CN109136120A - Microorganism and application thereof - Google Patents

Microorganism and application thereof Download PDF

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CN109136120A
CN109136120A CN201710464916.6A CN201710464916A CN109136120A CN 109136120 A CN109136120 A CN 109136120A CN 201710464916 A CN201710464916 A CN 201710464916A CN 109136120 A CN109136120 A CN 109136120A
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fermentation
gene
lycopene
microorganism
seq
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CN109136120B (en
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刘天罡
叶紫玲
沈佳
刘然
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Wuhan Hesheng Technology Co ltd
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Wuhan Zhenzhi Biological Science & Technology Co Ltd
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Priority to PCT/CN2018/091113 priority patent/WO2018233531A1/en
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Abstract

The invention proposes a kind of microorganism and application thereof and the method for obtaining lycopene, which is overexpressed PaCrtE, PagCrtB and BtCrtI gene.Using microorganism according to an embodiment of the present invention, the yield for producing lycopene is significantly improved, with short production cycle, high production efficiency.

Description

Microorganism and application thereof
Technical field
The present invention relates to bioengineering fields, specifically, the present invention relates to microorganism and application thereof, more specifically, this hair The bright method for being related to microorganism, obtaining lycopene and microorganism are preparing the purposes in lycopene.
Background technique
The development and production of lycopene mainly have the methods of natural product extraction, chemical synthesis, microbial fermentation.It is natural to produce Object, which extracts, mainly obtains lycopene by the abstraction purification of ripening fruits, however this production method climate, kind, Managing many uncontrollable factors such as position, maturity influences, and has apparent seasonal, content unstability, and large area kind It plants, the higher cost of cultivation, content is generally also relatively low.In addition, the lycopene of high-purity in abstraction purification technology very Difficulty, the finished product that these cause lycopene jointly are sufficiently expensive.There is chemical synthesis raw material to be easy to get inexpensive, reaction condition Mildly, reaction rate is fast, product is easy the advantages of separating with reaction system, but since double bond stereoselectivity is uncontrollable and not With the chemical agent residue of degree, quality, safety and the use scope of product are rather limited.Microbe fermentation method master It is this if converting lycopene for cheap raw materials such as glucose, starch, soybean cake powders using the biological metabolism of microorganism Method is not influenced by factors such as season, region, weathers, and raw material easily obtains, is with short production cycle, technological operation is simple, at low cost Honest and clean, product quality is controllable, product easy purification, highly-safe, and environmental pollution is less, not only solves due tos planting plants etc. The drawbacks of occupying a large amount of the problem of ploughing, also solving chemical synthesis not environmentally.Most of all, the tomato red of fermentation method production Element belongs to natural type product, and activity is consistent with the active constituent of extracted form natural plant, it is considered to be lycopene production most has The method of prospect.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.
The mainly trispore Bruce mould of more lycopene producing strains is studied at present.But trispore Bruce mould Degradation phenomena is easy to happen in succeeding generations, so that yield of lycopene is reduced, separately with toxic components such as aflatoxin Synthesis, on the other hand, the growth cycle of both microorganisms is longer compared to yeast and Escherichia coli, greatly reduces its life Efficiency is produced, so that the difficulty for causing production lycopene increases.
For this purpose, an object of the present invention is to provide a kind of microorganism, the yield of the micro-organisms lycopene Height, with short production cycle, high production efficiency.
In the first aspect of the present invention, the invention proposes a kind of microorganisms.According to an embodiment of the invention, micro- life Object is overexpressed PaCrtE, PagCrtB and BtCrtI gene.Using microorganism according to an embodiment of the present invention, lycopene is produced Yield significantly improve, with short production cycle, high production efficiency.
According to an embodiment of the invention, mentioned microorganism can further include following additional technical feature at least it One:
According to an embodiment of the invention, it includes being selected from tHMG1, INO2, yap1 that the microorganism, which is further overexpressed, Spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, EUTE, ERG12, At least one of IDI1, ERG10, MVD1, ERG13, ERG8 gene.Inventors have found that the microorganism is further overexpressed At least one of gene is stated, the yield of lycopene further increases.
