaggtgaa catcgaagtg aatcacgcca ccctggccgg ccacacattc 840 gagcacgagc tggcatgcgc agtggacgca ggaatgctgg gaagcatcga cgccaacagg 900 ggcgattacc agaatggctg ggacaccgat cagtttccta tcgatcagta tgagctggtg 960 caggcatgga tggagatcat caggggagga ggattcgtga ccggaggaac aaactttgac 1020 gccaagaccc ggagaaatag cacagacctg gaggatatca tcatcgccca cgtgtccgga 1080 atggatgcaa tggcacgggc cctggagaac gcagcaaagc tgctgcagga gtccccatac 1140 accaagatga agaaggagag atatgcctct ttcgacagcg gcatcggcaa ggactttgag 1200 gatggcaagc tgacactgga gcaggtgtac gagtatggca agaagaatgg cgagcctaag 1260 cagacctcag ggaagcagga gctgtacgaa gccatcatcg caatgtatca gtga 1314 <210> 2 <211> 1643 <212> DNA <213> Artificial Sequence <220> <223> ylXK sequence <400> 2 atgtatctcg gactggatct ttcgactcaa cagctcaagg gcatcattct ggacacaaaa 60 acgctggaca cggtcacaca agtccatgtg gactttgagg acgacttgcc gcagttcaac 120 accgaaaagg gcgtctttca cagctctaca gtggccggag aaatcaatgc tcctgtggca 180 atgtgggggg cagctgtgga cttgctgata gagcgtctgt caaaggaaat agacctttcc 240 acgatcaagt ttgtgtcggg ctcgtgccag caacacggct ctgtttatct caacagcagc 300 tacaaggagg gcctgggttc tctggacaaa cacaaagact tgtctacagg agtgtcatcc 360 ttactggcgc tcgaagtcag ccccaattgg caggatgcaa gcacggagaa ggagtgtgcg 420 cagtttgagg ctgcagtcgg cggtcccgag cagctggctg agatcactgg ctctcgagca 480 catactcgtt tcaccgggcc ccagattctc aaggtcaagg aacgcaaccc caaggtattc 540 aaggccacgt cacgggtcca gctcatatcc aactttctag catctctgtt tgccggcaag 600 gcgtgcccct ttgatcttgc tgacgcctgt ggaatgaatc tgtgggacat ccagaatggc 660 cagtggtgca agaaactcac agatctcatc accgatgaca cccactcggt cgagtccctc 720 cttggagacg tggaaacaga ccccaaggct ctactgggca aaatctcgcc ctatttcgtc 780 tccaagggct tctctccctc ttgtcaggtg gcacagttca caggcgacaa cccaggcact 840 atgctggctc tccccttaca ggccaatgac gtgattgtgt ctttgggaac atctacgacc 900 gccctcgtcg taacaaacaa gtacatgccc gaccccggat accatgtgtt caaccacccc 960 atggagggat acatgggcat gctgtgctac tgcaacggag gtctagcacg agagaagatc 1020 cgagacgagc ttggaggctg ggacgagttt aatgaggcgg ccgagaccac caacacagtg 1080 tctgctgacg atgtccatgt tggcatctac tttccactac gagaaatcct tcctcgagca 1140 ggtccctttg aacgacgttt catctacaac agacaaagtg aacagcttac agagatggct 1200 tctccagagg actcactggc aaccgaacac aaaccgcagg ctcaaaatct caaggacacg 1260 tggccgccac aaatggacgc cactgccatc attcaaagcc aggccctcag tatcaaaatg 1320 agactccaac gcatgatgca tggcgatatt ggaaaggtgt attttgtggg aggcgcctcg 1380 gtcaacactg ctatctgcag cgtaatgtct gccatcttaa aaccaacaaa gggcgcttgg 1440 agatgtggtc tggaaatggc aaacgcttgt gccattggaa gtgcccatca cgcctggctt 1500 tgcgacccca acaagacagg ccaggtacag gttcacgaag aagaggtcaa atacaagaat 1560 gtggacacag acgtgctact caaggcgttc aagctggccg aaaacgcctg cctggagaaa 1620 taactaacta ccacaccaac aca 1643 <210> 3 <211> 9688 <212> DNA <213> Artificial Sequence <220> <223> pMCS-UAS1B16-TEF-XylA3*-CYC1t <400> 3 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgctttt 420 cgtagataat ggaatacaaa tggatatcca gagtatacac atggatagta tacactgaca 480 cgacaattct gtatctcttt atgttaacta ctgtgaggcg ttaaatagag cttgatatat 540 aaaatgttac atttcacagt ctgaactttt gcagattacc taatttggta agatattaat 600 tatgaactga aagttgatgg catccctaaa tttgatgaaa gggcgcggga tccggcccgg 660 gaattcgaag gtaccaagga agcatgcggt acccgaattc ctgaggtgtc tcacaagtgc 720 cgtgcagtcc cgcccccact tgcttctctt tgtgtgtagt gtacgtacat tatcgagacc 780 gttgttcccg cccacctcga tccggtctag actgaggtgt ctcacaagtg ccgtgcagtc 840 ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc 900 gcccacctcg atccggggat ccctgaggtg tctcacaagt gccgtgcagt cccgccccca 960 cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc 1020 gatccgggtc gacctgaggt gtctcacaag tgccgtgcag tcccgccccc acttgcttct 1080 ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct cgatccggga 1140 gctcctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg 1200 tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggt ctagactgag 1260 gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg 1320 tacattatcg agaccgttgt tcccgcccac ctcgatccgg ggatccctga ggtgtctcac 1380 aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac gtacattatc 1440 gagaccgttg ttcccgccca cctcgatccg ggtcgacctg aggtgtctca caagtgccgt 1500 gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat cgagaccgtt 1560 gttcccgccc acctcgatcc gggcatgcgg tacccgaatt cctgaggtgt ctcacaagtg 1620 ccgtgcagtc ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac 1680 cgttgttccc gcccacctcg atccggtcta gactgaggtg tctcacaagt gccgtgcagt 1740 cccgccccca cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc 1800 cgcccacctc gatccgggga tccctgaggt gtctcacaag tgccgtgcag tcccgccccc 1860 acttgcttct ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct 1920 cgatccgggt cgacctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc 1980 tctttgtgtg tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggg 2040 agctcctgag gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt 2100 gtagtgtacg tacattatcg agaccgttgt tcccgcccac ctcgatccgg tctagactga 2160 ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac 2220 gtacattatc gagaccgttg ttcccgccca cctcgatccg gggatccctg aggtgtctca 2280 caagtgccgt gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat 2340 cgagaccgtt gttcccgccc acctcgatcc gggtcgacct gaggtgtctc acaagtgccg 2400 tgcagtcccg cccccacttg cttctctttg tgtgtagtgt acgtacatta tcgagaccgt 2460 tgttcccgcc cacctcgatc cgggcatgcc tgcagaagct tagagaccgg gttggcggcg 2520 tatttgtgtc ccaaaaaaca gccccaattg ccccaattga ccccaaattg acccagtagc 2580 gggcccaacc ccggcgagag cccccttcac cccacatatc aaacctcccc cggttcccac 2640 acttgccgtt aagggcgtag ggtactgcag tctggaatct acgcttgttc agactttgta 2700 ctagtttctt tgtctggcca tccgggtaac ccatgccgga cgcaaaatag actactgaaa 2760 atttttttgc tttgtggttg ggactttagc caagggtata aaagaccacc gtccccgaat 2820 tacctttcct cttcttttct ctctctcctt gtcaactcac acccgaaatc gttaagcatt 2880 tccttctgag tataagaatc attcaaaggc gcgccatggc taaggagtat ttccctcaga 2940 ttcagaagat caagttcgac ggcaaggaca gcaaaaaccc cctggctttc cattattacg 3000 acgccgagaa ggaagtgatg ggcaagaaga tgaaggactg gctgcggttc gcaatggcat 3060 ggtggcacac cctgtgcgca gagggagcag atcagttcgg aggaggcacc aagtcttttc 3120 catggaacga gggcacagac gccatcgaga tcgccaagca gaaggtggat gccggcttcg 3180 agatcatgca gaagctgggc atcccctact attgttttca cgacgtggat ctggtgtccg 3240 agggcaactc tatcggcgag tacgagtcca atctgaaggc cgtggtggcc tatctgaagg 3300 acaagcagaa ggagaccggc atcaagctgc tgtggagctc cgccaacgtg ttcggccaca 3360 agaggtacat gaacggcgcc tctaccaatc ccgactttga tgtggtggcc cgcgccatcg 3420 tgcagatcaa gaacgccatc gatacaggca tcgagctggg cgccgagaat tacgtgtttt 3480 ggggcggccg ggagggctat atgtctctgc tgaatacaga ccagaagaga gagaaggagc 3540 acatggccac catgctgaca atggccaggg attacgcccg cagcaagggc ttcaagggca 3600 cctttctgat cgagcccaag cctatggagc ctacaaagca ccagtatgac gtggataccg 3660 agacagccat cggcttcctg aaggcccaca atctggacaa ggacttcaag gtgaacatcg 3720 aagtgaatca cgccaccctg gccggccaca cattcgagca cgagctggca tgcgcagtgg 3780 acgcaggaat gctgggaagc atcgacgcca acaggggcga ttaccagaat ggctgggaca 3840 ccgatcagtt tcctatcgat cagtatgagc tggtgcaggc atggatggag atcatcaggg 3900 gaggaggatt cgtgaccgga ggaacaaact ttgacgccaa gacccggaga aatagcacag 3960 acctggagga tatcatcatc gcccacgtgt ccggaatgga tgcaatggca cgggccctgg 4020 agaacgcagc aaagctgctg caggagtccc catacaccaa gatgaagaag gagagatatg 4080 cctctttcga cagcggcatc ggcaaggact ttgaggatgg caagctgaca ctggagcagg 4140 tgtacgagta tggcaagaag aatggcgagc ctaagcagac ctcagggaag caggagctgt 4200 acgaagccat catcgcaatg tatcagtgaa tgcataccta gttaattaag gcacgtgcct 4260 aaaaaggatc gataccgtcg acctcgagtc atgtaattag