CN108949601A - Utilize the recombinant Saccharomyces cerevisiae bacterium and construction method of xylose production dammarendiol and protopanoxadiol - Google Patents

Utilize the recombinant Saccharomyces cerevisiae bacterium and construction method of xylose production dammarendiol and protopanoxadiol Download PDF

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CN108949601A
CN108949601A CN201810781608.0A CN201810781608A CN108949601A CN 108949601 A CN108949601 A CN 108949601A CN 201810781608 A CN201810781608 A CN 201810781608A CN 108949601 A CN108949601 A CN 108949601A
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saccharomyces cerevisiae
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卢文玉
高晓
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Tianjin University
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Abstract

It is promoter P by the promoter replacement of saccharomyces cerevisiae xylulokinase gene XKS1 that the invention discloses a kind of recombinant Saccharomyces cerevisiae bacterium and construction method using xylose production dammarendiol and protopanoxadiol by the method for homologous recombinationFBA1, Xylose reductase XYL1 is imported again, xylitol dehydrogenase XYL2 expression cassette, transketolase TKL1 and transaldolase TAL1 activity are improved again, obtain recombinant bacterium 1, Farnesyl-diphosphate farnesyltransferase gene ERG9 is imported into recombinant bacterium 1 again, squalene monooxygenase gene ERG1 and dammarendiol synthase gene DS, obtain recombinant bacterium 2, nicotinamide adenine dinucleotide -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr is imported into recombinant bacterium 2, farnesyl pyrophosphate synthase ERG20, protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1, obtain recombinant bacterium 3, recombinant Saccharomyces cerevisiae bacterium of the invention utilizes the artificial synthesized dammarendiol of xylose and protopanoxadiol.

Description

Using xylose production dammarendiol and protopanoxadiol recombinant Saccharomyces cerevisiae bacterium and Construction method
Technical field
The present invention relates to field of biotechnology, more particularly to utilize the weight of xylose production dammarendiol and protopanoxadiol Group S. cervisiae and construction method and application.
Background technique
Xylose is a kind of pentose, and natural D- xylose is present in plant in the form of polysaccharide, in nature so far not It was found that the xylose of free state.Main comprise material of the lignocellulosic as plant cell wall mainly includes cellulose, half fiber Dimension element and the big component of lignin three.100,000,000,000 are up to by the lignocellulose-like biomass that photosynthesis generates every year on the earth Ton or more, it is reserves renewable energy substance the most abundant on the earth.And hydrolyzed by lignocellulosic, it is available abundant Glucose and the monosaccharide substance such as xylose.The monosaccharide of glucose is only second to as content in ligno-cellulose hydrolysate, xylose Utilize the extensive concern for causing researcher.
Xylose can naturally be utilized in spite of various bacteria and yeast, but saccharomyces cerevisiae have by comparison it is apparent excellent Gesture has robustness, such as low pH, hyperosmosis, high ethano concentration and phage-infect under a variety of Environmental pressure of industry.Cause This, saccharomyces cerevisiae is widely used for the essential industries chemicals such as production alcohol fuel, 2,3-butanediol, hexadecanol.However, by Largely the study found that the efficiency of xylose production ethyl alcohol is still not as good as glucose.Saccharomyces cerevisiae metabolism wood is assumed in the propositions such as Kwak Sugar more suitable for producing non-ethyl alcohol substance, and verified with amorphadiene and squalene (Kwak S, et al.2017, Biotechnology&Bioengineering,114(11).).The study found that ethyl alcohol assimilation and cytosolic acetyl when wood-sugar fermentation The expression that coacetylase synthesizes relevant enzyme is higher than glucose fermentation.Will gene relevant to amorphadiene and squalene synthesis into After the necessary disturbance of row, the discovery titre of two kinds of products and yield under the conditions of wood-sugar fermentation are above glucose.
Currently, xylose has been used to produce multi-chemical, such as safe sweetener-xylitol, bio-fuel-fuel second Alcohol, Fuel butanol, biodiesel, hydrogen and methane, the raw material of industry-lactic acid, 2,3-butanediol, 3-hydroxy-2-butanone, 3,4- dihydroxy fourth Acid, succinic acid, fumaric acid, polysaccharide-pulullan polysaccharide, bioabsorbable polymer material-PHA, PHB.Therefore, xylose utilization engineering is developed Bacterial strain, the metabolic mechanism for promoting and accelerating xylose is studied and the type important in inhibiting of abundant biological-based chemicals.
Summary of the invention
The first purpose of this invention is to overcome the deficiencies of the prior art and provide a kind of utilization xylose production dammarendiol With the recombinant Saccharomyces cerevisiae bacterium of protopanoxadiol.
A second object of the present invention is to provide a kind of recombinations using xylose production dammarendiol and protopanoxadiol The construction method of S. cervisiae.
Third object of the present invention is to provide a kind of recombinations using xylose production dammarendiol and protopanoxadiol The application of S. cervisiae fermenting xylose.
Fourth object of the present invention is to provide second of weight using xylose production dammarendiol and protopanoxadiol Group S. cervisiae.
Fifth object of the present invention is to provide second of weights using xylose production dammarendiol and protopanoxadiol The construction method of group S. cervisiae.
Sixth object of the present invention is to provide second of weights using xylose production dammarendiol and protopanoxadiol The application of group S. cervisiae fermenting xylose.
Technical solution of the present invention is summarized as follows:
A kind of construction method for the recombinant Saccharomyces cerevisiae bacterium that can utilize xylose production dammarendiol and protopanoxadiol, Include the following steps: the method by homologous recombination, the promoter of the endogenous xylulokinase gene XKS1 of saccharomyces cerevisiae is set It is changed to strong promoter PFBA1, Xylose reductase gene XYL1 expression cassette then is imported to the site δ of saccharomyces cerevisiae and xylitol is de- Hydrogenase gene XYL2 expression cassette, obtains recombinant bacterium；Transaldolase gene in pentose phosphate pathway is imported to the site rDNA of recombinant bacterium TAL1 expression cassette and tkt gene TKL1 expression cassette, obtain the recombinant Saccharomyces cerevisiae bacterium 1 of tachymetabolism xylose；By homologous heavy The method of group imports Farnesyl-diphosphate farnesyltransferase gene ERG9 expression cassette, shark to the site rDNA of recombinant bacterium 1 Alkene monooxygenase gene ERG1 expression cassette and dammarendiol synthase gene DS expression cassette are utilized xylose production up to Ma alkene two The recombinant Saccharomyces cerevisiae bacterium 2 of alcohol；By the method for homologous recombination, two nucleoside of nicotinamide adenine is imported to the site δ of recombinant bacterium 2 Acid -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr expression cassette, farnesyl phosphate synthase gene ERG20 expression Box, protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 expression cassette, are utilized xylose Produce the recombinant Saccharomyces cerevisiae bacterium 3 of dammarendiol and protopanoxadiol；
Promoter PFBA1Nucleotide sequence as shown in SEQ ID NO.1；
The nucleotide sequence of Xylose reductase gene XYL1 is as shown in SEQ ID NO.2；
The nucleotide sequence of xylitol dehydrogenase gene XYL2 is as shown in SEQ ID NO.3；
The nucleotide sequence of transaldolase gene TAL1 is as shown in SEQ ID NO.4；
The nucleotide sequence of tkt gene TKL1 is as shown in SEQ ID NO.5.
The nucleotide sequence of Farnesyl-diphosphate farnesyltransferase gene ERG9 is as shown in SEQ ID NO.6；
The nucleotide sequence of squalene monooxygenase gene ERG1 is as shown in SEQ ID NO.7；
The nucleotide sequence of dammarendiol synthase gene DS is as shown in SEQ ID NO.8.
Nicotinamide adenine dinucleotide -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr nucleotide sequence As shown in SEQ ID NO.9；
The nucleotide sequence of farnesyl phosphate synthase gene ERG20 is as shown in SEQ ID NO.10；
Protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 nucleotide sequence is such as Shown in SEQ ID NO.11.
The recombinant Saccharomyces cerevisiae bacterium 3. using xylose production dammarendiol and protopanoxadiol of above method building
The recombinant Saccharomyces cerevisiae bacterium 3 that can utilize xylose production dammarendiol and protopanoxadiol of above method building Utilize the application of xylose production dammarendiol and protopanoxadiol.
The construction method of second of recombinant Saccharomyces cerevisiae bacterium using xylose production dammarendiol and protopanoxadiol, packet It includes following steps: being marked using his3, ura3 of Cre/loxP system recycling recombinant bacterium 3, pass through the method for homologous recombination, Xiang Chong The site δ of group bacterium 3 imports acetyl-CoA acyltransferase gene ERG10 expression cassette, Hydroxymethylglutaryl-CoA synthase gene ERG13 expression cassette and mevalonate kinase gene ERG12 expression cassette obtain and utilize xylose production dammarendiol and protoplast's ginseng two The recombinant Saccharomyces cerevisiae bacterium 4 of alcohol；
The nucleotide sequence of acetyl-CoA acyltransferase gene ERG10 is as shown in SEQ ID NO.12；
The nucleotide sequence of Hydroxymethylglutaryl-CoA synthase gene ERG13 is as shown in SEQ ID NO.13；
The nucleotide sequence of mevalonate kinase gene ERG12 is as shown in SEQ ID NO.14.
The recombinant Saccharomyces cerevisiae using xylose production dammarendiol and protopanoxadiol of above-mentioned second method building Bacterium 4.
The recombinant Saccharomyces cerevisiae using xylose production dammarendiol and protopanoxadiol of above-mentioned second method building Bacterium 4 utilizes xylose production dammarendiol and protopanoxadiol application.
It is demonstrated experimentally that by the method for homologous recombination, the utilization xylose production dammarendiol and protopanoxadiol of acquisition Recombinant Saccharomyces cerevisiae bacterium can use xylose production dammarendiol and protopanoxadiol, yield reaches 17.91mg/L up to Ma Enediol and 14.49mg/L protopanoxadiol, method of the invention are to be joined using the artificial synthesized dammarendiol of xylose and protoplast Glycol provides foundation.
Detailed description of the invention
Fig. 1 is the metabolic pathway figure that saccharomyces cerevisiae utilizes xylose production dammarendiol and protopanoxadiol.
Fig. 2 is the liquid chromatographic detection analysis result of dammarendiol and protopanoxadiol.
Fig. 3 is the LC-MS detection and analysis result of dammarendiol and protopanoxadiol.