According to an embodiment of the invention, the further silencing of microorganism includes being selected from GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef1 At least one of gene.Inventors have found that at least one of described further silencing said gene of microorganism, lycopene Yield further increases.
According to an embodiment of the invention, the microorganism is saccharomycete.The growth cycle of saccharomycete is short, real using the present invention The efficiency for applying the micro-organisms lycopene of example further increases.
According to an embodiment of the invention, the microorganism further comprises that can operate regulation ERG9 gene.According to the present invention Embodiment, it is described operate regulation ERG9 gene include but is not limited to replace ERG9 gene promoter so that ERG9 The expression of gene can according to need, and further increase the efficiency of micro-organisms lycopene.
In the second aspect of the present invention, the invention proposes a kind of methods for obtaining lycopene.Reality according to the present invention Apply example, which comprises mentioned-above microorganism is subjected to fermentation process；And fermentation process product is carried out at extraction Reason, to obtain the lycopene.Using it is according to an embodiment of the present invention obtain lycopene method, can high yield, High efficiency obtains lycopene, and the purity is high of lycopene.
According to an embodiment of the invention, the method for above-mentioned acquisition lycopene can further include following supplementary technology At least one feature:
According to an embodiment of the invention, using the method according to an embodiment of the present invention for obtaining lycopene, kind of acquisition Lycopene yield is higher, more efficient, and purity is also higher.
According to an embodiment of the invention, the fermentation process is realized in the following way: the microorganism is carried out Basal fermentation processing and the processing of two-stage fed batch fermentation, the basal fermentation processing is carried out in basic fermentation medium , the two-stage fed batch fermentation processing is by successively adding the first feed supplement training on the basis of the basic fermentation medium What feeding base and the second supplemented medium were realized, wherein the basic fermentation medium is containing containing 24g/L peptone, 12g/L The YPD culture medium of yeast extract and 12g/L glucose；First supplemented medium is to contain 500g/L glucose and 15g/ The YPD culture medium of L yeast extract；Second supplemented medium is 75% ethyl alcohol or 50% glycerol.Inventor passes through experiment It was found that the rate of amplification height of microorganism, the yield of lycopene also further increase using above-mentioned fermentation process mode.
According to an embodiment of the invention, the extraction processing includes: to carry out the fermentation process product at ultrasonication Reason and organic extraction processing.Inventor find during extraction processing to tunning, product after fermentation process is carried out Ultrasonication processing, compared to the method using salt acid cooking, the degradation of lycopene is greatly reduced, obtains after extraction processing The yield of lycopene further increases.
In the third aspect of the present invention, the invention proposes microorganisms noted earlier to prepare the purposes in lycopene. As previously mentioned, the yield for producing lycopene is high, with short production cycle, high production efficiency using the microorganism of the embodiment of the present invention.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of according to embodiments of the present invention 1 knockout box segment 1；
Fig. 2 is the structural schematic diagram of according to embodiments of the present invention 1 knockout box segment 2；
Fig. 3 is the structural schematic diagram of according to embodiments of the present invention 1 knockout box segment 3；
Fig. 4 is second generation engineered strain shake flask fermentation result；
Fig. 5 is the structural schematic diagram of according to embodiments of the present invention 4 knockout box segment 4；And
Fig. 6 is the selection result figure of fermentation medium according to an embodiment of the present invention.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair It is bright, and be not considered as limiting the invention.
Microorganism
In the first aspect of the present invention, the invention proposes a kind of microorganisms.According to an embodiment of the invention, the microorganism It is overexpressed PaCrtE, PagCrtB and BtCrtI gene.Using microorganism according to an embodiment of the present invention, lycopene is produced Yield is high, with short production cycle, high production efficiency.
According to a particular embodiment of the invention, the microorganism can also further be overexpressed including being selected from tHMG1, INO2, yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, At least one of EUTE, ERG12, IDI1, ERG10, MVD1, ERG13, ERG8 gene.
