CN107723252A - Produce the restructuring Yarrowia lipolytica and construction method of valencia orange alkene and nootkatone - Google Patents

Produce the restructuring Yarrowia lipolytica and construction method of valencia orange alkene and nootkatone Download PDF

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CN107723252A
CN107723252A CN201710864583.6A CN201710864583A CN107723252A CN 107723252 A CN107723252 A CN 107723252A CN 201710864583 A CN201710864583 A CN 201710864583A CN 107723252 A CN107723252 A CN 107723252A
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nootkatone
valencia orange
yarrowia lipolytica
alkene
orange alkene
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卢文玉
郭校燕
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Tianjin University
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    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
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Abstract

The invention discloses a kind of construction method for the restructuring Yarrowia lipolytica for producing valencia orange alkene and nootkatone, step：Pass through the method for homologous recombination, the encoding gene AtCPR expression cassettes of valencia orange alkene synthasee code gene CVS expression cassettes, nootkatone synthasee code gene CYP706M1 expression cassettes and cytochrome P450 reductase are imported to the rDNA sites of Yarrowia lipolytica, obtain producing the restructuring Yarrowia lipolytica 1 of valencia orange alkene and nootkatone；Experiment proves, pass through the method for homologous recombination, the production valencia orange alkene of acquisition and the restructuring Yarrowia lipolytica of nootkatone can make the output increased of valencia orange alkene and nootkatone, it is at least valencia orange alkene 6.5mg/L and the μ g/L of nootkatone 30, preferably at most 18mg/L and 0.5mg/L, method of the invention provide foundation for artificial synthesized valencia orange alkene and nootkatone.

Description

Produce the restructuring Yarrowia lipolytica and structure of valencia orange alkene and nootkatone Method
Technical field
The present invention relates to biological technical field, more particularly to the restructuring solution fat Ye Shi of production valencia orange alkene and nootkatone Yeast and its construction method.
Background technology
Nootkatone ((+)-nootkatone) belongs to sesquiterpenoids, separates t first from Nu Teka cypresses and obtains, Also it is present on a small quantity in the fruit such as grape, shaddock.Nootkatone has a kind of unique fragrance and threshold odour number is very low, this characteristic Nootkatone is turned into a kind of rather well received spices and be widely used in foods and cosmetics field as additive.To Nu Tekabai The constituent analysis and research of trees matter show that nootkatone also has the performance of desinsection, mite killing and antibacterial, and this performance can make to exert Spy card cypress is in the time of decades to anticorrosive.In recent years, the application of nootkatone in medicine has obtained more and more Concern.Research shows that nootkatone has anti-platelet aggregation effect, inhibiting tumor cell propagation, improves the effect such as energetic supersession.
Nootkatone is extracted from natural plants such as citrus, is limited by weather, environment, harvest season many factors, And the yield extracted is extremely low.Chemical synthesis further relates to use some poisonous heavy metals, inflammable compound and potent oxidation The catalyst unfavorable to environment such as agent or reactant (Salvador J, et al.2002, Green Chem.4,352-356.), So a kind of more efficient, more environmentally friendly method is found to produce the target that nootkatone turns into people and more paid close attention to.
Katarina Cankar et al. have found and identified in a Nu Teka cypress that valencia orange alkene can be catalyzed The P450 genes of nootkatone are generated, CYP706M1 [GenBanK databases, sequence number JX518290], the P450 has of a relatively high Selectivity and catalytic efficiency (Katarina Cankar, et al.2014, FEBS Letters 588,1001-1007).
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided a kind of weight for producing valencia orange alkene and nootkatone Group Yarrowia lipolytica.
Second object of the present invention is to provide a kind of restructuring solution fat Ye Shi ferment for producing valencia orange alkene and nootkatone The construction method of female bacterium.
Third object of the present invention is to provide the restructuring solution fat Ye Shi ferment of above-mentioned production valencia orange alkene and nootkatone The application of female bacterium fermenting and producing valencia orange alkene and nootkatone.
Fourth object of the present invention is to provide the restructuring solution fat Ye Shi of second of production valencia orange alkene and nootkatone Saccharomycete.
The 5th purpose of the present invention is to provide the restructuring solution fat Ye Shi of second of production valencia orange alkene and nootkatone The construction method of saccharomycete.
The 6th purpose of the present invention is to provide the restructuring solution fat Ye Shi of second of production valencia orange alkene and nootkatone Saccharomycetes to make fermentation produces the application of valencia orange alkene and nootkatone.
The 7th purpose of the present invention is to provide the restructuring solution fat Ye Shi of the third production valencia orange alkene and nootkatone Saccharomycete.
The 8th purpose of the present invention is to provide the restructuring solution fat Ye Shi of the third production valencia orange alkene and nootkatone The construction method of saccharomycete.
The 9th purpose of the present invention is to provide the restructuring solution fat Ye Shi of the third production valencia orange alkene and nootkatone Saccharomycetes to make fermentation produces the application of valencia orange alkene and nootkatone.
Technical scheme is summarized as follows：
A kind of construction method for the restructuring Yarrowia lipolytica for producing valencia orange alkene and nootkatone, including following step Suddenly：By the method for homologous recombination, valencia orange alkene synthasee code gene CVS is imported to the rDNA sites of Yarrowia lipolytica The encoding gene AtCPR tables of expression cassette, nootkatone synthasee code gene CYP706M1 expression cassettes and cytochrome P450 reductase Up to box, the restructuring Yarrowia lipolytica 1 of production valencia orange alkene and nootkatone is obtained；
Valencia orange alkene synthasee code gene CVS nucleotide sequence is as shown in SEQ ID NO.1；
Nootkatone synthasee code gene CYP706M1 nucleotide sequence is as shown in SEQ ID NO.2；
The encoding gene AtCPR of cytochrome P450 reductase nucleotide sequence is as shown in SEQ ID NO.3.
The production valencia orange alkene of above method structure and the restructuring Yarrowia lipolytica 1 of nootkatone.
The fermenting and producing valencia orange alkene of restructuring Yarrowia lipolytica 1 of above-mentioned production valencia orange alkene and nootkatone With the application of nootkatone.
The construction method of the restructuring Yarrowia lipolytica of second of production valencia orange alkene and nootkatone, including it is as follows Step：By the method for homologous recombination, valencia orange alkene synthasee code gene is imported to the rDNA sites of Yarrowia lipolytica CVS expression cassettes, nootkatone synthasee code gene and the cytochrome P450 reductase encoding gene of N-terminal 46 amino acid of truncation Fusion CYP706M1-tAtCPR expression cassettes, obtain producing the restructuring Yarrowia lipolytica of valencia orange alkene and nootkatone Bacterium 2；
Valencia orange alkene synthasee code gene CVS nucleotide sequence is as shown in SEQ ID NO.1；
Fusion CYP706M1-tAtCPR nucleotide sequence is as shown in SEQ ID NO.4.
The production valencia orange alkene of above-mentioned second method structure and the restructuring Yarrowia lipolytica 2 of nootkatone.
Produce valencia orange alkene and the fermenting and producing valencia orange alkene of restructuring Yarrowia lipolytica 2 of nootkatone and promise The application of card ketone.
The construction method of the restructuring Yarrowia lipolytica of the third production valencia orange alkene and nootkatone, including it is as follows Step：(1) by the method for homologous recombination, valencia orange alkene synthase coding base is imported to the rDNA sites of Yarrowia lipolytica Because of CVS expression cassettes, the cytochrome P450 reductase encoding gene of nootkatone synthasee code gene and N-terminal 46 amino acid of truncation Fusion CYP706M1-tAtCPR expression cassettes, obtain producing the restructuring solution fat Ye Shi ferment of valencia orange alkene and nootkatone Female bacterium 2；
(2) 3-hydroxy-3-methylglutaryl-coenzyme A is reduced into enzyme coding gene tHMG1 insertion plasmid PINA1269, obtained Expression plasmid ptHMG1；The expression plasmid ptHMG1 is imported to the restructuring solution fat Ye Shi of production valencia orange alkene and nootkatone Saccharomycete 2, obtain producing the restructuring Yarrowia lipolytica 3 of valencia orange alkene and nootkatone；
Valencia orange alkene synthasee code gene CVS nucleotide sequence is as shown in SEQ ID NO.1；
Fusion CYP706M1-tAtCPR nucleotide sequence is as shown in SEQ ID NO.4.
