CN108949601B - Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose and construction method - Google Patents

Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose and construction method Download PDF

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CN108949601B
CN108949601B CN201810781608.0A CN201810781608A CN108949601B CN 108949601 B CN108949601 B CN 108949601B CN 201810781608 A CN201810781608 A CN 201810781608A CN 108949601 B CN108949601 B CN 108949601B
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xylose
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卢文玉
高晓
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Tianjin University
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Abstract

The invention discloses a recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by utilizing xylose and a construction method thereof, wherein a promoter of a saccharomyces cerevisiae xylulokinase gene XKS1 is replaced by a promoter P through a homologous recombination methodFBA1Then introducing a xylose reductase XYL1 and a xylitol dehydrogenase XYL2 expression cassette, then improving the activities of transketolase TKL1 and transaldolase TAL1 to obtain a recombinant bacterium 1, then introducing a farnesyl diphosphate farnesyl transferase gene ERG9, a squalene monooxygenase gene ERG1 and a dammarenediol synthase gene DS into the recombinant bacterium 1 to obtain a recombinant bacterium 2, and introducing a nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase gene NADH-HMGr, a farnesyl pyrophosphate synthase ERG20 and a protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 into the recombinant bacterium 2 to obtain a recombinant bacterium 3.

Description

Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose and construction method
Technical Field
The invention relates to the technical field of biology, in particular to recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by utilizing xylose, a construction method and application thereof.
Background
Xylose is a pentose, and natural D-xylose is present in plants in the form of polysaccharides, and xylose in a free state has not been found in nature. Lignocellulose is used as a main component substance of plant cell walls and mainly comprises three main components, namely cellulose, hemicellulose and lignin. Lignocellulosic biomass produced by photosynthesis on earth is more than 1000 hundred million tons per year, and is the most abundant renewable energy resource on earth. And monosaccharide substances such as glucose, xylose and the like can be obtained by hydrolyzing lignocellulose. The utilization of xylose, a monosaccharide second only to glucose in the content of lignocellulose hydrolysate, has attracted extensive attention by researchers.
Although there are many bacteria and yeasts that naturally utilize xylose, Saccharomyces cerevisiae has distinct advantages over it, being robust under various industrial environmental stresses, such as low pH, high osmotic pressure, high ethanol concentration, and phage infection. Therefore, saccharomyces cerevisiae is widely used to produce important industrial chemicals such as fuel ethanol, 2, 3-butanediol, hexadecanol, etc. However, it has been found through extensive studies that xylose is still less efficient in producing ethanol than glucose. Kwak et al proposed that Saccharomyces cerevisiae metabolizes xylose more suitable for the production of non-ethanolic substances and verified with amorphadiene and squalene (Kwak S, et al 2017, Biotechnology & Bioengineering,114 (11)). Researches show that the expression level of enzymes related to ethanol assimilation and cytoplasmic acetyl coenzyme A synthesis is higher than that of glucose fermentation during xylose fermentation. After the necessary perturbation of the genes involved in amorphadiene and squalene synthesis, both product titers and yields were found to be higher than glucose under xylose fermentation conditions.
At present, xylose has been used to produce various chemicals such as safe sweetener-xylitol, biofuel-fuel ethanol, fuel butanol, biodiesel, hydrogen and methane, industrial raw materials-lactic acid, 2, 3-butanediol, acetoin, 3, 4-dihydroxybutyric acid, succinic acid, fumaric acid, polysaccharide-pullulan, biopolymer materials-PHA, PHB. Therefore, the development of xylose utilization engineering strains has important significance for promoting and accelerating the metabolic mechanism research of xylose and enriching the types of bio-based chemicals.
Disclosure of Invention
The first purpose of the invention is to overcome the defects of the prior art and provide a recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose.
The second purpose of the invention is to provide a construction method of the recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose.
The third purpose of the invention is to provide the application of the recombinant saccharomyces cerevisiae for producing the dammarenediol and the protopanoxadiol by utilizing xylose to metabolize the xylose.
The fourth object of the present invention is to provide a second recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose.
The fifth object of the present invention is to provide a second method for constructing recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol using xylose.
The sixth purpose of the present invention is to provide the second use of the recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose to metabolize xylose.