Fig. 4 is the recombinant Saccharomyces cerevisiae bacterium dammarendiol and original using xylose production dammarendiol and protopanoxadiol The yield of panoxadiol.
Specific embodiment
Below by specific embodiment, the present invention is further illustrated.
Test method used in following example is conventional method unless otherwise specified.
Material used in following example, reagent etc., are commercially available unless otherwise specified.
The Wine brewing yeast strain is Saccharomyces cerevisiae W303-1A (U.S., ATCC208352), Hereinafter referred to as saccharomyces cerevisiae.
The amplification and preparation of embodiment 1, Genetic elements
The Xylulokinase XKS1 promoter replacement is promoter PFBA1(promoter PFBA1；Nucleotide sequence such as SEQ ID Shown in NO.1；It simply is labeled as SEQ ID NO.1, similarly hereinafter)；
The Xylose reductase gene XYL1 expression cassette is by promoter PCCW12(SEQ ID NO.15), Xylose reductase gene XYL1 (SEQ ID NO.2) and terminator THSP26(SEQ ID NO.16) composition；
The xylitol dehydrogenase gene XYL2 expression cassette is by promoter PHXT7(SEQ ID NO.17), xylitol dehydrogenase Gene XYL2 (SEQ ID NO.3) and terminator THXT7(SEQ ID NO.18) composition；
The transaldolase gene TAL1 expression cassette is by promoter PHXT7, transaldolase gene TAL1 (SEQ ID NO.4) and eventually Only sub- THXT7Composition；
The tkt gene TKL1 expression cassette is by promoter PCCW12, tkt gene TKL1 (SEQ ID NO.5) and eventually Only sub- THSP26Composition；
The Farnesyl-diphosphate farnesyltransferase gene ERG9 expression cassette is by promoter PTPI1(SEQ ID NO.19), Farnesyl-diphosphate farnesyltransferase gene ERG9 (SEQ ID NO.6) and terminator TADH2(SEQ ID NO.20 it) forms；
The squalene monooxygenase gene ERG1 expression cassette is by promoter PTEF1(SEQ ID NO.21), squalene monooxygenase Gene ERG1 (SEQ ID NO.7) and terminator TADH1(SEQ ID NO.22) composition；
The dammarendiol synthase gene DS expression cassette is by promoter PPGK1(SEQ ID NO.23), dammarendiol close Enzyme gene DS (SEQ ID NO.8) and terminator TCYC1(SEQ ID NO.24) composition；
The nicotinamide adenine dinucleotide -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr expression cassette by Promoter PPGK1, nicotinamide adenine dinucleotide -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr (SEQ ID ) and terminator T NO.9CYC1Composition；
The farnesyl phosphate synthase gene ERG20 expression cassette is by promoter PTPI1, farnesyl phosphate synthase gene ERG20 (SEQ ID NO.10) (containing endogenous terminator) composition；
The protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 expression cassette is by opening Mover PTDH3(SEQ ID NO.25), protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 (SEQ ID NO.11) and terminator TADH1(SEQ ID NO.26) composition；
The acetyl-CoA acyltransferase gene ERG10 expression cassette is by promoter PHXT7, acetyl coenzyme A acyl group transfer Enzyme gene ERG10 (SEQ ID NO.12) (containing endogenous terminator) composition；
The Hydroxymethylglutaryl-CoA synthase gene ERG13 expression cassette is by promoter PTEF1, hydroxymethyl glutaryl-CoA Synthase gene ERG13 (SEQ ID NO.13) (containing endogenous terminator) composition；
The mevalonate kinase gene ERG12 expression cassette is by promoter PPGK1, mevalonate kinase gene ERG12 (SEQ ID NO.14) (containing endogenous terminator) composition.
Xylose reductase gene XYL1 (SEQ ID NO.2), it xylitol dehydrogenase gene XYL2 (SEQ ID NO.3), reaches Ma enediol synthase gene DS (SEQ ID NO.8), nicotinamide adenine dinucleotide -3-hydroxy-3-methylglutaryl coenzyme A reductase Gene NADH-HMGr (SEQ ID NO.9) and protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 (SEQ ID NO.11) is artificial synthesized sequence.
Using saccharomyces cerevisiae W303-1A genome as template, XKS1t2-F (SEQ ID NO.27) is leading, XKS1t2- R-his3 (SEQ ID NO.28) draws after being, amplifies XKS1t2 segment；His3-pFBA1-F (SEQ ID NO.31) is leading, PFBA1-R-XKS1t1 (SEQ ID NO.32) draws after being, expands PFBA1Segment (SEQ ID NO.1)；pFBA1-XKS1t1-F (SEQ ID NO.33) is leading, and XKS1t1-R (SEQ ID NO.34) draws after being, expands XKS1t1 segment.With plasmid pXP320 For template, XKS1t2-his3-F (SEQ ID NO.29) is leading, and his3-R-pFBA1 (SEQ ID NO.30) draws after being, is expanded Increase his3 segment.Above-mentioned segment is merged, XKS1 promoter replacement expression cassette XKS1t2-his3-P is obtainedFBA1- XKS1t1。
Using the genome of saccharomyces cerevisiae W303-1A as template, δ 2-F (SEQ ID NO.35) is leading, δ 2-R-pCCW12 (SEQ ID NO.36) draws after being, expands 2 segment of δ；PCCW12-F (SEQ ID NO.37) is leading, pCCW12-R (SEQ ID NO.38 draw after) being, expand PCCW12Segment；XYL1-tHSP26-F (SEQ ID NO.41) is leading, tHSP26-R-pHXT7 (SEQ ID NO.42) draws after being, expands THSP26Segment；THSP26-pHXT7-F (SEQ ID NO.43) is leading, pHXT7-R- XYL2 (SEQ ID NO.44) draws after being, expands PHXT7Segment；THXT7-F (SEQ ID NO.47) is leading, tHXT7-R (SEQ ID NO.48) be after draw, expand THXT7Segment；Leu2- δ 1-F (SEQ ID NO.51) is leading, δ 1-R (SEQ ID NO.52) After draw, expand 1 segment of δ.Using artificial sequence XYL1 as template, pCCW12-XYL1-F (SEQ ID NO.39) is leading, XYL1- R-tHSP26 (SEQ ID NO.40) draws after being, expands XYL1 segment；Using artificial sequence XYL2 as template, pHXT7-XYL2-F (SEQ ID NO.45) is leading, and XYL2-R-tHXT7 (SEQ ID NO.46) draws after being, expands XYL2 segment.With plasmid PRS415 is template, and tHXT7-leu2-F (SEQ ID NO.49) is leading, and leu2-R- δ 1 (SEQ ID NO.50) draws after being, Expand leu2 segment.By above-mentioned segment composition, gene XYL1 expression cassette P is obtainedCCW12-XYL1-THSP26, gene XYL2 expression cassette PHXT7-XYL2-THXT7。
Using saccharomyces cerevisiae W303-1A genome as template, with rDNA2-F (SEQ ID NO.53) for leading, rDNA2- R-pCCW12 (SEQ ID NO.54) draws after being, expands rDNA2 segment；PCCW12-TKL1-F (SEQ ID NO.55) is leading, TKL1-R-tHSP26 (SEQ ID NO.56) draws after being, expands TKL1 segment；Before pHXT7-TAL1-F (SEQ ID NO.57) is Draw, TAL1-R-tHXT7 (SEQ ID NO.58) draws after being, expands TAL1 segment；Before δ 1-rDNA1-F (SEQ ID NO.61) is Draw, rDNA1-R (SEQ ID NO.62) draws after being, expands rDNA1 segment.Using plasmid pXP218 as template, tHXT7-ura3-F (SEQ ID NO.59) is leading, and ura3-R- δ 1 (SEQ ID NO.60) draws after being, expands ura3 segment.Above-mentioned segment is melted It closes, obtains gene TKL1 expression cassette PCCW12-TKL1-THSP26, gene TAL1 expression cassette PHXT7-TAL1-THXT7。
Using plasmid pSH47 as template, pSH47-F (SEQ ID NO.63) is leading, and pSH47-R (SEQ ID NO.64) is After draw, expand pSH47v segment；Using plasmid pXP320 as template, pSH47-ura3-F (SEQ ID NO.65) is leading, ura3- R-pSH47 (SEQ ID NO.66) draws after being, expands ura3 segment.