3-hydroxy-3-methylglutaryl-coenzyme A reduction enzyme coding gene tHMG1 nucleotide sequence such as SEQ ID NO.5 It is shown.
The production valencia orange alkene of above-mentioned the third method structure and the restructuring Yarrowia lipolytica 3 of nootkatone.
Produce valencia orange alkene and the fermenting and producing valencia orange alkene of restructuring Yarrowia lipolytica 3 of nootkatone and promise The application of card ketone.
It is demonstrated experimentally that by the method for homologous recombination, the production valencia orange alkene of acquisition and the restructuring solution fat of nootkatone Ye Shi saccharomycete can make the output increased of valencia orange alkene and nootkatone, be at least valencia orange alkene 6.5mg/L and promise Card ketone 30 μ g/L, preferably at most 18mg/L and 0.5mg/L, method of the invention are that artificial synthesized valencia orange alkene and nootkatone carry Foundation is supplied.
Brief description of the drawings
Fig. 1 is the metabolic pathway figure that Yarrowia lipolytica produces valencia orange alkene and nootkatone.
Fig. 2A is that the GC-MS of valencia orange alkene tests and analyzes result.
Fig. 2 B are that the GC-MS of nootkatone tests and analyzes result.
Fig. 3 is the restructuring Yarrowia lipolytica valencia orange alkene and nootkatone of production valencia orange alkene and nootkatone Yield comparison.
Embodiment
Below by specific embodiment, the present invention is further illustrated.
Experimental method used in example below is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The Yarrowia lipolytica is Yarrowia Lipolytica ATCC 201249 (U.S. ATCC201249).
Plasmid PINA1269 is purchased from BioVector NTCC China plasmid vector bacterium cell gene collection.
The amplification and preparation of embodiment 1, Genetic elements
According to the valencia orange alkene synthasee code gene CVS and nootkatone synthasee code gene provided on NCBI CYP706M1 DNA sequence dna, balun is synthesized by the method (synthesis of Wuhan Jin Kairui bioengineering Co., Ltd) of chemical synthesis West Asia tangerine alkene synthase gene coding CVS (SEQ ID NO.1), nootkatone synthasee code gene CYP706M1 (SEQ ID NO.2), The encoding gene AtCPR (SEQ ID NO.3) of cytochrome P450 reductase.
The valencia orange alkene synthasee code gene expression cassette is by promoter PTEF1, valencia orange alkene synthase coding base Because of CVS and terminator Txpr2Composition；
The nootkatone synthasee code gene expression cassette is by promoter PEXP1, nootkatone synthasee code gene CYP706M1 and Terminator Tmig1Composition；
The cytochrome P450 reductase encoding gene expression cassette is by promoter PGPD1, cytochrome P450 reductase compile Code Gene A tCPR and terminator Tlip2Composition；
The nootkatone synthasee code gene truncates the cytochrome P450 reductase encoding gene of 46 amino acid with N-terminal The fusion CYP706M1-tAtCPR expression cassettes formed are merged by promoter PEXP1, fusion CYP706M1-tAtCPR and Terminator Tlip2Composition；
Fusion CYP706M1-tAtCPR nucleotide sequence is as shown in SEQ ID NO.4.
3-hydroxy-3-methylglutaryl-coenzyme A reduction enzyme coding gene tHMG1 nucleotide sequence such as SEQ ID NO.5 It is shown.
The promoter PTEF1Nucleotide sequence as shown in SEQ ID NO.6；
The terminator Txpr2Nucleotide sequence as shown in SEQ ID NO.9；
The promoter PEXP1Nucleotide sequence as shown in SEQ ID NO.7；
The terminator Tmig1Nucleotide sequence as shown in SEQ ID NO.10.
The promoter PGPD1Nucleotide sequence as shown in SEQ ID NO.8.
The terminator Tlip2Nucleotide sequence as shown in SEQ ID NO.11.
RDNA site upstream homology arms rDNAup (SEQ ID NO.12) and the downstream homology arm ura- with ura marks RDNAdown (SEQ ID NO.13) sequence is artificial synthesized.
Yarrowia lipolytica produces valencia orange alkene and the metabolic pathway figure of nootkatone is shown in Fig. 1.(Glucose in Fig. 1：Portugal Grape sugar；Acetyl-COA：Acetyl coenzyme A；HMG-COA：3-hydroxy-3-methylglutaryl-coenzyme A；Mevalonate：First hydroxyl penta Acid；IPP：Isopentenyl pyrophosphate；DMAPP：Dimethylallyl diphosphate；FPP：Farnesyl pyrophosphate；(+)- valencene：Valencia orange alkene；(+)-nootkatone：Nootkatone；IDI1：Isopentenyl diphosphate isomerase；ERG20： Farnesyl pyrophosphate synthase；CVS：Valencia orange alkene synthase；CYP706M1：Nootkatone synthase.)
Using the genomes of Yarrowia lipolytica Yarrowia Lipolytica ATCC 201249 as template, with rDNAup-F (SEQ ID NO.14) and rDNAup-R-TEF1p (SEQ ID NO.15) are primer, amplification homology arm rDNAup；
It is that primer amplification is opened with rDNAup-TEF1p-F (SEQ ID NO.16) and TEF1p-R-CVS (SEQ ID NO.17) Mover PTEF1；
Expanded with xpr2-EXP1p-F (SEQ ID NO.18) and EXP1p-R-CYP706M1 (SEQ ID NO.19) for primer Increase promoter PEXP1；
It is that primer amplification is opened with mig1-GPD1p-F (SEQ ID NO.20) and GPD1p-R-AtCPR (SEQ ID NO.21) Mover PGPD1；
It is that primer amplification terminates with CVS-xpr2-F (SEQ ID NO.22) and xpr2-R-EXP1p (SEQ ID NO.23) Sub- Txpr2；
Expanded with CYP706M1-mig1-F (SEQ ID NO.24) and mig1-R-GPDp (SEQ ID NO.25) for primer Terminator Tmig1；
It is that primer amplification terminates with AtCPR-lip2-F (SEQ ID NO.26) and lip2-R-ura (SEQ ID NO.27) Sub- Tlip2；
Using ura-rDNAdown DNA fragmentations as template, with lip2-ura-F (SEQ ID NO.28) and rDNAdown-R (SEQ ID NO.29) is primer, downstream homology arm ura-rDNAdown of the amplification with ura marks；
Using the plasmid PUC57-CVS of the CVS genes with synthesis as template, with TEF1p-CVS-F (SEQ ID NO.30) It is primer with CVS-R-xpr2 (SEQ ID NO.31), expands CVS genes；
Using the plasmid PUC57-CYP706M1 of the CYP706M1 genes with synthesis as template, with EXP1p-CYP706M1-F (SEQ ID NO.32) and CYP706M1-R-mig1 (SEQ ID NO.33) are primer, expand CYP706M1 genes；
Using the plasmid PUC57-CYP706M1 of the CYP706M1 genes with synthesis as template, with EXP1p-CYP706M1-F (SEQ ID NO.32) and CYP706M1-R-tAtCPR (SEQ ID NO.34) are primer, what amplification was merged with tAtCPR CYP706M1 genes；
Using the plasmid PUC57-AtCPR of the AtCPR genes with synthesis as template, with GPD1p-AtCPR-F (SEQ ID NO.35) and AtCPR-R-lip2 (SEQ ID NO.36) is primer, expands AtCPR genes；
Using the plasmid PUC57-AtCPR of the AtCPR genes with synthesis as template, with CYP706M1-tAtCPR-F (SEQ ID NO.37) and tAtCPR-R-lip2 (SEQ ID NO.36) be primer, amplification truncate 46 amino acid tAtCPR genes.