The technical scheme of the invention is summarized as follows:
a construction method of recombinant saccharomyces cerevisiae capable of producing dammarenediol and protopanoxadiol by using xylose comprises the following steps: by the homologous recombination method, the promoter of xylulokinase gene XKS1 endogenous to the saccharomyces cerevisiae is replaced by a strong promoter PFBA1Then introducing a xylose reductase gene XYL1 expression cassette and a xylitol dehydrogenase gene XYL2 expression cassette into a delta site of the saccharomyces cerevisiae to obtain a recombinant strain; introducing a transaldolase gene TAL1 expression cassette and a transketolase gene TKL1 expression cassette into rDNA sites of the recombinant bacteria to obtain recombinant saccharomyces cerevisiae 1 capable of rapidly metabolizing xylose; introducing a farnesyl diphosphate farnesyl transferase gene ERG9 expression cassette, a squalene monooxygenase gene ERG1 expression cassette and a dammarenediol synthase gene DS expression cassette into an rDNA locus of a recombinant bacterium 1 by a homologous recombination method to obtain a recombinant saccharomyces cerevisiae 2 for producing the dammarenediol by utilizing xylose; introducing a nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase gene NADH-HMGr expression cassette, a farnesyl pyrophosphate synthase gene ERG20 expression cassette and a protopanoxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 expression cassette into a delta site of a recombinant bacterium 2 by a homologous recombination method to obtain recombinant saccharomyces cerevisiae 3 for producing dammarenediol and protopanoxadiol by utilizing xylose;
promoter PFBA1The nucleotide sequence of (A) is shown as SEQ ID NO. 1;
the nucleotide sequence of the xylose reductase gene XYL1 is shown in SEQ ID NO. 2;
the nucleotide sequence of the xylitol dehydrogenase gene XYL2 is shown in SEQ ID NO. 3;
the nucleotide sequence of the transaldolase gene TAL1 is shown in SEQ ID NO. 4;
the nucleotide sequence of the transketolase gene TKL1 is shown in SEQ ID NO. 5.
The nucleotide sequence of farnesyl diphosphate farnesyl transferase gene ERG9 is shown in SEQ ID NO. 6;
the nucleotide sequence of the squalene monooxygenase gene ERG1 is shown in SEQ ID NO. 7;
the nucleotide sequence of the dammarenediol synthase gene DS is shown as SEQ ID NO. 8.
The nucleotide sequence of the nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase gene NADH-HMGr is shown as SEQ ID NO. 9;
the nucleotide sequence of the farnesyl pyrophosphate synthase gene ERG20 is shown in SEQ ID NO. 10;
the nucleotide sequence of the protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 is shown in SEQ ID NO. 11.
The recombinant saccharomyces cerevisiae 3 for producing dammarenediol and protopanoxadiol by using xylose constructed by the method.
The recombinant saccharomyces cerevisiae 3 which is constructed by the method and can produce dammarenediol and protopanoxadiol by utilizing xylose is applied to producing the dammarenediol and the protopanoxadiol by utilizing xylose.
The second construction method of the recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by utilizing xylose comprises the following steps: utilizing a Cre/loxP system to recover his3 and ura3 marks of the recombinant bacteria 3, and introducing an acetyl coenzyme A acyl transferase gene ERG10 expression cassette, a hydroxymethyl glutaryl-CoA synthase gene ERG13 expression cassette and a mevalonate kinase gene ERG12 expression cassette into a delta site of the recombinant bacteria 3 by a homologous recombination method to obtain recombinant saccharomyces cerevisiae 4 for producing dammarenediol and protopanoxadiol by utilizing xylose;
the nucleotide sequence of the acetyl coenzyme A acyltransferase gene ERG10 is shown as SEQ ID NO. 12;
the nucleotide sequence of hydroxymethyl glutaryl-CoA synthase gene ERG13 is shown in SEQ ID NO. 13;
the nucleotide sequence of the mevalonate kinase gene ERG12 is shown in SEQ ID NO. 14.
The recombinant saccharomyces cerevisiae 4 which is constructed by the second method and used for producing dammarenediol and protopanoxadiol by utilizing xylose.
The recombinant saccharomyces cerevisiae 4 for producing dammarenediol and protopanoxadiol by using xylose, which is constructed by the second method, is used for producing dammarenediol and protopanoxadiol by using xylose.
Experiments prove that the obtained recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose can produce the dammarenediol and the protopanoxadiol by using xylose through a homologous recombination method, the yield reaches 17.91mg/L of the dammarenediol and 14.49mg/L of the protopanoxadiol, and the method provides a basis for artificially synthesizing the dammarenediol and the protopanoxadiol by using xylose.
Drawings
FIG. 1 is a diagram showing the metabolic pathways for producing dammarenediol and protopanoxadiol by Saccharomyces cerevisiae using xylose.
