KR20200053394A - Yeast strain with glucose and xylose co-utilization capacity - Google Patents

Abstract

The present specification describes a transforming yeast strain can simultaneously use xylose and glucose as carbon sources, a method for manufacturing the same, and a method for producing a biofuel using the same. The transforming yeast strain can produce a high yield biofuel by transforming a wild-type yeast strain which cannot use xylose as a carbon source to simultaneous convert glucose and xylose. By providing a strain which can be easily converted into a high yield biofuel / material production strain through additional modification, such that economic efficiency and sustainability of the biofuel and biomaterial production process can be highly improved.

Description

Simultaneous conversion of glucose and xylose yeast strain {Yeast strain with glucose and xylose co-utilization capacity}

This specification describes a yeast strain capable of simultaneously converting glucose and xylose and a method for producing biofuel and biomaterial using the same.

As a sustainable and economical method for producing fuel / material for the defossil fuel era, biofuel / material production technology using wood-based biomass has been developed. Woody biomass is the most abundant carbon source, accounting for more than 90% of the total biomass produced on the planet, and is an environmentally friendly renewable resource, including herbal and woody biomass. Wood-based biomass includes waste wood, agricultural and forestry by-products, and energy crops. When the biofuel is produced using the wood-based biomass, the carbon reduction effect is greater than that of fossil fuels and first-generation biofuels.

Biological biofuel / material production technology using microbial fermentation process is an economical and eco-friendly technology. Saccharomyces yeast bio-ethanol production processes as the owner with Mrs. Celebi Asia (Saccharomyces cerevisiae) is being operated in the world. In order to further increase the economic efficiency and sustainability of the bioethanol, a fuel / material production process using a non-edible biomass such as wood-based biomass has been proposed, but Saccharomyces cerevisiae has a biomass-derived hexasaccharide (representatively glucose , There is a problem in that conversion efficiency is lowered because only 50% of the biomass is used and pentose sugar (typically xylose, containing up to 25% of the biomass) is not used. If this is overcome, it is expected that it is possible to greatly improve the maximum conversion rate of biomass from 50% to 75%. Accordingly, the development of a xylose conversion strain is underway.

The yeast strain as described above is endogenous and does not have a xylose metabolic pathway, so xylose metabolism is impossible. In order to impart the ability to use xylose to such yeast, development of a transforming strain in which a xylose metabolic pathway based on oxidase-reductase was introduced has been reported. However, the ability to use xylose has not yet reached a satisfactory level. In particular, the xylose metabolic pathway based on oxidoreductase has a problem of low ethanol production yield due to a problem of imbalance of cofactor. In addition, when glucose and xylose, which are the main carbon sources derived from woody biomass, exist simultaneously, the ability to use xylose deteriorates rapidly, and there is a problem in that productivity is deteriorated because xylose is consumed only after all glucose is consumed. In addition, xylose strains that have been developed to date have been limited in that further improvement is limited, so that the product is limited to bioethanol.

 Sun-Mi Lee et al., Systematic and evolutionary engineering of xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields, Biotechnol. Biofuel., 2014 1: 122  Verhoeven MD et al., Mutations in PMR1 stimulated xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis, Sci. Rep. 2017 7: 46155

In one aspect, the problem to be solved by the present invention is to provide a transformed yeast strain capable of simultaneously converting glucose / xylose and having improved xylose availability.

In one aspect, the problem to be solved by the present invention is to provide a method for producing a transformed yeast strain capable of simultaneously converting glucose / xylose and improving xylose availability.

In one aspect, the problem to be solved by the present invention is to provide a method for producing biofuels and materials using transformed yeast strains capable of simultaneous conversion of glucose / xylose and improved xylose utilization.

In order to achieve the above object, one embodiment of the invention is mainly transformed yeast, the variation of the PMR1 represented by the nucleotide sequence of SEQ ID NO: 1 or a deletion of a Saccharomyces three Levy cyano (Saccharomyces cerevisiae), and ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 contains one or more of the mutations or deletions,

A gene encoding xylose isomerase; And a gene encoding a xylulose phosphatase gene and a gene encoding an aldol transaldolase,

Variation of the PMR1 represented by the base sequence of said SEQ ID NO: 1 will be mutated to PMR1 ^G681A represented by the nucleotide sequence of SEQ ID NO: 2,

The variation of ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 provides a transformed yeast strain, which is a variation of ASC1 ^{Q237 *} represented by the nucleotide sequence of SEQ ID NO: 4.

In order to achieve the above object, an embodiment of the present invention to the MRS three Levy cyano (Saccharomyces cerevisiae) with the above transgenic Yeast production method, a saccharide parent strain,

A gene encoding xylose isomerase in the strain ; And inserting one or more of a gene encoding a xylulose phosphatase gene and a gene encoding an aldol transaldolase; And

Mutation or deletion of PMR1 represented by the nucleotide sequence of SEQ ID NO: 1 of the strain, and Comprising the steps of performing one or more of the mutation or deletion of ASC1 represented by the nucleotide sequence of SEQ ID NO: 3,

Variation steps of the PMR1 or ASC1 provides a production method which comprises passages adapted to evolution (adaptive evolution) in a minimal medium for xylose the strain with the only carbon source.

In order to solve the above problems, an embodiment of the present invention is a step of fermenting the transformed yeast strain in a medium containing xylose or glucose as the only carbon source, or in a medium containing glucose and xylose as a mixed carbon source. It provides a method for producing biofuels and materials comprising a.

According to the present invention, xylose metabolism based on xylose isomerase can be imparted by transforming a wild-type yeast strain that does not use xylose as a carbon source, and it is possible to improve xylose utilization capacity by applying an evolutionary method. The present invention does not introduce a redox-based xylose metabolic pathway, so it is possible to produce high-yield ethanol by simultaneously converting glucose and xylose without the problem of cofactor imbalance. Since the transformed yeast strain according to the present invention is easily converted into a high-yield biofuel / material producing strain through additional improvement, other biofuels or biomaterials such as butanol other than bioethanol can be produced without additional processing. Therefore, it is possible to greatly improve the economic efficiency and sustainability of the biofuel and biomaterial production process.

Figure 1a is a comparative example of Saccharomyces cerevisiae SXA-R2P-E strain (Comparative Example 1) of the present invention and xylose using an xylose strain (XUSE; Example 1) of one embodiment of the present invention as a single carbon source It is a diagram showing the xylose utilization curve in the present medium.
Figure 1b is a comparative example of the present invention, Saccharomyces cerevisiae SXA-R2P-E strain (Comparative Example 1) and xylose using one embodiment of the present invention (XUSE; Example 1) xylose as a single carbon source It is a diagram showing the ethanol production curve in the present medium.
Figure 1c is a comparative example of the present invention, Saccharomyces cerevisiae SXA-R2P-E strain (Comparative Example 1) and xylose using one embodiment of the present invention (XUSE; Example 1) xylose as a single carbon source It is a diagram showing the strain growth curve in the present medium.
Figure 2a xylose transforming strain (Example 2) and PMR1 deletion strain (Example 3) according to the expression of the PMR1 mutant, an embodiment of the present invention, and the wild species PMR1 expression strain (Comparative Example 2) of the present invention Is a diagram showing a xylose utilization curve when cultured in a medium having only carbon source.
Figure 2b xylose transforming strain (Example 2) and PMR1 deletion strain (Example 3) according to the expression of the PMR1 mutant, which is an embodiment of the present invention, and the wild type PMR1 expressing strain (Comparative Example 2), which is a comparative example of the present invention It is a diagram showing the ethanol production curve when cultured in a medium using only carbon source.
Figure 3a xylose transforming strain (Example 4) and ACS1- deficient strain (Example 5) according to the expression of the ACS1 mutant, which is an embodiment of the present invention, and the wild type ACS1 expressing strain (Comparative Example 3) of the present invention Is a diagram showing a xylose utilization curve when cultured in a medium using only carbon source.
Figure 3b xylose transforming strain (Example 4) and ACS1 deletion strain (Example 5) according to the expression of the ACS1 mutant, which is an embodiment of the present invention, and the wild type ACS1 expression strain (Comparative Example 3), which is a comparative example of the present invention It is a diagram showing the ethanol production curve when cultured in a medium using only carbon source.
Figure 4a is a transforming strain (XUSE; Example 1) according to an embodiment of the present invention when using xylose alone or in the presence of xylose and glucose incorporating glucose (glucose / xylose) availability of glucose utilization It is a diagram shown in a curve.
Figure 4b xylose transforming strain according to an embodiment of the present invention (XUSE; Example 1) xylose in the presence of xylose alone or in the presence of xylose and glucose (glucose / xylose) It is a diagram represented by the utilization curve.
Figure 4c is a transforming strain according to an embodiment of the present invention (XUSE; Example 1) ethanol production when cultivated in the presence of xylose alone or in the presence of xylose and glucose (glucose / xylose) ethanol production It is a diagram shown in a curve.
Figure 5 is a modified strain according to an embodiment of the present invention (XUSE A) improved strain introduced butanol production path (Example 6) glucose (Glucose) or xylose (Xylose) alone or xylose and glucose It is a diagram showing the butanol production capacity when cultured in the presence of (Glucose + Xylose) compared to the wild species Saccharomyces cerevisiae strain (WT).

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

One embodiment of the present invention A saccharide having an ability of simultaneous conversion of glucose (glucose) and xylose (xylose) provides a transformed yeast strains in MRS three Levy cyano (Saccharomyces cerevisiae).

As Saccharomyces Celebi Asia (Saccharomyces cerevisiae) yeast strain is a typical bio-ethanol production strain, metabolic engineering, but improved easy to use as a platform strain for a variety of bio-fuels, and materials production, Celebi Asian strain Mrs into wild-type Saccharomyces is Giles It is known that Ross cannot be used as a carbon source. Of the available carbon sources in the wood-based biomass, the carbon source available to yeast strains such as wild-type Saccharomyces cerevisiae is glucose, which accounts for about 50% of the total wood-based biomass. When woody biomass is used as a raw material that provides a carbon source necessary for producing biofuels and materials to yeast strains, the yeast strain uses only glucose as a carbon source, and thus has a problem of low conversion to ethanol.

Accordingly, the present inventor transforms a strain of Saccharomyces cerevisiae , a yeast strain that can use only glucose derived from xylem-based biomass as a technology capable of producing biofuels and materials, thereby conferring the ability to use xylose. By doing so, a yeast strain capable of simultaneously using integrating sugar (glucose / xylose) derived from woody biomass was developed.

As an embodiment, the Saccharomyces cerevisiae transforming yeast strain having the ability to co-convert glucose and xylose is a variant or deletion of PMR1 expressed by the base sequence of SEQ ID NO: 1, and A gene comprising at least one of ASC1 mutations or deletions represented by the nucleotide sequence of SEQ ID NO: 3, and encoding xylose isomerase; And a gene encoding xylulose phosphatase gene and a gene encoding aldol transaldolase.

In this case, an embodiment of the parent strain MRS three Levy cyano (Saccharomyces cerevisiae) a saccharide which is transformed with the three Mrs. Levy in wild-type Saccharomyces can not use xylose as a carbon source as a cyano yeast strains. The wild-type yeast strain may be commercially available or it may be confirmed that it is preserved in a credible preservation agency and can be freely distributed by a catalog issued by the preservation agency. In one embodiment may be a MRS three Levy cyano yeast Saccharomyces three Levy cyano BY4741 (Saccharomyces cerevisiae BY4741) strains MRS to that in the wild-type Saccharomyces, may be included in all, if a wild-type Saccharomyces three Levy cyano yeast strains not limited to this. The strain accession number of Saccharomyces cerevisia BY4741 is ATCC 201388.

In one embodiment, the PMR1 is a gene expressed by the base sequence of SEQ ID NO: 1 encoding Golgi Ca ²⁺ / Mg ²⁺ ATPase. In one embodiment variation of the PMR1 represented by the base sequence of said SEQ ID NO: 1 may be one that is mutated to PMR1 ^G681A represented by the nucleotide sequence of SEQ ID NO: 2. The PMR1 ^G681A is the 681 amino acids of the entire gene sequence of the parental strain in MRS three Levy cyano, wherein the saccharide with a mutant type of the PMR1 gene is converted from G (glycine) to A (alanine).

In one embodiment, the ASC1 is a gene expressed by the nucleotide sequence of SEQ ID NO: 3 encoding a guanine dissociation inhibitor of Gpa2p. The variation of ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 may be a variation of ASC1 ^{Q237 *} represented by the nucleotide sequence of SEQ ID NO: 4. The ASC1 ^{Q237 *} is a mutant form of the ASC1 gene, wherein 237 Q (glutamine) of the entire amino acid sequence of the Saccharomyces cerevisiae parent strain is converted to a stop codon.

One embodiment of the present invention may increase the activity of the xylose isomerase by including the PMR1 ^G681A and ASC1 ^{Q237 *} one or more mutations of the gene deletion to one or more of the PMR1 and ASC1 or.

In one embodiment, the gene encoding the xylose isomerase is a gene encoding an enzyme that converts D-xylose and D-xylulose to isomerization of xylose. If possible, it is not limited to this and can be included in all. One embodiment, as the xylose gene coding for isomerase is xylA3 represented by the nucleotide sequence of SEQ ID NO: 5 *, fatigue My process species (Piromyces sp.) XylA, chloride represented by the base sequence of SEQ ID NO: 6, isolated from host lithium pie stopper men Tansu comprise xylA represented by the base sequence of SEQ ID NO: 8 separated from the xylA or Clostridium Thermo surfer Jeans (Clostridium thermosulfurgenes) represented by the base sequence of the isolated SEQ ID NO: 7 from (Clostridium phytofermentans) Can be. Preferably, in terms of isomerization efficiency, xylA3 * represented by the nucleotide sequence of SEQ ID NO: 5 may be included.

In one embodiment, the gene encoding the xylulose phosphatase is an enzyme that produces D-xylulose-5-phosphate from D-xylulose. A gene that encodes, If phosphorylation of xylose is possible, it is not limited thereto and may be included. In one embodiment, a gene encoding the xylene rule Los kinases are xyl3 represented by the base sequence of the XKS1 or three peoso My process styryl blood teeth of SEQ ID NO: 10 isolated from (Scheffersomyces stipites) represented by the nucleotide sequence of SEQ ID NO: 9 May be included.

In one embodiment, the gene encoding aldol transaldolase converts cedoheptulose-7-phosphate and glyceraldehyde-3-phosphate to fructose-6-phosphate and erythrose-4-phosphate Any enzyme that catalyzes the reaction is not limited and may be included. As an example, the aldol transferase may include TAL1 represented by the nucleotide sequence of SEQ ID NO: 11 or tal1 represented by the nucleotide sequence of SEQ ID NO: 12 isolated from Sephomyces stipitis .

In one embodiment, the Saccharomyces cerevisiae transformed yeast strain may be one or more of genes encoding aldose reductase and genes encoding phosphatase.

As an embodiment, the gene encoding the aldose reductase may include GRE3 represented by the nucleotide sequence of SEQ ID NO: 13. As an example, the gene encoding phosphatase may include PHO13 represented by the nucleotide sequence of SEQ ID NO: 14.

In one embodiment, the transformed yeast is to ASC1 represented by the base sequence of the PMR1 and SEQ ID NO: 3, represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, xylose gene coding for isomerase, xylene rule Los kinase gene And a gene encoding an aldol transferase, and a gene encoding an aldose reductase and a gene encoding a phosphatase may be a transformed yeast strain.

In another exemplary embodiment, including, and encoding a xylose isomerase for ASC1 ^{Q237 *} represented by the nucleotide sequence of which is represented by the base sequence of SEQ ID NO: 2 that the transformed yeast PMR1 ^G681A and SEQ ID NO: 4 gene, xylene rule It includes a gene encoding a los phosphatase and a gene encoding an aldol transferase, and may be a transformed yeast strain in which a gene encoding an aldose reductase and a gene encoding a phosphatase are deleted.

As an example, the Saccharomyces cerevisiae transformed yeast strain is a strain deposited with accession number KCTC13614BP.

The mutation herein can be introduced by treating the microorganism with any chemical and / or physical means known to be capable of causing mutation in the art. The chemical means may include chemical substances such as NTG (nitrosoguanidine), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), benzopyrene, etc., which are effective guanidine derivatives as a mutagenic substance (mutant). , 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, specifically, genetic scissors, for example, CRISPR-Cas9 may be used, but is not limited thereto.

As an example, since the Saccharomyces cerevisiae transformed yeast strain is introduced into the xylose isomerase-based xylose metabolic pathway by transformation, as described above, the xylose fertilization efficiency is Saccharomyces cerevis , the parent strain. It may be increased compared to Sia . Specifically, it may be increased by 30% or more or 50% or more, and more specifically, may be increased by 30 to 150%.

In addition, as one embodiment, the Saccharomyces cerevisiae transformed yeast strain can ferment glucose and xylose at the same time and convert them into ethanol, so that the ethanol conversion from glucose and xylose is the parent strain, Saccharomyces cerevisiae. It may be increased compared to. Specifically, it may be increased to 50% or more, or 100% or more, and more specifically, 50 to 300%.

Since xylose accounts for up to about 30% in the xylem-based biomass and approximately 50% glucose, the transformed yeast strain according to one embodiment of the present invention converts the glucose and xylose in the xylem-based biomass. Up to 80% of available resources can be used. Accordingly, the yield of ethanol production for the unit biomass can be greatly improved, and the economic efficiency and sustainability of biofuel and biomaterial production using wood-based biomass can be greatly improved.