PCR enzymes used in the present invention are Nanjing Vazyme Biotechnology Co., Ltd.Max Super- Fidelity polymerases.50 μ L PCR amplification system is as follows：
Amplification program is set in PCR instrument.Amplification condition is 95 DEG C of pre-degeneration 4min (1 circulation)；95 DEG C of denaturation 15sec, 60 DEG C of 15sec of annealing, 72 DEG C of extension 1min (34 circulations)；72 DEG C of extension 5min (1 circulation).
Fusion DNA vaccine system is as follows used in the present invention：
Amplification program is set in PCR instrument.Amplification condition is 95 DEG C of pre-degeneration 4min (1 circulation)；95 DEG C of denaturation 15sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 2-4min (11 circulations), 72 DEG C of extension 5min (1 circulation).
Finally turned with Ago-Gel DNA QIAquick Gel Extraction Kits (Tiangeng biochemical technology Beijing Co., Ltd) purifying recovery Change the DNA fragmentation used in Yarrowia lipolytica, respectively upstream homology arm rDNAup, gene C VS expression cassettes PTEF1-CVS-Txpr2、 Gene C YP706M1 expression cassettes PEXP1-CYP706M1-Tmig1, Gene A tCPR expression cassettes PGPD1-AtCPR-Tlip2、CYP706M1- TAtCPR track fusion boxes PEXP1-CYP706M1-tAtCPR-Tlip2And the downstream containing riddled basins ura is homologous Arm ura-rDNAdown.
The primer sequence is listed in primer table row 1.
Primer table row 1
The conversion of the restructuring Yarrowia lipolytica of embodiment 2, production valencia orange alkene and nootkatone and structure
By DNA fragmentation rDNAup, PTEF1-CVS-Txpr2、PEXP1-CYP706M1-Tmig1、PGPD1-AtCPR-Tlip2And ura- RDNAdown is imported in Yarrowia lipolytica ATCC 201249 and is obtained recombinant bacterium 1；
By DNA fragmentation rDNAup, PTEF1-CVS-Txpr2、PEXP1-CYP706M1-tAtCPR-Tlip2And ura- RDNAdown is imported in Yarrowia lipolytica ATCC201249 and is obtained recombinant bacterium 2.Method for transformation is as follows：
By Yarrowia lipolytica ATCC 201249 in YPD culture mediums (1% yeast extract, 2% peptone, 2% grape Sugar) in culture 12h after take 300 μ L add 3mL fresh YPD mediums in, cultivate 5h.6000rpm normal temperature centrifugation 5min collects bacterium Body, supernatant is abandoned, with the ddH2O washing thallines of sterilizing, 5min collection thalline are centrifuged under 6000rpm normal temperature, abandon supernatant.Then will 1mL 100mM lithium acetate is added in thalline, and gently piping and druming is that cell suspends uniformly, at room temperature in 6000rpm after placement 5min 5min is centrifuged under normal temperature and collects thalline, prepares competent yeast cells.Conversion mixed system includes 240 μ L PEG (50%W/V), 36 μ L 1.0M lithium acetates, 10 μ L ss-DNA (2.0mg/ml), each 300ng of DNA fragmentation of conversion.Finally with the ddH2O of sterilizing Polishing is to 360 μ L.Items are sequentially added in the Saccharomyces cerevisiae competent cell just prepared by said sequence, blown and beaten with liquid-transfering gun Uniformly, 30min is stood in 30 DEG C of incubators, places 30min in 42 DEG C of water-baths, 6000rpm normal temperature centrifugation 3min, add after abandoning supernatant Enter 1mL YPD fluid nutrient mediums, 30 DEG C of 220rpm cultivate 2h.Then 6000rpm normal temperature centrifugation 2min, supernatant discarding, sterilized water Washing 2 times, cell finally is resuspended with 100 μ L sterilized waters, the flat board for applying missing histidine is screened.Screening and culturing condition is 30 DEG C, cultivate more than 48h.
Embodiment 3, expression plasmid ptHMG1 are prepared and linearisation
Using saccharomyces cerevisiae genome as template, tHMG1-BamHI-F (SEQ ID NO.38) and tHMG1-BamHI-R are to draw Thing (SEQ ID NO.39) (sequence is shown in list of primers 2), expand tHMG1.With the small extraction reagent kit of plasmid, (Tiangeng biochemical technology is limited Company) extraction plasmid PINA1269.With restriction enzyme BamHI digested plasmid PINA1269 and tHMG1, Ago-Gel electricity Plasmid PINA1269 and tHMG1 after swimming recovery digestion.
Primer table row 2
The plasmid PINA1269 and tHMG1 after digestion are connected with T4 ligases, system is as follows：
22 DEG C of reaction 30min.
5 μ L are taken to be used to convert Escherichia coli Trans T1 competence the system connected.Method for transformation is as follows：
(1) from -80 DEG C of refrigerators take out Escherichia coli Trans T1 competent cells, in ice bath place 10min to its Thaw completely, in the centrifuge tube that the competent cell separating device 1.5ml of 50 μ L dissolvings is sterilized.
(2) 5 μ L linked system solution are added into 50 μ L competent cells, gently mixes, should not blow and beat, placed on ice 30min。
(3) 42 DEG C of water-bath heat shock 30s, taking-up centrifuge tube is put at once stands 2min on ice, during which not rock centrifuge tube.
(4) the LB fluid nutrient mediums that 500 μ L are free of antibiotic are added into centrifuge tube, 37 DEG C, 200rpm culture 2h, make bacterium Body is recovered.
(5) 4000rpm centrifuges 5min, suctions out 350 μ L of supernatant liquid, thalline is resuspended in remaining culture medium.
(6) aseptically, the cell of resuspension is coated on the LB solid plates containing corresponding antibiotic, 37 DEG C, Inversion is incubated overnight.
(7) whether the plasmid of bacterium colony PCR and digestion verification structure is correct.
(8) by the plasmid PINA1269-tHMG1 built restriction enzyme NotI digestions, then Ago-Gel is used Electrophoresis reclaims, that is, the PINA1269-tHMG1 plasmids linearized.
(9) according to the method for transformation of embodiment 2, the PINA1269-tHMG1 plasmids conversion of linearisation is imported into recombinant bacterium 2. Obtain recombinant bacterium 3.
The restructuring Yarrowia lipolytica fermentation production valencia orange of embodiment 4, production valencia orange alkene and nootkatone Alkene and nootkatone
The single bacterium colony of picking recombinant bacterium 1, recombinant bacterium 2 and recombinant bacterium 3 carries out shake flask fermentation, and 10% (volume is added in shaking flask Than) n-dodecane.Fermentation condition is 30 DEG C, 220rpm/min, concussion and cultivate 5 days.Fermentation medium is YPD Liquid Cultures Base, wherein each component and its final concentration are as follows：The glucose (glucose) of final concentration of 2% (mass percent), it is final concentration of The peptone (peptone) of 2% (mass percent), the yeast extract (yeast of final concentration of 1% (mass percent) Soak powder), supply volume with water.
After fermentation ends, take n-dodecane mutually to carry out GC-MS detections, it is found that all recombinant bacteriums can produce Valencia Tangerine alkene；Recombinant bacterium 1 can produce micro nootkatone, be 30 μ g/L, and recombinant bacterium 2 can produce 200 μ g/L nootkatones, recombinant bacterium 3 can produce 500 μ g/L nootkatones.Nootkatone in the GC-MS testing results and recombinant bacterial strain of valencia orange alkene and nootkatone Yield comparison see accompanying drawing 2 and accompanying drawing 3.