FIG. 2 shows the results of liquid chromatography analysis of dammarenediol and protopanoxadiol.
FIG. 3 shows the results of LC-MS detection and analysis of dammarenediol and protopanoxadiol.
FIG. 4 shows the yields of dammarenediol and protopanoxadiol from the recombinant Saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol from xylose.
Detailed Description
The present invention will be further illustrated by the following specific examples.
The test methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Saccharomyces cerevisiae strain is Saccharomyces cerevisiae W303-1A (American No. ATCC208352), and is hereinafter referred to as Saccharomyces cerevisiae.
Example 1 amplification and preparation of Gene elements
The xylulokinase XKS1 promoter is replaced by a promoter PFBA1(promoter P)FBA1(ii) a The nucleotide sequence is shown as SEQ ID NO. 1; simply labeled as SEQ ID NO.1, the same applies below);
the expression cassette of the xylose reductase gene XYL1 consists of a promoter PCCW12(SEQ ID NO.15), xylose reductase gene XYL1(SEQ ID NO.2) and terminator THSP26(SEQ ID NO. 16);
the expression cassette of the xylitol dehydrogenase gene XYL2 consists of a promoter PHXT7(SEQ ID NO.17), xylitol dehydrogenase gene XYL2(SEQ ID NO.3) and terminator THXT7(SEQ ID NO. 18);
the transaldolase gene TAL1 expression cassette is composed of promoter PHXT7Transaldolase gene TAL1(SEQ ID NO.4) and terminator THXT7Composition is carried out;
the expression cassette of the transketolase gene TKL1 is composed of a promoter PCCW12Transketolase gene TKL1(SEQ ID NO.5) and terminator THSP26Composition is carried out;
the expression cassette of farnesyl diphosphate farnesyl transferase gene ERG9 is composed of promoter PTPI1(SEQ ID NO.19), farnesyl diphosphate farnesyl transferase gene ERG9(SEQ ID NO.6) and terminator TADH2(SEQ ID NO. 20);
the expression cassette of squalene monooxygenase gene ERG1 is composed of promoter PTEF1(SEQ ID NO.21), squalene monooxygenase gene ERG1(SEQ ID NO.7) and terminator TADH1(SEQ ID NO. 22);
the dammarenediol synthase gene DS expression box consists of a promoter PPGK1(SEQ ID NO.23), dammarendiol synthase gene DS (SEQ ID NO.8) and terminator TCYC1(SEQ ID NO. 24);
the expression cassette of the nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase gene NADH-HMGr is composed of a promoter PPGK1Nicotinamide adenine dinucleotide-hydroxymethylglutaryl-coenzyme A reductase gene NADH-HMGr (SEQ ID NO.9) and terminator TCYC1Composition is carried out;
the expression cassette of the farnesyl pyrophosphate synthase gene ERG20 consists of a promoter PTPI1Farnesyl pyrophosphate synthase gene ERG20(SEQ ID NO.10) (containing an endogenous terminator);
the protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 expression cassette is composed of a promoter PTDH3(SEQ ID NO.25), protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1(SEQ ID NO.11), and terminator TADH1(SEQ ID NO. 26);
the expression cassette of acetyl coenzyme A acyltransferase gene ERG10 is composed of promoter PHXT7acetyl-CoA acyltransferase gene ERG10(SEQ ID NO.12) (containing an endogenous terminator);
the expression cassette of the hydroxymethyl glutaryl-CoA synthase gene ERG13 is composed of a promoter PTEF1Hydroxymethyl glutaryl-CoA synthase gene ERG13(SEQ ID NO.13) (containing endogenous terminator);
the mevalonate kinase gene ERG12 expression cassette consists of promoter PPGK1And mevalonate kinase gene ERG12(SEQ ID NO.14) (containing an endogenous terminator).
Xylose reductase gene XYL1(SEQ ID NO.2), xylitol dehydrogenase gene XYL2(SEQ ID NO.3), dammarenediol synthase gene DS (SEQ ID NO.8), nicotinamide adenine dinucleotide-hydroxymethylglutaryl coenzyme A reductase gene NADH-HMGr (SEQ ID NO.9) and protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1(SEQ ID NO.11) are all artificially synthesized sequences.