Still another embodiment according to the present invention, the microorganism can also silencing include further being selected from GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, At least one of Yor292C, Sfk1, Mef1 gene.Inventors have found that the further silencing said gene of microorganism is at least One of, the yield of lycopene further increases.
Inventors have found that the microorganism be further overexpressed or silencing said gene at least one, lycopene Yield further significantly improves.
Microorganism described herein is for producing lycopene, and compared with the prior art, yield has obtained significantly mentioning Height can be at least up to 2.5g/L.
According to a particular embodiment of the invention, the microorganism, which can further include, can operate regulation ERG9 gene. The regulation ERG9 gene described herein that operates refers to the original promoter of replacement ERG, is regulated and controled to realize by glucose The expression of ERG9, to increase the yield of lycopene.
It should be noted that discovery: inventor is overexpressed PaCrtE, PagCrtB, BtCrtI gene can by experiment Achieve the purpose that efficiently synthesize lycopene in yeast；Silencing GAL1,7,10 or GAL1,7,10,80 can reach utilization Galactolipin induction or the purpose that production lycopene is adjusted using concentration of glucose；It is overexpressed INO2, yap1, spt15-5, Taf25-3 gene is adversity gene, can increase the yield of lycopene；It is overexpressed GapN, PYC2, SMAE1, MDH2, POS5, One or more genes in pntAB can balance the intracorporal reducing power of yeast, can increase the yield of lycopene；It is overexpressed One or more genes in ADH2, ALD6, EUTE can increase the supply of the precursor substance of synthesis lycopene, increase tomato The yield of red pigment；It is overexpressed ERG12, IDI1, ERG10, MVD1, ERG13, one or more genes in ERG8 can balance MVA approach in related lycopene synthesis, increases the yield of lycopene；Silencing ROX1, VBA5, DOS2, Ypl062W, At least one of Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef1 gene, can be from whole Yeast system is adjusted on body, to increase the yield of lycopene.Utilize microorganism according to an embodiment of the present invention, lycopene Yield be significantly improved.
The method for obtaining lycopene
In the second aspect of the present invention, the invention proposes a kind of methods for obtaining lycopene.Reality according to the present invention Example is applied, this method comprises: mentioned-above microorganism is carried out fermentation process；And fermentation process product is carried out at extraction Reason, to obtain the lycopene.Using it is according to an embodiment of the present invention obtain lycopene method, can high yield, High efficiency obtains lycopene, and the purity is high of lycopene.
According to a particular embodiment of the invention, the fermentation process is to be handled by basal fermentation and two-stage batch feeding hair Ferment processing carries out, described in detail below:
115 degree of progress skies go out 30 minutes after fermentor is packed into a small amount of pure water.Prepare the fermentation liquid and benefit of batch culture Feed liquid calibrates the pH electrode of fermentor, clamps each pipeline of fermentor, and 115 degree sterilize 30 minutes.Take out fermentor, connection Air pipe line, ventilated (0.1vvm or so) on a small quantity, is opened simultaneously chilled water unit and is cooled down.50 are down to temperature When spending left and right, revolving speed is opened to 100rpm.Calibrate dissolved oxygen electrode.It is 0% that the state of not connected route, which set oxygen dissolving value, subsequent general Revolving speed rises to 600rpm, while ventilatory capacity is adjusted to 2vvm, connects dissolved oxygen electrode route, set after dissolved oxygen stable the value as 100%.Ventilatory capacity and revolving speed are then reduced to fermentation desirable value (ventilatory capacity 1.5vvm, revolving speed 300rpm-600rpm).First certainly Right pH, the temperature was then adjusted to 30 degree.Start to be inoculated with, initial inoculation OD is adjusted to 0.5, and required seed liquor volume is counted according to formula It calculates.(such as fermentating liquid volume is 2500mL, and seed liquor OD value is n, then being inoculated with seed liquor volume is 2500x0.5/n mL).Start It is 5.5 that culture medium, which controls pH value with 2M NaOH, after fermentation, when upper tank, and initial ventilatory capacity is 1.5vvm, and initial stirring rates are set It is set to 300rpm, dissolved oxygen maintains 30% or more (association stirring rate, 300-600rpm).