Using the genome of saccharomyces cerevisiae W303-1A as template, rDNA2-F (SEQ ID NO.67) is leading, rDNA2-R- His3 (SEQ ID NO.68) draws after being, expands rDNA2 segment；His3-pPGK1-F (SEQ ID NO.71) is leading, PPGK1-R-DS (SEQ ID NO.72) draws after being, expands PPGK1Segment；DS-tCYC1-F (SEQ ID NO.75) is leading, TCYC1-R-pTEF1 (SEQ ID NO.76) draws after being, expands TCYC1Segment；TCYC1-pTEF1-F (SEQ ID NO.77) is Leading, pTEF1-R-ERG1 (SEQ ID NO.78) draw after being, expand PTEF1Segment；Before ERG1-F (SEQ ID NO.79) is Draw, ERG1-R (SEQ ID NO.80) draws after being, expands ERG1 segment；ERG1-tADH1-F (SEQ ID NO.81) is leading, TADH1-R-pTPI1 (SEQ ID NO.82) draws after being, expands TADH1Segment；TADH1-pTPI1-F (SEQ ID NO.83) is Leading, pPTI1-R-ERG9 (SEQ ID NO.84) draw after being, expand PTPI1Segment；Before ERG9-F (SEQ ID NO.85) is Draw, ERG9-R (SEQ ID NO.86) draws after being, expands ERG9 segment；ERG9-tADH2-F (SEQ ID NO.87) is leading, TADH2-R-rDNA1 (SEQ ID NO.88) draws after being, expands TADH2Segment；TADH2-rDNA1-F (SEQ ID NO.89) is Leading, rDNA1-R (SEQ ID NO.90) draw after being, expand rDNA1 segment.Using plasmid pXP320 as template, rDNA2-his3- F (SEQ ID NO.69) is leading, and his3-R-pPGK1 (SEQ ID NO.70) draws after being, expands his3 segment.With artificial sequence Column DS is template, and DS-F (SEQ ID NO.73) is leading, and DS-R (SEQ ID NO.74) draws after being, expands DS segment.It will be upper Segment composition is stated, gene DS expression cassette P is obtainedPGK1-DS-TCYC1, gene ERG1 expression cassette PTEF1-ERG1-TADH1, gene ERG9 Expression cassette PTPI1-ERG9-TADH2。
Using the genome of saccharomyces cerevisiae W303-1A as template, δ 2'-F (SEQ ID NO.91) is leading, δ 2'-R (SEQ ID NO.92) be after draw, expand δ 2' segment；δ 2'-pTPI1-F (SEQ ID NO.93) is leading, pTPI1-R-ERG20 (SEQ ID NO.94) be after draw, expand PTPI1Segment；PTPI1-ERG20-F (SEQ ID NO.95) is leading, ERG20-R-pPGK1 (SEQ ID NO.96) draws after being, expands ERG20 segment；PPGK1-F (SEQ ID NO.97) is leading, pPGK1-R (SEQ ID NO.98 draw after) being, expand PPGK1Segment；TCYC1-F (SEQ ID NO.101) is leading, tCYC1-R (SEQ ID NO.102) Draw for after, expands TCYC1Segment；TCYC1-pTDH3-F (SEQ ID NO.103) is leading, pTDH3-R-PPDS (SEQ ID NO.104 draw after) being, expand PTDH3Segment；ATR1-tADH1-F (SEQ ID NO.107) is leading, tADH1-R-ura3 (SEQ ID NO.108) be after draw, expand TADH1Segment；Ura3- δ 1'-F (SEQ ID NO.111) is leading, δ 1'-R (SEQ ID NO.112 draw after) being, expand δ 1' segment.Using artificial sequence NADH-HMGr as template, before HMGr-F (SEQ ID NO.99) is Draw, HMGr-R (SEQ ID NO.100) draws after being, expands HMGr segment；Using artificial sequence PPDS-ATR1 as template, PPDS-F (SEQ ID NO.105) is leading, and ATR1-R (SEQ ID NO.106) draws after being, expands PPDS-ATR1 segment；With plasmid PXP218 is template, and tADH1-ura3-F (SEQ ID NO.109) is leading, ura3-R- δ 1'(SEQ ID NO.110) be after Draw, expands ura3 segment.By above-mentioned segment composition, gene ERG20 expression cassette P is obtainedTPI1- ERG20, gene NADH-HMGr table Up to box PPGK1-NADH-HMGr-TCYC1, gene PPDS-ATR1 expression cassette PTDH3-PPDS-ATR1-TADH1。
Using saccharomyces cerevisiae W303-1A genome as template, δ 2-pHXT7-F (SEQ ID NO.113) is leading, pHXT7- R-ERG10 (SEQ ID NO.114) draws after being, expands PHXT7Segment；ERG10-F (SEQ ID NO.115) is leading, ERG10- R (SEQ ID NO.116) draws after being, expands ERG10 segment；ERG10-pPGK1-F (SEQ ID NO.117) is leading, PPGK1-R-ERG12 (SEQ ID NO.118) draws after being, expands PPGK1Segment；ERG12-F (SEQ ID NO.119) is leading, ERG12-R (SEQ ID NO.120) draws after being, expands ERG12 segment；Before ERG12-pTEF1-F (SEQ ID NO.121) is Draw, pTEF1-R-ERG13 (SEQ ID NO.122) draws after being, expands PTEF1Segment；Before ERG13-F (SEQ ID NO.123) is Draw, ERG13-R (SEQ ID NO.124) draws after being, expands ERG13 segment；δ 1-F (SEQ ID NO.127) is leading, δ 1-R (SEQ ID NO.128) draws after being, expands 1 segment of δ.Using saccharomyces cerevisiae BY4741 genome as template, ERG13-ade2-F (SEQ ID NO.125) is leading, and ade2-R- δ 1 (SEQ ID NO.126) draws after being, expands ade2 segment.By above-mentioned segment Fusion, obtains gene ERG10 expression cassette PHXT7- ERG10, gene ERG12 expression cassette PPGK1- ERG12 and gene ERG13 expression cassette PTEF1-ERG13。
PCR enzyme used in the present invention is the Phanta Super-Fidelity of Nanjing Vazyme Biotechnology Co., Ltd. DNA Polymerase.The PCR amplification system of 50 μ L is as shown in table 1:
Table 1
Amplification program is set in PCR instrument.Amplification condition is 95 DEG C of initial denaturation 4min (1 circulation)；95 DEG C of denaturation 15sec, 55 DEG C of annealing 15sec, 72 DEG C of extension 1min (35 circulations)；72 DEG C of extension 5min (1 circulation).
Fusion DNA vaccine system used in the present invention is as shown in table 2:
Table 2
The setting fusion program in PCR instrument.Fusion conditions are 95 DEG C of initial denaturation 4min (1 circulation)；95 DEG C of denaturation 15sec, 58 DEG C of annealing 15sec, 72 DEG C of extension 1min (11 circulations)；72 DEG C of extension 5min (1 circulation).
Segment obtained by PCR amplification is pure with Ago-Gel DNA QIAquick Gel Extraction Kit (Tiangeng biochemical technology Beijing Co., Ltd) Change recycling and obtain DNA fragmentation used in transformed saccharomyces cerevisiae, DNA fragmentation is then subjected to fusion DNA vaccine, and with fusion DNA vaccine product For template, expression casette, respectively XKS1t2-his3-P used in transformed saccharomyces cerevisiae are expandedFBA1- XKS1t1, gene XYL1 Expression cassette PCCW12-XYL1-THSP26, gene XYL2 expression cassette PHXT7-XYL2-THXT7, gene TKL1 expression cassette PCCW12-TKL1- THSP26, gene TAL1 expression cassette PHXT7-TAL1-THXT7, gene DS expression cassette PPGK1-DS-TCYC1, gene ERG1 expression cassette PTEF1- ERG1-TADH1, gene ERG9 expression cassette PTPI1-ERG9-TADH2, gene ERG20 expression cassette PTPI1- ERG20, gene NADH-HMGr Expression cassette PPGK1-NADH-HMGr-TCYC1, gene PPDS-ATR1 expression cassette PTDH3-PPDS-ATR1-TADH1, gene ERG10 expression Box PHXT7- ERG10, gene ERG12 expression cassette PPGK1- ERG12 and gene ERG13 expression cassette PTEF1-ERG13。
Embodiment 2 utilizes the conversion and building of the recombinant Saccharomyces cerevisiae bacterium of xylose production dammarendiol and protopanoxadiol
By DNA fragmentation XKS1t2-his3-PFBA1-XKS1t1、PCCW12-XYL1-THSP26、PHXT7-XYL2-THXT7、PCCW12- TKL1-THSP26And PHXT7-TAL1-THXT7It imports saccharomyces cerevisiae and obtains recombinant bacterium 1；
By DNA fragmentation PPGK1-DS-TCYC1PTEF1-ERG1-TADH1And PTPI1-ERG9-TADH2Recombinant bacterium 1 is imported to be recombinated Bacterium 2；
By DNA fragmentation PTPI1-ERG20、PPGK1-NADH-HMGr-TCYC1And PTDH3-PPDS-ATR1-TADH1Import recombinant bacterium 2 Obtain recombinant bacterium 3；
By DNA fragmentation PHXT7-ERG10、PPGK1- ERG12 and PTEF1- ERG13 imports recombinant bacterium 3 and obtains recombinant bacterium 4.
Method for transformation is as follows:
After saccharomyces cerevisiae W303-1A is cultivated 12h in YPD culture medium, take 300 μ L that 3mL fresh YPD medium is added In, cultivate 5h.Cell culture fluid is added in 1.5mL centrifuge tube, 4000rpm is centrifuged 5min and collects thallus, abandons supernatant.With Cell is resuspended in 1mL sterile water, and 4000rpm is centrifuged 5min again, abandons supernatant.Cell is resuspended with 1mL 100mM lithium acetate, it is static 5min.Then 3000rpm is centrifuged 3min, abandons supernatant.SsDNA boils 5min, cooling rapidly on ice.Converting mixed liquor includes 24 μ L PEG 3350 (50%w/v), 36 μ L 1.0M lithium acetates, 10 μ L ssDNA, DNA fragmentation 800ng, extremely with sterile water polishing finally 360μL.Centrifuge tube vortex 1min, is sufficiently mixed each ingredient.42 DEG C of water-bath thermal shock 30min.4000rpm is centrifuged 5min, micro Pipettor removes supernatant, and 1mLYPD culture medium, 30 DEG C of culture 2h are added.4000rpm is centrifuged 5min, abandons supernatant, sterile washing two Secondary thallus.Cell is resuspended in 100 μ L sterile waters, applies corresponding auxotroph plate and is screened.Condition of culture is 30 DEG C, culture 48h。
Embodiment 3 reaches Ma using the recombinant Saccharomyces cerevisiae bacterium fermenting and producing of xylose production dammarendiol and protopanoxadiol Enediol and protopanoxadiol
The single colonie of picking recombinant bacterium 2, recombinant bacterium 3 and recombinant bacterium 4 carries out shake flask fermentation.Fermentation condition is 30 DEG C, 220rpm is cultivated 3 days.Fermentation medium is YPX fluid nutrient medium, and wherein each component and its final concentration are as follows: final concentration of 4% The xylose of (mass percent), the peptone of 2% (mass percent), the yeast extract of 1% (mass percent) are supplied with water Volume.
After fermentation, it is extracted with n-butanol, n-butanol and fermentating liquid volume ratio are 1:4.N-butanol is taken mutually to carry out LC-MS detection, discovery recombinant bacterium 2 can produce 10.69mg/L dammarendiol, and recombinant bacterium 3 can produce 3.17mg/L up to Ma Enediol and 11.16mg/L protopanoxadiol, recombinant bacterium 4 can produce 5.57mg/L dammarendiol and 14.48mg/L protoplast Join glycol.Dammarendiol and protoplast's ginseng two in the LC-MS testing result and recombinant bacterial strain of dammarendiol and protopanoxadiol The yield comparison of alcohol is shown in attached drawing 2,3 and attached drawing 4.
A is 2 tunning of recombinant bacterium in Fig. 2, and B is dammarendiol standard items, and C is 3 tunning of recombinant bacterium, and D is original Panoxadiol standard items.
E is 3 tunning liquid chromatography mass spectrometric figure of recombinant bacterium in Fig. 3, and F is dammarendiol standard items liquid chromatography mass spectrometric figure, and G attaches most importance to Group 3 tunning liquid chromatography mass spectrometric figure of bacterium, H are protopanoxadiol standard items liquid chromatography mass spectrometric figure.