An XKS1t2 fragment is amplified by taking a saccharomyces cerevisiae W303-1A genome as a template, XKS1t2-F (SEQ ID NO.27) as a front guide and XKS1t2-R-his3(SEQ ID NO.28) as a rear guide; using his3-pFBA1-F (SEQ ID NO.31) as a front guide and pFBA1-R-XKS1t1(SEQ ID NO.32) as a rear guide to amplify PFBA1A fragment (SEQ ID NO. 1); pFBA1-XKS1t1-F (SEQ ID NO.33) is used as a front guide, XKS1t1-R (SEQ ID NO.34) is used as a rear guide, and an XKS1t1 fragment is amplified. Plasmid pXP320 is used as a template, XKS1t2-his3-F (SEQ ID NO.29) is used as a front guide, and his3-R-pFBA1(SEQ ID NO.30) is used as a rear guide, so that a his3 fragment is amplified. The fragments are fused to obtain an XKS1 promoter replacement expression cassette XKS1t2-his3-PFBA1-XKS1t1。
The genome of the saccharomyces cerevisiae W303-1A is taken as a template, delta 2-F (SEQ ID NO.35) is taken as a front lead, delta 2-R-pCCW12(SEQ ID NO.36) is taken as a rear lead, and delta 2 is amplifiedA fragment; pCCW12-F (SEQ ID NO.37) as a front guide and pCCW12-R (SEQ ID NO.38) as a rear guide, and P is amplifiedCCW12A fragment; XYL1-tHSP26-F (SEQ ID NO.41) is used as a front guide, tHSP26-R-pHXT7(SEQ ID NO.42) is used as a rear guide, and T is amplifiedHSP26A fragment; tHSP26-pHXT7-F (SEQ ID NO.43) is used as a front guide, pHXT7-R-XYL2(SEQ ID NO.44) is used as a rear guide, and P is amplifiedHXT7A fragment; amplifying T by using tHXT7-F (SEQ ID NO.47) as a front guide and tHXT7-R (SEQ ID NO.48) as a rear guideHXT7A fragment; leu 2-delta 1-F (SEQ ID NO.51) is used as a front guide, delta 1-R (SEQ ID NO.52) is used as a rear guide, and a delta 1 fragment is amplified. Amplifying a XYL1 fragment by taking an artificial sequence XYL1 as a template, pCCW12-XYL1-F (SEQ ID NO.39) as a front guide and XYL1-R-tHSP26(SEQ ID NO.40) as a rear guide; the artificial sequence XYL2 is used as a template, pHXT7-XYL2-F (SEQ ID NO.45) is used as a front guide, XYL2-R-tHXT7(SEQ ID NO.46) is used as a rear guide, and the XYL2 fragment is amplified. A leu2 fragment was amplified using plasmid pRS415 as a template, tHXT7-leu2-F (SEQ ID NO.49) as a front primer, and leu 2-R-delta 1(SEQ ID NO.50) as a rear primer. Fusing the above fragments to obtain gene XYL1 expression cassette PCCW12-XYL1-THSP26Gene XYL2 expression cassette PHXT7-XYL2-THXT7。
Amplifying rDNA2 fragment by taking a saccharomyces cerevisiae W303-1A genome as a template, rDNA2-F (SEQ ID NO.53) as a front primer and rDNA2-R-pCCW12(SEQ ID NO.54) as a rear primer; pCCW12-TKL1-F (SEQ ID NO.55) is used as a front guide, TKL1-R-tHSP26(SEQ ID NO.56) is used as a rear guide, and a TKL1 fragment is amplified; pHXT7-TAL1-F (SEQ ID NO.57) is used as a front lead, TAL1-R-tHXT7(SEQ ID NO.58) is used as a rear lead, and TAL1 segments are amplified; delta 1-rDNA1-F (SEQ ID NO.61) is used as a front guide, rDNA1-R (SEQ ID NO.62) is used as a rear guide, and an rDNA1 fragment is amplified. Plasmid pXP218 was used as a template, tHXT7-ura3-F (SEQ ID NO.59) was used as a leader, ura 3-R-delta 1(SEQ ID NO.60) was used as a back leader, and the ura3 fragment was amplified. Fusing the above fragments to obtain the gene TKL1 expression cassette PCCW12-TKL1-THSP26Gene TAL1 expression box PHXT7-TAL1-THXT7。
Amplifying a pSH47v fragment by taking a plasmid pSH47 as a template, pSH47-F (SEQ ID NO.63) as a front guide and pSH47-R (SEQ ID NO.64) as a rear guide; plasmid pXP320 was used as a template, pSH47-ura3-F (SEQ ID NO.65) was used as a leader, ura3-R-pSH47(SEQ ID NO.66) was used as a postleader, and ura3 fragment was amplified.