ttatgtcacg cttacattca 4320 cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag 4380 gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt 4440 tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 4500 gagaaggttt tgggacgctc ggctaacttg tttaaacaac tgcaggcatg caagcttggc 4560 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 4620 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 4680 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4740 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 4800 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 4860 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 4920 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 4980 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 5040 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5100 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 5160 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 5220 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 5280 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 5340 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 5400 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 5460 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 5520 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 5580 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 5640 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 5700 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 5760 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 5820 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 5880 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 5940 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6000 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 6060 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6120 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 6180 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 6240 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 6300 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 6360 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 6420 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 6480 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 6540 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 6600 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 6660 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 6720 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 6780 gcccagatcc tctagagtcg aagcggccgc tatgtctgat aaaaggatgt aacataggca 6840 agctgctcgt gagtgttgag tacgaacctt agatccaaat cacccgcacc cacggatata 6900 cttgcttgaa tatacagtag tatgcggccg cttcgacacc atatcatata aaactaacaa 6960 tgcattgctt attacgaaga ctacccgttg ctatctccac accgttatct ccacggtcca 7020 aaggctgctc aatgtgctgc atacgtaacg tggggtgcaa ccttgagcac atagtacttt 7080 tccgaaaacc ggcgataatt aagtgtgcac tccaactttt cacactgagc gtaaaatgtg 7140 gagaagaaat cggcactaaa aagtcaggta gactggaaaa tgcgccatga aatgaatatc 7200 tcttgctaca gtaatgccca gcatcgaggg gtattgtgtc accaacacta tagtggcagc 7260 tgaagcgctc gtgattgtag tatgagtctt tattggtgat gggaagagtt cactcaatat 7320 tctcgttact gccaaaacac cacggtaatc ggccagacac catggatgta gatcaccaag 7380 cctgtgaatg ttattcgagc taaaatgcac atggttggtg aaaggagtag ttgctgtcga 7440 attccgtcgt cgcctgagtc atcatttatt taccagttgg ccacaaaccc ttgacgatct 7500 cgtatgtccc ctccgacata ctcccggccg gctggggtac gttcgatagc gctatcggca 7560 tcgacaaggt ttgggtccct agccgatacc gcactacctg agtcacaatc ttcggaggtt 7620 tagtcttcca catagcacgg gcaaaagtgc gtatatatac aagagcgttt gccagccaca 7680 gattttcact ccacacacca catcacacat acaaccacac acatccacaa tggaacccga 7740 aactaagaag accaagactg actccaagaa gattgttctt ctcggcggcg acttctgtgg 7800 ccccgaggtg attgccgagg ccgtcaaggt gctcaagtct gttgctgagg cctccggcac 7860 cgagtttgtg tttgaggacc gactcattgg aggagctgcc attgagaagg agggcgagcc 7920 catcaccgac gctactctcg acatctgccg aaaggctgac tctattatgc tcggtgctgt 7980 cggaggcgct gccaacaccg tatggaccac tcccgacgga cgaaccgacg tgcgacccga 8040 gcagggtctc ctcaagctgc gaaaggacct gaacctgtac gccaacctgc gaccctgcca 8100 gctgctgtcg cccaagctcg ccgatctctc ccccatccga aacgttgagg gcaccgactt 8160 catcattgtc cgagagctcg tcggaggtat ctactttgga gagcgaaagg aggatgacgg 8220 atctggcgtc gcttccgaca ccgagaccta ctccgttcct gaggttgagc gaattgcccg 8280 aatggccgcc ttcctggccc ttcagcacaa cccccctctt cccgtgtggt ctcttgacaa 8340 ggccaacgtg ctggcctcct ctcgactttg gcgaaagact gtcactcgag tcctcaagga 8400 cgaattcccc cagctcgagc tcaaccacca gctgatcgac tcggccgcca tgatcctcat 8460 caagcagccc tccaagatga atggtatcat catcaccacc aacatgtttg gcgatatcat 8520 ctccgacgag gcctccgtca tccccggttc tctgggtctg ctgccctccg cctctctggc 8580 ttctctgccc gacaccaacg aggcgttcgg tctgtacgag ccctgtcacg gatctgcccc 8640 cgatctcggc aagcagaagg tcaaccccat tgccaccatt ctgtctgccg ccatgatgct 8700 caagttctct cttaacatga agcccgccgg tgacgctgtt gaggctgccg tcaaggagtc 8760 cgtcgaggct ggtatcacta ccgccgatat cggaggctct tcctccacct ccgaggtcgg 8820 agacttgttg ccaacaaggt caaggagctg ctcaagaagg agtaagtcgt ttctacgacg 8880 cattgatgga aggagcaaac tgacgcgcct gcgggttggt ctaccggcag ggtccgctag 8940 tgtataagac tctataaaaa gggccctgcc ctgctaatga aatgatgatt tataatttac 9000 cggtgtagca accttgacta gaagaagcag attgggtgtg tttgtagtgg aggacagtgg 9060 tacgttttgg aaacagtctt cttgaaagtg tcttgtctac agtatattca ctcataacct 9120 caatagccaa gggtgtagtc ggtttattaa aggaagggag ttgtggctga tgtggataga 9180 tatctttaag ctggcgactg cacccaacga gtgtggtggt agcttgttac tgtatattcg 9240 gtaagatata ttttgtgggg ttttagtggt gtttggtagg ttagtgcttg gtatatgagt 9300 tgtaggcatg acaatttgga aaggggtgga ctttgggaat attgtgggat ttcaatacct 9360 tagtttgtac agggtaattg ttacaaatga tacaaagaac tgtatttctt ttcatttgtt 9420 ttaattggtt gtatatcaag tccgttagac gagctcagtg ccatggcttt tggcactgta 9480 tttcattttt agaggtacac tacattcagt gaggtatggt aaggttgagg gcataatgaa 9540 ggcaccttgt actgacagtc acagacctct caccgagaat tttatgagat atactcgggt 9600 tcattttagg ctccgattcg attcaaatta ttactgtcga aatcggttga gcatccgttg 9660 atttccgaac agatctggcc ctttcgtc 9688 <210> 4 <211> 8566 <212> DNA <213> Artificial Sequence <220> <223> pMCSu-UAS1B12-TEF-XK-CYC1t <400> 4 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgctttt 420 cgtagataat ggaatacaaa tggatatcca gagtatacac atggatagta tacactgaca 480 cgacaattct gtatctcttt atgttaacta ctgtgaggcg ttaaatagag cttgatatat 540 aaaatgttac atttcacagt ctgaactttt gcagattacc taatttggta agatattaat 600 tatgaactga aagttgatgg catccctaaa tttgatgaaa gggcgcggga tccggcccgg 660 gaattcgaag gtaccaagga agcatgcggt acccgaattc ctgaggtgtc tcacaagtgc 720 cgtgcagtcc cgcccccact tgcttctctt tgtgtgtagt gtacgtacat tatcgagacc 780 gttgttcccg cccacctcga tccggtctag actgaggtgt ctcacaagtg ccgtgcagtc 840 ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc 900 gcccacctcg atccggggat ccctgaggtg tctcacaagt gccgtgcagt cccgccccca 960 cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc 1020 gatccgggtc gacctgaggt gtctcacaag tgccgtgcag tcccgccccc acttgcttct 1080 ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct cgatccggga 1140 gctcctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg 1200 tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggt ctagactgag 1260 gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg 1320 tacattatcg agaccgttgt tcccgcccac ctcgatccgg ggatccctga ggtgtctcac 1380 aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac gtacattatc 1440 gagaccgttg ttcccgccca cctcgatccg ggtcgacctg aggtgtctca caagtgccgt 1500 gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat cgagaccgtt 1560 gttcccgccc acctcgatcc gggcatgcgg tacccgaatt cctgaggtgt ctcacaagtg 1620 ccgtgcagtc ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac 1680 cgttgttccc gcccacctcg atccggtcta gactgaggtg tctcacaagt gccgtgcagt 1740 cccgccccca cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc 1800 cgcccacctc gatccgggga tccctgaggt gtctcacaag tgccgtgcag tcccgccccc 1860 acttgcttct ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct 1920 cgatccgggt cgacctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc 1980 tctttgtgtg tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggg 2040 catgcctgca gaagcttaga gaccgggttg gcggcgtatt tgtgtcccaa aaaacagccc 2100 caattgcccc aattgacccc aaattgaccc agtagcgggc ccaaccccgg cgagagcccc 2160 cttcacccca catatcaaac ctcccccggt tcccacactt gccgttaagg gcgtagggta 2220 ctgcagtctg gaatctacgc ttgttcagac tttgtactag tttctttgtc tggccatccg 2280 ggtaacccat gccggacgca aaatagacta ctgaaaattt ttttgctttg tggttgggac 2340 tttagccaag ggtataaaag accaccgtcc ccgaattacc tttcctcttc ttttctctct 2400 ctccttgtca actcacaccc gaaatcgtta agcatttcct tctgagtata agaatcattc 2460 aaaggcgcgc catgtatctc ggactggatc tttcgactca acagctcaag ggcatcattc 2520 tggacacaaa aacgctggac acggtcacac aagtccatgt ggactttgag gacgacttgc 2580 cgcagttcaa caccgaaaag ggcgtctttc acagctctac agtggccgga gaaatcaatg 2640 ctcctgtggc aatgtggggg gcagctgtgg acttgctgat agagcgtctg tcaaaggaaa 2700 tagacctttc cacgatcaag tttgtgtcgg gctcgtgcca gcaacacggc tctgtttatc 2760 tcaacagcag ctacaaggag ggcctgggtt ctctggacaa acacaaagac ttgtctacag 2820 gagtgtcatc cttactggcg ctcgaagtca gccccaattg gcaggatgca agcacggaga 2880 aggagtgtgc gcagtttgag gctgcagtcg gcggtcccga gcagctggct gagatcactg 2940 gctctcgagc acatactcgt ttcaccgggc cccagattct caaggtcaag gaacgcaacc 3000 ccaaggtatt caaggccacg tcacgggtcc agctcatatc caactttcta gcatctctgt 3060 ttgccggcaa ggcgtgcccc tttgatcttg ctgacgcctg tggaatgaat ctgtgggaca 3120 tccagaatgg ccagtggtgc aagaaactca cagatctcat caccgatgac acccactcgg 3180 tcgagtccct ccttggagac gtggaaacag accccaaggc tctactgggc aaaatctcgc 3240 cctatttcgt ctccaagggc ttctctccct cttgtcaggt ggcacagttc acaggcgaca 3300 acccaggcac tatgctggct ctccccttac aggccaatga cgtgattgtg tctttgggaa 3360 catctacgac cgccctcgtc gtaacaaaca agtacatgcc cgaccccgga taccatgtgt 3420 tcaaccaccc catggaggga tacatgggca tgctgtgcta ctgcaacgga ggtctagcac 3480 gagagaagat ccgagacgag cttggaggct gggacgagtt taatgaggcg gccgagacca 3540 ccaacacagt gtctgctgac gatgtccatg ttggcatcta ctttccacta cgagaaatcc 3600 ttcctcgagc aggtcccttt gaacgacgtt tcatctacaa cagacaaagt gaacagctta 3660 cagagatggc ttctccagag gactcactgg caaccgaaca caaaccgcag gctcaaaatc 3720 tcaaggacac gtggccgcca caaatggacg ccactgccat cattcaaagc caggccctca 3780 gtatcaaaat gagactccaa cgcatgatgc atggcgatat tggaaaggtg tattttgtgg 3840 gaggcgcctc ggtcaacact gctatctgca gcgtaatgtc tgccatctta aaaccaacaa 3900 agggcgcttg gagatgtggt ctggaaatgg caaacgcttg tgccattgga agtgcccatc 3960 acgcctggct ttgcgacccc aacaagacag gccaggtaca ggttcacgaa gaagaggtca 4020 aatacaagaa tgtggacaca gacgtgctac tcaaggcgtt caagctggcc gaaaacgcct 4080 gcctggagaa ataactaact accacaccaa cacattaatt aaggcacgtg cctaaaaagg 4140 atcgataccg tcgacctcga gtcatgtaat tagttatgtc acgcttacat tcacgccctc 4200 cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 4260 tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 4320 tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 4380 ttttgggacg ctcggctaac ttgtttaaac aactgcaggc atgcaagctt ggcgtaatca 4440 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4500 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4560 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4620 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 4680 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4740 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4800 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4860 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4920 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4980 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa 5040 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 5100 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 5160 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 5220 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 5280 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 5340 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 5400 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5460 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5520 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5580 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5640 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5700 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5760 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5820 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5880 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 5940 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 6000 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 6060 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 6120 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 6180 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 6240 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 6300 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 6360 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6420 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6480 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6540 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6600 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccaga 6660 tcctctagag tcgaagcggc cgctatgtct gataaaagga tgtaacatag gcaagctgct 6720 cgtgagtgtt gagtacgaac cttagatcca aatcacccgc acccacggat atacttgctt 6780 gaatatacag tagtatgcgg ccgcttcgac accatatcat ataaaactaa caatgcatgt 6840 cgacgagtat ctgtctgact cgtcattgcc gcctttggag tacgactcca actatgagtg 6900 tgcttggatc actttgacga tacattcttc gttggaggct gtgggtctga cagctgcgtt 6960 ttcggcgcgg ttggccgaca acaatatcag ctgcaacgtc attgctggct ttcatcatga 7020 tcacattttt gtcggcaaag gcgacgccca gagagccatt gacgttcttt ctaatttgga 7080 ccgatagccg tatagtccag tctatctata agttcaacta actcgtaact attaccataa 7140 catatacttc actgccccag ataaggttcc gataaaaagt tctgcagact aaatttattt 7200 cagtctcctc ttcaccacca aaatgccctc ctacgaagct cgagctaacg tccacaagtc 7260 cgcctttgcc gctcgagtgc tcaagctcgt ggcagccaag aaaaccaacc tgtgtgcttc 7320 tctggatgtt accaccacca aggagctcat tgagcttgcc gataaggtcg gaccttatgt 7380 gtgcatgatc aagacccata tcgacatcat tgacgacttc acctacgccg gcactgtgct 7440 ccccctcaag gaacttgctc ttaagcacgg tttcttcctg ttcgaggaca gaaagttcgc 7500 agatattggc aacactgtca agcaccagta caagaacggt gtctaccgaa tcgccgagtg 7560 gtccgatatc accaacgccc acggtgtacc cggaaccgga atcattgctg gcctgcgagc 7620 tggtgccgag gaaactgtct ctgaacagaa gaaggaggac gtctctgact acgagaactc 7680 ccagtacaag gagttcctgg tcccctctcc caacgagaag ctggccagag gtctgctcat 7740 gctggccgag ctgtcttgca agggctctct ggccactggc gagtactcca agcagaccat 7800 tgagcttgcc cgatccgacc ccgagtttgt ggttggcttc attgcccaga accgacctaa 7860 gggcgactct gaggactggc ttattctgac ccccggggtg ggtcttgacg acaagggaga 7920 cgctctcgga cagcagtacc gaactgttga ggatgtcatg tctaccggaa cggatatcat 7980 aattgtcggc cgaggtctgt acggccagaa ccgagatcct attgaggagg ccaagcgata 8040 ccagaaggct ggctgggagg cttaccagaa gattaactgt tagaggttag actatggata 8100 tgtcatttaa ctgtgtatat agagagcgtg caagtatgga gcgcttgttc agcttgtatg 