Sequence table
<110>University Of Tianjin
<120>Produce the restructuring Yarrowia lipolytica and construction method of valencia orange alkene and nootkatone
<141> 2017-09-20
<160> 39
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1770
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atggcggaga tgtttaacgg taacagcagc aacgacggca gcagctgcat gccggtgaaa 60
gatgcgctgc gtcgtaccgg taaccaccac ccgaacctgt ggaccgacga tttcatccaa 120
agcctgaaca gcccgtacag cgacagcagc tatcacaagc accgtgagat cctgattgac 180
gaaattcgtg atatgttcag caacggcgag ggcgatgaat ttggtgtgct ggaaaacatc 240
tggttcgttg acgtggttca acgtctgggc attgatcgtc actttcagga agagattaaa 300
accgcgctgg actacatcta taagttctgg aaccacgaca gcatctttgg tgatctgaac 360
atggtggcgc tgggcttccg tattctgcgt ctgaaccgtt acgtggcgag cagcgatgtt 420
ttcaagaaat ttaaaggcga ggaaggccaa ttcagcggtt ttgagagcag cgaccaggat 480
gcgaaactgg aaatgatgct gaacctgtat aaggcgagcg agctggactt tccggacgag 540
gatatcctga aggaagcgcg tgcgttcgcg agcatgtacc tgaagcacgt tattaaagag 600
tatggcgata tccaagaaag caaaaacccg ctgctgatgg agatcgaata cacctttaag 660
tatccgtggc gttgccgtct gccgcgtctg gaagcgtgga acttcatcca cattatgcgt 720
cagcaagact gcaacattag cctggcgaac aacctgtaca agatcccgaa gatctacatg 780
aagaaaatcc tggagctggc gattctggat tttaacatcc tgcagagcca acaccagcac 840
gaaatgaagc tgattagcac ctggtggaaa aacagcagcg cgatccagct ggacttcttt 900
cgtcaccgtc acattgagag ctacttctgg tgggcgagcc cgctgttcga gccggaattt 960
agcacctgcc gtatcaactg caccaagctg agcaccaaaa tgtttctgct ggacgatatt 1020
tacgacacct atggtaccgt tgaggaactg aaaccgttca ccaccaccct gacccgttgg 1080
gatgtgagca ccgttgacaa ccacccggat tacatgaaaa tcgcgttcaa ctttagctac 1140
gagatctata aggaaattgc gagcgaggcg gagcgtaagc acggtccgtt tgtgtacaaa 1200
tatctgcaaa gctgctggaa gagctacatc gaggcgtata tgcaggaagc ggaatggatt 1260
gcgagcaacc acatcccggg cttcgacgaa tatctgatga acggtgttaa aagcagcggc 1320
atgcgtatcc tgatgattca cgcgctgatc ctgatggaca ccccgctgag cgatgagatt 1380
ctggaacaac tggatatccc gagcagcaaa agccaggcgc tgctgagcct gatcacccgt 1440
ctggtggacg atgttaagga cttcgaggat gaacaagcgc acggcgagat ggcgagcagc 1500
attgaatgct acatgaagga caaccacggc agcacccgtg aggatgcgct gaactatctg 1560
aaaatccgta ttgagagctg cgtgcaggaa ctgaacaagg agctgctgga accgagcaac 1620
atgcacggta gctttcgtaa cctgtacctg aacgtgggca tgcgtgttat cttctttatg 1680
ctgaacgacg gcgacctgtt cacccacagc aaccgtaaag agatccagga cgcgattacc 1740
aagttctttg ttgaaccgat cattccgtaa 1770
<210> 2
<211> 1512
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atggacatgt ccaccatctg gtactactgg gtctccatca tcctgggcgt cttcatcttc 60
ctgatcgtcg gcatccagaa gtggcgatcc aagaagctgc cccctggtcc ctttgctctg 120
cccctgctgg gccacctgca cctgctggag cccaacgtcc acgagtgcct gtccaagatc 180
tccgagaagt tcggccccct gatgtccttc aagttcggca tgaagacctc catcatcgtc 240
tcctcccccg ctatggctaa ggagatcctg cgagagaacg accagatctt cgccaaccga 300
tccatccccg tcgtcgcccg atgcatcgcc tacgacgcct ccgacattct gtggtctccc 360
aacggtcccc gatggcgact gctgcgaaag atttgcgtca aggagctgtt ctcccccaag 420
tccaccgagg ccctgcagcc cctgcgacga gaggaggtcc gacgaaccat gggcaacatc 480
tacaaggact ccatcaacgg cgtctccgtc gatgtcggtg ctaaggcctt catcacctcc 540
ctgaacctga tcaccaacat gatgtggtcc acctccaccg agaccggtga gcgaggcggc 600
gagttcaagg acctggtcgg cgagctggtt cacgttctgg gcgtccccaa cgcctccgac 660
ctgttcccct tcctggagcg attcgacgtc cagggcctgt accgacgaat ggagaaggtc 720
ttcgtccgat tcgacaagat gttcgacggc atcatcgagg acaagctgtc cggtaagtcc 780
aaggagaagg acttcctgca gtccctgctg gacctggttg agcgaggcgt cgatgagcag 840
gaccctgact ccgtccagct gaccatgaag gacgtcaagg tcctgctgat ggacatggtc 900
accggctcta ccgacaccac ttccaacacc gtcgagtggg ctatggccga gctgctgcag 960
cagcccgaga tcatgaagcg agctcagaag gagctggagg aggtcgttgg tctggacaac 1020
atggtcgagg agtgccacct gtcccagctg ccctacctgg acattatcgt caaggaggtc 1080
ctgcgactgc accctgccct gcccctgctg gccccccacc gacctgagcg agagtgcgag 1140
atcggtggtt acatcatccc caaggacacc caggtcctga tcaacgtctg gtccatccag 1200
cgaaacccca aggtctggaa ggagcctctg ctgttcgacc ctgagcgatt ctccgactcc 1260
aagtgggact acaacggccg agacttcgac tacttcccct tcggttccgg ccgacgaatc 1320
tgtgctggtc tgtctatggc caagatcatg gtccactact ccctggcctc cctgctgcac 1380
tcctttgatt ggtccctgcc cgtcgctgag aagctgaaca tggacgagaa gtacggcatc 1440
gtcctgcgaa aggccgtccc tctggttgct ctgcctaagc ctcgactgct gtaccctaac 1500
ctgtacgagt aa 1512
<210> 3
<211> 2079
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atgacctccg ctctgtacgc ttccgacctg ttcaagcagc tgaagtccat catgggcacc 60
gactccctgt ccgatgacgt tgttctggtc atcgccacca cctccctggc cctggtcgcc 120
ggcttcgtcg tcctgctgtg gaagaagacc accgctgacc gatccggtga gctgaagccc 180
ctgatgatcc ctaagtccct gatggccaag gacgaggacg acgatctgga cctgggttct 240
ggtaagaccc gagtctccat cttcttcggc acccagaccg gtactgccga gggcttcgcc 300
aaggccctgt ccgaggagat caaggcccga tacgagaagg ccgctgtcaa ggttatcgac 360
ctggacgact acgccgccga tgatgatcag tacgaggaga agctgaagaa ggagaccctg 420
gccttcttct gcgtcgctac ttacggcgac ggcgagccca ccgacaacgc cgcccgattc 480
tacaagtggt tcaccgagga gaacgagcga gacatcaagc tgcagcagct ggcttacggc 540
gtcttcgccc tgggcaaccg acagtacgag catttcaaca agatcggcat cgtcctggac 600
gaggagctgt gtaagaaggg cgctaagcga ctgatcgagg tcggtctggg tgacgatgac 660
cagtctatcg aggacgactt caacgcctgg aaggagtccc tgtggtccga gctggataag 720
ctgctgaagg acgaggacga caagtccgtc gctaccccct acactgctgt catccctgag 780
taccgagtcg tcacccacga cccccgattt actacccaga agtccatgga gtccaacgtc 840
gccaacggca acaccactat cgacatccac cacccctgcc gagtcgatgt tgctgttcag 900
aaggagctgc acacccacga gtccgatcga agctgtatcc acctggagtt cgacatctcc 960
cgaaccggta tcacctacga gaccggtgac cacgtcggtg tttacgctga gaaccacgtc 1020
gagatcgtcg aggaggctgg taagctgctg ggtcattccc tggacctggt cttctccatc 1080
cacgctgaca aggaggacgg ctctcctctg gagtctgctg ttcctcctcc cttccctggt 1140
ccttgtaccc tgggcaccgg cctggcccga tacgccgacc tgctgaaccc ccctcgaaag 1200
tccgctctgg ttgctctggc tgcttacgct accgagcctt ctgaggctga gaagctgaag 1260
cacctgacct cccctgacgg taaggacgag tactcccagt ggatcgtcgc ctctcagcga 1320
tctctgctgg aggttatggc cgctttcccc tctgctaagc cccctctggg tgttttcttc 1380
gccgctatcg cccctcgact gcagcctcga tactactcta tctcctcctc cccccgactg 1440
gctccttccc gagtccacgt cacctccgct ctggtctacg gtcctactcc taccggtcga 1500
atccacaagg gcgtctgttc cacctggatg aagaacgccg tccccgctga gaagtcccat 1560
gagtgctctg gtgcccctat cttcatccga gcctccaact tcaagctgcc ctccaaccct 1620