In addition, the Saccharomyces cerevisiae transformed yeast strain according to the present invention can produce other biofuels or biomaterials such as butanol other than bioethanol without additional processing through additional improvement of the strain itself. The additional improvement may include expressing the strain by introducing a gene combination constituting the production path of the production material. For example, the Saccharomyces cerevisiae transformed yeast strain can be further improved to produce butanol by further including a gene constituting the butanol production pathway. In one embodiment the gene constituting the butanol production routes are Clostridium acetonitrile beauty rikum (Clostridium acetobutyricum) a Hbd (β-hydroxybutyryl-CoA dehydrogenase ) ( SEQ ID NO: 17), Crt (3-hydroxybutyryl -CoA dehydratase) derived from the ( SEQ ID NO: 18) and BdhB (butanol dehydergenase) (SEQ ID NO: 19); Erg10 (acetoacetyl-CpA thiolase) (SEQ ID NO: 20) and Etr1 (enoyl thioester reductase) (SEQ ID NO: 21) derived from Saccharomyces cerevisiae ; It may include EutE (butyraldehyde dehydrogenase) (SEQ ID NO: 22) derived from Escherichia coli . As an example, the Saccharomyces cerevisiae transformed yeast strain may be introduced into the plasmid form of a gene constituting the butanol production pathway. As an example, the Saccharomyces cerevisiae transformed yeast strain may be introduced into the form of genomic DNA inserted into the gene constituting the butanol production pathway. Specifically, the additionally improved Saccharomyces cerevisiae transformed yeast strain is a plasmid represented by the nucleotide sequence of SEQ ID NO: 23 (p423-GPDp- Ca Hbd-PRM9t-TEF1p- Ca Crt-CPS1t), the nucleotide sequence of SEQ ID NO: 24 Plasmid represented by (p426-PGKp- Ec Eute-CYC1t-CYC1p- Ca Bdhb-SPG5t) and plasmid represented by the nucleotide sequence of SEQ ID NO: 25 (p425-HXT7p- Sc cytoEtr1-TPI7t-TEF1p- Sc Erg10-CYC1t) It may be introduced. The improved Saccharomyces cerevisiae transformed yeast strain as described above can ferment glucose and xylose simultaneously to produce butanol separately from the production of ethanol.

In one embodiment, the present invention may include a culture of the Saccharomyces cerevisiae transformed yeast strain described above. The culture may contain ethanol, and may further contain other biofuels such as butanol by further refinement.

"Cultivation" in the present specification is applicable without limitation as long as it is by a culture method known in the art. As an example, the culture of the yeast strain may be cultured 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 the culture inoculated with the microorganism while shaking, and the stationary culture means a method of culturing the liquid culture inoculated with the microorganism without shaking. The batch culture means a method of culturing in a state in which the culture volume is fixed and no culture is newly added from the outside, and a device capable of realizing such a culture method is called a batch reactor. The fed-batch culture refers to a method of culturing by adding a small amount of elements first and adding a small amount of raw materials to it as a counterpart to a single culture in which all the raw materials are first put into the culture tank and 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 at the same time, the culture containing cells and products is continuously removed, and an apparatus capable of realizing such a culture method is called a continuous reactor. Specifically, the culture may be performed in a batch reactor, a continuous reactor, or a fed-batch reactor.

In one embodiment, the culture is achieved by satisfying the culture requirement of the yeast strain in an appropriate manner while controlling temperature, pH, etc. in a normal medium containing one or more selected from the group consisting of a suitable carbon source, nitrogen source, amino acid, vitamin, etc. Can be. Examples of 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, and the like. Fatty acids, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, organic acids such as acetic acid, acetic acid, butyric acid, isobutyric acid, propionic acid, valeric acid, isovaleric acid, caproic acid, etc. acid, VFA). These materials may be included individually or in combination. Examples of 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 and 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 soy cake or its degradation products Can be. These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate, and corresponding sodium-containing salts as personnel. Personnel that can be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, substances such as amino acids and vitamins can be used.

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

One embodiment according to the present invention can provide a method for producing the Saccharomyces cerevisiae transformed yeast strain.

The production method according to an exemplary embodiment is to the Mrs three Levy cyano (Saccharomyces cerevisiae) as a parent strain to a saccharide, a gene encoding the xylose isomerase in the isolates; And inserting one or more of a gene encoding a xylulose phosphatase gene and a gene encoding an aldol transaldolase; And the variant or deletion of PMR1 represented by the nucleotide sequence of SEQ ID NO: 1 of the strain, and ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 may include performing one or more of mutation or deletion. In one embodiment, the method is a gene encoding an aldose reductase and a gene encoding a phosphatase It may further include the step of deleting one or more of.

As an embodiment, the insertion or deletion of the genes may include all of the sequences of genes inserted or deleted without being limited to the order described herein.

In addition, as an embodiment, the manufacturing method may include a gene encoding the xylose isomerase ; And inserting at least one of a gene encoding a xylulose phosphatase gene and a gene encoding an aldol transaldolase, and / or a gene encoding a aldose reductase and a phosphatase. gene encoding (phosphatase) After the step of deleting one or more of, the variant or deletion of PMR1 represented by the nucleotide sequence of SEQ ID NO: 1 of the strain and ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 may include a gene mutation step of performing one or more of mutation or deletion.

In another embodiment, the method of manufacturing the mutant or deletion of PMR1 expressed by the nucleotide sequence of SEQ ID NO: 1 of the strain and A gene encoding the xylose isomerase after the gene mutation step of performing one or more of mutation or deletion of ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 ; And inserting at least one of a gene encoding a xylulose phosphatase gene and a gene encoding an aldol transaldolase, and / or a gene encoding a aldose reductase and a phosphatase. gene encoding (phosphatase) It may include the step of deleting one or more of.

In one embodiment, the variation step of the PMR1 or ASC1, but can include the sub-cultured in a minimal medium for the xylose the strain as the only carbon source for adapting evolution (adaptive evolution), the PMR1 to PMR1 ^G681A, ASC1 If it is possible to mutate ASC1 ^{Q237 *} , it is not limited to the above method and may be included in the scope of the present invention.

As an example, the adaptive evolution is a method of obtaining a strain having 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 screening a strain having good xylose utilization ability after screening the strain in a medium containing xylose as a carbon source, and selecting a strain having improved xylose utilization capacity. One embodiment a transgenic stage one or more of the PMR1 and ASC1 is a concentration of the strain of strains of the exponential growth period (exponential phase) were cultured in xylose medium in a new xylose medium, for example, about 0.5% (v / v ) When inoculating and cultivating and reaching the exponential growth phase, the method may further include subculturing by inoculating a new medium with a predetermined concentration, for example, about 0.5%. Specifically, the minimal medium may include xylose as a carbon source, and a medium 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 the xylose isomerase-based xylose metabolic pathway to the yeast strain that has been adaptively evolved through metabolic engineering and evolutionary engineering, a transformed yeast strain having improved xylose availability can be prepared. As an embodiment, the adaptive evolution may include adaptive evolution to have xylose availability by subculturing 2, 3, 4, 5 or more times in a medium containing xylose as the only carbon source, It is not limited thereto.

As an embodiment, the insertion or deletion of the gene may be performed using CRISPR / Cas9, but any method that can insert or delete 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 fuel / material production metabolic pathway other than bioethanol.

For example, the manufacturing method may further include introducing a gene constituting a butanol production pathway as another biofuel into the Saccharomyces cerevisiae transformed yeast strain. In one embodiment the gene constituting the above-butanol production routes are Clostridium acetonitrile beauty rikum (Clostridium Hbd (β-hydroxybutyryl-CoA dehydrogenase) derived from acetobutyricum ) (SEQ ID NO: 17), Crt (3-hydroxybutyryl-CoA dehydratase) (SEQ ID NO: 18) and BdhB (butanol dehydergenase) (SEQ ID NO: 19); Erg10 (acetoacetyl-CpA thiolase) (SEQ ID NO: 20) and Etr1 (enoyl thioester reductase) (SEQ ID NO: 21) derived from Saccharomyces cerevisiae ; And EutE (butyraldehyde dehydrogenase) (SEQ ID NO: 22) derived from Escherichia coli . As an embodiment, it may include the step of introducing a gene constituting the butanol production path in a plasmid form. As a more specific embodiment, the preparation method is a plasmid expressed in the nucleotide sequence of SEQ ID NO: 23 in Saccharomyces cerevisiae transformed yeast strain (p423-GPDp- Ca Hbd-PRM9t-TEF1p- Ca Crt-CPS1t), SEQ ID NO: Plasmid represented by the nucleotide sequence of 24 (p426-PGKp- Ec Eute-CYC1t-CYC1p- Ca Bdhb-SPG5t) and plasmid represented by the nucleotide sequence of SEQ ID NO: 25 (p425-HXT7p- Sc cytoEtr1-TPI7t-TEF1p- Sc Erg10 -CYC1t). As an embodiment, the additional step may be a method of introducing the plasmid into a strain through a transformation method, but any method capable of introducing the plasmid form may be included without being limited thereto. As another embodiment, the additional step may include introducing a gene constituting the butanol production pathway into the Saccharomyces cerevisiae transformed yeast strain in a form inserted into genomic DNA. In this case, the insertion of the gene may be performed using CRISPR / Cas9, but any method that can insert the gene is not limited thereto and may be included.

One embodiment according to the present invention, it is possible to provide a method for producing biofuels and materials comprising culturing the Saccharomyces cerevisiae transformed yeast strain in a medium containing xylose as the only carbon source. . Specifically, the culturing step is 24-168 hours at 28-32 ° C. after inoculating the Saccharomyces cerevisiae transformed yeast strain into a minimal medium (YSC medium: Yeast Synthetic Complete medium) having xylose as a single carbon source. It may include the step of culturing during.

Another embodiment may provide a method for producing biofuels and materials, comprising culturing the Saccharomyces cerevisiae transformed yeast strain in a medium containing glucose and xylose as a carbon source and simultaneously fermenting it. Specifically, the culture step is inoculated into a minimal medium (YSC medium: Yeast Synthetic Complete medium) having xylose and glucose as a carbon source for the Saccharomyces cerevisiae transformed yeast strain, 24-168 at 28-32 ° C. And culturing for a period of time.

As an embodiment, the medium may include a woody biomass, and the ethanol production method decomposes the woody biomass before culturing the yeast strain in the medium to produce one or more of glucose and xylose. It may further include a step. At this time, the step of decomposing the woody biomass to produce one or more of glucose and xylose may include, for example, strong acid saccharification, weak acid saccharification, organic solvent pretreatment, etc., but glucose and xylose from the woody biomass Any method that can produce one or more of the above is not limited thereto, and is all included.

As an embodiment, the method may further include a biofuel and material production step of producing biofuels and materials using the culture or fermentation product of the strain. The "fermented substance" is not limited to a substance obtained by culturing, ie, fermenting a transformed yeast strain according to the embodiment of the present invention, as well as a substance obtained by culturing a genetic recombinant microorganism using the transformed yeast strain as a parent strain. It is the meaning of Choi Guang-in to include. Examples of the fermentation product are raw materials capable of producing biofuels and materials. As an embodiment, the culture or fermentation product of the strain may include ethanol and butanol, but is not limited thereto.

The "bio fuel" means a fuel obtained by using biomass as a raw material, and may include ethanol obtained by a method according to one embodiment of the present invention, but is not limited thereto. As another embodiment, the method may further produce one or more biofuels or materials selected from butanol, fatty acid ethyl ester, and isoprenoid-based compounds through the introduction of an additional metabolic pathway to the transformed yeast strain.

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] Preparation of transformed yeast strains

As an example of the present invention, a Saccharomyces cerevisiae transformed yeast strain having xylose availability was prepared by the following method.

First, using the CRISPR / Cas9 system, the wild-type Saccharomyces cerevisiae yeast strain Saccharomyces cerevisia BY4741 (strain number: ATCC 201388) is a nucleotide sequence of SEQ ID NO: 5, a gene encoding xylose isomerase. Insert the XKS1 expressed as the nucleotide sequence of SEQ ID NO: 9, which is the gene encoding the xylA3 * and xylulose phosphatase gene, and at the same time, the nucleotide sequence of SEQ ID NO: 13, which is the gene encoding the aldose reductase. The expressed GRE3 was deleted. The strain additionally xylose the gene encoding the isomerase xylA3 * and aldol transferase (transaldolase) for coding the same time Phosphatase (phosphatase) to insert TAL1 represented by the base sequence of the gene of SEQ ID NO: 11 encoding The gene, PHO13 expressed by the nucleotide sequence of SEQ ID NO: 14, was deleted.

Then, in order to improve the xylose availability of the transformed strain, the xylose was cultured in a minimal medium (YSC medium: Yeast Synthetic Complete medium) as the only carbon source, and the strain of the exponential phase was used as a new xylose medium. Was inoculated with about 0.5% (v / v) and cultured, and then, when reaching the exponential growth phase, it was subcultured by inoculating with fresh medium at about 0.5% again, and the result was adapted evolution. As a result, the base sequence of SEQ ID NO: 1 the PMR1 ASC1 that the expression of the nucleotide sequence of the PMR1 ^G681A, SEQ ID NO: 3, represented by the base sequence of SEQ ID NO: 2 expressed in It was mutated to ASC1 ^{Q237 *} represented by the nucleotide sequence of SEQ ID NO: 4.

The strain was named XUSE and deposited with accession number KCTC13614BP.

[Test Example 1]

In order to evaluate the xylose utilization ability of the XUSE strain (Example 1) prepared in the above example, the xylose was inoculated into a minimal medium (YSC medium: Yeast Synthetic Complete medium) having a single carbon source and stirred at 30 ° C at 200 rpm. By culturing, the use of xylose, ethanol production, and growth of the strain were confirmed.

Sun-Mi Lee et al., Systematic and evolutionary engineering of xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields, Biotechnol., Incorporated herein by reference in its entirety. Biofuel., 2014, SXA-R2P-E (Comparative Example 1), a previously reported xylose-using strain disclosed in 1: 122, was also cultured under the same conditions as the XUSE strain to compare xylose utilization ability.

As a result, as shown in FIGS. 1A to 1C, the XUSE strain (Example 1), which is an embodiment of the present invention, consumes all 20 g / L xylose within 72 hours of culture to yield a yield of 0.43 g ethanol / g xylose. Ethanol was produced. Comparative Example SXA-R2P-E strain (Comparative Example 1), while consuming the same amount of xylose, while growing a small amount of strain and producing more ethanol (0.43 vs 0.4). SXA-R2P-E strain is known as the strain with the highest yield of ethanol converted to xylose among the previously reported transformed yeast strains, which means that the transformed yeast strain according to the present invention can produce ethanol with a higher yield from xylose. It means that the high yield xylose conversion strain. In addition, the comparative example SXA-R2P-E strain is not easy to improve additionally, whereas the production material is limited to ethanol, whereas the transformed yeast strain according to the present invention facilitates additional engineering by using CRISPR-Cas9 to diversify the product. It has the advantage of being applicable to the development of fuel and material production strains other than ethanol.

[Test Example 2]

And confirming that the transformed yeast strains according to one embodiment of the present invention full-length do the dielectric analysis (whole genome sequencing) to PMR1 and ACS1 gene is mutated to PMR1 ^G681A, ASC1 ^{Q237 *,} the higher xylose utilization capability increases the mutation The following experiment was carried out to confirm that it was due to.

A transformed yeast strain was prepared from the wild-type Saccharomyces cerevisiae strain in the same manner as the XUSE strain of Example 1 (Example 1), but the following genetic constructs were different.

Vector that PMR1 is the wild type gene expression in three Levy cyano strains MRS with a deletion saccharide (Comparative Example 2), SEQ ID NO vectors 15 is mutated gene PMR1 ^G681A according to the present invention represented by the nucleotide sequence the expression of (Example 2) , PMR1 deleted vector, that is, a blank vector (Example 3) was introduced.

Vector in which wild species gene is expressed in Saccharomyces cerevisiae strain in which ACS1 is deleted (Comparative Example 3), a vector in which the mutant gene ASC1 ^{Q237 *} according to the present invention expressed by the nucleotide sequence of SEQ ID NO: 16 is expressed (Example 4 ), A vector in which ACS1 was deleted, that is, an empty vector (Example 5) was introduced.

Each of the vectors was confirmed by inoculating each strain into a minimal medium (YSC medium: Yeast Synthetic Complete medium) having xylose as a single carbon source, and then culturing with stirring at 200 rpm at 30 ° C to confirm xylose availability and ethanol production capacity. .

As a result, in the case of Figure 2a, Figure 2b, embodiments of a saccharide with a PMR1 or ACS1 deletion, as shown in Figures 3a and 3b are three Levy cyano strains with mutations PMR1 or ACS1 MRS expressed Examples 2 to 5 xylose utilization and It was confirmed that the ethanol productivity was significantly increased.

In the case of the ^{PMR1 G681} expressing strain (Example 2), the xylose utilization increased 115% and the ethanol production increased 196% compared to the strain (Comparative Example 2) in which wild PMR1 was expressed. In the case of ASC1 ^{Q237 *} expression strain (Example 4), it was confirmed that xylose utilization increased by 60% and ethanol production increased by 104% compared to the strain (Comparative Example 3) in which wild species ASC1 was expressed. Similar results were obtained in the case where the PMR1 or ACS1 caused a mutation, even if the fruit, of ASC1 is believed to be because the loss of its function is altered amino acid No. 237 to the stop codon.

This means that a deletion or mutation in the PMR1 or ACS1 according to one embodiment of the present invention plays an important role in xylose utilization efficiency increases.