8160 atggtcagac gacctgtctg atcgagtatg tatgatactg cacaacctgt gtatccgcat 8220 gatctgtcca atggggcatg ttgttgtgtt tctcgatacg gagatgctgg gtacaagtag 8280 ctaatacgat tgaactactt atacttatat gaggcttgaa gaaagctgac ttgtgtatga 8340 cttattctca actacatccc cagtcacaat accaccactg cactaccact acaccaaaac 8400 catgatcaaa ccacccatgg acttcctgga ggcagaagaa cttgttatgg aaaagctcaa 8460 gagagagaag ccaagatact atcaagacat gtgtcgcaac ttcaaggagg accaagctct 8520 gtacaccgag aaacaggcct ttgtcgacag atctggccct ttcgtc 8566 <210> 5 <211> 10457 <212> DNA <213> Artificial Sequence <220> <223> pMCSu-UAS1B12-TEF-ylXK-CYC1t-TEFint-XylA3*-Syn7t <400> 5 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgag agaccgggtt ggcggcgtat ttgtgtccca aaaaacagcc ccaattgccc 240 caattgaccc caaattgacc cagtagcggg cccaaccccg gcgagagccc ccttcacccc 300 acatatcaaa cctcccccgg ttcccacact tgccgttaag ggcgtagggt actgcagtct 360 ggaatctacg cttgttcaga ctttgtacta gtttctttgt ctggccatcc gggtaaccca 420 tgccggacgc aaaatagact actgaaaatt tttttgcttt gtggttggga ctttagccaa 480 gggtataaaa gaccaccgtc cccgaattac ctttcctctt cttttctctc tctccttgtc 540 aactcacacc cgaaatcgtt aagcatttcc ttctgagtat aagaatcatt caaaatggtg 600 agtttcagag gcagcagcaa ttgccacggg ctttgagcac acggccgggt gtggtcccat 660 tcccatcgac acaagacgcc acgtcatccg accagcactt tttgcagtac taaccgcagg 720 ctaaggagta tttccctcag attcagaaga tcaagttcga cggcaaggac agcaaaaacc 780 ccctggcttt ccattattac gacgccgaga aggaagtgat gggcaagaag atgaaggact 840 ggctgcggtt cgcaatggca tggtggcaca ccctgtgcgc agagggagca gatcagttcg 900 gaggaggcac caagtctttt ccatggaacg agggcacaga cgccatcgag atcgccaagc 960 agaaggtgga tgccggcttc gagatcatgc agaagctggg catcccctac tattgttttc 1020 acgacgtgga tctggtgtcc gagggcaact ctatcggcga gtacgagtcc aatctgaagg 1080 ccgtggtggc ctatctgaag gacaagcaga aggagaccgg catcaagctg ctgtggagct 1140 ccgccaacgt gttcggccac aagaggtaca tgaacggcgc ctctaccaat cccgactttg 1200 atgtggtggc ccgcgccatc gtgcagatca agaacgccat cgatacaggc atcgagctgg 1260 gcgccgagaa ttacgtgttt tggggcggcc gggagggcta tatgtctctg ctgaatacag 1320 accagaagag agagaaggag cacatggcca ccatgctgac aatggccagg gattacgccc 1380 gcagcaaggg cttcaagggc acctttctga tcgagcccaa gcctatggag cctacaaagc 1440 accagtatga cgtggatacc gagacagcca tcggcttcct gaaggcccac aatctggaca 1500 aggacttcaa ggtgaacatc gaagtgaatc acgccaccct ggccggccac acattcgagc 1560 acgagctggc atgcgcagtg gacgcaggaa tgctgggaag catcgacgcc aacaggggcg 1620 attaccagaa tggctgggac accgatcagt ttcctatcga tcagtatgag ctggtgcagg 1680 catggatgga gatcatcagg ggaggaggat tcgtgaccgg aggaacaaac tttgacgcca 1740 agacccggag aaatagcaca gacctggagg atatcatcat cgcccacgtg tccggaatgg 1800 atgcaatggc acgggccctg gagaacgcag caaagctgct gcaggagtcc ccatacacca 1860 agatgaagaa ggagagatat gcctctttcg acagcggcat cggcaaggac tttgaggatg 1920 gcaagctgac actggagcag gtgtacgagt atggcaagaa gaatggcgag cctaagcaga 1980 cctcagggaa gcaggagctg tacgaagcca tcatcgcaat gtatcagtga ctgggcgcgc 2040 ctatataact gtctagaaat aaattttttc aaagctagcc ggtgtgaaat accgcacaga 2100 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg 2160 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 2220 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 2280 gccagtgaat tcgagctcgg tacccgcttt tcgtagataa tggaatacaa atggatatcc 2340 agagtataca catggatagt atacactgac acgacaattc tgtatctctt tatgttaact 2400 actgtgaggc gttaaataga gcttgatata taaaatgtta catttcacag tctgaacttt 2460 tgcagattac ctaatttggt aagatattaa ttatgaactg aaagttgatg gcatccctaa 2520 atttgatgaa agggcgcggg atccggcccg ggaattcgaa ggtaccaagg aagcatgcgg 2580 tacccgaatt cctgaggtgt ctcacaagtg ccgtgcagtc ccgcccccac ttgcttctct 2640 ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc gcccacctcg atccggtcta 2700 gactgaggtg tctcacaagt gccgtgcagt cccgccccca cttgcttctc tttgtgtgta 2760 gtgtacgtac attatcgaga ccgttgttcc cgcccacctc gatccgggga tccctgaggt 2820 gtctcacaag tgccgtgcag tcccgccccc acttgcttct ctttgtgtgt agtgtacgta 2880 cattatcgag accgttgttc ccgcccacct cgatccgggt cgacctgagg tgtctcacaa 2940 gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt acattatcga 3000 gaccgttgtt cccgcccacc tcgatccggg agctcctgag gtgtctcaca agtgccgtgc 3060 agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg tacattatcg agaccgttgt 3120 tcccgcccac ctcgatccgg tctagactga ggtgtctcac aagtgccgtg cagtcccgcc 3180 cccacttgct tctctttgtg tgtagtgtac gtacattatc gagaccgttg ttcccgccca 3240 cctcgatccg gggatccctg aggtgtctca caagtgccgt gcagtcccgc ccccacttgc 3300 ttctctttgt gtgtagtgta cgtacattat cgagaccgtt gttcccgccc acctcgatcc 3360 gggtcgacct gaggtgtctc acaagtgccg tgcagtcccg cccccacttg cttctctttg 3420 tgtgtagtgt acgtacatta tcgagaccgt tgttcccgcc cacctcgatc cgggcatgcg 3480 gtacccgaat tcctgaggtg tctcacaagt gccgtgcagt cccgccccca cttgcttctc 3540 tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc gatccggtct 3600 agactgaggt gtctcacaag tgccgtgcag tcccgccccc acttgcttct ctttgtgtgt 3660 agtgtacgta cattatcgag accgttgttc ccgcccacct cgatccgggg atccctgagg 3720 tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg tagtgtacgt 3780 acattatcga gaccgttgtt cccgcccacc tcgatccggg tcgacctgag gtgtctcaca 3840 agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg tacattatcg 3900 agaccgttgt tcccgcccac ctcgatccgg gcatgcctgc agaagcttag agaccgggtt 3960 ggcggcgtat ttgtgtccca aaaaacagcc ccaattgccc caattgaccc caaattgacc 4020 cagtagcggg cccaaccccg gcgagagccc ccttcacccc acatatcaaa cctcccccgg 4080 ttcccacact tgccgttaag ggcgtagggt actgcagtct ggaatctacg cttgttcaga 4140 ctttgtacta gtttctttgt ctggccatcc gggtaaccca tgccggacgc aaaatagact 4200 actgaaaatt tttttgcttt gtggttggga ctttagccaa gggtataaaa gaccaccgtc 4260 cccgaattac ctttcctctt cttttctctc tctccttgtc aactcacacc cgaaatcgtt 4320 aagcatttcc ttctgagtat aagaatcatt caaaggcgcg ccatgtatct cggactggat 4380 ctttcgactc aacagctcaa gggcatcatt ctggacacaa aaacgctgga cacggtcaca 4440 caagtccatg tggactttga ggacgacttg ccgcagttca acaccgaaaa gggcgtcttt 4500 cacagctcta cagtggccgg agaaatcaat gctcctgtgg caatgtgggg ggcagctgtg 4560 gacttgctga tagagcgtct gtcaaaggaa atagaccttt ccacgatcaa gtttgtgtcg 4620 ggctcgtgcc agcaacacgg ctctgtttat ctcaacagca gctacaagga gggcctgggt 4680 tctctggaca aacacaaaga cttgtctaca ggagtgtcat ccttactggc gctcgaagtc 4740 agccccaatt ggcaggatgc aagcacggag aaggagtgtg cgcagtttga ggctgcagtc 4800 ggcggtcccg agcagctggc tgagatcact ggctctcgag cacatactcg tttcaccggg 4860 ccccagattc tcaaggtcaa ggaacgcaac cccaaggtat tcaaggccac gtcacgggtc 4920 cagctcatat ccaactttct agcatctctg tttgccggca aggcgtgccc ctttgatctt 4980 gctgacgcct gtggaatgaa tctgtgggac atccagaatg gccagtggtg caagaaactc 5040 acagatctca tcaccgatga cacccactcg gtcgagtccc tccttggaga cgtggaaaca 5100 gaccccaagg ctctactggg caaaatctcg ccctatttcg tctccaaggg cttctctccc 5160 tcttgtcagg tggcacagtt cacaggcgac aacccaggca ctatgctggc tctcccctta 5220 caggccaatg acgtgattgt gtctttggga acatctacga ccgccctcgt cgtaacaaac 5280 aagtacatgc ccgaccccgg ataccatgtg ttcaaccacc ccatggaggg atacatgggc 5340 atgctgtgct actgcaacgg aggtctagca cgagagaaga tccgagacga gcttggaggc 5400 tgggacgagt ttaatgaggc ggccgagacc accaacacag tgtctgctga cgatgtccat 5460 gttggcatct actttccact acgagaaatc cttcctcgag caggtccctt tgaacgacgt 5520 ttcatctaca acagacaaag tgaacagctt acagagatgg cttctccaga ggactcactg 5580 gcaaccgaac acaaaccgca ggctcaaaat ctcaaggaca cgtggccgcc acaaatggac 5640 gccactgcca tcattcaaag ccaggccctc agtatcaaaa tgagactcca acgcatgatg 5700 catggcgata ttggaaaggt gtattttgtg ggaggcgcct cggtcaacac tgctatctgc 5760 agcgtaatgt ctgccatctt aaaaccaaca aagggcgctt ggagatgtgg tctggaaatg 5820 gcaaacgctt gtgccattgg aagtgcccat cacgcctggc