Sequence table
<110>University Of Tianjin
<120>recombinant Saccharomyces cerevisiae bacterium and the construction method of xylose production dammarendiol and protopanoxadiol are utilized
<141> 2018-06-12
<160> 128
<170> SIPOSequenceListing 1.0
<210> 1
<211> 710
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 1
ccatgtttcc aatgcccttc atgcctccaa cggctactat cacaaatcct catcaagctg 60
acgcaagccc taagaaatga ataacaatac tgacagtact aaataattgc ctacttggct 120
tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg attatcgtaa 180
tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg 240
ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt ggcatcctct 300
ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat atctaacaac 360
tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag tattggatgg 420
ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa aatctacaat 480
caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt cgcccacgtt 540
aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat aaaaagacaa 600
agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat tcttctgttc 660
ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa 710
<210> 2
<211> 957
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgccatcca tcaagttgaa ctctggttat gatatgccag ctgttggttt tggttgttgg 60
aaagttgatg ttgatacctg ttccgaacaa atctacagag ctattaagac cggttacaga 120
ttattcgatg gtgctgaaga ttacgccaac gaaaaattgg ttggtgctgg tgttaagaag 180
gctattgacg aaggtatcgt caagagagaa gatttgttct tgacctctaa gttgtggaac 240
aactaccatc atccagataa cgttgaaaag gctttgaaca gaaccttgtc tgacttgcaa 300
gttgattacg ttgacttgtt cttgatccat ttcccagtta ccttcaagtt cgttccattg 360
gaagaaaagt acccaccagg tttttactgt ggtaagggtg ataacttcga ctatgaagat 420
gtcccaattt tggaaacttg gaaggctttg gaaaagttgg ttaaggccgg taagattaga 480
tccattggtg tttctaattt cccaggtgct ttgttgttgg atttgttgag aggtgctacc 540
attaagccat ccgttttaca agttgaacac catccatact tgcaacaacc tagattgatc 600
gaatttgctc aatccagagg tattgctgtt actgcttact cttcttttgg tccacaatcc 660
ttcgtcgaat tgaatcaagg tagagctttg aacacctctc ctttgtttga aaacgaaacc 720
attaaggcta ttgctgctaa gcacggtaaa tctccagctc aagttttgtt gagatggtca 780
tctcaaagag gtattgccat tattccaaag tctaacaccg tcccaagatt attggaaaac 840
aaggatgtta actccttcga cttggatgaa caagatttcg ctgatattgc caagttggac 900
atcaacttga gattcaatga tccatgggat tgggataaga tcccaatttt cgtctaa 957
<210> 3
<211> 1092
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgactgcta acccttcctt ggtgttgaac aagatcgacg acatttcgtt cgaaacttac 60
gatgccccag aaatctctga acctaccgat gtcctcgtcc aggtcaagaa aaccggtatc 120
tgtggttccg acatccactt ctacgcccat ggtagaatcg gtaacttcgt tttgaccaag 180
ccaatggtct tgggtcacga atccgccggt actgttgtcc aggttggtaa gggtgtcacc 240
tctcttaagg ttggtgacaa cgtcgctatc gaaccaggta ttccatccag attctccgac 300
gaatacaaga gcggtcacta caacttgtgt cctcacatgg ccttcgccgc tactcctaac 360
tccaaggaag gcgaaccaaa cccaccaggt accttatgta agtacttcaa gtcgccagaa 420
gacttcttgg tcaagttgcc agaccacgtc agcttggaac tcggtgctct tgttgagcca 480
ttgtctgttg gtgtccacgc ctctaagttg ggttccgttg ctttcggcga ctacgttgcc 540
gtctttggtg ctggtcctgt tggtcttttg gctgctgctg tcgccaagac cttcggtgct 600
aagggtgtca tcgtcgttga cattttcgac aacaagttga agatggccaa ggacattggt 660
gctgctactc acaccttcaa ctccaagacc ggtggttctg aagaattgat caaggctttc 720
ggtggtaacg tgccaaacgt cgttttggaa tgtactggtg ctgaaccttg tatcaagttg 780
ggtgttgacg ccattgcccc aggtggtcgt ttcgttcaag tcggtaacgc tgctggtcca 840
gtcagcttcc caatcaccgt tttcgccatg aaggaattga ctttgttcgg ttctttcaga 900
tacggattca acgactacaa gactgctgtt ggaatctttg acactaacta ccaaaacggt 960
agagaaaatg ctccaattga ctttgaacaa ttgatcaccc acagatacaa gttcaaggac 1020
gctattgaag cctacgactt ggtcagagcc ggtaagggtg ctgtcaagtg tctcattgac 1080
ggccctgagt aa 1092
<210> 4
<211> 1008
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 4
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> 5
<211> 2043
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 5
atgactcaat tcactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60
gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120
gcacacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180
gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240
actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300
ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360
ggtatctcca acgctgttgg tatggccatg gctcaagcta acctggctgc cacttacaac 420
aagccgggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480
caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540
attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600
gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660
aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720
ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780
tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840
ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900
attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag cgaataccaa 960
aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020
aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080
ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140
gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200
tcttccggtt caggtaacta ctctggtaga tacattaggt acggtattag agaacacgct 1260
atgggtgcca taatgaacgg tatttcagct ttcggtgcca actacaaacc atacggtggt 1320
actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380
cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440
catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500
ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560
ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620
agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680
gctactggtt ccgaagtgtc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740
atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800
tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860
acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920
aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980
caaaagacca ttgcattcta taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040
taa 2043
<210> 6
<211> 1335
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 6
atgggaaagc tattacaatt ggcattgcat ccggtcgaga tgaaggcagc tttgaagctg 60
aagttttgca gaacaccgct attctccatc tatgatcagt ccacgtctcc atatctcttg 120
cactgtttcg aactgttgaa cttgacctcc agatcgtttg ctgctgtgat cagagagctg 180
catccagaat tgagaaactg tgttactctc ttttatttga ttttaagggc tttggatacc 240
atcgaagacg atatgtccat cgaacacgat ttgaaaattg acttgttgcg tcacttccac 300
gagaaattgt tgttaactaa atggagtttc gacggaaatg cccccgatgt gaaggacaga 360
gccgttttga cagatttcga atcgattctt attgaattcc acaaattgaa accagaatat 420
caagaagtca tcaaggagat caccgagaaa atgggtaatg gtatggccga ctacatctta 480
gatgaaaatt acaacttgaa tgggttgcaa accgtccacg actacgacgt gtactgtcac 540
tacgtagctg gtttggtcgg tgatggtttg acccgtttga ttgtcattgc caagtttgcc 600
aacgaatctt tgtattctaa tgagcaattg tatgaaagca tgggtctttt cctacaaaaa 660
accaacatca tcagagatta caatgaagat ttggtcgatg gtagatcctt ctggcccaag 720
gaaatctggt cacaatacgc tcctcagttg aaggacttca tgaaacctga aaacgaacaa 780
ctggggttgg actgtataaa ccacctcgtc ttaaacgcat tgagtcatgt tatcgatgtg 840
ttgacttatt tggccggtat ccacgagcaa tccactttcc aattttgtgc cattccccaa 900
gttatggcca ttgcaacctt ggctttggta ttcaacaacc gtgaagtgct acatggcaat 960
gtaaagattc gtaagggtac tacctgctat ttaattttga aatcaaggac tttgcgtggc 1020
tgtgtcgaga tttttgacta ttacttacgt gatatcaaat ctaaattggc tgtgcaagat 1080
ccaaatttct taaaattgaa cattcaaatc tccaagatcg aacagtttat ggaagaaatg 1140
taccaggata aattacctcc taacgtgaag ccaaatgaaa ctccaatttt cttgaaagtt 1200
aaagaaagat ccagatacga tgatgaattg gttccaaccc aacaagaaga agagtacaag 1260
ttcaatatgg ttttatctat catcttgtcc gttcttcttg ggttttatta tatatacact 1320
ttacacagag cgtga 1335
<210> 7
<211> 1491
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 7
atgtctgctg ttaacgttgc acctgaattg attaatgccg acaacacaat tacctacgat 60
gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg ctactggtct agcaagaaag 120
ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc ctgatagaat tgttggtgaa 180
ttgatgcaac caggtggtgt tagagcattg agaagtctgg gtatgattca atctatcaac 240
aacatcgaag catatcctgt taccggttat accgtctttt tcaacggcga acaagttgat 300
attccatacc cttacaaggc cgatatccct aaagttgaaa aattgaagga cttggtcaaa 360
gatggtaatg acaaggtctt ggaagacagc actattcaca tcaaggatta cgaagatgat 420
gaaagagaaa ggggtgttgc ttttgttcat ggtagattct tgaacaactt gagaaacatt 480
actgctcaag agccaaatgt tactagagtg caaggtaact gtattgagat attgaaggat 540
gaaaagaatg aggttgttgg tgccaaggtt gacattgatg gccgtggcaa ggtggaattc 600
aaagcccact tgacatttat ctgtgacggt atcttttcac gtttcagaaa ggaattgcac 660
ccagaccatg ttccaactgt cggttcttcg tttgtcggta tgtctttgtt caatgctaag 720
aatcctgctc ctatgcacgg tcacgttatt cttggtagtg atcatatgcc aatcttggtt 780
taccaaatca gtccagaaga aacaagaatc ctttgtgctt acaactctcc aaaggtccca 840
gctgatatca agagttggat gattaaggat gtccaacctt tcattccaaa gagtctacgt 900
ccttcatttg atgaagccgt cagccaaggt aaatttagag ctatgccaaa ctcctacttg 960
ccagctagac aaaacgacgt cactggtatg tgtgttatcg gtgacgctct aaatatgaga 1020
catccattga ctggtggtgg tatgactgtc ggtttgcatg atgttgtctt gttgattaag 1080
aaaataggtg acctagactt cagcgaccgt gaaaaggttt tggatgaatt actagactac 1140
catttcgaaa gaaagagtta cgattccgtt attaacgttt tgtcagtggc tttgtattct 1200
ttgttcgctg ctgacagcga taacttgaag gcattacaaa aaggttgttt caaatatttc 1260
caaagaggtg gcgattgtgt caacaaaccc gttgaatttc tgtctggtgt cttgccaaag 1320
cctttgcaat tgaccagggt tttcttcgct gtcgcttttt acaccattta cttgaacatg 1380
gaagaacgtg gtttcttggg attaccaatg gctttattgg aaggtattat gattttgatc 1440
acagctatta gagtattcac cccatttttg tttggtgagt tgattggtta a 1491
<210> 8
<211> 2310
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atgtggaagt tgaaggttgc tcagggaaat gacccttatt tatattctac aaataatttc 60
gtcggtagac agtactggga atttcagcca gacgctggta caccagagga gagggaggaa 120
gtcgagaagg caaggaaaga ctacgttaac aacaaaaagt tacatggtat acatccatgc 180
tcagacatgt taatgagaag acagttgatt aaagagtctg gaatagattt gttatcaata 240
ccaccattaa gattggacga gaacgaacag gtcaactacg acgcagtcac aacagcagtc 300
aagaaagcat tgagattgaa cagagcaata caggctcacg atggtcattg gcctgcagag 360
aacgcaggtt ctttgttata cacacctcct ttaattatag ctttatatat atctggtaca 420
atagacacaa tattaacaaa acaacacaag aaggaattga ttaggtttgt ttacaaccac 480