[Test Example 3]

In order to confirm the glucose / xylose-integrated sugar converting ability of the transformed yeast strain according to an embodiment of the present invention, the XUSE strain (Example 1) prepared in the above Example is the least of which xylose is present as glucose and mixed sugar. After inoculation in a medium (YSC medium: Yeast Synthetic Complete medium), the mixture was stirred and cultured at 30 ° C at 200 rpm to confirm the tendency of xylose / glucose use and ethanol production.

Most of the xylose strains reported so far have a tendency to use xylose sequentially after consuming all of the glucose in an environment where glucose and xylose exist simultaneously, resulting in poor efficiency of glucose / xylose conversion derived from woody biomass. There is this. However, in the case of the transformed yeast strain according to an embodiment of the present invention, as shown in FIGS. 4A to 4C, it can be confirmed that co-fermentation is possible per unit of ethanol production using xylose even in the presence of glucose. In particular, even when glucose is present in the same amount as xylose, xylose utilization performance is maintained high without glucose inhibition, so that an integrated sugar (glucose / xylose) conversion yield superior to the existing technology can be achieved.

[Test Example 4]

The following experiment was conducted to confirm that it is easy to convert the transformed yeast strain according to the present invention into a biofuel producing strain other than bioethanol.

First, by introducing a gene constituting the butanol (1-butanol) production pathway into the XUSE strain (Example 1) prepared in the above example, an improved strain (Example 6) having butanol production capacity was prepared. Gene constituting the above-butanol production routes are Clostridium acetonitrile beauty rikum (Clostridium acetobutyricum) a Hbd (β-hydroxybutyryl-CoA dehydrogenase ) ( SEQ ID NO: 17), Crt (3-hydroxybutyryl -CoA dehydratase) ( SEQ ID NO: 18 derived from ) And BdhB (butanol dehydergenase) (SEQ ID NO: 19); Erg10 (acetoacetyl-CpA thiolase) (SEQ ID NO: 20) and Etr1 (enoyl thioester reductase) (SEQ ID NO: 21) derived from Saccharomyces cerevisiae ; EutE (butyraldehyde dehydrogenase) (SEQ ID NO: 22) derived from Escherichia coli . The gene was introduced into the following three plasmid forms through a transformation method.

-p423-GPDp- Ca Hbd-PRM9t-TEF1p- Ca Crt-CPS1t (SEQ ID NO: 23)

-p426-PGKp- Ec Eute-CYC1t-CYC1p- Ca Bdhb-SPG5t (SEQ ID NO: 24)

-p425-HXT7p- Sc cytoEtr1-TPI7t-TEF1p- Sc Erg10-CYC1t) (SEQ ID NO: 25)

As a comparative example of the present invention, the wild-type Saccharomyces cerevisiae yeast strain Saccharomyces cerevisiae BY4741 (strain number: ATCC 201388) also constitutes the butanol production pathway of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. The gene was introduced in the same way.

Next, the XUSE modified strain (Example 6) of the present invention into which the gene constituting the butanol production pathway was introduced and the wild-type strain were glucose, xylose, or the minimum medium in which glucose and xylose were present as mixed sugars (YSC medium) : Yeast Synthetic Complete medium), and incubated with stirring at 200 ° C at 30 ° C., and produced butanol contained in the culture after 96 hours.

As a result, as shown in FIG. 5, in the case of wild species strain (WT), when xylose was used as a single carbon source, butanol was not produced at all, whereas the XUSE modified strain (Example 6) of the present invention was about 14.2 g / L. Butanol was produced. It was confirmed that under conditions where glucose and xylose exist simultaneously, the XUSE modified strain of the present invention (Example 6) produced about 4 times or more of butanol (27.3 mg / L) compared to the wild-type strain (WT) using glucose alone. . The above results indicate that the transformed yeast strain according to the present invention is easy to improve to other biofuel production strains such as butanol with simple additional improvement by introducing genes having different biofuel production paths, and also has excellent production capacity.