Amplifying rDNA2 fragment by taking a genome of saccharomyces cerevisiae W303-1A as a template, rDNA2-F (SEQ ID NO.67) as a front primer and rDNA2-R-his3(SEQ ID NO.68) as a rear primer; using his3-pPGK1-F (SEQ ID NO.71) as a front guide and pPGK1-R-DS (SEQ ID NO.72) as a rear guide to amplify PPGK1A fragment; DS-tCYC1-F (SEQ ID NO.75) as a front guide and tCYC1-R-pTEF1(SEQ ID NO.76) as a rear guide, and T is amplifiedCYC1A fragment; tCYC1-pTEF1-F (SEQ ID NO.77) as front guide and pTEF1-R-ERG1(SEQ ID NO.78) as rear guide to amplify PTEF1A fragment; ERG1-F (SEQ ID NO.79) is used as a front guide, ERG1-R (SEQ ID NO.80) is used as a rear guide, and an ERG1 fragment is amplified; ERG1-tADH1-F (SEQ ID NO.81) is used as a front guide, tADH1-R-pTPI1(SEQ ID NO.82) is used as a rear guide, and T is amplifiedADH1A fragment; amplifying P by taking tADH1-pTPI1-F (SEQ ID NO.83) as a front guide and pPTI1-R-ERG9(SEQ ID NO.84) as a rear guideTPI1A fragment; ERG9-F (SEQ ID NO.85) is used as a front guide, ERG9-R (SEQ ID NO.86) is used as a rear guide, and an ERG9 fragment is amplified; ERG9-tADH2-F (SEQ ID NO.87) is used as a front guide, tADH2-R-rDNA1(SEQ ID NO.88) is used as a rear guide, and T is amplifiedADH2A fragment; taking tEDH 2-rDNA1-F (SEQ ID NO.89) as a front guide and rDNA1-R (SEQ ID NO.90) as a rear guide, and amplifying an rDNA1 fragment. Plasmid pXP320 is used as a template, rDNA2-his3-F (SEQ ID NO.69) is used as a front guide, and his3-R-pPGK1(SEQ ID NO.70) is used as a rear guide, so that a his3 fragment is amplified. And (3) amplifying the DS fragment by taking the artificial sequence DS as a template, DS-F (SEQ ID NO.73) as a front guide and DS-R (SEQ ID NO.74) as a rear guide. Fusing the fragments to obtain a gene DS expression cassette PPGK1-DS-TCYC1Gene ERG1 expression box PTEF1-ERG1-TADH1Gene ERG9 expression box PTPI1-ERG9-TADH2。
Amplifying a delta 2' segment by taking a genome of saccharomyces cerevisiae W303-1A as a template, delta 2' -F (SEQ ID NO.91) as a front lead and delta 2' -R (SEQ ID NO.92) as a rear lead; delta 2' -pTPI1-F (SEQ ID NO.93) as the front lead and pTPI1-R-ERG20(SEQ ID NO.94) as the back lead, and P is amplifiedTPI1A fragment; pTPI1-ERG20-F (SEQ ID NO.95) is used as a front guide, ERG20-R-pPGK1(SEQ ID NO.96) is used as a rear guide, and ERG20 fragments are amplified; amplifying P by taking pPGK1-F (SEQ ID NO.97) as a front guide and pPGK1-R (SEQ ID NO.98) as a rear guidePGK1A fragment; tCYC1-F (SEQ ID NO.101) as a front guide and tCYC1-R (SEQ ID NO.102) as a rear guide, and T is amplifiedCYC1A fragment; tCYC1-pTDH3-F (SEQ ID NO.103) as a front guide and pTDH3-R-PPDS (SEQ ID NO.104) as a rear guide, and P is amplifiedTDH3A fragment; ATR1-tADH1-F (SEQ ID NO.107) is used as a front guide, tADH1-R-ura3(SEQ ID NO.108) is used as a rear guide, and T is amplifiedADH1A fragment; ura 3-delta 1' -F (SEQ ID NO.111) is used as a front guide, delta 1' -R (SEQ ID NO.112) is used as a rear guide, and a delta 1' fragment is amplified. Using an artificial sequence NADH-HMGr as a template, HMGr-F (SEQ ID NO.99) as a front guide and HMGr-R (SEQ ID NO.100) as a rear guide to amplify an HMGr fragment; amplifying a PPDS-ATR1 fragment by taking an artificial sequence PPDS-ATR1 as a template, PPDS-F (SEQ ID NO.105) as a front guide and ATR1-R (SEQ ID NO.106) as a rear guide; the ura3 fragment was amplified using plasmid pXP218 as a template, tADH1-ura3-F (SEQ ID NO.109) as a leader, ura 3-R-delta 1' (SEQ ID NO.110) as a back leader. Fusing the fragments to obtain the gene ERG20 expression cassette PTPI1ERG20, Gene NADH-HMGr expression cassette PPGK1-NADH-HMGr-TCYC1Gene PPDS-ATR1 expression box PTDH3-PPDS-ATR1-TADH1。