cagaacgagg atggaggttg gggttcatac atagagggtc actcaacaat gattggatct 540
gttttgtctt atgttatgtt gaggttgtta ggagagggat tggctgagtc agacgacgga 600
aacggtgcag ttgagagggg aaggaagtgg attttggacc atggaggtgc agctggtata 660
ccatcttggg gtaagactta cttggcagtc ttaggtgttt acgagtggga gggttgtaac 720
ccattgccac cagagttctg gttgtttcca tcttcttttc cattccatcc tgctaaaatg 780
tggatatact gcaggtgtac ttatatgcca atgtcatatt tgtatggaaa gaggtaccac 840
ggtccaatta cagacttggt cttatcatta aggcaggaaa tttataacat tccatacgaa 900
caaattaagt ggaaccaaca gaggcacaac tgctgcaaag aggacttgta ttacccacac 960
acattggtcc aggacttggt ctgggacggt ttacactact tctctgaacc attcttgaag 1020
aggtggccat ttaataagtt gaggaaaagg ggattgaaga gggtcgtcga gttgatgagg 1080
tacggtgcta cagagactag gttcataaca acaggaaacg gagaaaaggc attacagatt 1140
atgtcatggt gggctgagga ccctaacggt gacgagttta agcaccactt ggcaagaata 1200
cctgatttct tgtggatagc agaggacgga atgacagtcc agtctttcgg atcacagttg 1260
tgggactgta ttttggcaac tcaggcaata attgctacta atatggttga agaatatggt 1320
gactcattga agaaggcaca cttttttatt aaagagtcac agataaagga gaacccaaga 1380
ggtgattttt tgaagatgtg cagacagttc acaaagggag cttggacttt ctcagaccag 1440
gaccacggat gtgtcgtttc agattgcaca gcagaggcat taaaatgttt gttgttgttg 1500
tcacagatgc cacaggacat tgtcggtgag aagccagagg tcgagaggtt atacgaagca 1560
gtcaacgtct tattgtactt acaatcaagg gtctctggag gttttgctgt ttgggagcct 1620
ccagttccta agccatactt ggagatgtta aacccatcag aaatatttgc agacattgtc 1680
gtcgagagag agcacattga gtgtactgca tctgttataa agggattgat ggctttcaag 1740
tgcttacacc ctggtcacag gcaaaaggaa attgaagatt ctgtcgcaaa ggctattagg 1800
tacttggaaa ggaatcagat gccagacgga tcatggtacg gattctgggg tatttgcttt 1860
ttgtatggta ctttctttac tttgtctggt ttcgcatctg ctggtaggac ttacgacaac 1920
tctgaagctg tcaggaaggg tgttaagttt ttcttgtcaa cacaaaacga ggagggtggt 1980
tggggtgagt ctttagagtc atgcccatca gagaagttca ctccattgaa gggaaataga 2040
actaacttag tccagacatc ttgggctatg ttgggtttga tgttcggagg tcaggctgag 2100
agagatccta ctcctttgca cagagcagct aagttgttaa ttaatgctca gatggacaac 2160
ggtgatttcc cacagcaaga gattactggt gtctactgca aaaattctat gttacactac 2220
gctgagtaca ggaacatttt cccattgtgg gcattgggtg agtataggaa gagggtctgg 2280
ttgccaaagc accaacagtt aaagatataa 2310
<210> 9
<211> 1302
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atgactggta agaccggtca tattgatggt ttgaactcca gaatcgaaaa gatgagagat 60
ttggatccag ctcaaagatt ggttagagtt gctgaagctg ctggtttgga accagaagct 120
atttctgctt tggctggtaa tggtgctttg ccattgtctt tggctaatgg tatgatcgaa 180
aacgtcatcg gtaagttcga attgccattg ggtgttgcta ctaatttcac tgttaacggt 240
agagactact tgattccaat ggctgttgaa gaaccatctg ttgttgctgc tgcttcttat 300
atggctagaa ttgctagaga aaacggtggt tttactgctc atggtactgc tccattgatg 360
agagcacaaa ttcaagttgt tggtttgggt gatccagaag gtgctagaca aagattattg 420
gctcataagg ctgcttttat ggaagctgca gatgctgttg atccagtttt ggttggttta 480
ggtggtggtt gtagagatat cgaagttcac gtttttagag atactccagt tggtgccatg 540
gttgtcttgc atttgatagt tgatgttaga gatgctatgg gtgctaacac tgttaatacc 600
atggctgaaa gattggctcc agaagttgaa agaattgctg gtggtactgt tagattgagg 660
atcttgtcta atttggccga tttgagatta gttagagcca gagttgaatt ggctcctgaa 720
actttgacta ctcaaggtta tgatggtgct gatgttgcta gaggtatggt tgaagcttgt 780
gctttagcta tcgttgatcc atatagagct gctactcata acaagggtat tatgaacggt 840
atcgatccag ttgttgttgc cactggtaat gattggagag ctattgaagc tggtgcacat 900
gcttatgctg ctagaactgg tcattatact tcattgacca gatgggaatt agccaacgat 960
ggtagattgg ttggtactat tgaattgcct ttggccttgg gtttagtagg tggtgctaca 1020
aaaactcatc caactgctag agctgcattg gctttgatgc aagttgaaac tgctactgaa 1080
ttggcacaag ttactgctgc tgtaggtttg gctcaaaaca tggctgctat tagagctttg 1140
gctactgaag gtattcaaag gggtcacatg actttacatg ctagaaacat tgctattatg 1200
gctggtgcta ctggtgcaga tattgataga gttactagag ttattgtcga agccggtgat 1260
gtttctgttg caagagctaa acaagttttg gagaacacct aa 1302
<210> 10
<211> 1059
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 10
atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60
gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120
gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180
gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240
aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttacttctt ggtcgccgat 300
gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360
gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420
aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480
accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540
gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600
tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660
gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720
gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780
gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840
aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900
attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960
gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020
actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059
<210> 11
<211> 3411
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atggttttat ttttctcttt atcattgttg ttattacctt tattgttgtt gtttgcttac 60
ttctcataca caaagagaat tccacaaaag gaaaatgatt ctaaagctcc attgccacca 120
ggtcaaactg gttggccatt gattggtgaa actttgaact atttatcttg tgtcaaatct 180
ggtgtctctg agaatttcgt taagtacagg aaagaaaaat actcaccaaa ggttttcaga 240
acttcattat taggtgaacc aatggcaata ttgtgcggtc cagaaggtaa caaatttttg 300
tactctactg agaaaaaatt agttcaagtt tggtttcctt cttcagttga aaaaatgttc 360
ccaagatctc acggtgagtc aaacgctgat aacttttcta aggttagggg taaaatgatg 420
ttcttgttga aagttgatgg aatgaaaaaa tacgttggtt tgatggatag agttatgaag 480
caatttttgg aaacagattg gaatagacaa caacaaatta acgttcataa tactgtcaaa 540
aagtatactg tcactatgtc ttgtagagtt tttatgtcaa ttgatgatga agaacaagtt 600
acaaggttgg gttcttcaat tcaaaacatt gaagcaggtt tgttagcagt tccaataaat 660
atacctggta cagctatgaa cagagctata aagactgtta aattattgac tagagaagtt 720
gaggctgtta ttaaacaaag aaaggttgat ttgttagaaa acaagcaagc atctcaacca 780
caagacttgt tgtctcattt gttattaaca gctaatcaag atggtcaatt cttgtctgag 840
tctgatattg cttctcattt aattggtttg atgcaaggag gttacactac tttaaacggt 900
acaattactt tcgtcttgaa ttatttggca gaatttccag atgtttacaa tcaagttttg 960
aaagaacaag ttgaaattgc aaattctaag cacccaaaag aattgttgaa ttgggaggat 1020
ttaaggaaaa tgaagtattc ttggaacgtt gctcaagaag tcttgagaat tattccacca 1080
ggtgttggta ctttcaggga ggctattact gatttcactt atgcaggtta cttgattcca 1140
aaaggttgga aaatgcattt gattccacat gatactcata agaatccaac ttattttcca 1200
tctccagaaa aattcgatcc aactagattt gagggaaacg gtccagctcc atacactttt 1260
acacctttcg gtggtggtcc aagaatgtgc ccaggtattg agtacgcaag gttggttatt 1320
ttaattttca tgcataatgt cgtcacaaat tttaggtggg aaaaattgat tccaaacgaa 1380
aagattttaa ctgatccaat tccaagattt gcacatggtt tacctattca tttgcatcca 1440
cataacggtt ctacttcttc aggttcaggt tggaaaaaga caacagctga taggtctggt 1500
gaattaaagc cattaatgat acctaaatct ttaatggcta aggacgagga cgacgacttg 1560
gatttaggat caggaaagac tagagtctct atatttttcg gaactcagac aggaacagct 1620
gagggattcg caaaggcttt atcagaagag attaaagcaa ggtacgagaa ggctgctgtc 1680
aaagttatag atttggatga ctacgcagct gatgacgacc agtacgagga aaagttgaaa 1740
aaggaaactt tggcattttt ctgtgttgca acatacggtg acggtgagcc aactgacaac 1800
gctgctaggt tctacaaatg gttcacagag gaaaatgaga gagacattaa attgcagcag 1860
ttggcttacg gtgtcttcgc attgggaaac aggcaatatg aacatttcaa taagattgga 1920
attgtcttgg acgaagaatt atgcaaaaaa ggagctaaga ggttgataga ggtcggtttg 1980
ggtgacgatg accagtcaat agaggacgac ttcaatgcat ggaaagagtc attgtggtca 2040
gagttagata agttattaaa agacgaagac gacaagtcag tcgcaacacc ttacacagca 2100
gtcatacctg agtatagggt cgtcactcac gacccaagat tcactactca aaagtcaatg 2160
gagtcaaatg tcgcaaacgg aaatactact attgacattc atcacccatg cagggttgac 2220
gtcgctgtcc agaaagagtt acacactcac gagtctgaca ggtcatgcat tcacttggag 2280
ttcgatattt caagaactgg tattacttac gaaacaggtg accacgttgg tgtctacgct 2340
gagaaccacg tcgagattgt cgaggaagct ggaaagttgt tgggacattc tttagatttg 2400
gtcttctcaa ttcatgctga caaagaggac ggttcaccat tggagtctgc tgttccacca 2460
ccattccctg gaccatgcac tttaggtact ggtttggcaa ggtacgcaga cttattgaac 2520
ccacctagga agtcagcttt agttgcattg gctgcatatg caacagaacc atctgaggca 2580
gagaaattaa agcacttgac ttctcctgac ggtaaggacg agtactcaca gtggatagtc 2640
gcatctcaga ggtcattgtt ggaggtcatg gcagcatttc catcagcaaa gccaccttta 2700
ggtgttttct tcgcagctat agcacctaga ttgcagccta ggtattattc aatatcttct 2760
tcacctaggt tggctccatc tagggtccac gtcacatcag ctttggttta cggacctact 2820
cctacaggaa ggatacataa aggagtctgc tctacttgga tgaagaacgc tgtcccagca 2880
gagaagtctc atgagtgctc aggagctcct atttttatta gggcatcaaa tttcaaattg 2940
ccttcaaacc catctactcc aatagtcatg gtcggaccag gaacaggttt ggctcctttc 3000
aggggatttt tgcaggagag gatggctttg aaggaggatg gtgaggaatt gggatcatct 3060
ttgttgttct ttggttgtag gaataggcaa atggacttca tttatgagga cgaattgaac 3120
aactttgttg atcaaggagt catatcagag ttaattatgg ctttctcaag ggagggtgca 3180
caaaaggaat acgtccaaca caagatgatg gaaaaggctg cacaggtctg ggacttgatt 3240
aaggaggagg gatacttata tgtctgcggt gacgcaaagg gtatggcaag agacgtccac 3300
aggactttgc acacaattgt ccaggaacag gagggtgttt cttcatctga agcagaggct 3360
attgttaaaa agttgcaaac tgaaggtagg tacttgaggg acgtctggta a 3411
<210> 12
<211> 1197
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 12
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> 13
<211> 1476
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 13
atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60
caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120
gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180
caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240
attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300
tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360
agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420
atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480
ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540
agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca 600
accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660
tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc 720
gaatatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa 780
gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt 840
tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt 900
aaattggtca caaaatcata cggtagatta ctatataacg atttcagagc caatcctcaa 960
ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat 1020
aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc 1080
caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc 1140
tttgcatctc tattaaacta tgttggatct gacgacttac aaggcaagcg tgttggttta 1200
ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260
caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320
ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380
aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440
gacaaattta gaagatctta cgatgttaaa aaataa 1476
<210> 14
<211> 1332
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 14
atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60
gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120
ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180
cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240
ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tcttttggat 300
ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360
atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420
cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480
gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540
catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600
atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660
ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720
ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780
gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840
ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900
gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960
ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020
gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080
ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140
gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200
gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260
aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320
tggacttcat aa 1332
<210> 15
<211> 496
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 15
ggcgtctgat ttccgttttg ggaatccttt gccgcgcgcc cctctcaaaa ctccgcacaa 60
gtcccagaaa gcgggaaaga aataaaacgc caccaaaaaa aaaaaaataa aagccaatcc 120
tcgaagcgtg ggtggtaggc cctggattat cccgtacaag tatttctcag gagtaaaaaa 180
accgtttgtt ttggaattcc ccatttcgcg gccacctacg ccgctatctt tgcaacaact 240
atctgcgata actcagcaaa ttttgcatat tcgtgttgca gtattgcgat aatgggagtc 300
ttacttccaa cataacggca gaaagaaatg tgagaaaatt ttgcatcctt tgcctccgtt 360
caagtatata aagtcggcat gcttgataat ctttctttcc atcctacatt gttctaatta 420
ttcttattct cctttattct ttcctaacat accaagaaat taatcttctg tcattcgctt 480
aaacactata tcaata 496
<210> 16
<211> 513
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 16
agtgacctgg ctctatagtg ttgtccctct cgcgaggacc attgttgctt gcatatgggc 60
ttgaaacata tggtcatcac atctgagcga ttttacctct tagaattagt ttagatatat 120
atgagttgat gaataaatag ttataaaaac ttgctttggc ttcgatatat gaccgttatt 180
tttgactaag ttttaacgaa ggaatctaac ctcgttcttg taattaccaa aatcttcaac 240
aacgcgctgt tggaggtatc tctatggatg tggcttgaaa tatggatgtc ttgcctactt 300
ctacttctgg gaaaggcatt tttactcgat cgcgttaata tatgcatcaa gaaaataaaa 360
aataaaacgc gaagagctaa aaaaaaaaaa gaaaacctac tataaataac cgattagaat 420
cgagtttttg tattgaaatg gcggtaataa gcgttaaacc tcgacgaaga gagaagatcc 480
tacaggaggt aaaaaacagc tcggtatatc aaa 513
<210> 17
<211> 700
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 17
tctagtttct gccttaaaca aagccgcagc cagagccgtt tttccgccat atttatccag 60
gattgttcca tacggctccg tcagaggctg ctacgggatg tttttttttt accccgtgga 120
aatgaggggt atgcaggaat ttgtgcgggg taggaaatct tttttttttt taggaggaac 180
aactggtgga agaatgccca cacttctcag aaatgcatgc agtggcagca cgctaattcg 240
aaaaaattct ccagaaaggc aacgcaaaat tttttttcca gggaataaac tttttatgac 300
ccactacttc tcgtaggaac aatttcgggc ccctgcgtgt tcttctgagg ttcatctttt 360
acatttgctt ctgctggata attttcagag gcaacaagga aaaattagat ggcaaaaagt 420
cgtctttcaa ggaaaaatcc ccaccatctt tcgagatccc ctgtaactta ttggcaactg 480
aaagaatgaa aaggaggaaa atacaaaata tactagaact gaaaaaaaaa aagtataaat 540
agagacgata tatgccaata cttcacaatg ttcgaatcta ttcttcattt gcagctattg 600
taaaataata aaacatcaag aacaaacaag ctcaacttgt cttttctaag aacaaagaat 660
aaacacaaaa acaaaaagtt tttttaattt taatcaaaaa 700
<210> 18
<211> 616
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 18
tttgcgaaca cttttattaa ttcatgatca cgctctaatt tgtgcatttg aaatgtactc 60
taattctaat tttatatttt taatgatatc ttgaaaagta aatacgtttt taatatatac 120
aaaataatac agtttaattt tcaagttttt gatcatttgt tctcagaaag ttgagtggga 180
cggagacaaa gaaactttaa agagaaatgc aaagtgggaa gaagtcagtt gtttaccgac 240
cgcactgtta ttcacaaata ttccaatttt gcctgcagac ccacgtctac aaattttggt 300
tagtttggta aatggtaagg atatagtaga gcctttttga aatgggaaat atcttctttt 360
tctgtatccc gcttcaaaaa gtgtctaatg agtcagttat ttctttctta ctcatcgccc 420
gtcacttaaa agaagaaaaa ttactttcat gatgcgaagc gaaaaaaatt tttagcttca 480
attttcacaa tgcatctatg gagaggatat tataaggtta cgaaataaat tcttgagtgt 540
tgtaaattct gttaatcaaa gaaaaagcaa tagctcgttt ttctacagaa tggctagcac 600
agcaaatatg atttct 616
<210> 19
<211> 505
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 19
gcgggattta aactgtgagg accttaatac attcagacac ttctgcggta tcaccctact 60
tattcccttc gagattatat ctaggaaccc atcaggttgg tggaagatta cccgttctaa 120
gacttttcag cttcctctat tgatgttaca cctggacacc ccttttctgg catccagttt 180
ttaatcttca gtggcatgtg agattctccg aaattaatta aagcaatcac acaattctct 240
cggataccac ctcggttgaa actgacaggt ggtttgttac gcatgctaat gcaaaggagc 300
ctatatacct ttggctcggc tgctgtaaca gggaatataa agggcagcat aatttaggag 360
tttagtgaac ttgcaacatt tactattttc ccttcttacg taaatatttt tctttttaat 420
tctaaatcaa tctttttcaa ttttttgttt gtattctttt cttgcttaaa tctataacta 480
caaaaaacac atacataaac taaaa 505
<210> 20
<211> 476
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 20
gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60
tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120
ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180
attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240
acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300
tttccttcgc atgccagcat tgaaatgatc gaagttcaat gatgaaacgg taattcttct 360
gtcatttact catctcatct catcaagtta tataattcta tacggatgta atttttcact 420
tttcgtcttg acgtccaccc tataatttca attattgaac cctcacaaat gatgca 476
<210> 21
<211> 464
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 21
ccgcgaatcc ttacatcaca cccaatcccc cacaagtgat cccccacaca ccatagcttc 60
aaaatgtttc tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta 120
ccacttcaaa acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt 180
cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc 240
ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt tttcttgaaa attttttttt 300
ttgatttttt tctctttcga tgacctccca ttgatattta agttaataaa cggtcttcaa 360
tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgctca 420
ttagaaagaa agcatagcaa tctaatctaa gttttaatta caaa 464
<210> 22
<211> 286
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 22
gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60
tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120
ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180
tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata 240
tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttatg 286
<210> 23
<211> 778
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 23
tattttagat tcctgacttc aactcaagac gcacagatat tataacatct gcataatagg 60
catttgcaag aattactcgt gagtaaggaa agagtgagga actatcgcat acctgcattt 120
aaagatgccg atttgggcgc gaatccttta ttttggcttc accctcatac tattatcagg 180
gccagaaaaa ggaagtgttt ccctccttct tgaattgatg ttaccctcat aaagcacgtg 240
gcctcttatc gagaaagaaa ttaccgtcgc tcgtgatttg tttgcaaaaa gaacaaaact 300
gaaaaaaccc agacacgctc gacttcctgt cttcctattg attgcagctt ccaatttcgt 360
cacacaacaa ggtcctagcg acggctcaca ggttttgtaa caagcaatcg aaggttctgg 420
aatggcggga aagggtttag taccacatgc tatgatgccc actgtgatct ccagagcaaa 480
gttcgttcga tcgtactgtt actctctctc tttcaaacag aattgtccga atcgtgtgac 540
aacaacagcc tgttctcaca cactcttttc ttctaaccaa gggggtggtt tagtttagta 600
gaacctcgtg aaacttacat ttacatatat ataaacttgc ataaattggt caatgcaaga 660
aatacatatt tggtcttttc taattcgtag tttttcaagt tcttagatgc tttctttttc 720
tcttttttac agatcatcaa ggaagtaatt atctactttt tacaacaaat ataaaaca 778
<210> 24
<211> 305
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 24
gcttacattc acgccctcct cccacatccg ctctaaccga aaaggaagga gttagacaac 60
ctgaagtcta ggtccctatt tatttttttt aatagttatg ttagtattaa gaacgttatt 120
tatatttcaa atttttcttt tttttctgta caaacgcgtg tacgcatgta acattatact 180
gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgca agcttcgcag 240
tttacactct catcgtcgct ctcatcatcg cttccgttgt tgttttcctt agtagcgtct 300
gcttc 305
<210> 25
<211> 680
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 25
cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 60
agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 120
acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 180
acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 240
aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 300
ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 360
ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 420
atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 480
agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 540
atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 600
tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 660
aacacacata aacaaacaaa 680
<210> 26
<211> 285
<212> DNA
<213>saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 26
gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60
tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120
ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 180
tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgtagata 240
tgctaactcc agcaatgagt tgatgaatct cggtgtgtat tttat 285
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ggctcaatta acagggtcca 20
<210> 28
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cggagagggg tgatacttat 20
<210> 29
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ctggtcaccg ataagtatca cccctctccg tgtaaaacga cggccagt 48