Korea Research Institute of Bioscience and Biotechnology KCTC13614BP 20180807

<110> Korea Institute of Science of Technology <120> Yeast strain with glucose and xylose co-utilization capacity <130> 19P096IND <150> KR 2018-0136647 <151> 2018-11-08 <160> 25 <170> KopatentIn 2.0 <210> 1 <211> 2853 <212> DNA <213> Artificial Sequence <220> <223> PMR1 <400> 1 atgagtgaca atccatttaa tgcgagtctt cttgacgagg actcaaaccg tgagagagaa 60 atactagatg ccacagcaga ggccctttcg aaaccaagcc cttctttaga gtattgtact 120 ttatccgtgg acgaagctct agaaaaactg gacactgaca aaaacggtgg tttacgatca 180 tctaacgagg ccaacaatag gagatcactt tatggcccca atgaaataac cgtagaagat 240 gatgaaagtc ttttcaagaa gttcttgtca aatttcattg aggatcgaat gattctactt 300 ttaataggat ccgcagtggt ctctcttttt atgggtaaca ttgatgatgc tgttagtatc 360 acactggcca ttttcatagt tgtcactgtc ggttttgtcc aagaatatag gtctgaaaaa 420 tctctagaag cgttgaataa attggttcct gctgaatgtc acttaatgag atgtggtcaa 480 gagagtcatg tactggcttc caccttggtt cctggtgatt tagtgcactt cagaataggt 540 gacagaatcc ccgcagacat tagaattatt gaagcaatcg atttatccat cgatgaaagt 600 aatttaactg gtgaaaatga accggtacat aaaacctcac aaacgatcga aaaatcttcc 660 tttaacgatc agcctaattc aattgtaccg atttctgaga gatcttgtat agcttatatg 720 ggtacattag tcaaggaagg tcatggtaag ggtatcgtcg taggaacagg tacaaacaca 780 tcctttggtg ccgtttttga aatgatgaat aatattgaaa aaccgaagac tccattgcag 840 ttaacaatgg acaaattggg aaaggacttg tcactggtta gcttcatagt tattggtatg 900 atttgtttag ttggtatcat acaaggtaga tcttggttag aaatgttcca aatatcggta 960 tccttagcgg ttgctgctat tccagaaggg ttaccaatta ttgtcactgt tactttggca 1020 ttgggtgttc tgagaatggc caagcgtaaa gccatcgtga gaaggttacc aagtgtcgaa 1080 actttaggct ctgtcaacgt tatctgctcc gacaaaacag gtacactaac ctcaaaccac 1140 atgaccgtat ctaaactttg gtgcttggac agtatgtcca ataagctaaa cgtcctctca 1200 ttagacaaaa ataagaagac taaaaattct aatggaaatt tgaaaaacta tttgactgaa 1260 gacgttaggg aaactctaac tatcggtaat ctctgtaata atgcatcttt ctctcaagaa 1320 catgccatat ttctgggaaa tcctactgat gtagctcttt tagagcaatt ggcaaacttt 1380 gaaatgcctg atatcagaaa caccgttcaa aaagttcagg aacttccatt taactcgaaa 1440 agaaaattaa tggcaaccaa gattctcaac cctgtcgaca ataagtgtac agtttatgtt 1500 aaaggtgcat ttgaaagaat tcttgagtac tccacaagtt atttgaaatc aaagggtaaa 1560 aaaactgaaa agttgactga agcccaaaaa gctacgataa atgagtgcgc aaattctatg 1620 gcatctgaag gtttgcgtgt ctttggattt gctaaactaa ctttgtctga ttcatcaact 1680 cctctaaccg aagacctaat caaagattta acctttactg gtttaatcgg tatgaatgac 1740 ccaccaagac cgaacgttaa atttgccatc gaacaattac tacaaggtgg tgtccatatt 1800 attatgatca ctggtgattc tgagaatacc gcagtaaaca ttgcaaaaca aattggtatt 1860 ccagttattg atccaaagct ttccgtttta tccggtgata aattagatga aatgtcagat 1920 gatcaactgg ccaatgtcat cgaccacgtt aatatttttg ctcgtgctac gcctgagcat 1980 aagttaaaca ttgttcgtgc attaagaaag aggggtgatg tggtagcaat gactggtgat 2040 ggtgttaacg acgctcctgc gttgaaactt tcagatattg gtgtttctat gggtagaatt 2100 ggtacagatg tagccaaaga agcctcagat atggtcttaa ctgatgatga cttcagtact 2160 attttaactg ccattgaaga gggtaaaggt atctttaata atattcagaa tttcctgact 2220 tttcaattgt ctacttctgt tgccgcacta tcattagttg cactatctac agcgtttaaa 2280 ctacccaatc cactgaacgc aatgcaaatt ctttggataa atattttaat ggatgggcca 2340 ccagctcaat ccttaggtgt ggaacctgtt gatcatgaag ttatgaaaaa acctccaaga 2400 aaacgtaccg ataaaatttt gacccatgat gtaatgaaac gtttactaac caccgcggcc 2460 tgtatcatcg ttgggacagt ttacattttt gttaaagaga tggccgaaga tggtaaagta 2520 actgctagag atactactat gacatttact tgttttgttt tttttgatat gtttaatgct 2580 ttggcctgca gacataacac aaagtcaatc ttcgaaatcg gctttttcac gaacaaaatg 2640 ttcaactacg ccgttggact gtctctgtta ggtcaaatgt gcgctatata tataccattt 2700 ttccaaagta tctttaaaac tgagaaactt ggtatctctg atatactatt gttattgctc 2760 atcagcagta gcgttttcat cgttgatgaa ttgagaaaat tgtggacgag gaaaaagaat 2820 gaagaagact caacgtattt ctcaaatgtt tga 2853 <210> 2 <211> 2853 <212> DNA <213> Artificial Sequence <220> <223> PMR1 mutant <400> 2 atgagtgaca atccatttaa tgcgagtctt cttgacgagg actcaaaccg tgagagagaa 60 atactagatg ccacagcaga ggccctttcg aaaccaagcc cttctttaga gtattgtact 120 ttatccgtgg acgaagctct agaaaaactg gacactgaca aaaacggtgg tttacgatca 180 tctaacgagg ccaacaatag gagatcactt tatggcccca atgaaataac cgtagaagat 240 gatgaaagtc ttttcaagaa gttcttgtca aatttcattg aggatcgaat gattctactt 300 ttaataggat ccgcagtggt ctctcttttt atgggtaaca ttgatgatgc tgttagtatc 360 acactggcca ttttcatagt tgtcactgtc ggttttgtcc aagaatatag gtctgaaaaa 420 tctctagaag cgttgaataa attggttcct gctgaatgtc acttaatgag atgtggtcaa 480 gagagtcatg tactggcttc caccttggtt cctggtgatt tagtgcactt cagaataggt 540 gacagaatcc ccgcagacat tagaattatt gaagcaatcg atttatccat cgatgaaagt 600 aatttaactg gtgaaaatga accggtacat aaaacctcac aaacgatcga aaaatcttcc 660 tttaacgatc agcctaattc aattgtaccg atttctgaga gatcttgtat agcttatatg 720 ggtacattag tcaaggaagg tcatggtaag ggtatcgtcg taggaacagg tacaaacaca 780 tcctttggtg ccgtttttga aatgatgaat aatattgaaa aaccgaagac tccattgcag 840 ttaacaatgg acaaattggg aaaggacttg tcactggtta gcttcatagt tattggtatg 900 atttgtttag ttggtatcat acaaggtaga tcttggttag aaatgttcca aatatcggta 960 tccttagcgg ttgctgctat tccagaaggg ttaccaatta ttgtcactgt tactttggca 1020 ttgggtgttc tgagaatggc caagcgtaaa gccatcgtga gaaggttacc aagtgtcgaa 1080 actttaggct ctgtcaacgt tatctgctcc gacaaaacag gtacactaac ctcaaaccac 1140 atgaccgtat ctaaactttg gtgcttggac agtatgtcca ataagctaaa cgtcctctca 1200 ttagacaaaa ataagaagac taaaaattct aatggaaatt tgaaaaacta tttgactgaa 1260 gacgttaggg aaactctaac tatcggtaat ctctgtaata atgcatcttt ctctcaagaa 1320 catgccatat ttctgggaaa tcctactgat gtagctcttt tagagcaatt ggcaaacttt 1380 gaaatgcctg atatcagaaa caccgttcaa aaagttcagg aacttccatt taactcgaaa 1440 agaaaattaa tggcaaccaa gattctcaac cctgtcgaca ataagtgtac agtttatgtt 1500 aaaggtgcat ttgaaagaat tcttgagtac tccacaagtt atttgaaatc aaagggtaaa 1560 aaaactgaaa agttgactga agcccaaaaa gctacgataa atgagtgcgc aaattctatg 1620 gcatctgaag gtttgcgtgt ctttggattt gctaaactaa ctttgtctga ttcatcaact 1680 cctctaaccg aagacctaat caaagattta acctttactg gtttaatcgg tatgaatgac 1740 ccaccaagac cgaacgttaa atttgccatc gaacaattac tacaaggtgg tgtccatatt 1800 attatgatca ctggtgattc tgagaatacc gcagtaaaca ttgcaaaaca aattggtatt 1860 ccagttattg atccaaagct ttccgtttta tccggtgata aattagatga aatgtcagat 1920 gatcaactgg ccaatgtcat cgaccacgtt aatatttttg ctcgtgctac gcctgagcat 1980 aagttaaaca ttgttcgtgc attaagaaag aggggtgatg tggtagcaat gactggtgat 2040 gatgttaacg acgctcctgc gttgaaactt tcagatattg gtgtttctat gggtagaatt 2100 ggtacagatg tagccaaaga agcctcagat atggtcttaa ctgatgatga cttcagtact 2160 attttaactg ccattgaaga gggtaaaggt atctttaata atattcagaa tttcctgact 2220 tttcaattgt ctacttctgt tgccgcacta tcattagttg cactatctac agcgtttaaa 2280 ctacccaatc cactgaacgc aatgcaaatt ctttggataa atattttaat ggatgggcca 2340 ccagctcaat ccttaggtgt ggaacctgtt gatcatgaag ttatgaaaaa acctccaaga 2400 aaacgtaccg ataaaatttt gacccatgat gtaatgaaac gtttactaac caccgcggcc 2460 tgtatcatcg ttgggacagt ttacattttt gttaaagaga tggccgaaga tggtaaagta 2520 actgctagag atactactat gacatttact tgttttgttt tttttgatat gtttaatgct 2580 ttggcctgca gacataacac aaagtcaatc ttcgaaatcg gctttttcac gaacaaaatg 2640 ttcaactacg ccgttggact gtctctgtta ggtcaaatgt gcgctatata tataccattt 2700 ttccaaagta tctttaaaac tgagaaactt ggtatctctg atatactatt gttattgctc 2760 atcagcagta gcgttttcat cgttgatgaa ttgagaaaat tgtggacgag gaaaaagaat 2820 gaagaagact caacgtattt ctcaaatgtt tga 2853 <210> 3 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> ASC1 <400> 3 atggcatcta acgaagtttt agttttgaga ggtaccttgg aaggtcacaa cggttgggtc 60 acatctttgg ctacttctgc tggtcaacca aacctattgt tgtccgcttc ccgtgataag 120 actttgatct cctggaagtt gactggtgac gaccaaaagt ttggtgtccc agttagatct 180 ttcaagggtc acagtcacat tgtccaagac tgtactttga ctgctgacgg tgcttacgct 240 ttgtctgctt cttgggacaa gaccttgaga ttatgggatg ttgccaccgg tgaaacctac 300 caaagattcg tcggtcacaa gtccgatgtt atgtccgttg acattgacaa gaaggcttcc 360 atgattatct ctggttcccg tgacaagacc atcaaggtct ggaccatcaa aggtcaatgt 420 ttggccactt tgttgggtca caatgactgg gtttcccaag tcagagttgt tccaaacgaa 480 aaagctgatg atgactctgt caccatcatt tctgccggta acgacaaaat ggttaaggta 540 tgtgatatat tttctttcca tgatagaata tgatgacaat cgagtagaag aagaaaagtg 600 gatttgtgta tgccattcaa atgatgtaat aacatatttg ctacttcaga tggaactttg 660 agttccgaat gagacatacc aattatcacc aagatctctg atgaatggtt tagcattact 720 ctgctcttct ctttactcgt tatgtcaaaa tggaaacttt ttttttaaat taattttgtt 780 cccttactaa caaaaatgat ataatgacag gcttggaact taaaccaatt ccaaattgaa 840 gctgacttca tcggtcacaa ctccaacatc aacactttga ctgcttcccc agacggaact 900 ttgattgctt ccgctggtaa ggacggtgaa attatgttgt ggaacttggc tgctaagaag 960 gctatgtaca ctttgtctgc ccaagatgaa gttttctctt tggctttctc tccaaacaga 1020 tactggttgg ctgctgccac tgctaccggt attaaggtct tttctttgga cccacaatac 1080 ttggtcgatg acttgagacc agaatttgct ggttacagca aggccgctga accacatgct 1140 gtttctttgg cttggtctgc tgacggtcaa actttgtttg ccggttacac cgacaacgtc 1200 attagagttt ggcaagttat gactgctaac taa 1233 <210> 4 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> ASC1 mutant <400> 4 atggcatcta acgaagtttt agttttgaga ggtaccttgg aaggtcacaa cggttgggtc 60 acatctttgg ctacttctgc tggtcaacca aacctattgt tgtccgcttc ccgtgataag 120 actttgatct cctggaagtt gactggtgac gaccaaaagt ttggtgtccc agttagatct 180 ttcaagggtc acagtcacat tgtccaagac tgtactttga ctgctgacgg tgcttacgct 240 ttgtctgctt cttgggacaa gaccttgaga ttatgggatg ttgccaccgg tgaaacctac 300 caaagattcg tcggtcacaa gtccgatgtt atgtccgttg acattgacaa gaaggcttcc 360 atgattatct ctggttcccg tgacaagacc atcaaggtct ggaccatcaa aggtcaatgt 420 ttggccactt tgttgggtca caatgactgg gtttcccaag tcagagttgt tccaaacgaa 480 aaagctgatg atgactctgt caccatcatt tctgccggta acgacaaaat ggttaaggta 540 tgtgatatat tttctttcca tgatagaata tgatgacaat cgagtagaag aagaaaagtg 600 gatttgtgta tgccattcaa atgatgtaat aacatatttg ctacttcaga tggaactttg 660 agttccgaat gagacatacc aattatcacc aagatctctg atgaatggtt tagcattact 720 ctgctcttct ctttactcgt tatgtcaaaa tggaaacttt ttttttaaat taattttgtt 780 cccttactaa caaaaatgat ataatgacag gcttggaact taaaccaatt ccaaattgaa 840 gctgacttca tcggtcacaa ctccaacatc aacactttga ctgcttcccc agacggaact 900 ttgattgctt ccgctggtaa ggacggtgaa attatgttgt ggaacttggc tgctaagaag 960 gctatgtaca ctttgtctgc ctaagatgaa gttttctctt tggctttctc tccaaacaga 1020 tactggttgg ctgctgccac tgctaccggt attaaggtct tttctttgga cccacaatac 1080 ttggtcgatg acttgagacc agaatttgct ggttacagca aggccgctga accacatgct 1140 gtttctttgg cttggtctgc tgacggtcaa actttgtttg ccggttacac cgacaacgtc 1200 attagagttt ggcaagttat gactgctaac taa 1233 <210> 5 <211> 1314 <212> DNA <213> Artificial Sequence <220> <223> xylA3 * <400> 5 atggctaaag aatatttccc tcaaattcaa aagataaaat tcgatggtaa agatagcaag 60 aacccattgg cctttcatta ctatgatgcc gaaaaagagg tcatgggtaa aaaaatgaaa 120 gactggttaa gatttgccat ggcttggtgg cacaccttgt gtgctgaagg agcagatcaa 180 tttggaggag ggactaaaag tttcccatgg aacgaaggta cagacgcaat tgaaatagca 240 aagcaaaaag ttgatgctgg tttcgagata atgcaaaaat taggtattcc ttactattgt 300 tttcatgacg tggatttagt atctgaaggt aactctattg gggaatatga atccaacttg 360 aaggcagttg tcgcgtactt gaaagataaa cagaaggaaa ctgggattaa gttattatgg 420 tcatctgcta atgtatttgg tcataaaagg tatatgaacg gagcttcgac taatcctgat 480 tttgatgtgg ttgcgagggc tattgtacaa attaaaaacg caatcgacac aggcatcgaa 540 ttgggcgcag aaaattacgt gttttggggc ggtagagaag gttatatgtc attacttaat 600 actgatcaaa aaagagaaaa agaacatatg gcaaccatgt tgaccatggc tagggactat 660 gcacgtagta aaggctttaa aggtaccttc cttattgaac ctaagcctat ggaacctact 720 aaacaccaat acgacgtaga cactgagact gcaatcggtt tcttgaaagc tcataatcta 780 gataaagact tcaaagtcaa tattgaagtt aatcacgcta ctctagccgg tcatacgttc 840 gagcatgagt tggcttgcgc tgtagacgca gggatgttag gtagcataga tgccaatagg 900 ggtgactatc aaaatggctg ggacactgac cagtttccta tcgaccagta cgaattggta 960 caggcttgga tggaaattat aaggggtggc ggttttgtaa ccggtggaac aaacttcgac 1020 gctaagacaa gacgtaattc gactgactta gaagatatta ttatcgctca tgtgtctggt 1080 atggatgcca tggctcgtgc attggagaat gcagctaaat tgttacagga gtctccatac 1140 acaaaaatga agaaggaaag atatgcttca ttcgattctg gaataggaaa agattttgaa 1200 gatgggaaac tgacattgga gcaagtctat gaatatggaa agaagaatgg agaaccgaaa 1260 caaacctcag gaaaacaaga actatacgag gctattatcg cgatgtatca ataa 1314 <210> 6 <211> 1314 <212> DNA <213> Artificial Sequence <220> <223> xylA from Piromyces sp. <400> 6 atggctaagg aatacttccc acaaatccaa aagatcaagt tcgaaggtaa ggactctaag 60 aacccattgg ctttccacta ctacgacgct gaaaaggaag ttatgggtaa gaagatgaag 120 gactggttga gattcgctat ggcttggtgg cacaccttgt gtgctgaagg tgctgaccaa 180 ttcggtggtg gtaccaagtc tttcccatgg aacgaaggta ccgacgctat cgaaatcgct 240 aagcaaaagg ttgacgctgg tttcgaaatc atgcaaaagt tgggtatccc atactactgt 300 ttccacgacg tcgacttggt ttctgaaggt aactctatcg aagaatacga atctaacttg 360 aaggctgttg ttgcttactt gaaggaaaag caaaaggaaa ctggtatcaa gttgttgtgg 420 tctaccgcta acgttttcgg tcacaagaga tacatgaacg gtgcttctac caacccagac 480 ttcgacgttg ttgctagagc tatcgttcaa atcaagaacg ctatcgacgc tggtatcgaa 540 ttgggtgctg aaaactacgt tttctggggt ggtagagaag gttacatgtc tttgttgaac 600 accgaccaaa agagagaaaa ggaacacatg gctaccatgt tgaccatggc tagagactac 660 gctagatcta agggtttcaa gggtaccttc ttgatcgaac caaagccaat ggaaccaacc 720 aagcaccaat acgacgttga caccgaaacc gctatcggtt tcttgaaggc tcacaacttg 780 gacaaggact tcaaggtcaa catcgaagtt aaccacgcta ccttggctgg tcacaccttc 840 gaacacgaat tggcttgtgc tgttgacgct ggtatgttgg gttctatcga cgctaacaga 900 ggtgactacc aaaacggttg ggacaccgac caattcccaa tcgaccaata cgaattggtt 960 caagcctgga tggaaatcat cagaggtggt ggtttcgtta ctggtggtac caacttcgac 1020 gctaagacca gaagaaactc taccgacttg gaagacatca tcatcgctca cgtttctggt 1080 atggacgcta tggctagagc tttggaaaac gctgctaagt tgttgcaaga atctccatac 1140 accaagatga agaaggaaag atacgcttct ttcgactctg gtatcggtaa ggacttcgaa 1200 gacggtaagt tgaccttgga acaagtttac gaatacggta agaagaacgg tgaaccaaag 1260 caaacctctg gtaagcaaga attgtacgaa gctatcgttg ctatgtacca ataa 1314 <210> 7 <211> 1317 <212> DNA <213> Artificial Sequence <220> <223> xylA from Clostridium phytofermentans <400> 7 atgaaaaatt actttccaaa tgttccagaa gtaaaatacg aaggcccaaa ttcaacgaat 60 ccatttgctt ttaaatatta tgacgcaaat aaagttgtag cgggtaaaac aatgaaagag 120 cactgtcgtt ttgcattatc ttggtggcat actctttgtg caggtggtgc tgatccattc 180 ggtgtaacaa ctatggatag aacctacgga aatatcacag atccaatgga acttgctaag 240 gcaaaagttg acgctggttt cgaattaatg actaaattag gaattgaatt cttctgtttc 300 catgacgcag atattgctcc agaaggtgat acttttgaag agtcaaagaa gaatcttttt 360 gaaatcgttg attacatcaa agagaagatg gatcagactg gtatcaagtt attatggggt 420 actgctaata actttagtca tccaagattt atgcatggtg cttccacatc ttgcaacgca 480 gacgtatttg catatgctgc tgctaagatt aagaatgcat tagatgcaac aattaaatta 540 ggcggtaaag gttatgtatt ctggggtggt cgtgaaggtt atgaaacact tcttaataca 600 gatttaggac ttgagcttga taatatggct agacttatga agatggctgt agagtatggc 660 cgtgcaaatg gttttgatgg cgacttctat attgagccaa agccaaagga accaaccaag 720 catcaatatg attttgatac agcaaccgta cttgctttcc ttcgcaaata tggcttagaa 780 aaagatttca agatgaacat tgaagcaaac catgctactc ttgcaggtca tacctttgaa 840 catgaacttg caatggctag agttaatggt gcatttggtt ctgtagatgc aaaccagggt 900 gatccaaacc ttggatggga tacggatcaa ttcccaactg atgttcatag tgcaactctt 960 gcaatgcttg aagtacttaa ggctggtgga ttcactaacg gcggacttaa ctttgatgca 1020 aaggtaagac gtggttcctt cgaatttgat gatattgcat acggttatat tgcaggaatg 1080 gatacttttg cacttggttt aattaaggct gctgagatta tcgacgatgg tagaatcgca 1140 aaatttgtag atgatcgtta tgcaagctat aaaacaggaa ttggtaaagc aattgtggat 1200 ggaactacat ctcttgaaga attagagcag tatgttttaa cacatagtga accagtaatg 1260 cagagtggtc gtcaggaagt tcttgaaaca atcgtaaata atattttatt tagataa 1317 <210> 8 <211> 1320 <212> DNA <213> Artificial Sequence <220> <223> xylA from Clostridium thermosulfurgenes <400> 8 atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaacaat 60 ccttattctt ttaaatttta caatcctgag gaagtaatcg atggtaagac gatggaggag 120 catcttcgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180 ggcaaagcta ccatgcaaag gccatggaat cactatacag atcctatgga catagctaaa 240 gcaagggtag aggcagcatt tgagtttttt gataagataa atgcaccgta tttctgcttc 300 catgatagag atattgcccc tgaaggagac actcttagag agacgaacaa aaatttagat 360 acaatagttg ctatgataaa ggattacttg aagaccagca agacgaaagt tttgtggggt 420 actgcgaatc ttttctccaa tccaagattt gtgcatggtg catcaacgtc ttgcaatgcc 480 gatgttttcg catattctgc agcgcaagtc aaaaaagcac ttgagattac taaggagctt 540 ggtggcgaaa actacgtatt ctggggtgga agagaaggat atgagacact tctcaataca 600 gatatggagt ttgagcttga taattttgca agatttttgc acatggctgt tgattatgca 660 aaggaaatcg gctttgaagg ccagttcttg attgagccga agccaaagga gcctacaaag 720 catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgatcttgac 780 aaatatttca aagttaatat cgaagcaaat catgcaacat tagcattcca tgatttccag 840 catgagctaa gatacgccag aataaacggt gtattaggat cgattgacgc aaatacgggt 900 gatatgctat taggatggga tacagatcag ttccctacag atatacgcat gacaacactt 960 gctatgtatg aagttataaa gatgggtgga tttgacaaag gcggactcaa cttcgatgcg 1020 aaagtaagac gtgcttcatt tgagccagaa gatcttttct tgggtcacat agcaggaatg 1080 gatgcttttg caaaaggctt caaagtggct tacaagcttg taaaagatag ggtatttgac 1140 aagttcatcg aagaaagata tgcaagctac aaagatggca taggtgcaga tattgtaagt 1200 gggaaagctg attttagaag tcttgaaaag tatgcattag agcgcagcca gattgtcaac 1260 aaatcaggaa gacaagagct attagagtca atcctaaatc agtatttgtt tgcagaataa 1320                                                                         1320 <210> 9 <211> 1803 <212> DNA <213> Artificial Sequence <220> <223> XKS1 <400> 9 atgttgtgtt cagtaattca gagacagaca agagaggttt ccaacacaat gtctttagac 60 tcatactatc ttgggtttga tctttcgacc caacaactga aatgtctcgc cattaaccag 120 gacctaaaaa ttgtccattc agaaacagtg gaatttgaaa aggatcttcc gcattatcac 180 acaaagaagg gtgtctatat acacggcgac actatcgaat gtcccgtagc catgtggtta 240 gaggctctag atctggttct ctcgaaatat cgcgaggcta aatttccatt gaacaaagtt 300 atggccgtct cagggtcctg ccagcagcac gggtctgtct actggtcctc ccaagccgaa 360 tctctgttag agcaattgaa taagaaaccg gaaaaagatt tattgcacta cgtgagctct 420 gtagcatttg caaggcaaac cgcccccaat tggcaagacc acagtactgc aaagcaatgt 480 caagagtttg aagagtgcat aggtgggcct gaaaaaatgg ctcaattaac agggtccaga 540 gcccatttta gatttactgg tcctcaaatt ctgaaaattg cacaattaga accagaagct 600 tacgaaaaaa caaagaccat ttctttagtg tctaattttt tgacttctat cttagtgggc 660 catcttgttg aattagagga ggcagatgcc tgtggtatga acctttatga tatacgtgaa 720 agaaaattca gtgatgagct actacatcta attgatagtt cttctaagga taaaactatc 780 agacaaaaat taatgagagc acccatgaaa aatttgatag cgggtaccat ctgtaaatat 840 tttattgaga agtacggttt caatacaaac tgcaaggtct ctcccatgac tggggataat 900 ttagccacta tatgttcttt acccctgcgg aagaatgacg ttctcgtttc cctaggaaca 960 agtactacag ttcttctggt caccgataag tatcacccct ctccgaacta tcatcttttc 1020 attcatccaa ctctgccaaa ccattatatg ggtatgattt gttattgtaa tggttctttg 1080 gcaagggaga ggataagaga cgagttaaac aaagaacggg aaaataatta tgagaagact 1140 aacgattgga ctctttttaa tcaagctgtg ctagatgact cagaaagtag tgaaaatgaa 1200 ttaggtgtat attttcctct gggggagatc gttcctagcg taaaagccat aaacaaaagg 1260 gttatcttca atccaaaaac gggtatgatt gaaagagagg tggccaagtt caaagacaag 1320 aggcacgatg ccaaaaatat tgtagaatca caggctttaa gttgcagggt aagaatatct 1380 cccctgcttt cggattcaaa cgcaagctca caacagagac tgaacgaaga tacaatcgtg 1440 aagtttgatt acgatgaatc tccgctgcgg gactacctaa ataaaaggcc agaaaggact 1500 ttttttgtag gtggggcttc taaaaacgat gctattgtga agaagtttgc tcaagtcatt 1560 ggtgctacaa agggtaattt taggctagaa acaccaaact catgtgccct tggtggttgt 1620 tataaggcca tgtggtcatt gttatatgac tctaataaaa ttgcagttcc ttttgataaa 1680 tttctgaatg acaattttcc atggcatgta atggaaagca tatccgatgt ggataatgaa 1740 aattgggatc gctataattc caagattgtc cccttaagcg aactggaaaa gactctcatc 1800 taa 1803 <210> 10 <211> 1871 <212> DNA <213> Artificial Sequence <220> <223> xyl3 from Scheffersomyces stipitis <400> 10 atgaccacta ccccatttga tgctccagat aagctcttcc tcgggttcga tctttcgact 60 cagcagttga agatcatcgt caccgatgaa aacctcgctg ctctcaaaac ctacaatgtc 120 gagttcgata gcatcaacag ctctgtccag aagggtgtca ttgctatcaa cgacgaaatc 180 agcaagggtg ccattatttc ccccgtttac atgtggttgg atgcccttga ccatgttttt 240 gaagacatga agaaggacgg attccccttc aacaaggttg ttggtatttc cggttcttgt 300 caacagcacg gttcggtata ctggtctaga acggccgaga aggtcttgtc cgaattggac 360 gctgaatctt cgttatcgag ccagatgaga tctgctttca ccttcaagca cgctccaaac 420 tggcaggatc actctaccgg taaagagctt gaagagttcg aaagagtgat tggtgctgat 480 gccttggctg atatctctgg ttccagagcc cattacagat tcacagggct ccagattaga 540 aagttgtcta ccagattcaa gcccgaaaag tacaacagaa ctgctcgtat ctctttagtt 600 tcgtcatttg ttgccagtgt gttgcttggt agaatcacct ccattgaaga agccgatgct 660 tgtggaatga acttgtacga tatcgaaaag cgcgagttca acgaagagct cttggccatc 720 gctgctggtg tccaccctga gttggatggt gtagaacaag acggtgaaat ttacagagct 780 ggtatcaatg agttgaagag aaagttgggt cctgtcaaac ctataacata cgaaagcgaa 840 ggtgacattg cctcttactt tgtcaccaga tacggcttca accccgactg taaaatctac 900 tcgttcaccg gagacaattt ggccacgatt atctcgttgc ctttggctcc aaatgatgct 960 ttgatctcat tgggtacttc tactacagtt ttaattatca ccaagaacta cgctccttct 1020 tctcaatacc atttgtttaa acatccaacc atgcctgacc actacatggg catgatctgc 1080 tactgtaacg gttccttggc cagagaaaag gttagagacg aagtcaacga aaagttcaat 1140 gtagaagaca agaagtcgtg ggacaagttc aatgaaatct tggacaaatc cacagacttc 1200 aacaacaagt tgggtattta cttcccactt ggcgaaattg tccctaatgc cgctgctcag 1260 atcaagagat cggtgttgaa cagcaagaac gaaattgtag acgttgagtt gggcgacaag 1320 aactggcaac ctgaagatga tgtttcttca attgtagaat cacagacttt gtcttgtaga 1380 ttgagaactg gtccaatgtt gagcaagagt ggagattctt ctgcttccag ctctgcctca 1440 cctcaaccag aaggtgatgg tacagatttg cacaaggtct accaagactt ggttaaaaag 1500 tttggtgact tgttcactga tggaaagaag caaacctttg agtctttgac cgccagacct 1560 aaccgttgtt actacgtcgg tggtgcttcc aacaacggca gcattatccc aagatgggtt 1620 ccatcttggc tcccgtcaac ggaaactaca aggttgacat tcctaacgcc tgtgcattgg 1680 gtggtgctta caaggccagt tggagttacg agtgtgaagc caagaaggaa tggatcggat 1740 acgatcagta tatcaacaga ttgtttgaag taagtgacga gatgaatctg ttcgaagtca 1800 aggataaatg gctcgaatat gccaacgggg ttggaatgtt ggccaagatg gaaagtgaat 1860 tgaaacacta