tttgcgaccc caacaagaca 5880 ggccaggtac aggttcacga agaagaggtc aaatacaaga atgtggacac agacgtgcta 5940 ctcaaggcgt tcaagctggc cgaaaacgcc tgcctggaga aataactaac taccacacca 6000 acacattaat taaggcacgt gcctaaaaag gatcgatacc gtcgacctcg agtcatgtaa 6060 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa 6120 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta 6180 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt 6240 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcggctaa cttgtttaaa 6300 caactgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 6360 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 6420 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 6480 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 6540 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 6600 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 6660 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 6720 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 6780 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 6840 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 6900 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt 6960 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 7020 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 7080 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 7140 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 7200 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 7260 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 7320 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 7380 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 7440 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 7500 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 7560 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 7620 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 7680 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 7740 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 7800 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 7860 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 7920 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 7980 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 8040 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 8100 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 8160 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 8220 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 8280 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 8340 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 8400 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 8460 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 8520 ctataaaaat aggcgtatca cgaggcccag atcctctaga gtcgaagcgg ccgctatgtc 8580 tgataaaagg atgtaacata ggcaagctgc tcgtgagtgt tgagtacgaa ccttagatcc 8640 aaatcacccg cacccacgga tatacttgct tgaatataca gtagtatgcg gccgcttcga 8700 caccatatca tataaaacta acaatgcatg tcgacgagta tctgtctgac tcgtcattgc 8760 cgcctttgga gtacgactcc aactatgagt gtgcttggat cactttgacg atacattctt 8820 cgttggaggc tgtgggtctg acagctgcgt tttcggcgcg gttggccgac aacaatatca 8880 gctgcaacgt cattgctggc tttcatcatg atcacatttt tgtcggcaaa ggcgacgccc 8940 agagagccat tgacgttctt tctaatttgg accgatagcc gtatagtcca gtctatctat 9000 aagttcaact aactcgtaac tattaccata acatatactt cactgcccca gataaggttc 9060 cgataaaaag ttctgcagac taaatttatt tcagtctcct cttcaccacc aaaatgccct 9120 cctacgaagc tcgagctaac gtccacaagt ccgcctttgc cgctcgagtg ctcaagctcg 9180 tggcagccaa gaaaaccaac ctgtgtgctt ctctggatgt taccaccacc aaggagctca 9240 ttgagcttgc cgataaggtc ggaccttatg tgtgcatgat caagacccat atcgacatca 9300 ttgacgactt cacctacgcc ggcactgtgc tccccctcaa ggaacttgct cttaagcacg 9360 gtttcttcct gttcgaggac agaaagttcg cagatattgg caacactgtc aagcaccagt 9420 acaagaacgg tgtctaccga atcgccgagt ggtccgatat caccaacgcc cacggtgtac 9480 ccggaaccgg aatcattgct ggcctgcgag ctggtgccga ggaaactgtc tctgaacaga 9540 agaaggagga cgtctctgac tacgagaact cccagtacaa ggagttcctg gtcccctctc 9600 ccaacgagaa gctggccaga ggtctgctca tgctggccga gctgtcttgc aagggctctc 9660 tggccactgg cgagtactcc aagcagacca ttgagcttgc ccgatccgac cccgagtttg 9720 tggttggctt cattgcccag aaccgaccta agggcgactc tgaggactgg cttattctga 9780 cccccggggt gggtcttgac gacaagggag acgctctcgg acagcagtac cgaactgttg 9840 aggatgtcat gtctaccgga acggatatca taattgtcgg ccgaggtctg tacggccaga 9900 accgagatcc tattgaggag gccaagcgat accagaaggc tggctgggag gcttaccaga 9960 agattaactg ttagaggtta gactatggat atgtcattta actgtgtata tagagagcgt 10020 gcaagtatgg agcgcttgtt cagcttgtat gatggtcaga cgacctgtct gatcgagtat 10080 gtatgatact gcacaacctg tgtatccgca tgatctgtcc aatggggcat gttgttgtgt 10140 ttctcgatac ggagatgctg ggtacaagta gctaatacga ttgaactact tatacttata 10200 tgaggcttga agaaagctga cttgtgtatg acttattctc aactacatcc ccagtcacaa 10260 taccaccact gcactaccac tacaccaaaa ccatgatcaa accacccatg gacttcctgg 10320 aggcagaaga acttgttatg gaaaagctca agagagagaa gccaagatac tatcaagaca 10380 tgtgtcgcaa cttcaaggag gaccaagctc tgtacaccga gaaacaggcc tttgtcgaca 10440 gatctggccc tttcgtc 10457 <210> 6 <211> 1134 <212> DNA <213> Artificial Sequence <220> <223> peroxisomal biogenesis factor 10 <400> 6 atgtggggaa gttcacatgc attcgctggt gaatctgatc tgacactaca actacacacc 60 aggtccaaca tgagcgacaa tacgacaatc aaaaagccga tccgacccaa accgatccgg 120 acggaacgcc tgccttacgc tggggccgca gaaatcatcc gagccaacca gaaagaccac 180 tactttgagt ccgtgcttga acagcatctc gtcacgtttc tgcagaaatg gaagggagta 240 cgatttatcc accagtacaa ggaggagctg gagacggcgt ccaagtttgc atatctcggt 300 ttgtgtacgc ttgtgggctc caagactctc ggagaagagt acaccaatct catgtacact 360 atcagagacc gaacagctct accgggggtg gtgagacggt ttggctacgt gctttccaac 420 actctgtttc catacctgtt tgtgcgctac atgggcaagt tgcgcgccaa actgatgcgc 480 gagtatcccc atctggtgga gtacgacgaa gatgagcctg tgcccagccc ggaaacatgg 540 aaggagcggg tcatcaagac gtttgtgaac aagtttgaca agttcacggc gctggagggg 600 tttaccgcga tccacttggc gattttctac gtctacggct cgtactacca gctcagtaag 660 cggatctggg gcatgcgtta tgtatttgga caccgactgg acaagaatga gcctcgaatc 720 ggttacgaga tgctcggtct gctgattttc gcccggtttg ccacgtcatt tgtgcagacg 780 ggaagagagt acctcggagc gctgctggaa aagagcgtgg agaaagaggc aggggagaag 840 gaagatgaaa aggaagcggt tgtgccgaaa aagaagtcgt caattccgtt cattgaggat 900 acagaagggg agacggaaga caagatcgat ctggaggacc ctcgacagct caagttcatt 960 cctgaggcgt ccagagcgtg cactctgtgt ctgtcataca ttagtgcgcc ggcatgtacg 1020 ccatgtggac actttttctg ttgggactgt atttccgaat gggtgagaga gaagcccgag 1080 tgtcccttgt gtcggcaggg tgtgagagag cagaacttgt tgcctatcag ataa 1134 <210> 7 <211> 1546 <212> DNA <213> Artificial Sequence <220> <223> Diacylglycerol acyltransferase 1 <400> 7 atgactatcg actcacaata ctacaagtcg cgagacaaaa acgacacggc acccaaaatc 60 gcgggaatcc gatatgcccc gctatcgaca ccattactca accgatgtga gaccttctct 120 ctggtctggc acattttcag cattcccact ttcctcacaa ttttcatgct atgctgcgca 180 attccactgc tctggccatt tgtgattgcg tatgtagtgt acgctgttaa agacgactcc 240 ccgtccaacg gaggagtggt caagcgatac tcgcctattt caagaaactt cttcatctgg 300 aagctctttg gccgctactt ccccataact ctgcacaaga cggtggatct ggagcccacg 360 cacacatact accctctgga cgtccaggag tatcacctga ttgctgagag atactggccg 420 cagaacaagt acctccgagc aatcatctcc accatcgagt actttctgcc cgccttcatg 480 aaacggtctc tttctatcaa cgagcaggag cagcctgccg agcgagatcc tctcctgtct 540 cccgtttctc ccagctctcc gggttctcaa cctgacaagt ggattaacca cgacagcaga 600 tatagccgtg gagaatcatc tggctccaac ggccacgcct cgggctccga acttaacggc 660 aacggcaaca atggcaccac taaccgacga cctttgtcgt ccgcctctgc