tccaccccta tcgttatggt cggccctggt actggcctgg ctcctttccg aggtttcctg 1680
caggagcgaa tggccctgaa ggaggatggt gaggagctgg gttcctccct gctgtttttc 1740
ggctgccgaa accgacagat ggacttcatc tacgaggacg agctgaacaa cttcgtcgat 1800
cagggcgtca tctccgagct gatcatggct ttctcccgag agggcgctca gaaggagtac 1860
gttcagcaca agatgatgga gaaggccgcc caggtctggg atctgattaa ggaggagggc 1920
tacctgtacg tctgcggtga tgctaagggc atggcccgag atgtccaccg aactctgcac 1980
actatcgtcc aggagcagga gggtgtctcc tcttctgagg ctgaggctat cgtcaagaag 2040
ctgcagaccg agggccgata cctgcgagac gtctggtaa 2079
<210> 4
<211> 3450
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atggacatgt ccaccatctg gtactactgg gtctccatca tcctgggcgt cttcatcttc 60
ctgatcgtcg gcatccagaa gtggcgatcc aagaagctgc cccctggtcc ctttgctctg 120
cccctgctgg gccacctgca cctgctggag cccaacgtcc acgagtgcct gtccaagatc 180
tccgagaagt tcggccccct gatgtccttc aagttcggca tgaagacctc catcatcgtc 240
tcctcccccg ctatggctaa ggagatcctg cgagagaacg accagatctt cgccaaccga 300
tccatccccg tcgtcgcccg atgcatcgcc tacgacgcct ccgacattct gtggtctccc 360
aacggtcccc gatggcgact gctgcgaaag atttgcgtca aggagctgtt ctcccccaag 420
tccaccgagg ccctgcagcc cctgcgacga gaggaggtcc gacgaaccat gggcaacatc 480
tacaaggact ccatcaacgg cgtctccgtc gatgtcggtg ctaaggcctt catcacctcc 540
ctgaacctga tcaccaacat gatgtggtcc acctccaccg agaccggtga gcgaggcggc 600
gagttcaagg acctggtcgg cgagctggtt cacgttctgg gcgtccccaa cgcctccgac 660
ctgttcccct tcctggagcg attcgacgtc cagggcctgt accgacgaat ggagaaggtc 720
ttcgtccgat tcgacaagat gttcgacggc atcatcgagg acaagctgtc cggtaagtcc 780
aaggagaagg acttcctgca gtccctgctg gacctggttg agcgaggcgt cgatgagcag 840
gaccctgact ccgtccagct gaccatgaag gacgtcaagg tcctgctgat ggacatggtc 900
accggctcta ccgacaccac ttccaacacc gtcgagtggg ctatggccga gctgctgcag 960
cagcccgaga tcatgaagcg agctcagaag gagctggagg aggtcgttgg tctggacaac 1020
atggtcgagg agtgccacct gtcccagctg ccctacctgg acattatcgt caaggaggtc 1080
ctgcgactgc accctgccct gcccctgctg gccccccacc gacctgagcg agagtgcgag 1140
atcggtggtt acatcatccc caaggacacc caggtcctga tcaacgtctg gtccatccag 1200
cgaaacccca aggtctggaa ggagcctctg ctgttcgacc ctgagcgatt ctccgactcc 1260
aagtgggact acaacggccg agacttcgac tacttcccct tcggttccgg ccgacgaatc 1320
tgtgctggtc tgtctatggc caagatcatg gtccactact ccctggcctc cctgctgcac 1380
tcctttgatt ggtccctgcc cgtcgctgag aagctgaaca tggacgagaa gtacggcatc 1440
gtcctgcgaa aggccgtccc tctggttgct ctgcctaagc ctcgactgct gtaccctaac 1500
ctgtacgagt ggaagaagac caccgctgac cgatccggtg agctgaagcc cctgatgatc 1560
cctaagtccc tgatggccaa ggacgaggac gacgatctgg acctgggttc tggtaagacc 1620
cgagtctcca tcttcttcgg cacccagacc ggtactgccg agggcttcgc caaggccctg 1680
tccgaggaga tcaaggcccg atacgagaag gccgctgtca aggttatcga cctggacgac 1740
tacgccgccg atgatgatca gtacgaggag aagctgaaga aggagaccct ggccttcttc 1800
tgcgtcgcta cttacggcga cggcgagccc accgacaacg ccgcccgatt ctacaagtgg 1860
ttcaccgagg agaacgagcg agacatcaag ctgcagcagc tggcttacgg cgtcttcgcc 1920
ctgggcaacc gacagtacga gcatttcaac aagatcggca tcgtcctgga cgaggagctg 1980
tgtaagaagg gcgctaagcg actgatcgag gtcggtctgg gtgacgatga ccagtctatc 2040
gaggacgact tcaacgcctg gaaggagtcc ctgtggtccg agctggataa gctgctgaag 2100
gacgaggacg acaagtccgt cgctaccccc tacactgctg tcatccctga gtaccgagtc 2160
gtcacccacg acccccgatt tactacccag aagtccatgg agtccaacgt cgccaacggc 2220
aacaccacta tcgacatcca ccacccctgc cgagtcgatg ttgctgttca gaaggagctg 2280
cacacccacg agtccgatcg aagctgtatc cacctggagt tcgacatctc ccgaaccggt 2340
atcacctacg agaccggtga ccacgtcggt gtttacgctg agaaccacgt cgagatcgtc 2400
gaggaggctg gtaagctgct gggtcattcc ctggacctgg tcttctccat ccacgctgac 2460
aaggaggacg gctctcctct ggagtctgct gttcctcctc ccttccctgg tccttgtacc 2520
ctgggcaccg gcctggcccg atacgccgac ctgctgaacc cccctcgaaa gtccgctctg 2580
gttgctctgg ctgcttacgc taccgagcct tctgaggctg agaagctgaa gcacctgacc 2640
tcccctgacg gtaaggacga gtactcccag tggatcgtcg cctctcagcg atctctgctg 2700
gaggttatgg ccgctttccc ctctgctaag ccccctctgg gtgttttctt cgccgctatc 2760
gcccctcgac tgcagcctcg atactactct atctcctcct ccccccgact ggctccttcc 2820
cgagtccacg tcacctccgc tctggtctac ggtcctactc ctaccggtcg aatccacaag 2880
ggcgtctgtt ccacctggat gaagaacgcc gtccccgctg agaagtccca tgagtgctct 2940
ggtgccccta tcttcatccg agcctccaac ttcaagctgc cctccaaccc ttccacccct 3000
atcgttatgg tcggccctgg tactggcctg gctcctttcc gaggtttcct gcaggagcga 3060
atggccctga aggaggatgg tgaggagctg ggttcctccc tgctgttttt cggctgccga 3120
aaccgacaga tggacttcat ctacgaggac gagctgaaca acttcgtcga tcagggcgtc 3180
atctccgagc tgatcatggc tttctcccga gagggcgctc agaaggagta cgttcagcac 3240
aagatgatgg agaaggccgc ccaggtctgg gatctgatta aggaggaggg ctacctgtac 3300
gtctgcggtg atgctaaggg catggcccga gatgtccacc gaactctgca cactatcgtc 3360
caggagcagg agggtgtctc ctcttctgag gctgaggcta tcgtcaagaa gctgcagacc 3420
gagggccgat acctgcgaga cgtctggtaa 3450
<210> 5
<211> 1578
<212> DNA
<213>Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 5
atggaccaat tggtgaaaac tgaagtcacc aagaagtctt ttactgctcc tgtacaaaag 60
gcttctacac cagttttaac caataaaaca gtcatttctg gatcgaaagt caaaagttta 120
tcatctgcgc aatcgagctc atcaggacct tcatcatcta gtgaggaaga tgattcccgc 180
gatattgaaa gcttggataa gaaaatacgt cctttagaag aattagaagc attattaagt 240
agtggaaata caaaacaatt gaagaacaaa gaggtcgctg ccttggttat tcacggtaag 300
ttacctttgt acgctttgga gaaaaaatta ggtgatacta cgagagcggt tgcggtacgt 360
aggaaggctc tttcaatttt ggcagaagct cctgtattag catctgatcg tttaccatat 420
aaaaattatg actacgaccg cgtatttggc gcttgttgtg aaaatgttat aggttacatg 480
cctttgcccg ttggtgttat aggccccttg gttatcgatg gtacatctta tcatatacca 540
atggcaacta cagagggttg tttggtagct tctgccatgc gtggctgtaa ggcaatcaat 600
gctggcggtg gtgcaacaac tgttttaact aaggatggta tgacaagagg cccagtagtc 660
cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat ggttagactc agaagaggga 720
caaaacgcaa ttaaaaaagc ttttaactct acatcaagat ttgcacgtct gcaacatatt 780
caaacttgtc tagcaggaga tttactcttc atgagattta gaacaactac tggtgacgca 840
atgggtatga atatgatttc taaaggtgtc gaatactcat taaagcaaat ggtagaagag 900
tatggctggg aagatatgga ggttgtctcc gtttctggta actactgtac cgacaaaaaa 960
ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg tcgtcgcaga agctactatt 1020
cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt ccgcattggt tgagttgaac 1080
attgctaaga atttggttgg atctgcaatg gctgggtctg ttggtggatt taacgcacat 1140