Amplifying P by using Saccharomyces cerevisiae W303-1A genome as template, delta 2-pHXT7-F (SEQ ID NO.113) as front guide and pHXT7-R-ERG10(SEQ ID NO.114) as rear guideHXT7A fragment; ERG10-F (SEQ ID NO.115) is used as a front guide, ERG10-R (SEQ ID NO.116) is used as a rear guide, and an ERG10 fragment is amplified; ERG10-pPGK1-F (SEQ ID NO.117) is used as a front guide, pPGK1-R-ERG12(SEQ ID NO.118) is used as a rear guide, and P is amplifiedPGK1A fragment; ERG12-F (SEQ ID NO.119) is used as a front guide, ERG12-R (SEQ ID NO.120) is used as a rear guide, and an ERG12 fragment is amplified; ERG12-pTEF1-F (SEQ ID NO.121) is used as a front guide, pTEF1-R-ERG13(SEQ ID NO.122) is used as a rear guide, and P is amplifiedTEF1A fragment; ERG13-F (SEQ ID NO.123) is used as a front guide, ERG13-R (SEQ ID NO.124) is used as a rear guide, and an ERG13 fragment is amplified; delta 1-F (SEQ ID NO.127) is used as a front guide, delta 1-R (SEQ ID NO.128) is used as a rear guide, and a delta 1 fragment is amplified. An ade2 fragment is amplified BY taking a saccharomyces cerevisiae BY4741 genome as a template, ERG13-ade2-F (SEQ ID NO.125) as a front lead and ade 2-R-delta 1(SEQ ID NO.126) as a rear lead. Fusing the fragments to obtain the gene ERG10 expression cassette PHXT7ERG10, Gene ERG12 expression cassette PPGK1ERG12 and gene ERG13 expression cassette PTEF1-ERG13。
The PCR enzyme used in the present invention is Phanta Super-Fidelity DNA Polymerase from Biotechnology Ltd of Nanjing Novowed. A50. mu.L PCR amplification system is shown in Table 1:
TABLE 1
Figure BDA0001732728860000061
An amplification program is set up on the PCR instrument. The amplification conditions were 95 ℃ pre-denaturation for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 55 ℃ for 15sec, and extension at 72 ℃ for 1min (35 cycles); extension at 72 ℃ for 5min (1 cycle).
The fusion PCR system used in the present invention is shown in Table 2:
TABLE 2
Figure BDA0001732728860000062
The fusion program was set up on a PCR instrument. The fusion conditions were pre-denaturation at 95 ℃ for 4min (1 cycle); denaturation at 95 ℃ for 15sec, annealing at 58 ℃ for 15sec, and extension at 72 ℃ for 1min (11 cycles); extension at 72 ℃ for 5min (1 cycle).
Purifying and recovering the fragment obtained by PCR amplification by using an agarose gel DNA recovery kit (Tiangen Biochemical technology Beijing, Ltd.) to obtain a DNA fragment used for transforming the saccharomyces cerevisiae, then carrying out fusion PCR on the DNA fragment, and amplifying gene expression cassettes used for transforming the saccharomyces cerevisiae by using a fusion PCR product as a template, wherein the gene expression cassettes are XKS1t2-his3-PFBA1-XKS1t1, Gene XYL1 expression cassette PCCW12-XYL1-THSP26Gene XYL2 expression cassette PHXT7-XYL2-THXT7The gene TKL1 expression cassette PCCW12-TKL1-THSP26Gene TAL1 expression box PHXT7-TAL1-THXT7Gene DS expression cassette PPGK1-DS-TCYC1Gene ERG1 expression box PTEF1-ERG1-TADH1Gene ERG9 expression box PTPI1-ERG9-TADH2Gene ERG20 expression box PTPI1ERG20, Gene NADH-HMGr expression cassette PPGK1-NADH-HMGr-TCYC1Gene PPDS-ATR1 expression box PTDH3-PPDS-ATR1-TADH1ERG10 expressionBox PHXT7ERG10, Gene ERG12 expression cassette PPGK1ERG12 and gene ERG13 expression cassette PTEF1-ERG13。
Example 2 transformation and construction of recombinant Saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol Using xylose
The DNA fragment XKS1t2-his3-PFBA1-XKS1t1、PCCW12-XYL1-THSP26、PHXT7-XYL2-THXT7、PCCW12-TKL1-THSP26And PHXT7-TAL1-THXT7Introducing saccharomyces cerevisiae to obtain a recombinant bacterium 1;
DNA fragment PPGK1-DS-TCYC1PTEF1-ERG1-TADH1And PTPI1-ERG9-TADH2Introducing the recombinant bacterium 1 to obtain a recombinant bacterium 2;
DNA fragment PTPI1-ERG20、PPGK1-NADH-HMGr-TCYC1And PTDH3-PPDS-ATR1-TADH1Introducing the recombinant bacterium 2 to obtain a recombinant bacterium 3;
DNA fragment PHXT7-ERG10、PPGK1ERG12 and PTEF1ERG13 is introduced into the recombinant bacterium 3 to obtain a recombinant bacterium 4.