a 1871 <210> 11 <211> 1008 <212> DNA <213> Artificial Sequence <220> <223> TAL1 <400> 11 atgtctgaac cagctcaaaa gaaacaaaag gttgctaaca actctctaga acaattgaaa 60 gcctccggca ctgtcgttgt tgccgacact ggtgatttcg gctctattgc caagtttcaa 120 cctcaagact ccacaactaa cccatcattg atcttggctg ctgccaagca accaacttac 180 gccaagttga tcgatgttgc cgtggaatac ggtaagaagc atggtaagac caccgaagaa 240 caagtcgaaa atgctgtgga cagattgtta gtcgaattcg gtaaggagat cttaaagatt 300 gttccaggca gagtctccac cgaagttgat gctagattgt cttttgacac tcaagctacc 360 attgaaaagg ctagacatat cattaaattg tttgaacaag aaggtgtctc caaggaaaga 420 gtccttatta aaattgcttc cacttgggaa ggtattcaag ctgccaaaga attggaagaa 480 aaggacggta tccactgtaa tttgactcta ttattctcct tcgttcaagc agttgcctgt 540 gccgaggccc aagttacttt gatttcccca tttgttggta gaattctaga ctggtacaaa 600 tccagcactg gtaaagatta caagggtgaa gccgacccag gtgttatttc cgtcaagaaa 660 atctacaact actacaagaa gtacggttac aagactattg ttatgggtgc ttctttcaga 720 agcactgacg aaatcaaaaa cttggctggt gttgactatc taacaatttc tccagcttta 780 ttggacaagt tgatgaacag tactgaacct ttcccaagag ttttggaccc tgtctccgct 840 aagaaggaag ccggcgacaa gatttcttac atcagcgacg aatctaaatt cagattcgac 900 ttgaatgaag acgctatggc cactgaaaaa ttgtccgaag gtatcagaaa attctctgcc 960 gatattgtta ctctattcga cttgattgaa aagaaagtta ccgcttaa 1008 <210> 12 <211> 970 <212> DNA <213> Artificial Sequence <220> <223> tal1 from Scheffersomyces stipitis <400> 12 atgtcctcca actcccttga acaattgaaa gccacaggta ccgtcatcgt caccgacacc 60 ggtgaattcg actcgattgc caagtacact ccacaagatg ccaccaccaa cccatcgttg 120 attttggctg ctgctaagaa gcctgaatac gccaaggtca ttgacgtcgc catgaatacg 180 ccaaggacaa gggttcctcc aagaaggaaa aggctgaaat cgccttggac cgtttgttga 240 ttgaattcgg taagaacatc ttggccattg ttccaggaag agtgtctacc gaagtcgacg 300 ccagattgtc tttcgacaaa gaggccacat caagaaggct cttgaattga ttgccttgta 360 cgaatcccaa ggtatctcca aggacagaat cttgatcaag atcgcctcca cttgggaagg 420 tatccaagct gccagagaat tggaagccaa gcacggtatt cactgtaact tgactttgtt 480 gttctctttc gttcaggcag ttgcctgtgc tgaagccaag gtcaccttga tctcgccatt 540 cgtcggcaga atcttggact ggtacaaggc ttctaccgga aagacctacg aaggtgacga 600 agacccaggt gtgatttctg tcagagccat ctacaactac tacaagaagt acggctacaa 660 aactattgtc atgggtgcct ctttcagaaa caccggtgaa atcaaggctt tggctggttg 720 cgactactta actgttgctc ctaagttgtt ggaagaattg ttgaactcca ctgaaccagt 780 tccacaagtg ttggacgctg cttctgcctc tgctactgat gtcgaaaagg tttcttacgt 840 tgatgacgaa gctaccttca gatacttgtt caacgaagac gccatggcta ccgaaaagtt 900 ggcccaaggt atcagagctt tcggcaagga cgctgtcacc ttgttggaac aattggaagc 960 cagattctaa 970 <210> 13 <211> 984 <212> DNA <213> Artificial Sequence <220> <223> GRE3 <400> 13 atgtcttcac tggttactct taataacggt ctgaaaatgc ccctagtcgg cttagggtgc 60 tggaaaattg acaaaaaagt ctgtgcgaat caaatttatg aagctatcaa attaggctac 120 cgtttattcg atggtgcttg cgactacggc aacgaaaagg aagttggtga aggtatcagg 180 aaagccatct ccgaaggtct tgtttctaga aaggatatat ttgttgtttc aaagttatgg 240 aacaattttc accatcctga tcatgtaaaa ttagctttaa agaagacctt aagcgatatg 300 ggacttgatt atttagacct gtattatatt cacttcccaa tcgccttcaa atatgttcca 360 tttgaagaga aataccctcc aggattctat acgggcgcag atgacgagaa gaaaggtcac 420 atcaccgaag cacatgtacc aatcatagat acgtaccggg ctctggaaga atgtgttgat 480 gaaggcttga ttaagtctat tggtgtttcc aactttcagg gaagcttgat tcaagattta 540 ttacgtggtt gtagaatcaa gcccgtggct ttgcaaattg aacaccatcc ttatttgact 600 caagaacacc tagttgagtt ttgtaaatta cacgatatcc aagtagttgc ttactcctcc 660 ttcggtcctc aatcattcat tgagatggac ttacagttgg caaaaaccac gccaactctg 720 ttcgagaatg atgtaatcaa gaaggtctca caaaaccatc caggcagtac cacttcccaa 780 gtattgctta gatgggcaac tcagagaggc attgccgtca ttccaaaatc ttccaagaag 840 gaaaggttac ttggcaacct agaaatcgaa aaaaagttca ctttaacgga gcaagaattg 900 aaggatattt ctgcactaaa tgccaacatc agatttaatg atccatggac ctggttggat 960 ggtaaattcc ccacttttgc ctga 984 <210> 14 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> PHO13 <400> 14 atgactgctc aacaaggtgt accaataaag ataaccaata aggagattgc tcaagaattc 60 ttggacaaat atgacacgtt tctgttcgat tgtgatggtg tattatggtt aggttctcaa 120 gcattaccat acaccctgga aattctaaac cttttgaagc aattgggcaa acaactgatc 180 ttcgttacga ataactctac caagtcccgt ttagcataca cgaaaaagtt tgcttcgttt 240 ggtattgatg tcaaagaaga acagattttc acctctggtt atgcgtcagc tgtttatatt 300 cgtgactttc tgaaattgca gcctggcaaa gataaggtat gggtatttgg agaaagcggt 360 attggtgaag aattgaaact aatggggtac gaatctctag gaggtgccga ttccagattg 420 gatacgccgt tcgatgcagc taaatcacca tttttggtga acggccttga taaggatgtt 480 agttgtgtta ttgctgggtt agacacgaag gtaaattacc accgtttggc tgttacactg 540 cagtatttgc agaaggattc tgttcacttt gttggtacaa atgttgattc tactttcccg 600 caaaagggtt atacatttcc cggtgcaggc tccatgattg aatcattggc attctcatct 660 aataggaggc catcgtactg tggtaagcca aatcaaaata tgctaaacag cattatatcg 720 gcattcaacc tggatagatc aaagtgctgt atggttggtg acagattaaa caccgatatg 780 aaattcggtg ttgaaggtgg gttaggtggc acactactcg ttttgagtgg tattgaaacc 840 gaagagagag ccttgaagat ttcgcacgat tatccaagac ctaaatttta cattgataaa 900 cttggtgaca tctacacctt aaccaataat gagttatag 939 <210> 15 <211> 9435 <212> DNA <213> Artificial Sequence <220> <223> TEFp- PMR1 mt- CYC1t <400> 15 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata 120 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt 180 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa 240 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata 300 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct 360 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata 420 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact 480 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa 540 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg 600 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 660 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 720 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 780 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 840 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 900 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 960 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1020 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1080 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1140 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1200 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1260 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1320 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1380 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1440 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1500 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1560 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 1620 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1680 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1740 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1800 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1860 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1920 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1980 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2040 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2100 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2160 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2220 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2280 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2340 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 2400 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 2460 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 2520 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 2580 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact 2640 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg 2700 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 2760 aaccctcact aaagggaaca aaagctggag ctcatagctt caaaatgttt ctactccttt 2820 tttactcttc cagatttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc 2880 acagcatact aaatttccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag 2940 gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa 3000 aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg 3060 acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3120 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3180 taatctaagt tttctagaac tagtatgagt gacaatccat ttaatgcgag tcttcttgac 3240 gaggactcaa accgtgagag agaaatacta gatgccacag cagaggccct ttcgaaacca 3300 agcccttctt tagagtattg tactttatcc gtggacgaag ctctagaaaa actggacact 3360 gacaaaaacg gtggtttacg atcatctaac gaggccaaca ataggagatc actttatggc 3420 cccaatgaaa taaccgtaga agatgatgaa agtcttttca agaagttctt gtcaaatttc 3480 attgaggatc gaatgattct acttttaata ggatccgcag tggtctctct ttttatgggt 3540 aacattgatg atgctgttag tatcacactg gccattttca tagttgtcac tgtcggtttt 3600 gtccaagaat ataggtctga aaaatctcta gaagcgttga ataaattggt tcctgctgaa 3660 tgtcacttaa tgagatgtgg tcaagagagt catgtactgg cttccacctt ggttcctggt 3720 gatttagtgc acttcagaat aggtgacaga atccccgcag acattagaat tattgaagca 3780 atcgatttat ccatcgatga aagtaattta actggtgaaa atgaaccggt acataaaacc 3840 tcacaaacga tcgaaaaatc ttcctttaac gatcagccta attcaattgt accgatttct 3900 gagagatctt gtatagctta tatgggtaca ttagtcaagg aaggtcatgg taagggtatc 3960 gtcgtaggaa caggtacaaa cacatccttt ggtgccgttt ttgaaatgat gaataatatt 4020 gaaaaaccga agactccatt gcagttaaca atggacaaat tgggaaagga cttgtcactg 4080 gttagcttca tagttattgg tatgatttgt ttagttggta tcatacaagg tagatcttgg 4140 ttagaaatgt tccaaatatc ggtatcctta gcggttgctg ctattccaga agggttacca 4200 attattgtca ctgttacttt ggcattgggt gttctgagaa tggccaagcg taaagccatc 4260 gtgagaaggt taccaagtgt cgaaacttta ggctctgtca acgttatctg ctccgacaaa 4320 acaggtacac taacctcaaa ccacatgacc gtatctaaac tttggtgctt ggacagtatg 4380 tccaataagc taaacgtcct ctcattagac aaaaataaga agactaaaaa ttctaatgga 4440 aatttgaaaa actatttgac tgaagacgtt agggaaactc taactatcgg taatctctgt 4500 aataatgcat ctttctctca agaacatgcc atatttctgg gaaatcctac tgatgtagct 4560 cttttagagc aattggcaaa ctttgaaatg cctgatatca gaaacaccgt tcaaaaagtt 4620 caggaacttc catttaactc gaaaagaaaa ttaatggcaa ccaagattct caaccctgtc 4680 gacaataagt gtacagttta tgttaaaggt gcatttgaaa gaattcttga gtactccaca 4740 agttatttga aatcaaaggg taaaaaaact gaaaagttga ctgaagccca aaaagctacg 4800 ataaatgagt gcgcaaattc tatggcatct gaaggtttgc gtgtctttgg atttgctaaa 4860 ctaactttgt ctgattcatc aactcctcta accgaagacc taatcaaaga tttaaccttt 4920 actggtttaa tcggtatgaa tgacccacca agaccgaacg ttaaatttgc catcgaacaa 4980 ttactacaag gtggtgtcca tattattatg atcactggtg attctgagaa taccgcagta 5040 aacattgcaa aacaaattgg tattccagtt attgatccaa agctttccgt tttatccggt 5100 gataaattag atgaaatgtc agatgatcaa ctggccaatg tcatcgacca cgttaatatt 5160 tttgctcgtg ctacgcctga gcataagtta aacattgttc gtgcattaag aaagaggggt 5220 gatgtggtag caatgactgg tgatgatgtt aacgacgctc ctgcgttgaa actttcagat 5280 attggtgttt ctatgggtag aattggtaca gatgtagcca aagaagcctc agatatggtc 5340 ttaactgatg atgacttcag tactatttta actgccattg aagagggtaa aggtatcttt 5400 aataatattc agaatttcct gacttttcaa ttgtctactt ctgttgccgc actatcatta 5460 gttgcactat ctacagcgtt taaactaccc aatccactga acgcaatgca aattctttgg 5520 ataaatattt taatggatgg gccaccagct caatccttag gtgtggaacc tgttgatcat 5580 gaagttatga aaaaacctcc aagaaaacgt accgataaaa ttttgaccca tgatgtaatg 5640 aaacgtttac taaccaccgc ggcctgtatc atcgttggga cagtttacat ttttgttaaa 5700 gagatggccg aagatggtaa agtaactgct agagatacta ctatgacatt tacttgtttt 5760 gttttttttg atatgtttaa tgctttggcc tgcagacata acacaaagtc aatcttcgaa 5820 atcggctttt tcacgaacaa aatgttcaac tacgccgttg gactgtctct gttaggtcaa 5880 atgtgcgcta tatatatacc atttttccaa agtatcttta aaactgagaa acttggtatc 5940 tctgatatac tattgttatt gctcatcagc agtagcgttt tcatcgttga tgaattgaga 6000 aaattgtgga cgaggaaaaa gaatgaagaa gactcaacgt atttctcaaa tgtttgactc 6060 gagattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa 6120 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt 6180 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc 6240 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat 6300 ttgggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt 6360 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 6420 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 6480 gcgcagcctg aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg 6540 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 6600 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 6660 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 6720 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 6780 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 6840 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 6900 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 6960 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatcgacg 7020 gtcgaggaga acttctagta tatccacata cctaatatta ttgccttatt aaaaatggaa 7080 tcccaacaat tacatcaaaa tccacattct cttcaaaatc aattgtcctg tacttccttg 7140 ttcatgtgtg ttcaaaaacg ttatatttat aggataatta tactctattt ctcaacaagt 7200 aattggttgt ttggccgagc ggtctaaggc gcctgattca agaaatatct tgaccgcagt 7260 taactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt 7320 atttttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa tcaaattcga 7380 tgactggaaa ttttttgtta atttcagagg tcgcctgacg catatacctt tttcaactga 7440 aaaattggga gaaaaaggaa aggtgagagg ccggaaccgg cttttcatat agaatagaga 7500 agcgttcatg actaaatgct tgcatcacaa tacttgaagt tgacaatatt atttaaggac 7560 ctattgtttt ttccaatagg tggttagcaa tcgtcttact ttctaacttt tcttaccttt 7620 tacatttcag caatatatat atatatttca aggatatacc attctaatgt ctgcccctat 7680 gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag aaatcacagc 7740 cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca agttcgattt 7800 cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgtcccac ttccagatga 7860 ggcgctggaa gcctccaaga aggttgatgc cgttttgtta ggtgctgtgg gtggtcctaa 7920 atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta aagaacttca 7980 attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag acttatctcc 8040 aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat tagtgggagg 8100 tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg atagtgaaca 8160 atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg ccctacaaca 8220 tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct cttcaagatt 8280 atggagaaaa actgtggagg aaaccatcaa gaacgaattc cctacattga aggttcaaca 8340 tcaattgatt gattctgccg ccatgatcct agttaagaac ccaacccacc taaatggtat 8400 tataatcacc agcaacatgt ttggtgatat catctccgat gaagcctccg ttatcccagg 8460 ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga acaccgcatt 8520 tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata aggttgaccc 8580 tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact tgcctgaaga 8640 aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca gaactggtga 8700 tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag aagttaagaa 8760 aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact ttataaatga 8820 aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg gtagacgaaa 8880 ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggata gtaaaggaat 8940 acaggtaagc aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa 9000 cattaatatt gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg 9060 aaactacgat tcctaatttg atattggagg attttctcta aaaaaaaaaa aatacaacaa 9120 ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc ttcttgaacc 9180 atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac ggccttacga 9240 cgtagtcgat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 9300 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 9360 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 9420 caccgaaacg cgcga 9435 <210> 16 <211> 7815 <212> DNA <213> Artificial Sequence <220> <223> TEFp- ASC1 mt- CYC1t (w. SpeI, XhoI) <400> 16 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata 120 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt 180 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa 240 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata 300 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct 360 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata 420 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact 480 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa 540 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg 600 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 660 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 720 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 780 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 840 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 900 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 960 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1020 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1080 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1140 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1200 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1260 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1320 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1380 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1440 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1500 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1560 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 1620 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1680 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1740 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1800 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1860 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1920 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1980 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2040 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2100 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2160 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2220 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2280 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2340 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 2400 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 2460 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 2520 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 2580 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact 2640 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg 2700 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 2760 aaccctcact aaagggaaca aaagctggag ctcatagctt caaaatgttt ctactccttt 2820 tttactcttc cagatttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc 2880 acagcatact aaatttccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag 2940 gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa 3000 aatttttatc acgtttcttt ttcttgaaaa tttttttttt gatttttttc tctttcgatg 3060 acctcccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3120 tttcttgttc tattacaact ttttttactt cttgctcatt agaaagaaag catagcaatc 3180 taatctaagt tttctagaac tagtatggca tctaacgaag ttttagtttt gagaggtacc 3240 ttggaaggtc acaacggttg ggtcacatct ttggctactt ctgctggtca accaaaccta 3300 ttgttgtccg cttcccgtga taagactttg atctcctgga agttgactgg tgacgaccaa 3360 aagtttggtg tcccagttag atctttcaag ggtcacagtc acattgtcca agactgtact 3420 ttgactgctg acggtgctta cgctttgtct gcttcttggg acaagacctt gagattatgg 3480 gatgttgcca ccggtgaaac ctaccaaaga ttcgtcggtc acaagtccga tgttatgtcc 3540 gttgacattg acaagaaggc ttccatgatt atctctggtt cccgtgacaa gaccatcaag 3600 gtctggacca tcaaaggtca atgtttggcc actttgttgg gtcacaatga ctgggtttcc 3660 caagtcagag ttgttccaaa cgaaaaagct gatgatgact ctgtcaccat catttctgcc 3720 ggtaacgaca aaatggttaa ggtatgtgat atattttctt tccatgatag aatatgatga 3780 caatcgagta gaagaagaaa agtggatttg tgtatgccat tcaaatgatg taataacata 3840 tttgctactt cagatggaac tttgagttcc gaatgagaca taccaattat caccaagatc 3900 tctgatgaat ggtttagcat tactctgctc ttctctttac tcgttatgtc aaaatggaaa 3960 cttttttttt aaattaattt tgttccctta ctaacaaaaa tgatataatg acaggcttgg 4020 aacttaaacc aattccaaat tgaagctgac ttcatcggtc acaactccaa catcaacact 4080 ttgactgctt ccccagacgg aactttgatt gcttccgctg gtaaggacgg tgaaattatg 4140 ttgtggaact tggctgctaa gaaggctatg tacactttgt ctgcctaaga tgaagttttc 4200 tctttggctt tctctccaaa cagatactgg ttggctgctg ccactgctac cggtattaag 4260 gtcttttctt tggacccaca atacttggtc gatgacttga gaccagaatt tgctggttac 4320 agcaaggccg ctgaaccaca tgctgtttct ttggcttggt ctgctgacgg tcaaactttg 4380 tttgccggtt acaccgacaa cgtcattaga gtttggcaag ttatgactgc taactaactc 4440 gagattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa 4500 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt 4560 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc 4620 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat 4680 ttgggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt 4740 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 4800 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 4860 gcgcagcctg aatggcgaat ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg 4920 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4980 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5040 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5100 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5160 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5220 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 5280 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 5340 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatcgacg 5400 gtcgaggaga acttctagta tatccacata cctaatatta ttgccttatt aaaaatggaa 5460 tcccaacaat tacatcaaaa tccacattct cttcaaaatc aattgtcctg tacttccttg 5520 ttcatgtgtg ttcaaaaacg ttatatttat aggataatta tactctattt ctcaacaagt 5580 aattggttgt ttggccgagc