Start feed supplement glucose when concentration of glucose is down to 2g/L or so when batch culture, initial feed rate is 10mL/L fermentation liquid/h, to maintain the residual concentration of glucose in fermentation liquid 2-3g/L or so (not be down to 0g/L), every two Glucose content of OD600 value of a hour sampling and measuring and detection, increases feed supplement when concentration of glucose is lower than 1g/L Rate.When the increase of OD value is slow, (initially entering stationary phase) stops feed supplement glucose, starts to monitor Residual ethanol at this time, when Concentration of alcohol starts feed supplement ethyl alcohol glycerol when being down to 5-10g/L, initial feed rate is 2mL/L fermentation liquid/h.Subsequent every 4h takes Sample detects an ethyl alcohol and glycerol content, when adjustment feed rate maintains ethyl alcohol and glycerol within the scope of 5-10g/L.Color has Start to extract the variation of product detection product after variation, terminates to ferment when lycopene concentration is not further added by.
Wherein, inventors have found that cultivating thallus in basic fermentation medium, culture medium compared with the prior art, bacterium The speed of growth and growth conditions of body are significantly better than the prior art, and thallus has been effectively ensured in the first supplemented medium added Quick and healthy growth, the second supplemented medium added effectively increases the yield of lycopene.It is according to the present invention Still another embodiment, the extraction processing include: that product after the fermentation process is carried out ultrasonication processing and organic Extraction processing.Screening is optimized to the extraction process after fermentation process in inventor, inventors have found that using ultrasonic treatment Compared to by the way of salt acid cooking, the degradation rate of lycopene is greatly reduced mode, the stable yield of lycopene.Separately Outside, it should be noted that " organic extraction processing " described herein, which refers to, takes organic solvent to handle product to ultrasonication It is extracted, the mode of organic extraction is not particularly limited, and such as according to a particular embodiment of the invention, can use acetone solvent Lycopene is extracted from ultrasonication processing product.
Purposes
In the third aspect of the present invention, the invention proposes mentioned-above microorganisms to prepare the use in lycopene On the way.Inventor passes through it is experimentally confirmed that microorganism described herein has significant advantage, tomato in terms of producing lycopene The yield of red pigment is high, the degradation rate of lycopene is low, with short production cycle.
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art It offers described technology or conditions or is carried out according to product description.Reagents or instruments used without specified manufacturer, For can be with conventional products that are commercially available.
The building of 1 first generation engineered strain J1011-C-3 of embodiment
In the present embodiment, inventor describes the building process of J1011-C-3 bacterial strain in detail.
Building Δ LEU2:pGAL1-PaCrtE (Pantoea ananatis) knocks out box first, i.e. knockout box segment 1, if It counts upstream and downstream primer PCR and expands each segment, making it between each other has the overlapping fragments of 60-80bp, then the side for passing through homologous recombination Formula recombinates all segments together, obtains Δ LEU2::pGAL1-PaCrtE (Pantoea by linearization for enzyme restriction Ananatis box) is knocked out, as knocked out box segment 1 as shown in Figure 1, knocking out box segment 1 has the nucleosides as shown in SEQ ID NO:1 Acid sequence.The segment is integrated into yeast by Li-acetate method yeast conversion respectively using the homologous recombination machinery of yeast itself On 30000B genome, integration site LEU2, since the LEU2 in false yeasts is without activity, and the homologous left arm after integrating contains There is LEU2 complete genome, Gu using SD-Leu solid panel, (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L are lacked after conversion The kilnitamin powder 1.3g/L of leucine, 2% agar powder) it is screened, obtained transformant after dividing pure culture by mentioning Yeast genome is taken to carry out PCR verifying, the Strain Designation of good authentication is J1011-C-1.
ATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGATGACTGGAAATTTTTTGTTAATTTCAGAGGTC GCCTGACGCATATACCTTTTTCAACTGAAAAATTGGGAGAAAAAGGAAAGGTGAGAGCGCCGGAACCGGCTTTTCAT ATAGAATAGAGAAGCGTTCATGACTAAATGCTTGCATCACAATACTTGAAGTTGACAATATTATTTAAGGACCTATT GTTTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTTCAGCAATATATATAT ATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAA GAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAA TCATTTAATTGGTGGTGCTGCTATCGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGG CTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTACTA AAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATC TCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTTGGTA AGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACA AGAATGGCCGCTTTCATGGCCCTACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGC CTCTTCAAGATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAGGTTCAACATCAAT TGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCAACATGTTT GGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTTGGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTT GCCAGACAAGAACACCGCATTTGGTTTGTACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCA ACCCTATCGCCACTATCTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATT GAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAGTACCACCGAAGT CGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAATTTAACTCCTTAAGTTACTTTAATGATTTAGTT TTTATTATTAATAATTCATGCTCATGACATCTCATATACACGTTTATAAAACTTAAATAGATTGAAAATGTATTAAA GATTCCTCAGGGATTCGATTTTTTTGGAAGTTTTTGTTTTTTTTTCCTTGAGATGCTGTAGTATTTGGGAACAATTA TACAATCGAAAGATATATGCTTACATTCGACCGTTTTAGCCGTGATCATTATCCTATAGTAACATAACCTGAAGCAT AACTGACACTACTATCATCAATACTTGTCACATGAGAACTCTGTGAATAATTAGGCCACTGAAATTTGATGCCTGAA GGACCGGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAATTGGCTTTTCGCGCAAATTAAAGCCTTCGAG CGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAA AATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTC TAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATA TCGACAAAGGAAAAGGGGCCTGTTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGT TTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAA AGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATAT TGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCA CCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTT TATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCAT AGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTA ACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTAT TCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAAT GACTGTTTGTGCTAAGAAACATGTTCATTTGACAAGAGATGCTGCAGAACAATTGTTGGCAGATATCGATAGAAGAT TGGATCAATTGTTACCAGTTGAAGGTGAAAGAGATGTTGTTGGTGCTGCAATGAGAGAAGGTGCTTTGGCACCAGGT AAAAGAATCAGACCAATGTTGTTGTTGTTGACTGCTAGAGATTTGGGTTGTGCAGTTTCTCATGATGGTTTGTTAGA TTTGGCTTGTGCAGTTGAAATGGTTCATGCTGCATCATTGATCTTGGATGATATGCCATGTATGGATGATGCTAAAT TGAGAAGAGGTAGACCAACTATTCATTCTCATTATGGTGAACATGTTGCTATTTTAGCTGCAGTTGCTTTGTTATCA AAAGCATTTGGTGTTATTGCTGATGCAGATGGTTTGACACCATTGGCTAAAAATAGAGCAGTTTCTGAATTGTCAAA CGCTATCGGTATGCAAGGTTTGGTTCAAGGTCAATTCAAAGATTTGTCTGAAGGTGACAAACCAAGATCAGCTGAAG