The transformation method comprises the following steps:
after Saccharomyces cerevisiae W303-1A was cultured in YPD medium for 12 hours, 300. mu.L of the medium was added to 3mL of fresh YPD medium and cultured for 5 hours. The cell culture solution was put into a 1.5mL centrifuge tube, centrifuged at 4000rpm for 5min to collect the cells, and the supernatant was discarded. The cells were resuspended in 1mL sterile water, centrifuged again at 4000rpm for 5min, and the supernatant discarded. Cells were resuspended in 1mL of 100mM lithium acetate and allowed to stand for 5 min. Then, the mixture was centrifuged at 3000rpm for 3min, and the supernatant was discarded. ssDNA was boiled for 5min and rapidly cooled on ice. The transformation mixture consisted of 24. mu.L of PEG 3350 (50% w/v), 36. mu.L of 1.0M lithium acetate, 10. mu.L of ssDNA, 800ng of DNA fragment, and was finally made up to 360. mu.L with sterile water. Vortex the tube for 1min to mix the ingredients thoroughly. Heating in 42 deg.C water bath for 30 min. Centrifuging at 4000rpm for 5min, removing supernatant by micropipette, adding 1mLYPD medium, and culturing at 30 deg.C for 2 hr. Centrifuging at 4000rpm for 5min, discarding the supernatant, and washing the thallus twice with sterile water. 100 μ L of sterile water resuspended cells and plated on corresponding auxotrophic plates for selection. The culture conditions were 30 ℃ and 48 h.
Example 3 fermentation of recombinant Saccharomyces cerevisiae for production of dammarenediol and Protopanaxadiol Using xylose
And (3) selecting single colonies of the recombinant bacteria 2, the recombinant bacteria 3 and the recombinant bacteria 4 for shake flask fermentation. The fermentation conditions were 30 ℃ and 220rpm, and the culture was carried out for 3 days. The fermentation medium is YPX liquid medium, wherein the components and the final concentration thereof are as follows: the final concentration is xylose of 4% (mass percent), peptone of 2% (mass percent), yeast extract powder of 1% (mass percent), and water to make up the volume.
After the fermentation is finished, n-butyl alcohol is used for extraction, and the volume ratio of the n-butyl alcohol to the fermentation liquor is 1: 4. LC-MS detection is carried out on the n-butanol phase, and the result shows that the recombinant bacterium 2 can produce 10.69mg/L dammarenediol, the recombinant bacterium 3 can produce 3.17mg/L dammarenediol and 11.16mg/L protopanoxadiol, and the recombinant bacterium 4 can produce 5.57mg/L dammarenediol and 14.48mg/L protopanaxadiol. LC-MS detection results of dammarane diol and protopanaxadiol and comparison of the yields of dammarane diol and protopanaxadiol in recombinant strains are shown in figures 2,3 and 4.
In the figure 2, A is a recombinant bacterium 2 fermentation product, B is a dammarenediol standard product, C is a recombinant bacterium 3 fermentation product, and D is a protopanaxadiol standard product.
In fig. 3, E is a liquid phase mass spectrogram of a fermentation product of the recombinant bacterium 3, F is a liquid phase mass spectrogram of a dammarenediol standard substance, G is a liquid phase mass spectrogram of a fermentation product of the recombinant bacterium 3, and H is a liquid phase mass spectrogram of a protopanaxadiol standard substance.