ggtctaaggc gcctgattca agaaatatct tgaccgcagt 5640 taactgtggg aatactcagg tatcgtaaga tgcaagagtt cgaatctctt agcaaccatt 5700 atttttttcc tcaacataac gagaacacac aggggcgcta tcgcacagaa tcaaattcga 5760 tgactggaaa ttttttgtta atttcagagg tcgcctgacg catatacctt tttcaactga 5820 aaaattggga gaaaaaggaa aggtgagagg ccggaaccgg cttttcatat agaatagaga 5880 agcgttcatg actaaatgct tgcatcacaa tacttgaagt tgacaatatt atttaaggac 5940 ctattgtttt ttccaatagg tggttagcaa tcgtcttact ttctaacttt tcttaccttt 6000 tacatttcag caatatatat atatatttca aggatatacc attctaatgt ctgcccctat 6060 gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag aaatcacagc 6120 cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca agttcgattt 6180 cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgtcccac ttccagatga 6240 ggcgctggaa gcctccaaga aggttgatgc cgttttgtta ggtgctgtgg gtggtcctaa 6300 atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta aagaacttca 6360 attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag acttatctcc 6420 aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat tagtgggagg 6480 tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg atagtgaaca 6540 atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg ccctacaaca 6600 tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct cttcaagatt 6660 atggagaaaa actgtggagg aaaccatcaa gaacgaattc cctacattga aggttcaaca 6720 tcaattgatt gattctgccg ccatgatcct agttaagaac ccaacccacc taaatggtat 6780 tataatcacc agcaacatgt ttggtgatat catctccgat gaagcctccg ttatcccagg 6840 ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga acaccgcatt 6900 tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata aggttgaccc 6960 tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact tgcctgaaga 7020 aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca gaactggtga 7080 tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag aagttaagaa 7140 aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact ttataaatga 7200 aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg gtagacgaaa 7260 ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggata gtaaaggaat 7320 acaggtaagc aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa 7380 cattaatatt gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg 7440 aaactacgat tcctaatttg atattggagg attttctcta aaaaaaaaaa aatacaacaa 7500 ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc ttcttgaacc 7560 atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac ggccttacga 7620 cgtagtcgat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 7680 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 7740 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 7800 caccgaaacg cgcga 7815 <210> 17 <211> 849 <212> DNA <213> Artificial Sequence <220> <223> Hbd <400> 17 atgaaaaagg tatgtgttat aggtgcaggt actatgggtt caggaattgc tcaggcattt 60 gcagctaaag gatttgaagt agtattaaga gatattaaag atgaatttgt tgatagagga 120 ttagatttta tcaataaaaa tctttctaaa ttagttaaaa aaggaaagat agaagaagct 180 actaaagttg aaatcttaac tagaatttcc ggaacagttg accttaatat ggcagctgat 240 tgcgatttag ttatagaagc agctgttgaa agaatggata ttaaaaagca gatttttgct 300 gacttagaca atatatgcaa gccagaaaca attcttgcat caaatacatc atcactttca 360 ataacagaag tggcatcagc aactaaaaga cctgataagg ttataggtat gcatttcttt 420 aatccagctc ctgttatgaa gcttgtagag gtaataagag gaatagctac atcacaagaa 480 acttttgatg cagttaaaga gacatctata gcaataggaa aagatcctgt agaagtagca 540 gaagcaccag gatttgttgt aaatagaata ttaataccaa tgattaatga agcagttggt 600 atattagcag aaggaatagc ttcagtagaa gacatagata aagctatgaa acttggagct 660 aatcacccaa tgggaccatt agaattaggt gattttatag gtcttgatat atgtcttgct 720 ataatggatg ttttatactc agaaactgga gattctaagt atagaccaca tacattactt 780 aagaagtatg taagagcagg atggcttgga agaaaatcag gaaaaggttt ctacgattat 840 tcaaaataa 849 <210> 18 <211> 786 <212> DNA <213> Artificial Sequence <220> <223> Crt <400> 18 atggaactaa acaatgtcat ccttgaaaag gaaggtaaag ttgctgtagt taccattaac 60 agacctaaag cattaaatgc gttaaatagt gatacactaa aagaaatgga ttatgttata 120 ggtgaaattg aaaatgatag cgaagtactt gcagtaattt taactggagc aggagaaaaa 180 tcatttgtag caggagcaga tatttctgag atgaaggaaa tgaataccat tgaaggtaga 240 aaattcggga tacttggaaa taaagtgttt agaagattag aacttcttga aaagcctgta 300 atagcagctg ttaatggttt tgctttagga ggcggatgcg aaatagctat gtcttgtgat 360 ataagaatag cttcaagcaa cgcaagattt ggtcaaccag aagtaggtct cggaataaca 420 cctggttttg gtggtacaca aagactttca agattagttg gaatgggcat ggcaaagcag 480 cttatattta ctgcacaaaa tataaaggca gatgaagcat taagaatcgg acttgtaaat 540 aaggtagtag aacctagtga attaatgaat acagcaaaag aaattgcaaa caaaattgtg 600 agcaatgctc cagtagctgt taagttaagc aaacaggcta ttaatagagg aatgcagtgt 660 gatattgata ctgctttagc atttgaatca gaagcatttg gagaatgctt ttcaacagag 720 gatcaaaagg atgcaatgac agctttcata gagaaaagaa aaattgaagg cttcaaaaat 780 agatag 786 <210> 19 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> BdhB <400> 19 atggttgatt tcgaatattc aataccaact agaatttttt tcggtaaaga taagataaat 60 gtacttggaa gagagcttaa aaaatatggt tctaaagtgc ttatagttta tggtggagga 120 agtataaaga gaaatggaat atatgataaa gctgtaagta tacttgaaaa aaacagtatt 180 aaattttatg aacttgcagg agtagagcca aatccaagag taactacagt tgaaaaagga 240 gttaaaatat gtagagaaaa tggagttgaa gtagtactag ctataggtgg aggaagtgca 300 atagattgcg caaaggttat agcagcagca tgtgaatatg atggaaatcc atgggatatt 360 gtgttagatg gctcaaaaat aaaaagggtg cttcctatag ctagtatatt aaccattgct 420 gcaacaggat cagaaatgga tacgtgggca gtaataaata atatggatac aaacgaaaaa 480 ctaattgcgg cacatccaga tatggctcct aagttttcta tattagatcc aacgtatacg 540 tataccgtac ctaccaatca aacagcagca ggaacagctg atattatgag tcatatattt 600 gaggtgtatt ttagtaatac aaaaacagca tatttgcagg atagaatggc agaagcgtta 660 ttaagaactt gtattaaata tggaggaata gctcttgaga agccggatga ttatgaggca 720 agagccaatc taatgtgggc ttcaagtctt gcgataaatg gacttttaac atatggtaaa 780 gacactaatt ggagtgtaca cttaatggaa catgaattaa gtgcttatta cgacataaca 840 cacggcgtag ggcttgcaat tttaacacct aattggatgg agtatatttt aaataatgat 900 acagtgtaca agtttgttga atatggtgta aatgtttggg gaatagacaa agaaaaaaat 960 cactatgaca tagcacatca agcaatacaa aaaacaagag attactttgt aaatgtacta 1020 ggtttaccat ctagactgag agatgttgga attgaagaag aaaaattgga cataatggca 1080 aaggaatcag taaagcttac aggaggaacc ataggaaacc taagaccagt aaacgcctcc 1140 gaagtcctac aaatattcaa aaaatctgtg taa 1173 <210> 20 <211> 1197 <212> DNA <213> Artificial Sequence <220> <223> Erg10 <400> 20 atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60 tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120 aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180 tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240 catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300 ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360 atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420 gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480 ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540 tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600 gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660 gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720 aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780 gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt ggctattatc 840 aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900 gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960 ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020 tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080 gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140 gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197 <210> 21 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> Etr1 <400> 21 atgtcgtcct cagctcatca gattcccaag cacttcaaat cgctcatcta ttcaactcat 60 gaagttgagg attgtaccaa ggttttgtca gtgaaaaatt atacgcctaa acaagactta 120 tctcaatcaa ttgtgttaaa aactttggcc tttcccataa acccttcgga tatcaatcag 180 ttgcaaggag tatacccgtc tcgtccagaa aagacatacg attactccac agatgagcca 240 gccgctatcg ccggtaatga gggtgtcttt gaagttgttt ctttaccttc gggaagttcc 300 aagggagatt tgaaattggg tgaccgagtt atcccattgc aggcaaatca agggacttgg 360 tccaattata gagttttctc tagtagttct gatttaatca aggtaaatga tttggatctg 420 ttttctgcgg caactgtatc tgttaatggt tgtaccggtt tccaattagt atcagactat 480 atcgactgga acagtaacgg taatgaatgg attatccaaa atgccggtac atctagtgta 540 tcaaaaatag ttacgcaagt agcaaaagct aaagggatca aaacattaag tgttatacgt 600 gaccgtgata attttgatga ggtagcaaaa gttttggagg ataagtatgg tgctacgaag 660 gttatttccg aatcgcaaaa caacgacaag acttttgcca aagaagtatt gtccaagatt 720 ttgggtgaaa atgcaagggt gaggcttgcc ttgaattctg ttggaggtaa atccagtgca 780 tcaatagcac gtaagttgga aaataatgct ttgatgctca cttatggagg aatgtcaaaa 840 caacctgtaa ctttaccaac atctctacac attttcaaag gcttgacatc caaagggtac 900 tgggtgactg aaaagaacaa aaaaaacccc caaagcaaga ttgacaccat cagtgatttt 960 atcaaaatgt ataattatgg tcacattatt tcaccaagag atgaaattga aactcttacc 1020 tggaatacta acactactac tgacgaacag ttactagaac tagtcaaaaa aggtataact 1080 gggaagggga agaaaaaaat ggttgtttta gaatggtaa 1119 <210> 22 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> EutE <400> 22 atgaatcaac aggatattga acaggtggtg aaagcggtac tgctgaaaat gcaaagcagt 60 gacacgccgt ccgccgccgt tcatgagatg ggcgttttcg cgtccctgga tgacgccgtt 120 gcggcagcca aagtcgccca gcaagggtta aaaagcgtgg caatgcgcca gttagccatt 180 gctgccattc gtgaagcagg cgaaaaacac gccagagatt tagcggaact tgccgtcagt 240 gaaaccggca tggggcgcgt tgaagataaa tttgcaaaaa acgtcgctca ggcgcgcggc 300 acaccaggcg ttgagtgcct ctctccgcaa gtgctgactg gcgacaacgg cctgacccta 360 attgaaaacg caccctgggg cgtggtggct tcggtgacgc cttccactaa cccggcggca 420 accgtaatta acaacgccat cagcctgatt gccgcgggca acagcgtcat ttttgccccg 480 catccggcgg cgaaaaaagt ctcccagcgg gcgattacgc tgctcaacca ggcgattgtt 540 gccgcaggtg ggccggaaaa cttactggtt actgtggcaa atccggatat cgaaaccgcg 600 caacgcttgt tcaagtttcc gggtatcggc ctgctggtgg taaccggcgg cgaagcggta 660 gtagaagcgg cgcgtaaaca caccaataaa cgtctgattg ccgcaggcgc tggcaacccg 720 ccggtagtgg tggatgaaac cgccgacctc gcccgtgccg ctcagtccat cgtcaaaggc 780 gcttctttcg ataacaacat catttgtgcc gacgaaaagg tactgattgt tgttgatagc 840 gtagccgatg aactgatgcg tctgatggaa ggccagcacg cggtgaaact gaccgcagaa 900 caggcgcagc agctgcaacc ggtgttgctg aaaaatatcg acgagcgcgg aaaaggcacc 960 gtcagccgtg actgggttgg tcgcgacgca ggcaaaatcg cggcggcaat cggccttaaa 1020 gttccgcaag aaacgcgcct gctgtttgtg gaaaccaccg cagaacatcc gtttgccgtg 1080 actgaactga tgatgccggt gttgcccgtc gtgcgcgtcg ccaacgtggc ggatgccatt 1140 gcgctagcgg tgaaactgga aggcggttgc caccacacgg cggcaatgca ctcgcgcaac 1200 atcgaaaaca tgaaccagat ggcgaatgct attgatacca gcattttcgt taagaacgga 1260 ccgtgcattg ccgggctggg gctgggcggg gaaggctgga ccaccatgac catcaccacg 1320 ccaaccggtg aaggggtaac cagcgcgcgt acgtttgtcc gtctgcgtcg ctgtgtatta 1380 gtcgatgcgt ttcgcattgt ttaa 1404 <210> 23 <211> 8657 <212> DNA <213> Artificial Sequence <220> <223> p423-GPDp-CaHbd-PRM9t-TEF1p-CaCrt-CPS1t <400> 23 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 120 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 180 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 240 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 300 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 360 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 420 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 480 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 540 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 600 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 660 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 720 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 780 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 840 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 900 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 960 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1020 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1080 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1140 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1200 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1260 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1320 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1380 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1440 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1500 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1560 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1620 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1680 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1740 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1800 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1860 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1920 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1980 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2040 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2100 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2160 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2220 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2280 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2340 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2400 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2460 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2520 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2580 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2640 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2700 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 2760 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2820 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2880 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2940 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3000 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3060 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3120 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3180 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3240 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3300 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3360 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3420 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc 3480 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa 3540 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3600 agggaacaaa agctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg 3660 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat 3720 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg 3780 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt 3840 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac 3900 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca 3960 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag 4020 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct 4080 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct 4140 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc 4200 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa 4260 cttagtttcg acggattcta gaactagtat gaaaaaggta tgtgttatag gtgcaggtac 4320 tatgggttca ggaattgctc aggcatttgc agctaaagga tttgaagtag tattaagaga 4380 tattaaagat gaatttgttg atagaggatt agattttatc aataaaaatc tttctaaatt 4440 agttaaaaaa ggaaagatag aagaagctac taaagttgaa atcttaacta gaatttccgg 4500 aacagttgac cttaatatgg cagctgattg cgatttagtt atagaagcag ctgttgaaag 4560 aatggatatt aaaaagcaga tttttgctga cttagacaat atatgcaagc cagaaacaat 4620 tcttgcatca aatacatcat cactttcaat aacagaagtg gcatcagcaa ctaaaagacc 4680 tgataaggtt ataggtatgc atttctttaa tccagctcct gttatgaagc ttgtagaggt 4740 aataagagga atagctacat cacaagaaac ttttgatgca gttaaagaga catctatagc 4800 aataggaaaa gatcctgtag aagtagcaga agcaccagga tttgttgtaa atagaatatt 4860 aataccaatg attaatgaag cagttggtat attagcagaa ggaatagctt cagtagaaga 4920 catagataaa gctatgaaac ttggagctaa tcacccaatg ggaccattag aattaggtga 4980 ttttataggt cttgatatat gtcttgctat aatggatgtt ttatactcag aaactggaga 5040 ttctaagtat agaccacata cattacttaa gaagtatgta agagcaggat ggcttggaag 5100 aaaatcagga aaaggtttct acgattattc aaaataagtc gacacagaag acgggagaca 5160 ctagcacaca actttaccag gcaaggtatt tgacgctagc atgtgtccaa ttcagtgtca 5220 tttatgattt tttgtagtag gatataaata tatacagcgc tccaaatagt gcggttgccc 5280 caaaaacacc acggaacctc atctgttctc gtactttgtt gtgacaaagt agctcactgc 5340 cttattatca cattttcatt atgcaacgct tcggaaaata cgatgttgaa aatcatagct 5400 tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg 5460 taccacttca aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt 5520 gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt 5580 tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt 5640 tttgattttt ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca 5700 atttctcaag tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc 5760 attagaaaga aagcatagca atctaatcta agtctagaac tagtatggaa ctaaacaatg 5820 tcatccttga aaaggaaggt aaagttgctg tagttaccat taacagacct aaagcattaa 5880 atgcgttaaa tagtgataca ctaaaagaaa tggattatgt tataggtgaa attgaaaatg 5940 atagcgaagt acttgcagta attttaactg gagcaggaga aaaatcattt gtagcaggag 6000 cagatatttc tgagatgaag gaaatgaata ccattgaagg tagaaaattc gggatacttg 6060 gaaataaagt gtttagaaga ttagaacttc ttgaaaagcc tgtaatagca gctgttaatg 6120 gttttgcttt aggaggcgga tgcgaaatag ctatgtcttg tgatataaga atagcttcaa 6180 gcaacgcaag atttggtcaa ccagaagtag gtctcggaat aacacctggt tttggtggta 6240 cacaaagact ttcaagatta gttggaatgg gcatggcaaa gcagcttata tttactgcac 6300 aaaatataaa ggcagatgaa gcattaagaa tcggacttgt aaataaggta gtagaaccta 6360 gtgaattaat gaatacagca aaagaaattg caaacaaaat tgtgagcaat gctccagtag 6420 ctgttaagtt aagcaaacag gctattaata gaggaatgca gtgtgatatt gatactgctt 6480 tagcatttga atcagaagca tttggagaat gcttttcaac agaggatcaa aaggatgcaa 6540 tgacagcttt catagagaaa agaaaaattg aaggcttcaa aaatagatag gtcgacgcgc 6600 aatgattgaa tagtcaaaga tttttttttt ttaatttttt ttttttaatt tttttttttt 6660 ttcatagaac tttttattta aataaatcac gtctatatat gtatcagtat aacgtaaaaa 6720 aaaaaacacc gtcagttaaa caaaacataa ataaaaaaaa aaagaagtgt caaatcaagt 6780 gtcaaatcgg ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc 6840 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 6900 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 6960 ccaacagttg cgcagcctga atggcgaatg gcgcgacgcg ccctgtagcg gcgcattaag 7020 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 7080 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 7140 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 7200 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 7260 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 7320 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 7380 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 7440 gtttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 7500 atagggtaat aactgatata attaaattga agctctaatt tgtgagttta gtatacatgc 7560 atttacttat aatacagttt tctacataag aacacctttg gtggagggaa catcgttggt 7620 accattgggc gaggtggctt ctcttatggc aaccgcaaga gccttgaacg cactctcact 7680 acggtgatga tcattcttgc ctcgcagaca atcaacgtgg agggtaattc tgctagcctc 7740 tgcaaagctt tcaagaaaat gcgggatcat ctcgcaagag agatctccta ctttctccct 7800 ttgcaaacca agttcgacaa ctgcgtacgg cctgttcgaa agatctacca ccgctctgga 7860 aagtgcctca tccaaaggcg caaatcctga tccaaacctt tttactccac gcgccagtag 7920 ggcctcttta aaagcttgac cgagagcaat cccgcagtct tcagtggtgt gatggtcgtc 7980 tatgtgtaag tcaccaatgc actcaacgat tagcgaccag ccggaatgct tggccagagc 8040 atgtatcata tggtccagaa accctatacc tgtgtggacg ttaatcactt gcgattgtgt 8100 ggcctgttct gctactgctt ctgcctcttt ttctgggaag atcgagtgct ctatcgctag 8160 gggaccaccc tttaaagaga tcgcaatctg aatcttggtt tcatttgtaa tacgctttac 8220 tagggctttc tgctctgtca tgatttatct tcgtttcctg caggtttttg ttctgtgcag 8280 ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac 8340 caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa 8400 aaaatttcaa agaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaattg 8460 aaaagctgtg gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 8520 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 8580 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 8640 atcaccgaaa cgcgcga 8657 <210> 24 <211> 9755 <212> DNA <213> Artificial Sequence <220> <223> p426-PGKp-EcEute-CYC1t-CYC1p-CaBdhb-SPG5t <400> 24 acgcacagat attataacat ctgcataata ggcatttgca agaattactc gtgagtaagg 60 aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120 tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180 cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240 gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300 gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360 caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420 gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480 tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540 tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600 atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660 agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720 ttatctactt tttacaacaa atataaaaca tctagaacta gtatgaatca acaggatatt 780 gaacaggtgg tgaaagcggt actgctgaaa atgcaaagca gtgacacgcc gtccgccgcc 840 gttcatgaga tgggcgtttt cgcgtccctg gatgacgccg ttgcggcagc caaagtcgcc 900 cagcaagggt taaaaagcgt ggcaatgcgc cagttagcca ttgctgccat tcgtgaagca 960 ggcgaaaaac acgccagaga tttagcggaa cttgccgtca gtgaaaccgg catggggcgc 1020 gttgaagata aatttgcaaa aaacgtcgct caggcgcgcg gcacaccagg cgttgagtgc 1080 ctctctccgc aagtgctgac tggcgacaac ggcctgaccc taattgaaaa cgcaccctgg 1140 ggcgtggtgg cttcggtgac gccttccact aacccggcgg caaccgtaat taacaacgcc 1200 atcagcctga ttgccgcggg caacagcgtc atttttgccc cgcatccggc ggcgaaaaaa 1260 gtctcccagc gggcgattac gctgctcaac caggcgattg ttgccgcagg tgggccggaa 1320 aacttactgg ttactgtggc aaatccggat atcgaaaccg