tggctccact 720 gcatctgatt ccacgcttct taacgggtcc ctcaactcct acgccaacca gatcattggc 780 gaaaacgacc cacagctgtc gcccacaaaa ctcaagccca ctggcagaaa atacatcttc 840 ggctaccacc cccacggcat tatcggcatg ggagcctttg gtggaattgc caccgaggga 900 gctggatggt ccaagctctt tccgggcatc cctgtttctc ttatgactct caccaacaac 960 ttccgagtgc ctctctacag agagtacctc atgagtctgg gagtcgcttc tgtctccaag 1020 aagtcctgca aggccctcct caagcgaaac cagtctatct gcattgtcgt tggtggagca 1080 caggaaagtc ttctggccag acccggtgtc atggacctgg tgctactcaa gcgaaagggt 1140 tttgttcgac ttggtatgga ggtcggaaat gtcgcccttg ttcccatcat ggcctttggt 1200 gagaacgacc tctatgacca ggttagcaac gacaagtcgt ccaagctgta ccgattccag 1260 cagtttgtca agaacttcct tggattcacc cttcctttga tgcatgcccg aggcgtcttc 1320 aactacgatg tcggtcttgt cccctacagg cgacccgtca acattgtggt tggttccccc 1380 attgacttgc cttatctccc acaccccacc gacgaagaag tgtccgaata ccacgaccga 1440 tacatcgccg agctgcagcg aatctacaac gagcacaagg atgaatattt catcgattgg 1500 accgaggagg gcaaaggagc cccagagttc cgaatgattg agtaag 1546 <210> 8 <211> 9908 <212> DNA <213> Artificial Sequence <220> <223> pMCS-16TEF-DGA1-CYC1t <400> 8 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgctttt 420 cgtagataat ggaatacaaa tggatatcca gagtatacac atggatagta tacactgaca 480 cgacaattct gtatctcttt atgttaacta ctgtgaggcg ttaaatagag cttgatatat 540 aaaatgttac atttcacagt ctgaactttt gcagattacc taatttggta agatattaat 600 tatgaactga aagttgatgg catccctaaa tttgatgaaa gggcgcggga tccggcccgg 660 gaattcgaag gtaccaagga agcatgcggt acccgaattc ctgaggtgtc tcacaagtgc 720 cgtgcagtcc cgcccccact tgcttctctt tgtgtgtagt gtacgtacat tatcgagacc 780 gttgttcccg cccacctcga tccggtctag actgaggtgt ctcacaagtg ccgtgcagtc 840 ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc 900 gcccacctcg atccggggat ccctgaggtg tctcacaagt gccgtgcagt cccgccccca 960 cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc 1020 gatccgggtc gacctgaggt gtctcacaag tgccgtgcag tcccgccccc acttgcttct 1080 ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct cgatccggga 1140 gctcctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg 1200 tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggt ctagactgag 1260 gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg 1320 tacattatcg agaccgttgt tcccgcccac ctcgatccgg ggatccctga ggtgtctcac 1380 aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac gtacattatc 1440 gagaccgttg ttcccgccca cctcgatccg ggtcgacctg aggtgtctca caagtgccgt 1500 gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat cgagaccgtt 1560 gttcccgccc acctcgatcc gggcatgcgg tacccgaatt cctgaggtgt ctcacaagtg 1620 ccgtgcagtc ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac 1680 cgttgttccc gcccacctcg atccggtcta gactgaggtg tctcacaagt gccgtgcagt 1740 cccgccccca cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc 1800 cgcccacctc gatccgggga tccctgaggt gtctcacaag tgccgtgcag tcccgccccc 1860 acttgcttct ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct 1920 cgatccgggt cgacctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc 1980 tctttgtgtg tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggg 2040 agctcctgag gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt 2100 gtagtgtacg tacattatcg agaccgttgt tcccgcccac ctcgatccgg tctagactga 2160 ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac 2220 gtacattatc gagaccgttg ttcccgccca cctcgatccg gggatccctg aggtgtctca 2280 caagtgccgt gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat 2340 cgagaccgtt gttcccgccc acctcgatcc gggtcgacct gaggtgtctc acaagtgccg 2400 tgcagtcccg cccccacttg cttctctttg tgtgtagtgt acgtacatta tcgagaccgt 2460 tgttcccgcc cacctcgatc cgggcatgcc tgcagaagct tagagaccgg gttggcggcg 2520 tatttgtgtc ccaaaaaaca gccccaattg ccccaattga ccccaaattg acccagtagc 2580 gggcccaacc ccggcgagag cccccttcac cccacatatc aaacctcccc cggttcccac 2640 acttgccgtt aagggcgtag ggtactgcag tctggaatct acgcttgttc agactttgta 2700 ctagtttctt tgtctggcca tccgggtaac ccatgccgga cgcaaaatag actactgaaa 2760 atttttttgc tttgtggttg ggactttagc caagggtata aaagaccacc gtccccgaat 2820 tacctttcct cttcttttct ctctctcctt gtcaactcac acccgaaatc gttaagcatt 2880 tccttctgag tataagaatc attcaaaggc gcgccatgac tatcgactca caatactaca 2940 agtcgcgaga caaaaacgac acggcaccca aaatcgcggg aatccgatat gccccgctat 3000 cgacaccatt actcaaccga tgtgagacct tctctctggt ctggcacatt ttcagcattc 3060 ccactttcct cacaattttc atgctatgct gcgcaattcc actgctctgg ccatttgtga 3120 ttgcgtatgt agtgtacgct gttaaagacg actccccgtc caacggagga gtggtcaagc 3180 gatactcgcc tatttcaaga aacttcttca tctggaagct ctttggccgc tacttcccca 3240 taactctgca caagacggtg gatctggagc ccacgcacac atactaccct ctggacgtcc 3300 aggagtatca cctgattgct gagagatact ggccgcagaa caagtacctc cgagcaatca 3360 tctccaccat cgagtacttt ctgcccgcct tcatgaaacg gtctctttct atcaacgagc 3420 aggagcagcc tgccgagcga gatcctctcc tgtctcccgt ttctcccagc tctccgggtt 3480 ctcaacctga caagtggatt aaccacgaca gcagatatag ccgtggagaa tcatctggct 3540 ccaacggcca cgcctcgggc tccgaactta acggcaacgg caacaatggc accactaacc 3600 gacgaccttt gtcgtccgcc tctgctggct ccactgcatc tgattccacg cttcttaacg 3660 ggtccctcaa ctcctacgcc aaccagatca ttggcgaaaa cgacccacag ctgtcgccca 3720 caaaactcaa gcccactggc agaaaataca tcttcggcta ccacccccac ggcattatcg 3780 gcatgggagc ctttggtgga attgccaccg agggagctgg atggtccaag ctctttccgg 3840 gcatccctgt ttctcttatg actctcacca acaacttccg agtgcctctc tacagagagt 3900 acctcatgag tctgggagtc gcttctgtct ccaagaagtc ctgcaaggcc ctcctcaagc 3960 gaaaccagtc tatctgcatt gtcgttggtg gagcacagga aagtcttctg gccagacccg 4020 gtgtcatgga cctggtgcta ctcaagcgaa agggttttgt tcgacttggt atggaggtcg 4080 gaaatgtcgc ccttgttccc atcatggcct ttggtgagaa cgacctctat gaccaggtta 4140 gcaacgacaa gtcgtccaag ctgtaccgat tccagcagtt tgtcaagaac ttccttggat 4200 tcacccttcc tttgatgcat gcccgaggcg tcttcaacta cgatgtcggt cttgtcccct 4260 acaggcgacc cgtcaacatt gtggttggtt cccccattga cttgccttat ctcccacacc 4320 ccaccgacga agaagtgtcc gaataccacg accgatacat cgccgagctg cagcgaatct 4380 acaacgagca caaggatgaa tatttcatcg attggaccga ggagggcaaa ggagccccag 4440 agttccgaat gattgagtaa gttaattaag gcacgtgcct aaaaaggatc gataccgtcg 4500 acctcgagtc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc 4560 tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat 4620 agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag 4680 acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc 4740 ggctaacttg tttaaacaac tgcaggcatg caagcttggc gtaatcatgg tcatagctgt 4800 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4860 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4920 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4980 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 5040 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 5100 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 5160 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5220 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5280 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5340 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5400 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5460 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5520 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5580 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5640 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5700 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5760 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5820 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5880 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5940 