gcagctaatt tagtgacagc tgttttcttg gcattaggac aagatcctgc acaaaatgtt 1200
gaaagttcca actgtataac attgatgaaa gaagtggacg gtgatttgag aatttccgta 1260
tccatgccat ccatcgaagt aggtaccatc ggtggtggta ctgttctaga accacaaggt 1320
gccatgttgg acttattagg tgtaagaggc ccgcatgcta ccgctcctgg taccaacgca 1380
cgtcaattag caagaatagt tgcctgtgcc gtcttggcag gtgaattatc cttatgtgct 1440
gccctagcag ccggccattt ggttcaaagt catatgaccc acaacaggaa acctgctgaa 1500
ccaacaaaac ctaacaattt ggacgccact gatataaatc gtttgaaaga tgggtccgtc 1560
acctgcatta aatcctaa 1578
<210> 6
<211> 406
<212> DNA
<213>Yarrowia lipolytica (Yarrowia lipolytica)
<400> 6
agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60
cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 120
aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180
cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240
gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300
aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360
cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaa 406
<210> 7
<211> 999
<212> DNA
<213>Yarrowia lipolytica (Yarrowia lipolytica)
<400> 7
gagtttggcg cccgtttttt cgagccccac acgtttcggt gagtatgagc ggcggcagat 60
tcgagcgttt ccggtttccg cggctggacg agagcccatg atgggggctc ccaccaccag 120
caatcagggc cctgattaca cacccacctg taatgtcatg ctgttcatcg tggttaatgc 180
tgctgtgtgc tgtgtgtgtg tgttgtttgg cgctcattgt tgcgttatgc agcgtacacc 240
acaatattgg aagcttatta gcctttctat tttttcgttt gcaaggctta acaacattgc 300
tgtggagagg gatggggata tggaggccgc tggagggagt cggagaggcg ttttggagcg 360
gcttggcctg gcgcccagct cgcgaaacgc acctaggacc ctttggcacg ccgaaatgtg 420
ccacttttca gtctagtaac gccttaccta cgtcattcca tgcatgcatg tttgcgcctt 480
ttttcccttg cccttgatcg ccacacagta cagtgcactg tacagtggag gttttggggg 540
ggtcttagat gggagctaaa agcggcctag cggtacacta gtgggattgt atggagtggc 600
atggagccta ggtggagcct gacaggacgc acgaccggct agcccgtgac agacgatggg 660
tggctcctgt tgtccaccgc gtacaaatgt ttgggccaaa gtcttgtcag ccttgcttgc 720
gaacctaatt cccaattttg tcacttcgca cccccattga tcgagcccta acccctgccc 780
atcaggcaat ccaattaagc tcgcattgtc tgccttgttt agtttggctc ctgcccgttt 840
cggcgtccac ttgcacaaac acaaacaagc attatatata aggctcgtct ctccctccca 900
accacactca cttttttgcc cgtcttccct tgctaacaca aaagtcaaga acacaaacaa 960
ccaccccaac ccccttacac acaagacata tctacagca 999
<210> 8
<211> 931
<212> DNA
<213>Yarrowia lipolytica (Yarrowia lipolytica)
<400> 8
cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg tgcttggaga 60
tggaagccgg tagaaccggg ctgcttgggg ggatttgggg ccgctgggct ccaaagaggg 120
gtaggcattt cgttggggtt acgtaattgc ggcatttggg tcctgcgcgc atgtcccatt 180
ggtcagaatt agtccggata ggagacttat cagccaatca cagcgccgga tccacctgta 240
ggttgggttg ggtgggagca cccctccaca gagtagagtc aaacagcagc agcaacatga 300
tagttggggg tgtgcgtgtt aaaggaaaaa aaaagaagct tgggttatat tcccgctcta 360
tttagaggtt gcgggataga cgccgacgga gggcaatggc gccatggaac cttgcggata 420
tcgatacgcc gcggcggact gcgtccgaac cagctccagc agcgtttttt ccgggccatt 480
gagccgactg cgaccccgcc aacgtgtctt ggcccacgca ctcatgtcat gttggtgttg 540
ggaggccact ttttaagtag cacaaggcac ctagctcgca gcaaggtgtc cgaaccaaag 600
aagcggctgc agtggtgcaa acggggcgga aacggcggga aaaagccacg ggggcacgaa 660
ttgaggcacg ccctcgaatt tgagacgagt cacggcccca ttcgcccgcg caatggctcg 720
ccaacgcccg gtcttttgca ccacatcagg ttaccccaag ccaaaccttt gtgttaaaaa 780
gcttaacata ttataccgaa cgtaggtttg ggcgggcttg ctccgtctgt ccaaggcaac 840
atttatataa gggtctgcat cgccggctca attgaatctt ttttcttctt ctcttctcta 900
tattcattct tgaattaaac acacatcaac a 931
<210> 9
<211> 411
<212> DNA
<213>Yarrowia lipolytica (Yarrowia lipolytica)
<400> 9
cctgtcccca cgttgccggt cttgcctcct actacctgtc catcaatgac gaggttctca 60
cccctgccca ggtcgaggct cttattactg agtccaacac cggtgttctt cccaccacca 120
acctcaaggg ctctcccaac gctgttgcct acaacggtgt tggcatttag gcaattaaca 180
gatagtttgc cggtgataat tctcttaacc tcccacactc ctttgacata acgatttatg 240
taacgaaact gaaatttgac cagatattgt tgtaaataga aaatctggct tgtaggtggc 300
aaaatcccgt ctttgttcat caattccctc tgtgactact cgtcatccct ttatgttcga 360
ctgtcgtatt tttattttcc atacatacgc aagtgagatg cccgtgtccg a 411
<210> 10
<211> 502
<212> DNA
<213>Yarrowia lipolytica (Yarrowia lipolytica)
<400> 10
cactggccgg tcgataattt aacgtgctga gctcagcaca cgcattgccc attggctgta 60
tatagatgaa tgtaatgata ccgtaagaga atgagagcac ggtattgtat tacaggggat 120
taagtacaca ttacttggag ttctgtacca gaagacacta ctatacatgg tatcacttac 180
attagagtcg gtgaccgtat tcgtctcgta tagacataat attttcctac cccacattgt 240
tcctgggcct tcggagcaca tctacagtga gtgactgttt cagttgagct tgaggggtta 300
agtaagtggg ggaagggttt gcgattctga aaaagagcat gactaatctc tctgtggagg 360
agcaatgaag tcacgtgatg caatcatacc ggtgtatcgg atctgcctgg gtgtctgatt 420
actaatcatt tactcacctg ttttccccag ctatctcatc catctcagag cctcggccca 480
gccttcggcc cttttgggtt tc 502
<210> 11
<211> 200
<212> DNA
<213>Yarrowia lipolytica (Yarrowia lipolytica)
<400> 11
gctatttatc actctttaca acttctacct caactatcta ctttaataaa tgaatatcgt 60
ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc agcatgcgat 120
cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc aggcagcttg 180
agaagattga cacaggtgga 200
<210> 12
<211> 700
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
tcgatcctaa ggggtggcat aactgtcgcg tacggcccga taagggcctt ctccaaaagg 60
gaagccggtt gaaattccgg cacttggatg tggattctcc acggcaacgt aactgaatgt 120
ggggacggtg gcacaagtct tggaaggagt tatcttttct ttttaacgga gtcaacaccc 180
tggaattagt ttgtctagag atagggtatc gttccggaag aggggggcag ctttgtcccc 240
tccgatgcac ttgtgacgcc ccttgaaaac ccgcaggaag gaatagtttt cacgccaagt 300
cgtactgata accgcagcag gtctccaagg tgaacagcct ctagttgata gaataatgta 360
gataagggaa gtcggcaaaa tagatccgta acttcgggat aaggattggc tctgggggtt 420
ggtggatgga agcgtgggag accccaaggg actggcggct gggcaactgg cagccggacc 480
cgcggcagac actgcgtcgc tccgtccaca tcatcaaccg ccccagaact ggtacggaca 540
aggggaatct gactgtctaa ttaaaacata gctttgcgat ggttgtaaaa caatgttgac 600
gcaaagtgat ttctgcccag tgctctgaat gtcaaagtga agaaattcaa ccaagcgcgg 660
gtaaacggcg ggagtaacta tgactctctt aaggtagcca 700
<210> 13
<211> 2506
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
ggtgtgttct gtggagcatt ctcacttttg gtaaacgaca ttgcttcaag tgcagcggaa 60
tcaaaaagta taaagtgggc agcgagtata cctgtacaga ctgtaggcga taactcaatc 120
caattacccc ccacaacatg actggccaaa ctgatctcaa gactttattg aaatcagcaa 180