cgcaacgctt gttcaagttt 1380 ccgggtatcg gcctgctggt ggtaaccggc ggcgaagcgg tagtagaagc ggcgcgtaaa 1440 cacaccaata aacgtctgat tgccgcaggc gctggcaacc cgccggtagt ggtggatgaa 1500 accgccgacc tcgcccgtgc cgctcagtcc atcgtcaaag gcgcttcttt cgataacaac 1560 atcatttgtg ccgacgaaaa ggtactgatt gttgttgata gcgtagccga tgaactgatg 1620 cgtctgatgg aaggccagca cgcggtgaaa ctgaccgcag aacaggcgca gcagctgcaa 1680 ccggtgttgc tgaaaaatat cgacgagcgc ggaaaaggca ccgtcagccg tgactgggtt 1740 ggtcgcgacg caggcaaaat cgcggcggca atcggcctta aagttccgca agaaacgcgc 1800 ctgctgtttg tggaaaccac cgcagaacat ccgtttgccg tgactgaact gatgatgccg 1860 gtgttgcccg tcgtgcgcgt cgccaacgtg gcggatgcca ttgcgctagc ggtgaaactg 1920 gaaggcggtt gccaccacac ggcggcaatg cactcgcgca acatcgaaaa catgaaccag 1980 atggcgaatg ctattgatac cagcattttc gttaagaacg gaccgtgcat tgccgggctg 2040 gggctgggcg gggaaggctg gaccaccatg accatcacca cgccaaccgg tgaaggggta 2100 accagcgcgc gtacgtttgt ccgtctgcgt cgctgtgtat tagtcgatgc gtttcgcatt 2160 gtttaaaagc ttatcgatac cgtcgacctc gagattagtt atgtcacgct tacattcacg 2220 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt 2280 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt 2340 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga 2400 gaaggttttg ggacgctcga aggctttaat ttgagctcat ttggcgagcg ttggttggtg 2460 gatcaagccc acgcgtaggc aatcctcgag cagatccgcc aggcgtgtat atatagcgtg 2520 gatggccagg caactttagt gctgacacat acaggcatat atatatgtgt gcgacgacac 2580 atgatcatat ggcatgcatg tgctctgtat gtatataaaa ctcttgtttt cttcttttct 2640 ctaaatattc tttccttata cattaggacc tttgcagcat aaattactat acttctatag 2700 acacgcaaac acaaatacac acactaatct agaaatggtt gatttcgaat attcaatacc 2760 aactagaatt tttttcggta aagataagat aaatgtactt ggaagagagc ttaaaaaata 2820 tggttctaaa gtgcttatag tttatggtgg aggaagtata aagagaaatg gaatatatga 2880 taaagctgta agtatacttg aaaaaaacag tattaaattt tatgaacttg caggagtaga 2940 gccaaatcca agagtaacta cagttgaaaa aggagttaaa atatgtagag aaaatggagt 3000 tgaagtagta ctagctatag gtggaggaag tgcaatagat tgcgcaaagg ttatagcagc 3060 agcatgtgaa tatgatggaa atccatggga tattgtgtta gatggctcaa aaataaaaag 3120 ggtgcttcct atagctagta tattaaccat tgctgcaaca ggatcagaaa tggatacgtg 3180 ggcagtaata aataatatgg atacaaacga aaaactaatt gcggcacatc cagatatggc 3240 tcctaagttt tctatattag atccaacgta tacgtatacc gtacctacca atcaaacagc 3300 agcaggaaca gctgatatta tgagtcatat atttgaggtg tattttagta atacaaaaac 3360 agcatatttg caggatagaa tggcagaagc gttattaaga acttgtatta aatatggagg 3420 aatagctctt gagaagccgg atgattatga ggcaagagcc aatctaatgt gggcttcaag 3480 tcttgcgata aatggacttt taacatatgg taaagacact aattggagtg tacacttaat 3540 ggaacatgaa ttaagtgctt attacgacat aacacacggc gtagggcttg caattttaac 3600 acctaattgg atggagtata ttttaaataa tgatacagtg tacaagtttg ttgaatatgg 3660 tgtaaatgtt tggggaatag acaaagaaaa aaatcactat gacatagcac atcaagcaat 3720 acaaaaaaca agagattact ttgtaaatgt actaggttta ccatctagac tgagagatgt 3780 tggaattgaa gaagaaaaat tggacataat ggcaaaggaa tcagtaaagc ttacaggagg 3840 aaccatagga aacctaagac cagtaaacgc ctccgaagtc ctacaaatat tcaaaaaatc 3900 tgtgtaacca aagacgttgt ttcatcgcgc tattaccaag aaggttactt tacttgttct 3960 tgcacatgga cgcacgttgt gtgttcatat atatatatat atatatatat atatatttgt 4020 gcttgttttc attgtctcta tagttaatac attctatttt tatcgttata tttgcattct 4080 cttcgcataa aaacttcatg aaaattcggc agaaaataag ccggccggta cccaattcgc 4140 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg 4200 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 4260 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 4320 aatggcgcga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 4380 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 4440 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 4500 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 4560 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 4620 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 4680 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 4740 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcctga tgcggtattt 4800 tctccttacg catctgtgcg gtatttcaca ccgcataggg taataactga tataattaaa 4860 ttgaagctct aatttgtgag tttagtatac atgcatttac ttataataca gttttttagt 4920 tttgctggcc gcatcttctc aaatatgctt cccagcctgc ttttctgtaa cgttcaccct 4980 ctaccttagc atcccttccc tttgcaaata gtcctcttcc aacaataata atgtcagatc 5040 ctgtagagac cacatcatcc acggttctat actgttgacc caatgcgtct cccttgtcat 5100 ctaaacccac accgggtgtc ataatcaacc aatcgtaacc ttcatctctt ccacccatgt 5160 ctctttgagc aataaagccg ataacaaaat ctttgtcgct cttcgcaatg tcaacagtac 5220 ccttagtata ttctccagta gatagggagc ccttgcatga caattctgct aacatcaaaa 5280 ggcctctagg ttcctttgtt acttcttctg ccgcctgctt caaaccgcta acaatacctg 5340 ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc tgctattctg tatacacccg 5400 cagagtactg caatttgact gtattaccaa tgtcagcaaa ttttctgtct tcgaagagta 5460 aaaaattgta cttggcggat aatgccttta gcggcttaac tgtgccctcc atggaaaaat 5520 cagtcaagat atccacatgt gtttttagta aacaaatttt gggacctaat gcttcaacta 5580 actccagtaa ttccttggtg gtacgaacat ccaatgaagc acacaagttt gtttgctttt 5640 cgtgcatgat attaaatagc ttggcagcaa caggactagg atgagtagca gcacgttcct 5700 tatatgtagc tttcgacatg atttatcttc gtttcctgca ggtttttgtt ctgtgcagtt 5760 gggttaagaa tactgggcaa tttcatgttt cttcaacact acatatgcgt atatatacca 5820 atctaagtct gtgctccttc cttcgttctt ccttctgttc ggagattacc gaatcaaaaa 5880 aatttcaaag aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa ttgaattgaa 5940 aagctgtggt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 6000 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 6060 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 6120 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 6180 tgataataat ggtttcttag tatgatccaa tatcaaagga aatgatagca ttgaaggatg 6240 agactaatcc aattgaggag tggcagcata tagaacagct aaagggtagt gctgaaggaa 6300 gcatacgata ccccgcatgg aatgggataa tatcacagga ggtactagac tacctttcat 6360 cctacataaa tagacgcata taagtacgca tttaagcata aacacgcact atgccgttct 6420 tctcatgtat atatatatac aggcaacacg cagatatagg tgcgacgtga acagtgagct 6480 gtatgtgcgc agctcgcgtt gcattttcgg aagcgctcgt tttcggaaac gctttgaagt 6540 tcctattccg aagttcctat tctctagaaa gtataggaac ttcagagcgc ttttgaaaac 6600 caaaagcgct ctgaagacgc actttcaaaa aaccaaaaac gcaccggact gtaacgagct 6660 actaaaatat tgcgaatacc gcttccacaa acattgctca aaagtatctc tttgctatat 6720 atctctgtgc tatatcccta tataacctac ccatccacct ttcgctcctt gaacttgcat 6780 ctaaactcga cctctacatt ttttatgttt atctctagta ttactcttta gacaaaaaaa 6840 ttgtagtaag aactattcat agagtgaatc gaaaacaata cgaaaatgta aacatttcct 6900 atacgtagta tatagagaca aaatagaaga aaccgttcat aattttctga ccaatgaaga 6960 atcatcaacg ctatcacttt ctgttcacaa agtatgcgca atccacatcg gtatagaata 7020 taatcgggga tgcctttatc ttgaaaaaat gcacccgcag cttcgctagt aatcagtaaa 7080 cgcgggaagt ggagtcaggc tttttttatg gaagagaaaa tagacaccaa agtagccttc 7140 ttctaacctt aacggaccta cagtgcaaaa agttatcaag agactgcatt atagagcgca 7200 caaaggagaa aaaaagtaat ctaagatgct ttgttagaaa aatagcgctc tcgggatgca 7260 tttttgtaga acaaaaaaga agtatagatt ctttgttggt aaaatagcgc tctcgcgttg 7320 catttctgtt ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg 7380 ttgcattttt gttttacaaa aatgaagcac agattcttcg ttggtaaaat agcgctttcg 7440 cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa attagcgctc 7500 tcgcgttgca tttttgttct acaaaatgaa gcacagatgc ttcgttcagg tggcactttt 7560 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 7620 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 7680 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 7740 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 7800 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 7860 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 7920 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 7980 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 8040 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 8100 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 8160 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 8220 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 8280 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 8340 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 8400 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 8460 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 8520 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 8580 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 8640 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 8700 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 8760 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 8820 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 8880 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 8940 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 9000 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 9060 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 9120 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 9180 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 9240 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 9300 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 9360 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 9420 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 9480 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 9540 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttacctc 9600 actcattagg caccccaggc tttacacttt atgcttccgg ctcctatgtt gtgtggaatt 9660 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca 9720 attaaccctc actaaaggga acaaaagctg gagct 9755 <210> 25 <211> 10552 <212> DNA <213> Artificial Sequence <220> <223> p425-HXT7p-SccytoEtr1-TPI7t-TEF1p-ScErg10-CYC1t <400> 25 catagcttca aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc 60 atcgccgtac cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct 120 ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt 180 ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaaa 240 tttttttttt gatttttttc tctttcgatg acctcccatt gatatttaag ttaataaacg 300 gtcttcaatt tctcaagttt cagtttcatt tttcttgttc tattacaact ttttttactt 360 cttgctcatt agaaagaaag catagcaatc taatctaagt ctagaactag tatgtctcag 420 aacgtttaca ttgtatcgac tgccagaacc ccaattggtt cattccaggg ttctctatcc 480 tccaagacag cagtggaatt gggtgctgtt gctttaaaag gcgccttggc taaggttcca 540 gaattggatg catccaagga ttttgacgaa attatttttg gtaacgttct ttctgccaat 600 ttgggccaag ctccggccag acaagttgct ttggctgccg gtttgagtaa tcatatcgtt 660 gcaagcacag ttaacaaggt ctgtgcatcc gctatgaagg caatcatttt gggtgctcaa 720 tccatcaaat gtggtaatgc tgatgttgtc gtagctggtg gttgtgaatc tatgactaac 780 gcaccatact acatgccagc agcccgtgcg ggtgccaaat ttggccaaac tgttcttgtt 840 gatggtgtcg aaagagatgg gttgaacgat gcgtacgatg gtctagccat gggtgtacac 900 gcagaaaagt gtgcccgtga ttgggatatt actagagaac aacaagacaa ttttgccatc 960 gaatcctacc aaaaatctca aaaatctcaa aaggaaggta aattcgacaa tgaaattgta 1020 cctgttacca ttaagggatt tagaggtaag cctgatactc aagtcacgaa ggacgaggaa 1080 cctgctagat tacacgttga aaaattgaga tctgcaagga ctgttttcca aaaagaaaac 1140 ggtactgtta ctgccgctaa cgcttctcca atcaacgatg gtgctgcagc cgtcatcttg 1200 gtttccgaaa aagttttgaa ggaaaagaat ttgaagcctt tggctattat caaaggttgg 1260 ggtgaggccg ctcatcaacc agctgatttt acatgggctc catctcttgc agttccaaag 1320 gctttgaaac atgctggcat cgaagacatc aattctgttg attactttga attcaatgaa 1380 gccttttcgg ttgtcggttt ggtgaacact aagattttga agctagaccc atctaaggtt 1440 aatgtatatg gtggtgctgt tgctctaggt cacccattgg gttgttctgg tgctagagtg 1500 gttgttacac tgctatccat cttacagcaa gaaggaggta agatcggtgt tgccgccatt 1560 tgtaatggtg gtggtggtgc ttcctctatt gtcattgaaa agatatgagt cgacctcgag 1620 attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga 1680 aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt 1740 aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg 1800 taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg 1860 ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca 1920 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 1980 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 2040 cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2100 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 2160 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 2220 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 2280 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 2340 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 2400 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 2460 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc 2520 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atcgacggtc 2580 gaggagaact tctagtatat ccacatacct aatattattg ccttattaaa aatggaatcc 2640 caacaattac atcaaaatcc acattctctt caaaatcaat tgtcctgtac ttccttgttc 2700 atgtgtgttc aaaaacgtta tatttatagg ataattatac tctatttctc aacaagtaat 2760 tggttgtttg gccgagcggt ctaaggcgcc tgattcaaga aatatcttga ccgcagttaa 2820 ctgtgggaat actcaggtat cgtaagatgc aagagttcga atctcttagc aaccattatt 2880 tttttcctca acataacgag aacacacagg ggcgctatcg cacagaatca aattcgatga 2940 ctggaaattt tttgttaatt tcagaggtcg cctgacgcat ataccttttt caactgaaaa 3000 attgggagaa aaaggaaagg tgagaggccg gaaccggctt ttcatataga atagagaagc 3060 gttcatgact aaatgcttgc atcacaatac ttgaagttga caatattatt taaggaccta 3120 ttgttttttc caataggtgg ttagcaatcg tcttactttc taacttttct taccttttac 3180 atttcagcaa tatatatata tatttcaagg atataccatt ctaatgtctg cccctatgtc 3240 tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa tcacagccga 3300 agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt tcgatttcga 3360 aaatcattta attggtggtg ctgctatcga tgctacaggt gtcccacttc cagatgaggc 3420 gctggaagcc tccaagaagg ttgatgccgt tttgttaggt gctgtggctg gtcctaaatg 3480 gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag aacttcaatt 3540 gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact tatctccaat 3600 caagccacaa tttgctaaag gtactgactt cgttgttgtc agagaattag tgggaggtat 3660 ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata gtgaacaata 3720 caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc tacaacatga 3780 gccaccattg cctatttggt ccttggataa agctaatctt ttggcctctt caagattatg 3840 gagaaaaact gtggaggaaa ccatcaagaa cgaattccct acattgaagg ttcaacatca 3900 attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa atggtattat 3960 aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta tcccaggttc 4020 cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca ccgcatttgg 4080 tttgtacgaa ccatgccacg gttctgctcc agatttgcca aagaataagg ttgaccctat 4140 cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc ctgaagaagg 4200 taaggccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa ctggtgattt 4260 aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag ttaagaaaat 4320 ccttgcttaa aaagattctc tttttttatg atatttgtac ataaacttta taaatgaaat 4380 tcataataga aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta 4440 tatacgcaat ctacatacat ttatcaagaa ggagaaaaag gaggatagta aaggaataca 4500 ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa aaagaattgc actttaacat 4560 taatattgac aaggaggagg gcaccacaca aaaagttagg tgtaacagaa aatcatgaaa 4620 ctacgattcc taatttgata ttggaggatt ttctctaaaa aaaaaaaaat acaacaaata 4680 aaaaacactc aatgacctga ccatttgatg gagtttaagt caataccttc ttgaagcatt 4740 tcccataatg gtgaaagttc cctcaagaat tttactctgt cagaaacggc cttacgacgt 4800 agtcgatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 4860 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 4920 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 4980 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 5040 taataatggt ttcttagtat gatccaatat caaaggaaat gatagcattg aaggatgaga 5100 ctaatccaat tgaggagtgg cagcatatag aacagctaaa gggtagtgct gaaggaagca 5160 tacgataccc cgcatggaat gggataatat cacaggaggt actagactac ctttcatcct 5220 acataaatag acgcatataa gtacgcattt aagcataaac acgcactatg ccgttcttct 5280 catgtatata tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta 5340 tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc 5400 tattccgaag ttcctattct ctagaaagta taggaacttc agagcgcttt tgaaaaccaa 5460 aagcgctctg aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact 5520 aaaatattgc gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc 5580 tctgtgctat atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta 5640 aactcgacct ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg 5700 tagtaagaac tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata 5760 cgtagtatat agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc 5820 atcaacgcta tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa 5880 tcggggatgc ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc 5940 gggaagtgga gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc 6000 taaccttaac ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa 6060 aggagaaaaa aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt 6120 ttgtagaaca aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat 6180 ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg 6240 catttttgtt ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt 6300 tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg 6360 cgttgcattt ttgttctaca aaatgaagca cagatgcttc gttcaggtgg cacttttcgg 6420 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 6480 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 6540 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 6600 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 6660 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 6720 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 6780 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 6840 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 6900 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 6960 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 7020 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 7080 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 7140 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 7200 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 7260 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 7320 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 7380 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 7440 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 7500 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 7560 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 7620 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 7680 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 7740 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 7800 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 7860 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 7920 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 7980 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 8040 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 8100 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 8160 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 8220 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 8280 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 8340 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 8400 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact 8460 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg 8520 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 8580 aaccctcact aaagggaaca aaagctggag ctcacttctc gtaggaacaa tttcgggccc 8640 ctgcgtgttc ttctgaggtt catcttttac atttgcttct gctggataat tttcagaggc 8700 aacaaggaaa aattagatgg caaaaagtcg tctttcaagg aaaaatcccc accatctttc 8760 gagatcccct gtaacttatt ggcaactgaa agaatgaaaa ggaggaaaat acaaaatata 8820 ctagaactga aaaaaaaaaa gtataaatag agacgatata tgccaatact tcacaatgtt 8880 cgaatctatt cttcatttgc agctattgta aaataataaa acatcaagaa caaacaagct 8940 caacttgtct tttctaagaa caaagaataa acacaaaaac aaaaagtttt tttaatttta 9000 atcaaaaatc tagaatgtcg tcctcagctc atcagattcc caagcacttc aaatcgctca 9060 tctattcaac tcatgaagtt gaggattgta ccaaggtttt gtcagtgaaa aattatacgc 9120 ctaaacaaga cttatctcaa tcaattgtgt taaaaacttt ggcctttccc ataaaccctt 9180 cggatatcaa tcagttgcaa ggagtatacc cgtctcgtcc agaaaagaca tacgattact 9240 ccacagatga gccagccgct atcgccggta atgagggtgt ctttgaagtt gtttctttac 9300 cttcgggaag ttccaaggga gatttgaaat tgggtgaccg agttatccca ttgcaggcaa 9360 atcaagggac ttggtccaat tatagagttt tctctagtag ttctgattta atcaaggtaa 9420 atgatttgga tctgttttct gcggcaactg tatctgttaa tggttgtacc ggtttccaat 9480 tagtatcaga ctatatcgac tggaacagta acggtaatga atggattatc caaaatgccg 9540 gtacatctag tgtatcaaaa atagttacgc aagtagcaaa agctaaaggg atcaaaacat 9600 taagtgttat acgtgaccgt gataattttg atgaggtagc aaaagttttg gaggataagt 9660 atggtgctac gaaggttatt tccgaatcgc aaaacaacga caagactttt gccaaagaag 9720 tattgtccaa gattttgggt gaaaatgcaa gggtgaggct tgccttgaat tctgttggag 9780 gtaaatccag tgcatcaata gcacgtaagt tggaaaataa tgctttgatg ctcacttatg 9840 gaggaatgtc aaaacaacct gtaactttac caacatctct acacattttc aaaggcttga 9900 catccaaagg gtactgggtg actgaaaaga acaaaaaaaa cccccaaagc aagattgaca 9960 ccatcagtga ttttatcaaa atgtataatt atggtcacat tatttcacca agagatgaaa 10020 ttgaaactct tacctggaat actaacacta ctactgacga acagttacta gaactagtca 10080 aaaaaggtat aactgggaag gggaagaaaa aaatggttgt tttagaatgg taagtcgacc 10140 tcgaggatta atataattat ataaaaatat tatcttcttt tctttatatc tagtgttatg 10200 taaaataaat tgatgactac ggaaagcttt tttatattgt ttctttttca ttctgagcca 10260 cttaaatttc gtgaatgttc ttgtaaggga cggtagattt acaagtgata caacaaaaag 10320 caaggcgctt tttctaataa aaagaagaaa agcatttaac aattgaacac ctctatatca 10380 acgaagaata ttactttgtc tctaaatcct tgtaaaatgt gtacgatctc tatatgggtt 10440 actcataagt gtaccgaaga ctgcattgaa agtttatgtt ttttcactgg aggcgtcatt 10500 ttcgcgttga gaagatgttc ttatccaaat ttcaactgtt atataggagc tc 10552