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 6000 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 6060 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 6120 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 6180 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 6240 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6300 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6360 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6420 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6480 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6540 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6600 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6660 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6720 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6780 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6840 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6900 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 6960 ttatcatgac attaacctat aaaaataggc gtatcacgag gcccagatcc tctagagtcg 7020 aagcggccgc tatgtctgat aaaaggatgt aacataggca agctgctcgt gagtgttgag 7080 tacgaacctt agatccaaat cacccgcacc cacggatata cttgcttgaa tatacagtag 7140 tatgcggccg cttcgacacc atatcatata aaactaacaa tgcattgctt attacgaaga 7200 ctacccgttg ctatctccac accgttatct ccacggtcca aaggctgctc aatgtgctgc 7260 atacgtaacg tggggtgcaa ccttgagcac atagtacttt tccgaaaacc ggcgataatt 7320 aagtgtgcac tccaactttt cacactgagc gtaaaatgtg gagaagaaat cggcactaaa 7380 aagtcaggta gactggaaaa tgcgccatga aatgaatatc tcttgctaca gtaatgccca 7440 gcatcgaggg gtattgtgtc accaacacta tagtggcagc tgaagcgctc gtgattgtag 7500 tatgagtctt tattggtgat gggaagagtt cactcaatat tctcgttact gccaaaacac 7560 cacggtaatc ggccagacac catggatgta gatcaccaag cctgtgaatg ttattcgagc 7620 taaaatgcac atggttggtg aaaggagtag ttgctgtcga attccgtcgt cgcctgagtc 7680 atcatttatt taccagttgg ccacaaaccc ttgacgatct cgtatgtccc ctccgacata 7740 ctcccggccg gctggggtac gttcgatagc gctatcggca tcgacaaggt ttgggtccct 7800 agccgatacc gcactacctg agtcacaatc ttcggaggtt tagtcttcca catagcacgg 7860 gcaaaagtgc gtatatatac aagagcgttt gccagccaca gattttcact ccacacacca 7920 catcacacat acaaccacac acatccacaa tggaacccga aactaagaag accaagactg 7980 actccaagaa gattgttctt ctcggcggcg acttctgtgg ccccgaggtg attgccgagg 8040 ccgtcaaggt gctcaagtct gttgctgagg cctccggcac cgagtttgtg tttgaggacc 8100 gactcattgg aggagctgcc attgagaagg agggcgagcc catcaccgac gctactctcg 8160 acatctgccg aaaggctgac tctattatgc tcggtgctgt cggaggcgct gccaacaccg 8220 tatggaccac tcccgacgga cgaaccgacg tgcgacccga gcagggtctc ctcaagctgc 8280 gaaaggacct gaacctgtac gccaacctgc gaccctgcca gctgctgtcg cccaagctcg 8340 ccgatctctc ccccatccga aacgttgagg gcaccgactt catcattgtc cgagagctcg 8400 tcggaggtat ctactttgga gagcgaaagg aggatgacgg atctggcgtc gcttccgaca 8460 ccgagaccta ctccgttcct gaggttgagc gaattgcccg aatggccgcc ttcctggccc 8520 ttcagcacaa cccccctctt cccgtgtggt ctcttgacaa ggccaacgtg ctggcctcct 8580 ctcgactttg gcgaaagact gtcactcgag tcctcaagga cgaattcccc cagctcgagc 8640 tcaaccacca gctgatcgac tcggccgcca tgatcctcat caagcagccc tccaagatga 8700 atggtatcat catcaccacc aacatgtttg gcgatatcat ctccgacgag gcctccgtca 8760 tccccggttc tctgggtctg ctgccctccg cctctctggc ttctctgccc gacaccaacg 8820 aggcgttcgg tctgtacgag ccctgtcacg gatctgcccc cgatctcggc aagcagaagg 8880 tcaaccccat tgccaccatt ctgtctgccg ccatgatgct caagttctct cttaacatga 8940 agcccgccgg tgacgctgtt gaggctgccg tcaaggagtc cgtcgaggct ggtatcacta 9000 ccgccgatat cggaggctct tcctccacct ccgaggtcgg agacttgttg ccaacaaggt 9060 caaggagctg ctcaagaagg agtaagtcgt ttctacgacg cattgatgga aggagcaaac 9120 tgacgcgcct gcgggttggt ctaccggcag ggtccgctag tgtataagac tctataaaaa 9180 gggccctgcc ctgctaatga aatgatgatt tataatttac cggtgtagca accttgacta 9240 gaagaagcag attgggtgtg tttgtagtgg aggacagtgg tacgttttgg aaacagtctt 9300 cttgaaagtg tcttgtctac agtatattca ctcataacct caatagccaa gggtgtagtc 9360 ggtttattaa aggaagggag ttgtggctga tgtggataga tatctttaag ctggcgactg 9420 cacccaacga gtgtggtggt agcttgttac tgtatattcg gtaagatata ttttgtgggg 9480 ttttagtggt gtttggtagg ttagtgcttg gtatatgagt tgtaggcatg acaatttgga 9540 aaggggtgga ctttgggaat attgtgggat ttcaatacct tagtttgtac agggtaattg 9600 ttacaaatga tacaaagaac tgtatttctt ttcatttgtt ttaattggtt gtatatcaag 9660 tccgttagac gagctcagtg ccatggcttt tggcactgta tttcattttt agaggtacac 9720 tacattcagt gaggtatggt aaggttgagg gcataatgaa ggcaccttgt actgacagtc 9780 acagacctct caccgagaat tttatgagat atactcgggt tcattttagg ctccgattcg 9840 attcaaatta ttactgtcga aatcggttga gcatccgttg atttccgaac agatctggcc 9900 ctttcgtc 9908

Claims (27)

It is an adaptive evolution strain that was passaged in a minimal medium using xylose as the only carbon source,
It includes a gene encoding a xylose isomerase and a gene encoding a xylulokinase, and the gene encoding the xylose isomerase is xylA3* represented by the nucleotide sequence of SEQ ID NO: 1. The gene encoding the xylulose kinase is ylXK (xylulokinase) represented by the nucleotide sequence of SEQ ID NO: 2,
The adaptive evolutionary strain contains a 495_500delCAACTT mutation in the YALI0_D07634g gene,
Yarrow subtotal Li poly urticae state transformation of yeast (Yarrowia lipolytica). The method of claim 1, wherein the adaptive evolution strain YALI0_A00374g, YALI0_A00935g, YALI0_A02497g, YALI0_A02937g, YALI0_A15642g, YALI0_A15796g, YALI0_A17578g, YALI0_A17776g, YALI0_A17853g, YALI0_A18744g, YALI0_A19646g, YALI0_B00748g, YALI0_B05676g, YALI0_B18194g, YALI0_C07722g, YALI0_C08327g, YALI0_C08349g, YALI0_C08473g, YALI0_C09031g, YALI0_C09614g , YALI0_C13002g, YALI0_C13728g, YALI0_C13970g, YALI0_C15532g, YALI0_C15741g, YALI0_C16148g, YALI0_C16247g, YALI0_D00627g, YALI0_D09647g, YALI0_D14542g, YALI0_D15752g, YALI0_D17820g, YALI0_D18678g, YALI0_D19360g, YALI0_D20064g, YALI0_D20526g, YALI0_D20790g, YALI0_D24849g, YALI0_D25058g, YALI0_D26972g, YALI0_E07832g, YALI0_E08008g, YALI0_E13948g, YALI0_E18766g, YALI0_E19767g , YALI0_E20053g, YALI0_E20449g, YALI0_E23969g, YALI0_E29623g, YALI0_E31999g, YALI0_E33891g, YALI0_E34089g, YALI0_E35046g, YALI0_F12573g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g, YALI0_F12771g
597_598insGGCTCCGCTGCCGCCTCC mutation in the YALI0_A00374g gene;
202_203insGCTC mutation in the YALI0_A00935g gene;
427_429dupAAC mutation in the YALI0_A02497g gene;
20_21insGGAGACCGTGGGGGAGACCGTGGA mutation in the YALI0_A02937g gene;
147G>A mutation in the YALI0_A15642g gene;
One or more of the 576T>G mutation and the 567_568insACA mutation in the YALI0_A15796g gene;
434_435insGGACCCGCC mutation in the YALI0_A17578g gene;
1810_1811insACAACAACACCC mutation in the YALI0_A17776g gene and
One or more of the 959_960insACAGCAGAT mutations;
1940_1942delAGG mutation in the YALI0_A17853g gene and
One or more of the 1990_1991insAGGAGGAGGCTAAGAAGA mutations;
2512_2513insCTGGAGGGGGTGGAAGCG mutation in the YALI0_A18744g gene;
1467delA mutation in the YALI0_A19646g gene;
522_523insAACTCC mutation in the YALI0_B00748g gene;
1369_1370insCAGCAGCGT mutation in the YALI0_B05676g gene;
1695_1697delCGG mutation in the YALI0_B18194g gene;
At least one of the 1553C>T mutation and 1552A>G mutation in the YALI0_C07722g gene;
1221_1250delTCCTCACGTGCAGCCTCCTCATGCGCAGCC mutation in the YALI0_C08327g gene;
2455_2456insCTCCCATCTCCTCCT mutation in the YALI0_C08349g gene and
One or more of the 2471_2472insTCCCATCTCCTCCGA mutations;
1326_1327insGCACCTGCGACTTCTTCG mutation in the YALI0_C08473g gene;