caccgattct caatgaaggc acatacttct tctgcaacat tcacttgacg cctaaagttg 240
gtgagaaatg gaccgacaag acatattctg ctatccacgg actgttgcct gtgtcggtgg 300
ctacaatacg tgagtcagaa gggctgacgg tggtggttcc caaggaaaag gtcgacgagt 360
atctgtctga ctcgtcattg ccgcctttgg agtacgactc caactatgag tgtgcttgga 420
tcactttgac gatacattct tcgttggagg ctgtgggtct gacagctgcg ttttcggcgc 480
ggttggccga caacaatatc agctgcaacg tcattgctgg ctttcatcat gatcacattt 540
ttgtcggcaa aggcgacgcc cagagagcca ttgacgttct ttctaatttg gaccgatagc 600
cgtatagtcc agtctatcta taagttcaac taactcgtaa ctattaccat aacatatact 660
tcactgcccc agataaggtt ccgataaaaa gttctgcaga ctaaatttat ttcagtctcc 720
tcttcaccac caaaatgccc tcctacgaag ctcgagctaa cgtccacaag tccgcctttg 780
ccgctcgagt gctcaagctc gtggcagcca agaaaaccaa cctgtgtgct tctctggatg 840
ttaccaccac caaggagctc attgagcttg ccgataaggt cggaccttat gtgtgcatga 900
tcaagaccca tatcgacatc attgacgact tcacctacgc cggcactgtg ctccccctca 960
aggaacttgc tcttaagcac ggtttcttcc tgttcgagga cagaaagttc gcagatattg 1020
gcaacactgt caagcaccag tacaagaacg gtgtctaccg aatcgccgag tggtccgata 1080
tcaccaacgc ccacggtgta cccggaaccg gaatcattgc tggcctgcga gctggtgccg 1140
aggaaactgt ctctgaacag aagaaggagg acgtctctga ctacgagaac tcccagtaca 1200
aggagttcct ggtcccctct cccaacgaga agctggccag aggtctgctc atgctggccg 1260
agctgtcttg caagggctct ctggccactg gcgagtactc caagcagacc attgagcttg 1320
cccgatccga ccccgagttt gtggttggct tcattgccca gaaccgacct aagggcgact 1380
ctgaggactg gcttattctg acccccgggg tgggtcttga cgacaaggga gacgctctcg 1440
gacagcagta ccgaactgtt gaggatgtca tgtctaccgg aacggatatc ataattgtcg 1500
gccgaggtct gtacggccag aaccgagatc ctattgagga ggccaagcga taccagaagg 1560
ctggctggga ggcttaccag aagattaact gttagaggtt agactatgga tatgtaattt 1620
aactgtgtat atagagagcg tgcaagtatg gagcgcttgt tcagcttgta tgatggtcag 1680
acgacctgtc tgatcgagta tgtatgatac tgcacaacct gtgtatccgc atgatctgtc 1740
caatggggca tgttgttgtg tttctcgata cggagatgct gggtacaagt agctaatacg 1800
attgaactac ttatacttat atgaggcttg aagaaagctg acttgtgtat gacttattct 1860
caactacatc cccagtcaca ataccaccac tgcactacca ctacaccaat gcctcgtcat 1920
ctaattagtg acgcgcatga atggattaac gagattccca ctgtccctat ctactatcta 1980
gcgaaaccac agccaaggga acgggcttgg cagaatcagc ggggaaagaa gaccctgttg 2040
agcttgactc tagtttgaca ttgtgaagag acataggggg tgtagaataa gtgggagctt 2100
cggcgccggt gaaataccac tacccttatc gtttctttac ttatttagta agtggaagtg 2160
gtttaacaac cattttctag cattcctttc caggctgaag acattgtcag gtggggagtt 2220
tggctggggc ggcacatctg ttaaaagata acgcagatgt cctaaggggg actcaatgag 2280
aacagaaatc tcatgtagaa caaaagggta aaagtcccct tgattttgat tttcagtgtg 2340
aatacaaacc atgaaagtgt ggcctatcga tcctttagtt gttcggagtt tgaacctaga 2400
ggtgccagaa aagttaccac agggataact ggcttgtggc agtcaagcgt tcatagcgac 2460
attgcttttt gatccttcga tgtcggctct tcctatcata ccgaag 2506
<210> 14
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
tcgatcctaa ggggtggcat aactgtcgc 29
<210> 15
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
acgccgccaa cccggtctct tggctacctt aagagagtca t 41
<210> 16
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
atgactctct taaggtagcc aagagaccgg gttggcggcg ta 42
<210> 17
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
accgttaaac atctccgcca ttttgaatga ttcttatact cag 43
<210> 18
<211> 66
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
tatttttatt ttccatacat acgcaagtga gatgcccgtg tccgagagtt tggcgcccgt 60
tttttc 66
<210> 19
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
ccagatggtg gacatgtcca ttgctgtaga tatgtcttgt gt 42
<210> 20
<211> 76
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
tcagagcctc ggcccagcct tcggcccttt tgggtttccg cagtaggatg tcctgcacgg 60
gtctttttgt ggggtg 76
<210> 21
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
agcgtacaga gcggaggtca ttgttgatgt gtgtttaatt c 41
<210> 22
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
gttgaaccga tcattccgta acctgtcccc acgttgccgg tc 42
<210> 23
<211> 65
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
tcaccgaaac gtgtggggct cgaaaaaacg ggcgccaaac tctcggacac gggcatctca 60
cttgc 65
<210> 24
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
taccctaacc tgtacgagta acactggccg gtcgataatt t 41
<210> 25
<211> 75
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
caccccacaa aaagacccgt gcaggacatc ctactgcgga aacccaaaag ggccgaaggc 60
tgggccgagg ctctg 75
<210> 26
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 26
tacctgcgag acgtctggta agctatttat cactctttac aac 43
<210> 27
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 27
aatgtcgttt accaaaagtg agaatgctcc acagaacaca cctccacctg tgtcaatctt 60
ctc 63
<210> 28
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 28
gagaagattg acacaggtgg aggtgtgttc tgtggagcat tctcactttt ggtaaacgac 60
att 63
<210> 29
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 29
cttcggtatg ataggaagag ccgacatcg 29
<210> 30
<211> 44
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 30
tctgagtata agaatcattc aaaatggcgg agatgtttaa cggt 44
<210> 31
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 31
gaccggcaac gtggggacag gttacggaat gatcggttca ac 42
<210> 32
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 32
acacaagaca tatctacagc aatggacatg tccaccatct gg 42
<210> 33
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 33
aaattatcga ccggccagtg ttactcgtac aggttagggt ac 42
<210> 34
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 34
gtcagcggtg gtcttcttcc actcgtacag gttagggtac ag 42
<210> 35
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 35
tgaattaaac acacatcaac aatgacctcc gctctgtacg ct 42
<210> 36
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 36
gttgtaaaga gtgataaata gcttaccaga cgtctcgcag gt 42
<210> 37
<211> 49
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 37
cctgtacgag ggttccacct cctccggttg gaagaagacc accgctgac 49
<210> 38
<211> 44
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 38
ggaacccgaa actaaggatc catggaccaa ttggtgaaaa ctga 44
<210> 39
<211> 44
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 39
acaagttccg tagttggatc cttaggattt aatgcaggtg acgg 44

Claims (9)