<210> 30
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ttggaggcat gaagggcatt ggaaacatgg gagtctttta catcttcgga 50
<210> 31
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ccatgtttcc aatgccctt 19
<210> 32
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tttgaatatg tattacttgg ttatggtt 28
<210> 33
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
atataaccat aaccaagtaa tacatattca aaatgttgtg ttcagtaatt ca 52
<210> 34
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
attttttcag gcccacctat gcactcttca aactc 35
<210> 35
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gcttcggtta cttctaagga agtccacaca aatcaagatc cgttaga 47
<210> 36
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
aaaggattcc caaaacggaa atcagacgcc ttggaaagtc attaggtgag 50
<210> 37
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
ggcgtctgat ttccgtttt 19
<210> 38
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tattgatata gtgtttaagc gaatgac 27
<210> 39
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
tctgtcattc gcttaaacac tatatcaata atgccatcca tcaagttgaa ct 52
<210> 40
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
agagggacaa cactatagag ccaggtcact tcattagacg aaaattggga tctt 54
<210> 41
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
aagtgacctg gctctatagt gttgtccctc tc 32
<210> 42
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
aaggcagaaa ctagatttga tataccgagc tgt 33
<210> 43
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
acagctcggt atatcaaatc tagtttctgc ctttctagtt tctgccttaa aca 53
<210> 44
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
tttttgatta aaattaaaaa aactttttgt ttttgtgttt attctttgtt cttag 55
<210> 45
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
caaaaacaaa aagttttttt aattttaatc aaaaaatgac tgctaaccct tccttggt 58
<210> 46
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
cgtgatcatg aattaataaa agtgttcgca aattactcag ggccgtcaat gag 53
<210> 47
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
tttgcgaaca cttttattaa ttcat 25
<210> 48
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
gagaaatcat atttgctgtg ct 22
<210> 49
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
gaatggctag cacagcaaat atgatttctc actgtgggaa tactcaggta 50
<210> 50
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
tagttagtag atgatagttg atttctattc caacattttc cttatcacgt tgagcc 56
<210> 51
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
tgttggaata gaaatcaact atcatctact 30
<210> 52
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
cacaggcgct accatgagaa 20
<210> 53
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
accaaaacca acggatatca tacattacac taccaccatt caaactt 47
<210> 54
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
aaaggattcc caaaacggaa atcagacgcc gagggacggt tgaaagtgga 50
<210> 55
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
tctgtcattc gcttaaacac tatatcaata atgactcaat tcactgacat t 51
<210> 56
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
agagggacaa cactatagag ccaggtcact ttagaaagct tttttcaaag gaga 54
<210> 57
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
caaaaacaaa aagttttttt aattttaatc aaaaaatgtc tgaaccagct caaaaga 57
<210> 58
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
cgtgatcatg aattaataaa agtgttcgca aattaagcgg taactttctt ttcaatc 57
<210> 59
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
gaatggctag cacagcaaat atgatttctc tgtaaaacga cggccagt 48
<210> 60
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
tagttagtag atgatagttg atttctattc caacactgtt ctatatgctg ccact 55
<210> 61
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
tgttggaata gaaatcaact atcatctact aactagtttc acggaatggt acgtt 55
<210> 62
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
gtgaggaaaa gtagttggga ggtacttcat gcgaaa 36
<210> 63
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
agtggcagca tatagaacag gagaaaatac cgcatcagga 40
<210> 64
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
actggccgtc gttttacatt cttccttctg ttcggaga 38
<210> 65
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
tctccgaaca gaaggaagaa tgtaaaacga cggccagt 38
<210> 66
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
tcctgatgcg gtattttctc ctgttctata tgctgccact 40
<210> 67
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
ccaacggata tcatacatta cactaccacc attcaaactt 40
<210> 68
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
ccgtcgtttt acagagggac ggttgaaagt gga 33
<210> 69
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
ctttcaaccg tccctctgta aaacgacggc cagt 34
<210> 70
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
gaagtcagga atctaaaata gagtctttta catcttcgga 40
<210> 71
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
cgaagatgta aaagactcta ttttagattc ctgacttcaa c 41
<210> 72
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
ccttcaactt ccacattgtt ttatatttgt tgtaaaaagt agataa 46
<210> 73
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
ctttttacaa caaatataaa acaatgtgga agttgaaggt tgct 44
<210> 74
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
gcgtgaatgt aagcttatat ctttaactgt tggtgctttg 40
<210> 75
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
caccaacagt taaagatata agcttacatt cacgccctcc 40
<210> 76
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
tgtaaggatt cgcgggaagc agacgctact aagga 35
<210> 77
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
agtagcgtct gcttcccgcg aatccttaca tcaca 35
<210> 78
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
cgttaacagc agacattttg taattaaaac ttagattaga ttgc 44
<210> 79
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
ctaatctaag ttttaattac aaaatgtctg ctgttaacgt tgca 44
<210> 80
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
cataaatcat aagaaattcg cttaaccaat caactcacca aacaa 45
<210> 81
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 81
ggtgagttga ttggttaagc gaatttctta tgatttatga ttt 43
<210> 82
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 82
cagtttaaat cccgccataa aatacacacc gagattcat 39
<210> 83
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 83
ctcggtgtgt attttatggc gggatttaaa ctgtgagga 39
<210> 84
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 84
gtaatagctt tcccattttt agtttatgta tgtgtttttt gtag 44
<210> 85
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 85
cacatacata aactaaaaat gggaaagcta ttacaattgg 40
<210> 86
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 86
gacataagag atccgctcac gctctgtgta aagtgta 37
<210> 87
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 87
tacacagagc gtgagcggat ctcttatgtc tttacg 36
<210> 88
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 88
gtaccattcc gtgaaacgtg catcatttgt gagggtt 37
<210> 89
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 89
cctcacaaat gatgcacgtt tcacggaatg gtacgtt 37
<210> 90
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 90
gtgaggaaaa gtagttggga ggtacttcat gcgaaa 36
<210> 91
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 91
gcttcggtta cttctaagga agtccacaca aatcaagat 39
<210> 92
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 92
gaatgtatta aggtcctcac agtttaaatc ccgcttggaa agtcattagg tgaggt 56
<210> 93
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 93
gcgggattta aactgtgagg acct 24
<210> 94
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 94
ttttagttta tgtatgtgtt ttttgtagtt atagatttaa gcaag 45
<210> 95
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 95
tgcttaaatc tataactaca aaaaacacat acataaacta aaaatggctt cagaaaaaga 60
aattaggaga ga 72
<210> 96
<211> 62
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 96
ttaggtgagg ttaacattgg tggtggtctg acatacaact tactcttaat cggacttgct 60
ca 62
<210> 97
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 97
tatgtcagac caccaccaat gttaacctca cctaatattt tagattcctg acttcaac 58
<210> 98
<211> 70
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 98
tggagttcaa accatcaata tgaccggtct taccagtcat tgttttatat ttgttgtaaa 60
aagtagataa 70
<210> 99
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 99
atgactggta agaccggtca tattgat 27
<210> 100
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 100
ttaggtgttc tccaaaactt gtttagctct 30
<210> 101
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 101
gcaagagcta aacaagtttt ggagaacacc taaatcatgt aattagttat gtcacgctta 60
cattca 66
<210> 102
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 102
ttaaagcctt cgagcgtccc aaaaccttct caagc 35
<210> 103
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 103
gaaggttttg ggacgctcga aggctttaac agttcgagtt tatcattatc aatactg 57
<210> 104
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 104
gagaaaaata aaaccatttt gtttgtttat gtgtgtttat tcga 44
<210> 105
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 105
cacacataaa caaacaaaat ggttttattt ttctctttat cattgt 46
<210> 106
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 106
cataaatcat aagaaattcg cttaccagac gtccctcaag t 41
<210> 107
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 107
gagggacgtc tggtaagcga atttcttatg atttatgatt t 41
<210> 108
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 108
cataaaatac acaccgagat tcat 24
<210> 109
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 109
tgtaaaacga cggccagtgc caagcttgca tgtaaaacga cggccagt 48
<210> 110
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 110
ctagttagta gatgatagtt gatttctatt ccaacactgt tctatatgct gccact 56
<210> 111
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 111
tgttggaata gaaatcaact atcatctact aactag 36
<210> 112
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 112
cacaggcgct accatgagaa ttg 23
<210> 113
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 113
acagctcggt atatcaaatc tagtttctgc ctttctagtt tctgccttaa aca 53
<210> 114
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 114
tacaatgtaa acgttctgag acattttttg attaaaatta aaaaaacttt ttgtttttg 59
<210> 115
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 115
ctaagaacaa agaataaaca caaaaacaaa aagttttttt aattttaatc aaaaaatgt 59
<210> 116
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 116
gagttgaagt caggaatcta aaatatagaa ttatataact tgatgagatg tt 52