Sequence listing
<110> Tianjin university
<120> recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose and construction method thereof
<141> 2018-06-12
<160> 128
<170> SIPOSequenceListing 1.0
<210> 1
<211> 710
<212> DNA
<213> 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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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
<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. The construction method of the recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by utilizing xylose is characterized by comprising the following steps:
(1) by the homologous recombination method, the promoter of xylulokinase gene XKS1 endogenous to Saccharomyces cerevisiae is replaced by promoter PFBA1Then brewing wineIntroducing a xylose reductase gene XYL1 expression cassette and a xylitol dehydrogenase gene XYL2 expression cassette into a delta site of the yeast to obtain a recombinant strain; then introducing a transaldolase gene TAL1 expression cassette and a transketolase gene TKL1 expression cassette in a pentose phosphate pathway into the rDNA locus of the recombinant strain to obtain recombinant saccharomyces cerevisiae 1 for metabolizing xylose;
the promoter PFBA1The nucleotide sequence of (A) is shown as SEQ ID NO. 1;
the nucleotide sequence of the xylose reductase gene XYL1 is shown in SEQ ID NO. 2;
the nucleotide sequence of the xylitol dehydrogenase gene XYL2 is shown in SEQ ID NO. 3;
the nucleotide sequence of the transaldolase gene TAL1 is shown in SEQ ID NO. 4;
the nucleotide sequence of the transketolase gene TKL1 is shown in SEQ ID NO. 5;
(2) introducing a farnesyl diphosphate farnesyl transferase gene ERG9 expression cassette, a squalene monooxygenase gene ERG1 expression cassette and a dammarenediol synthase gene DS expression cassette into the rDNA locus of the recombinant saccharomyces cerevisiae 1 for metabolizing xylose by a homologous recombination method to obtain a recombinant saccharomyces cerevisiae 2 for metabolizing xylose;
the nucleotide sequence of farnesyl diphosphate farnesyl transferase gene ERG9 is shown in SEQ ID NO. 6;
the nucleotide sequence of the squalene monooxygenase gene ERG1 is shown in SEQ ID NO. 7;
the nucleotide sequence of the dammarenediol synthase gene DS is shown as SEQ ID NO. 8;
(3) introducing a nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase gene NADH-HMGr expression box, a farnesyl pyrophosphate synthase gene ERG20 expression box and a protopanoxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 expression box into a delta site of the recombinant saccharomyces cerevisiae 2 for metabolizing xylose by a homologous recombination method to obtain a recombinant saccharomyces cerevisiae 3 for producing dammarenediol and protopanoxadiol by utilizing xylose;
the nucleotide sequence of the nicotinamide adenine dinucleotide-hydroxymethyl glutaryl coenzyme A reductase gene NADH-HMGr is shown as SEQ ID NO. 9;
the nucleotide sequence of the farnesyl pyrophosphate synthase gene ERG20 is shown in SEQ ID NO. 10;
the nucleotide sequence of the protopanaxadiol synthase-cytochrome P450 reductase fusion protein gene PPDS-ATR1 is shown in SEQ ID NO. 11.
2. The recombinant saccharomyces cerevisiae 3 for producing dammarenediol and protopanoxadiol using xylose constructed by the method of claim 1.
3. The use of the recombinant saccharomyces cerevisiae for the production of dammarenediol and protopanoxadiol using xylose according to claim 2 for the production of dammarenediol and protopanoxadiol by metabolizing xylose.
4. The construction method of the recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by utilizing xylose is characterized by comprising the following steps:
introducing an acetyl coenzyme A acyltransferase gene ERG10 expression cassette, a hydroxymethylglutaryl-CoA synthase gene ERG13 expression cassette and a mevalonate kinase gene ERG12 expression cassette into the delta site of the recombinant Saccharomyces cerevisiae 3 for producing dammarenediol and protopanoxadiol from xylose according to claim 2 by a homologous recombination method to obtain recombinant Saccharomyces cerevisiae 4 for metabolizing xylose;
the nucleotide sequence of the acetyl coenzyme A acyltransferase gene ERG10 is shown as SEQ ID NO. 12;
the nucleotide sequence of hydroxymethyl glutaryl-CoA synthase gene ERG13 is shown in SEQ ID NO. 13;
the nucleotide sequence of the mevalonate kinase gene ERG12 is shown in SEQ ID NO. 14.
5. The recombinant saccharomyces cerevisiae 4 for producing dammarenediol and protopanoxadiol using xylose constructed by the method of claim 4.
6. The use of the recombinant saccharomyces cerevisiae 4 for the production of dammarenediol and protopanoxadiol using xylose according to claim 5 for the production of dammarenediol and protopanoxadiol by metabolizing xylose.
CN201810781608.0A 2018-07-17 2018-07-17 Recombinant saccharomyces cerevisiae for producing dammarenediol and protopanoxadiol by using xylose and construction method Active CN108949601B (en)

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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
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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
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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
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