1. a kind of construction method for the restructuring Yarrowia lipolytica for producing valencia orange alkene and nootkatone, it is characterized in that including Following steps：By the method for homologous recombination, valencia orange alkene synthase coding is imported to the rDNA sites of Yarrowia lipolytica The encoding gene of gene C VS expression cassettes, nootkatone synthasee code gene CYP706M1 expression cassettes and cytochrome P450 reductase AtCPR expression cassettes, obtain producing the restructuring Yarrowia lipolytica 1 of valencia orange alkene and nootkatone；
Valencia orange alkene synthasee code gene CVS nucleotide sequence is as shown in SEQ ID NO.1；
Nootkatone synthasee code gene CYP706M1 nucleotide sequence is as shown in SEQ ID NO.2；
The encoding gene AtCPR of cytochrome P450 reductase nucleotide sequence is as shown in SEQ ID NO.3.
2. the production valencia orange alkene of method structure and the restructuring Yarrowia lipolytica 1 of nootkatone of claim 1.
3. the production valencia orange alkene of claim 2 and the fermenting and producing balun of restructuring Yarrowia lipolytica 1 west of nootkatone The application of sub- tangerine alkene and nootkatone.
4. a kind of construction method for the restructuring Yarrowia lipolytica for producing valencia orange alkene and nootkatone, it is characterized in that including Following steps：By the method for homologous recombination, valencia orange alkene synthase coding is imported to the rDNA sites of Yarrowia lipolytica Gene C VS expression cassettes, nootkatone synthasee code gene truncate the cytochrome P450 reductase coding base of 46 amino acid with N-terminal The fusion CYP706M1-tAtCPR expression cassettes of cause, obtain producing the restructuring solution fat Ye Shi of valencia orange alkene and nootkatone Saccharomycete 2；
Valencia orange alkene synthasee code gene CVS nucleotide sequence is as shown in SEQ ID NO.1；
Fusion CYP706M1-tAtCPR nucleotide sequence is as shown in SEQ ID NO.4.
5. the production valencia orange alkene of method structure and the restructuring Yarrowia lipolytica 2 of nootkatone of claim 4.
6. the production valencia orange alkene of claim 5 and the fermenting and producing balun of restructuring Yarrowia lipolytica 2 west of nootkatone The application of sub- tangerine alkene and nootkatone.
7. a kind of construction method for the restructuring Yarrowia lipolytica for producing valencia orange alkene and nootkatone, it is characterized in that including Following steps：
(1) by the method for homologous recombination, valencia orange alkene synthase coding base is imported to the rDNA sites of Yarrowia lipolytica Because of CVS expression cassettes, the cytochrome P450 reductase encoding gene of nootkatone synthasee code gene and N-terminal 46 amino acid of truncation Fusion CYP706M1-tAtCPR expression cassettes, obtain producing the restructuring solution fat Ye Shi ferment of valencia orange alkene and nootkatone Female bacterium 2；
(2) 3-hydroxy-3-methylglutaryl-coenzyme A is reduced into enzyme coding gene tHMG1 insertion plasmid PINA1269, expressed Plasmid ptHMG1；The expression plasmid ptHMG1 is imported to the restructuring Yarrowia lipolytica of production valencia orange alkene and nootkatone Bacterium 2, obtain producing the restructuring Yarrowia lipolytica 3 of valencia orange alkene and nootkatone；
Valencia orange alkene synthasee code gene CVS nucleotide sequence is as shown in SEQ ID NO.1；
Fusion CYP706M1-tAtCPR nucleotide sequence is as shown in SEQ ID NO.4.
3-hydroxy-3-methylglutaryl-coenzyme A reduction enzyme coding gene tHMG1 nucleotide sequence is as shown in SEQ ID NO.5.
8. the production valencia orange alkene of method structure and the restructuring Yarrowia lipolytica 3 of nootkatone of claim 7.
9. the production valencia orange alkene of claim 8 and the fermenting and producing balun of restructuring Yarrowia lipolytica 3 west of nootkatone The application of sub- tangerine alkene and nootkatone.
CN201710864583.6A 2017-09-22 2017-09-22 Produce the restructuring Yarrowia lipolytica and construction method of valencia orange alkene and nootkatone Pending CN107723252A (en)

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CN110117551A (en) * 2019-04-04 2019-08-13 华南理工大学 The saccharomyces cerevisiae engineered yeast and its construction method of production Valencia alkene 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
CN111235047A (en) * 2020-02-12 2020-06-05 天津大学 Recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method
CN111235045A (en) * 2020-01-19 2020-06-05 天津大学 Recombinant yarrowia lipolytica for heterologous synthesis of β -balsam stem and oleanolic acid and construction method thereof
CN112823668A (en) * 2019-11-20 2021-05-21 内蒙古伊利实业集团股份有限公司 Composition for reducing weight and preparation method thereof
CN115976118A (en) * 2022-06-14 2023-04-18 武汉合生科技有限公司 Method and carrier for biologically synthesizing nootkatone

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110117551A (en) * 2019-04-04 2019-08-13 华南理工大学 The saccharomyces cerevisiae engineered yeast and its construction method of production Valencia alkene and application
CN110117551B (en) * 2019-04-04 2023-02-10 华南理工大学 Saccharomyces cerevisiae engineering bacterium for producing valencene, and construction method and application thereof
CN112823668A (en) * 2019-11-20 2021-05-21 内蒙古伊利实业集团股份有限公司 Composition for reducing weight and preparation method thereof
CN112823668B (en) * 2019-11-20 2023-03-28 内蒙古伊利实业集团股份有限公司 Composition for reducing weight and preparation method thereof
CN110982720A (en) * 2019-12-13 2020-04-10 天津大学 Recombinant yarrowia lipolytica producing dammarane diol and protopanoxadiol and use thereof
CN111235045A (en) * 2020-01-19 2020-06-05 天津大学 Recombinant yarrowia lipolytica for heterologous synthesis of β -balsam stem and oleanolic acid and construction method thereof
CN111235047A (en) * 2020-02-12 2020-06-05 天津大学 Recombinant yarrowia lipolytica for heterogeneously synthesizing α -coumarol and ursolic acid and construction method
CN115976118A (en) * 2022-06-14 2023-04-18 武汉合生科技有限公司 Method and carrier for biologically synthesizing nootkatone
CN115976118B (en) * 2022-06-14 2024-01-23 武汉合生科技有限公司 Method and carrier for biosynthesis of nocardomperidone

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