<210> 117
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 117
catctcatca agttatataa ttctatattt tagattcctg acttcaactc 50
<210> 118
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 118
gttaagaacg gtaatgacat tgttttatat ttgttgtaaa aagtagataa 50
<210> 119
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 119
ctacttttta caacaaatat aaaacaatgt cattaccgtt cttaacttct 50
<210> 120
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 120
gtaaggattc gcggcgtaaa attcgcgggt gga 33
<210> 121
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 121
cgcgaatttt acgccgcgaa tccttacatc aca 33
<210> 122
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 122
aagtttagtt gagagtttca ttttgtaatt aaaacttaga ttagattgct atgc 54
<210> 123
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 123
gcaatctaat ctaagtttta attacaaaat gaaactctca actaaacttt gttg 54
<210> 124
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 124
caacgtcgtg actgggaaaa aacagcatac cctgaataca tggaaaacac gtc 53
<210> 125
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 125
ggtatgctgt tttttcccag tcacgacgtt gtaaaacggt tagctatttc gcccaa 56
<210> 126
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 126
tagttagtag atgatagttg atttctattc caacacgcta tcctcggttc tgcat 55
<210> 127
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 127
tgttggaata gaaatcaact atcatctact 30
<210> 128
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 128
cacaggcgct accatgagaa ttgggtgaat gttgagataa ttgtt 45

Claims (6)

1. using the recombinant Saccharomyces cerevisiae bacterium construction method of xylose production dammarendiol and protopanoxadiol, it is characterized in that including Following steps:
It (1) is to open by the promoter replacement of the endogenous xylulokinase gene XKS1 of saccharomyces cerevisiae by the method for homologous recombination Mover PFBA1, then to the site δ of saccharomyces cerevisiae import Xylose reductase gene XYL1 expression cassette and xylose dehydrogenase gene XYL2 expression cassette, obtains recombinant bacterium；Transaldolase gene TAL1 table in pentose phosphate pathway is imported to the site rDNA of recombinant bacterium again Up to box and tkt gene TKL1 expression cassette, the recombinant Saccharomyces cerevisiae bacterium 1 of fermenting xylose is obtained；
The promoter PFBA1Nucleotide sequence as shown in SEQ ID NO.1；
The nucleotide sequence of Xylose reductase gene XYL1 is as shown in SEQ ID NO.2；
The nucleotide sequence of xylitol dehydrogenase gene XYL2 is as shown in SEQ ID NO.3；
The nucleotide sequence of transaldolase gene TAL1 is as shown in SEQ ID NO.4；
The nucleotide sequence of tkt gene TKL1 is as shown in SEQ ID NO.5；
(2) by the method for homologous recombination, to the site the rDNA introductory technique acyl of the recombinant Saccharomyces cerevisiae bacterium 1 of the fermenting xylose Bisphosphate Farnesyltransferase gene ERG9 expression cassette, squalene monooxygenase gene ERG1 expression cassette and dammarendiol close Enzyme gene DS expression cassette obtains the recombinant Saccharomyces cerevisiae bacterium 2 of fermenting xylose；
The nucleotide sequence of Farnesyl-diphosphate farnesyltransferase gene ERG9 is as shown in SEQ ID NO.6；
The nucleotide sequence of squalene monooxygenase gene ERG1 is as shown in SEQ ID NO.7；
The nucleotide sequence of dammarendiol synthase gene DS is as shown in SEQ ID NO.8；
(3) by the method for homologous recombination, nicotinamide adenine is imported to the site δ of the recombinant Saccharomyces cerevisiae bacterium 2 of fermenting xylose Dinucleotides -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr expression cassette, farnesyl phosphate synthase gene ERG20 Expression cassette, protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 expression cassette, are utilized The recombinant Saccharomyces cerevisiae bacterium 3 of xylose production dammarendiol and protopanoxadiol；
Nicotinamide adenine dinucleotide -3-hydroxy-3-methylglutaryl coenzyme A reductase gene NADH-HMGr nucleotide sequence is such as Shown in SEQ ID NO.9；
The nucleotide sequence of farnesyl phosphate synthase gene ERG20 is as shown in SEQ ID NO.10；
Protopanoxadiol synthase-cytochrome P450 reductase antigen-4 fusion protein gene PPDS-ATR1 nucleotide sequence such as SEQ Shown in ID NO.11.
2. the recombinant Saccharomyces cerevisiae bacterium using xylose production dammarendiol and protopanoxadiol of method of claim 1 building 3。
3. claim 2 is raw using the recombinant Saccharomyces cerevisiae bacterium fermenting xylose of xylose production dammarendiol and protopanoxadiol Produce the application of dammarendiol and protopanoxadiol.
4. the construction method of the recombinant Saccharomyces cerevisiae bacterium using xylose production dammarendiol and protopanoxadiol, it is characterized in that packet Include following steps:
By the method for homologous recombination, to the recombinant Saccharomyces cerevisiae bacterium with xylose production dammarendiol and protopanoxadiol 3 site δ imports acetyl-CoA acyltransferase gene ERG10 expression cassette, Hydroxymethylglutaryl-CoA synthase gene ERG13 Expression cassette and mevalonate kinase gene ERG12 expression cassette, obtain the recombinant Saccharomyces cerevisiae bacterium 4 of fermenting xylose；
The nucleotide sequence of acetyl-CoA acyltransferase gene ERG10 is as shown in SEQ ID NO.12；
The nucleotide sequence of Hydroxymethylglutaryl-CoA synthase gene ERG13 is as shown in SEQ ID NO.13；
The nucleotide sequence of mevalonate kinase gene ERG12 is as shown in SEQ ID NO.14.
5. the recombinant Saccharomyces cerevisiae bacterium using xylose production dammarendiol and protopanoxadiol of method for claim 4 building 4。
6. 4 fermenting xylose of recombinant Saccharomyces cerevisiae bacterium using xylose production dammarendiol and protopanoxadiol of claim 5 Produce the application of dammarendiol and protopanoxadiol.
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CN110982720A (en) * 2019-12-13 2020-04-10 天津大学 Recombinant yarrowia lipolytica producing dammarane diol and protopanoxadiol and use thereof
CN111118052A (en) * 2020-01-19 2020-05-08 天津大学 Recombinant saccharomyces cerevisiae, construction method thereof and application of recombinant saccharomyces cerevisiae in production of hydroxy fatty acid
CN111378681A (en) * 2018-12-27 2020-07-07 中国医学科学院药物研究所 Recombinant bacterium for producing dammarenediol-II glucoside and application thereof
CN111440733A (en) * 2020-02-07 2020-07-24 天津大学 Recombinant saccharomyces cerevisiae for producing terpineol, construction method and application
CN111471704A (en) * 2019-01-23 2020-07-31 中国医学科学院药物研究所 Recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof
CN114150012A (en) * 2021-11-17 2022-03-08 天津大学 Recombinant saccharomyces cerevisiae for heterogeneously synthesizing ginsenoside F2 and construction method thereof
CN114807211A (en) * 2022-05-13 2022-07-29 天津大学 Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method
CN115261243A (en) * 2021-04-30 2022-11-01 中国科学院天津工业生物技术研究所 Recombinant saccharomyces cerevisiae as well as construction method and application thereof
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CN115340957A (en) * 2022-08-29 2022-11-15 广西科学院 Construction method and application of protopanoxadiol yeast cell factory
WO2023039518A1 (en) * 2021-09-09 2023-03-16 Manus Bio Inc. Enzymes, host cells, and methods for biosynthesis of dammarenediol and derivatives

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CN111378681A (en) * 2018-12-27 2020-07-07 中国医学科学院药物研究所 Recombinant bacterium for producing dammarenediol-II glucoside and application thereof
CN111378681B (en) * 2018-12-27 2023-01-17 中国医学科学院药物研究所 Recombinant bacterium for producing dammarenediol-II glucoside and application thereof
CN111471704A (en) * 2019-01-23 2020-07-31 中国医学科学院药物研究所 Recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof
CN111471704B (en) * 2019-01-23 2024-02-06 中国医学科学院药物研究所 Recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof
CN110484553A (en) * 2019-06-13 2019-11-22 叁爻生物科技(上海)有限公司 The construction method of recombinant yeast and application
CN110982720A (en) * 2019-12-13 2020-04-10 天津大学 Recombinant yarrowia lipolytica producing dammarane diol and protopanoxadiol and use thereof
CN111118052A (en) * 2020-01-19 2020-05-08 天津大学 Recombinant saccharomyces cerevisiae, construction method thereof and application of recombinant saccharomyces cerevisiae in production of hydroxy fatty acid
CN111440733A (en) * 2020-02-07 2020-07-24 天津大学 Recombinant saccharomyces cerevisiae for producing terpineol, construction method and application
CN115261243A (en) * 2021-04-30 2022-11-01 中国科学院天津工业生物技术研究所 Recombinant saccharomyces cerevisiae as well as construction method and application thereof
CN115261243B (en) * 2021-04-30 2024-02-06 中国科学院天津工业生物技术研究所 Recombinant saccharomyces cerevisiae as well as construction method and application thereof
CN115305254A (en) * 2021-05-08 2022-11-08 中国科学院天津工业生物技术研究所 Terpenoid chassis microorganism and engineering bacterium as well as construction method and application thereof
CN115305254B (en) * 2021-05-08 2024-03-26 中国科学院天津工业生物技术研究所 Terpenoid chassis microorganism and engineering bacterium as well as construction method and application thereof
WO2023039518A1 (en) * 2021-09-09 2023-03-16 Manus Bio Inc. Enzymes, host cells, and methods for biosynthesis of dammarenediol and derivatives
CN114150012A (en) * 2021-11-17 2022-03-08 天津大学 Recombinant saccharomyces cerevisiae for heterogeneously synthesizing ginsenoside F2 and construction method thereof
CN114807211A (en) * 2022-05-13 2022-07-29 天津大学 Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method
CN114807211B (en) * 2022-05-13 2023-06-27 天津大学 Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method
CN115340957A (en) * 2022-08-29 2022-11-15 广西科学院 Construction method and application of protopanoxadiol yeast cell factory
CN115340957B (en) * 2022-08-29 2023-09-29 广西科学院 Construction method and application of protopanaxadiol yeast cell factory

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