1789_1790insCTCCCGAGTCCTCTGCTGAGCCTACCAGCGAAGAGACTTCTTCCG mutation in the YALI0_C09031g gene;
1617_1622delACAACA mutation in the YALI0_C09614g gene;
815_816insTCTAA mutation in the YALI0_C13002g gene;
One or more of the 99_100insAAAAAGTGGCCGAAAGAGTGGCCA mutation and the 129_130insTGGCCGAAAAAGTGGCCAAAA mutation in the YALI0_C13728g gene;
1587_1588insGCT mutation in the YALI0_C13970g gene;
1611_1616delCAGCTT mutation in the YALI0_C15532g gene;
198_209delCTATTCAAACGA mutation in the YALI0_C15741g gene;
957_968delCAGCAGCAGCAG mutation in the YALI0_C16148g gene;
One or more of the 1292A>G mutation, 1412T>A mutation, 1441G>A mutation, 1513A>G mutation, 1534A>C mutation, 1544T>A mutation, and 1847C>T mutation in the YALI0_C16247g gene;
2603A>C mutation in the YALI0_D00627g gene;
491_492insAAAGCAGCAGCAGCA mutation in the YALI0_D09647g gene;
80_81insC mutation in the YALI0_D14542g gene;
One or more of the 1038_1039insCAG mutation and the 1038_1039insCGGCAG mutation in the YALI0_D15752g gene;
One or more of the 615_616insCCCCACCACCCGCAA mutation and the 641_642insGCAAACCTACCACCA mutation in the YALI0_D17820g gene;
1300_1323 delACCCAGAACCAGACCCAGAACCAG mutation in the YALI0_D18678g gene;
2132_2155delGGAGTGAGAGTGGGAGTGAGAGTG mutation in the YALI0_D19360g gene;
69_70delCT mutation in the YALI0_D20064g gene;
355_356insCCACCGGCG mutation in the YALI0_D20526g gene;
39_40insCGTGAAAGGAGCCGAACC mutation in the YALI0_D20790g gene;
103G>A mutation in the YALI0_D24849g gene;
592_594dupAAG mutation in the YALI0_D25058g gene;
36_41delACAGCA mutation in the YALI0_D26972g gene;
499_500insCCAAGCCCCCCGCTTCCAAGCCCACCGCTT mutation in the YALI0_E07832g gene;
794_795insCTCTTCCTCTTCCTCTTCCTCTTCCTCTTC mutation in the YALI0_E08008g gene;
825_830delCAGCAA mutation in the YALI0_E13948g gene;
1100_1101insTTCTGCCGCTTA mutation in the YALI0_E18766g gene;
2699A>G mutation in the YALI0_E19767g gene;
533_534insGGCCGAGGAGGC mutation in the YALI0_E20053g gene;
902_903insGGCCAA mutation in the YALI0_E20449g gene;
One or more of the 480_481insTCCGCTACCACCGAG mutation and 497A>C mutation in the YALI0_E23969g gene;
10A>G mutation in the YALI0_E29623g gene;
269_270insTGTGTATATATGCAGAAAAAAAAAAA mutation in the YALI0_E31999g gene;
One or more of the 237C>A mutation, and the 239G>C mutation in the YALI0_E33891g gene;
72_73insCG mutation in the YALI0_E34089g gene;
1926T>C mutation in the YALI0_E35046g gene;
75_76insTCCCCG mutation in the YALI0_F12573g gene;
1019_1021delAGT mutation in the YALI0_F12771g gene;
One or more of the 1855_1860delTCTTCT mutation and the 4102_4103insTAGCCG mutation in the YALI0_F12793g gene;
718_723delCAGCAG mutation in the YALI0_F18568g gene;
One or more of the 2099C>A mutation, 2098A>G mutation, 2065T>A mutation, and 1344_1345insCCTACTCCCTCCGAGGTT mutation in the YALI0_F19030g gene. delete delete The method of claim 1, wherein the strain comprises: a first vector including a gene encoding a xylose isomerase, and a second vector including a gene encoding a xylulose kinase; Or a third vector comprising a gene encoding a xylose isomerase and a gene encoding a xylulose kinase is introduced, a transforming yeast strain. The transformed yeast strain according to claim 5, wherein the genes introduced into the first vector, the second vector and the third vector are inserted into genomic DNA or expressed in a plasmid form. The transformed yeast strain of claim 5, wherein the first vector comprises a UAS1B enhancer, a translational elongation factor (TEF) promoter, and a gene encoding a xylose isomerase. The transformed yeast strain of claim 5, wherein the second vector comprises a UAS1B enhancer, a translational elongation factor (TEF) promoter, and a gene encoding a xylulose kinase. The transformation according to claim 5, wherein the third vector includes a UAS1B enhancer, a translational elongation factor (TEF) promoter, a xylose isomerase-encoding gene, and a xylulose kinase-encoding gene. Yeast strain. The transformed yeast strain according to claim 7, wherein the first vector consists of the nucleotide sequence of SEQ ID NO: 3. The transformed yeast strain of claim 8, wherein the second vector consists of the nucleotide sequence of SEQ ID NO: 4. The transformed yeast strain according to claim 9, wherein the third vector consists of the nucleotide sequence of SEQ ID NO: 5. According to claim 1, wherein the strain is a strain overexpressing diacylglycerol acyltransferase (Diacylglycerol acyltransferase), a transformed yeast strain. The method of claim 13, wherein the strain is a peroxisomal biogenesis factor 10 (peroxisomal biogenesis factor 10) is deleted, and diacylglycerol acyl transferase (Diacylglycerol acyltransferase) is overexpressed, transformed yeast strain. The method of claim 1, wherein the parent strain of Yarrow subtotal Li poly Mathematica for switching the wild type is transformed Yarrow subtotal Li poly Mathematica which, transformed yeast strains. The method of claim 15, wherein the wild-type Yarrowia lipolytica strain is accession number ATCC MYA-2613, a transformed yeast strain. The method of claim 1, wherein the adaptive evolution strain is accession number KCTC 13911BP, a transformed yeast strain. A culture of the transformed yeast strain according to any one of claims 1, 2 and 5 to 17. A method for producing a transformed yeast strain according to any one of claims 1, 2 and 5 to 17,
Wild type Yarrow subtotal Li poly urticae (Yarrowia lipolytica) and to the parent strain, sub-cultured by adaptive evolution in a minimal medium for the xylose as the only carbon source comprising: (adaptive evolution); And
A method comprising the step of inserting a gene encoding a xylose isomerase and a gene encoding a xylulose kinase into the adapted strain. The method of claim 19, wherein the step of adaptive evolution comprises:
Inserting a gene encoding a wild-type gene and the poly Yarrow subtotal Lee Giles rule Los kinases encoding the xylose isomerase in the Mathematica (Yarrowia lipolytica);
Subculturing the inserted strain in a minimal medium containing xylose as the sole carbon source; And
Removing a gene encoding a xylose isomerase and a gene encoding a xylulose kinase from the subcultured strain;
Containing, the manufacturing method. The method according to claim 19, wherein the gene is inserted using CRISPR/Cas9 or by introducing a plasmid. The method of claim 19, wherein the method further comprises the step of overexpressing diacylglycerol acyltransferase. The method of claim 22, wherein the step comprises deleting peroxisomal biogenesis factor 10 and overexpressing diacylglycerol acyltransferase. The transformed yeast strain according to any one of claims 1, 2 and 5 to 17 is cultivated in a medium containing xylose as the sole carbon source, or a medium containing glucose and xylose as a carbon source. Bio-oil production method comprising the step. The method of claim 24, wherein the bio-oil comprises a lipid. The method of claim 24, wherein the medium comprises lignocellulosic biomass,
Before the step of culturing the strain in a medium, further comprising the step of producing at least one of glucose and xylose by decomposing the lignocellulosic biomass. The method of claim 24, further comprising transesterifying the lipid obtained in the culturing step to obtain biodiesel.

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