Claims (31)

As a transforming yeast strain of Saccharomyces cerevisiae ,
Variation or deletion of PMR1 represented by the nucleotide sequence of SEQ ID NO: 1, and ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 contains one or more of the mutations or deletions,
A gene encoding xylose isomerase; And a gene encoding xylulose phosphatase and a gene encoding aldol transaldolase,
Variation of the PMR1 represented by the base sequence of said SEQ ID NO: 1 will be mutated to PMR1 ^G681A represented by the nucleotide sequence of SEQ ID NO: 2,
The variation of ASC1 represented by the nucleotide sequence of SEQ ID NO: 3 is a variation of ASC1 ^{Q237 *} represented by the nucleotide sequence of SEQ ID NO: 4,
Transgenic yeast strain. The transformed yeast strain according to claim 1, wherein at least one of genes encoding aldose reductase and genes encoding phosphatase is deleted. According to claim 1,
The ASC1 represented by the base sequence of the PMR1 and SEQ ID NO: 3, represented by the nucleotide sequence of SEQ ID NO: 1 is deleted,
It includes a gene encoding xylose isomerase, a gene encoding xylulose phosphatase and a gene encoding aldol transferase,
The gene encoding the aldose reductase and the gene encoding the phosphatase are deleted,
Transgenic yeast strain. According to claim 1,
Including ASC1 ^{Q237 *} represented by the base sequence of the PMR1 ^G681A and SEQ ID NO: 4, represented by the nucleotide sequence of SEQ ID NO: 2,
It includes a gene encoding xylose isomerase, a gene encoding xylulose phosphatase and a gene encoding aldol transferase,
The gene encoding aldose reductase and the gene encoding phosphatase are deleted,
Transgenic yeast strain. The method of claim 1, wherein the parent strain MRS three Levy cyano yeast host, yeast strains transformed with the wild-type saccharide of MRS three Levy cyano (Saccharomyces cerevisiae) as a saccharide to switch the trait. The transformed yeast strain according to claim 5, wherein the wild-type Saccharomyces cerevisiae yeast strain has accession number ATCC 201388. The method of claim 1 wherein the xylose isomerase gene encoding the enzyme xylA represented by xylA3 *, base sequence of the xylA, SEQ ID NO: 7 is represented by the base sequence of SEQ ID NO: 6 is represented by the base sequence of SEQ ID NO: 5 or A transgenic yeast strain , which is xylA represented by the nucleotide sequence of SEQ ID NO: 8. The transgenic yeast strain according to claim 1, wherein the gene encoding the xylulose phosphatase is XKS1 expressed by the nucleotide sequence of SEQ ID NO: 9 or xyl3 expressed by the nucleotide sequence of SEQ ID NO: 10. The transgenic yeast strain according to claim 1, wherein the gene encoding the aldol transferase is TAL1 expressed by the nucleotide sequence of SEQ ID NO: 11 or tal1 expressed by the nucleotide sequence of SEQ ID NO: 12. The transgenic yeast strain according to claim 2, wherein the gene encoding the aldose reductase is GRE3 represented by the nucleotide sequence of SEQ ID NO: 13. The transgenic yeast strain according to claim 2, wherein the gene encoding the phosphatase is PHO13 expressed by the nucleotide sequence of SEQ ID NO: 14. The transformed yeast strain according to claim 1, which has accession number KCTC13614BP. According to claim 1, wherein the transformed yeast strain is transformed into ethanol by fermenting glucose and xylose at the same time, transformed yeast strain. According to any one of claims 1 to 13, Hbd (β-hydroxybutyryl-CoA dehydrogenase) expressed by the nucleotide sequence of SEQ ID NO: 17, expressed by the nucleotide sequence of SEQ ID NO: 18 Crt (3-hydroxybutyryl-CoA dehydratase), BdhB (butanol dehydergenase) represented by the nucleotide sequence of SEQ ID NO: 19, Erg10 (acetoacetyl-CpA thiolase) represented by the nucleotide sequence of SEQ ID NO: 20, represented by the nucleotide sequence of SEQ ID NO: 21 Etr1 (enoyl thioester reductase) And A transformed yeast strain further comprising a gene constituting a butanol production pathway comprising EutE (butyraldehyde dehydrogenase) represented by the nucleotide sequence of SEQ ID NO: 22. The transforming yeast strain according to claim 14, wherein the gene constituting the butanol production pathway is introduced in a form inserted in genomic DNA or in a plasmid form. The method of claim 15, wherein the transformed yeast strain is a plasmid represented by the nucleotide sequence of SEQ ID NO: 24, a plasmid represented by the nucleotide sequence of SEQ ID NO: 25, and a plasmid represented by the nucleotide sequence of SEQ ID NO: 26 introduced. Conversion yeast strain. The transforming yeast strain according to claim 14, wherein the transforming yeast strain converts glucose and xylose into ethanol and butanol simultaneously. A culture of the transformed yeast strain according to any one of claims 1 to 13. Culture of the transformed yeast strain according to claim 14. Wherein the first to thirteenth any one transgenic Yeast production process according to one wherein, in the Saccharomyces three Levy cyano (Saccharomyces cerevisiae) as a parent strain,
A gene encoding xylose isomerase in the strain ; And inserting one or more of a gene encoding a xylulose phosphatase gene and a gene encoding an aldol transaldolase; And
Mutation or deletion of PMR1 represented by the nucleotide sequence of SEQ ID NO: 1 of the strain, and The method comprising the step of performing one or more of the mutation or deletion of ASC1 represented by the nucleotide sequence of SEQ ID NO: 3. The method of claim 20, wherein the method is a gene encoding a aldose reductase and a gene encoding a phosphatase. The method further comprising the step of deleting one or more of. In, step variation of the PMR1 or ASC1, the method comprising sub-cultured by adaptive evolution (adaptive evolution) in a minimal medium for xylose the strain with the only carbon source according to claim 20. The method of claim 20, wherein the insertion or deletion of the gene is made using CRISPR / Cas9. The method of claim 20, wherein the strain Hbd (β-hydroxybutyryl-CoA dehydrogenase), Crt (3-hydroxybutyryl-CoA dehydratase), BdhB (butanol dehydergenase), Erg10 (acetoacetyl-CpA thiolase), Etr1 (enoyl thioester reductase) And introducing a gene constituting a butanol production pathway including EutE (butyraldehyde dehydrogenase). The method of claim 24, wherein the step of introducing the gene constituting the butanol production path to the strain comprises introducing the gene constituting the butanol production path into a genomic DNA (genomic DNA) inserted form or plasmid form, Transgenic yeast strain. The method of claim 25, wherein the step of introducing a gene constituting the butanol production pathway into the strain is a plasmid represented by the nucleotide sequence of SEQ ID NO: 23, a plasmid represented by the nucleotide sequence of SEQ ID NO: 24 and a nucleotide sequence of SEQ ID NO: 25. A method of production, comprising introducing a expressed plasmid. Biofuel comprising the step of culturing and fermenting the transformed yeast strain according to any one of claims 1 to 13 in a medium containing xylose as the only carbon source, or a medium containing glucose and xylose as a carbon source. And material production method. 28. The method of claim 27, wherein the biofuel comprises ethanol. 28. The method of claim 27, wherein the medium comprises a wood-based biomass,
The method further comprising the step of producing at least one of glucose and xylose by decomposing the woody biomass prior to the step of culturing the transformed yeast strain in a medium. A method for producing biofuels and materials, comprising culturing and fermenting the transformed yeast strain according to claim 14 in a medium containing xylose or glucose as the only carbon source, or a medium containing glucose and xylose as a mixed carbon source. 31. The method of claim 30, wherein the biofuel comprises ethanol and butanol.

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