CAATTTTGATGACTAACCATTTCAAGACTTCTACATTATTTTGTGCTTCTATGCAAATGGCATCAATTGTTGCTAAT GCATCTTCAGAAGCTAGAGATTGTTTGCATAGATTTTCATTGGATTTGGGTCAAGCATTTCAATTGTTAGATGATTT GACTGATGGTATGACTGATACTGGTAAAGATTCTAATCAAGATGCTGGTAAATCAACATTGGTTAATTTGTTAGGTC CAAGAGCTGTTGAAGAAAGATTGAGACAACATTTGCAATTAGCATCTGAACATTTGTCAGCTGCATGTCAACATGGT CATGCTACACAACATTTCATCCAAGCATGGTTCGATAAGAAATTAGCTGCAGTTTCTTAAGTCTGAAGAATGAATGA TTTGATGATTTCTTTTTCCCTCCATTTTTCTTACTGAATATATCAATGATATAGACTTGTATAGTTTATTATTTCAA ATTAAGTAGCTATATATAGTCAAGATAACGTTTGTTTGACACGATTACATTATTCGTCGACATCTTTTTTCAGCCTG TCGTGGTAGCAATTTGAGGAGTATTATTAATTGAATAGGTTCATTTTGCGCTCGCATAAACAGTTTTCGTCAGGGAC AGTATGTTGGAATGAGTGGTAATTAATGGTGACATGACATGTTATAGCAATAACCTTGATGTTTACATCGTAGTTTA ATGTACACCCCGCGAATTCGTTCAAGTAGGAGTGCACCAATTGCAAAGGGAAAAGCTGAATGGGCAGTTCGAATAAA AGATTCTCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATAATAGAAACGACACGAAATTACAAA ATGGAATATGTTCATAGGGTAGACGAAACTATATACGCAATCTACATACATTTATCAAGAAGGAGAAAAAGGAGGAT GTAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAACGTGATAAGGAAAAAGAATTGCACTTTAACATTAAT ATTGACAAGGAGGAGGGCACCACACAAAAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATA TTGGAGGATTTTCTCTAAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCATTTGATGGAGTTTA AGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAATTTTACTCTGTCAGAAACGGCCTTA ACGACGTAGTCGACCTCCTCTTCAGTACTAAATCTACCAATACCAAATCTGATGGAAGAATGGGCTAATGCATCATC CTTACCCAGCGCATGTAAAACATAAGAAGGTTCTAGGGAAGCAGATGTACAGGCTGAACCCGAGGATAATGCGATAT CCCTTAGTGCCATCAATAAAGATTCTCCTTCCACGTAGGCGAAAGAAACGTTAACACACCCTGGATAACGATGATCT GGAGATCCGTTCAACGTGGTATGTTCAGCGGATAATAGACCTTTGACTAATTTATCGGATAGTCTTTTGATGTGAGC TTGGTCGTTGTC (SEQ ID NO:1).
Δ URA3:pGAL1-PagCrtB (Pantoea agglomerans) is constructed again；pGAL10-BtCrtI (Blakeslea trispora) knocks out box, i.e. knockout box segment 2, and knocking out box segment 2 has the core as shown in SEQ ID NO:2 Nucleotide sequence.It designs upstream and downstream primer PCR and expands each segment, making it between each other has the overlapping fragments of 60-80bp, then passes through The mode of homologous recombination recombinates all segments together, obtains Δ URA3::pGAL1-PagCrtB by linearization for enzyme restriction (Pantoea agglomerans)；PGAL10-BtCrtI (Blakeslea trispora) knocks out box, knocks out box segment 2 as schemed Shown in 2.The segment is integrated into yeast by Li-acetate method yeast conversion respectively using the homologous recombination machinery of yeast itself On J1011-C-1 genome, integration site URA3 uses SD-HIS solid panel (synthetic yeast nitrogen source YNB6.7g/ after conversion L, glucose 20g/L lack the kilnitamin powder 1.3g/L of histidine, 2% agar powder) it is screened, obtained transformant Yeast genome progress PCR verifying is extracted by dividing after pure culture, the Strain Designation of good authentication is J1011-C-2.
ACGCAGATAATTCCAGGTATTTTGAAGCAGAACTTTTATTTATCATCGAATTGACTATTGCATTATTTCTATTTTGC AAAGAGGAGAAAGAATTAGGAAAGTTCATACTTCAAAAAGTTTTCCAACTTTCTCACACGAAAGGCCTCACGAAAAG GACTGTTCGTAGAATGCTAACATACAAAATTTTGTTAATTTCGTTATGTGCGGATCAGACGGAGTACTTGTCCAAAT TAATAAACGATGAGCTGTTAAAAAAGGGGGATATTTTTACCCAAAAATTTTTTGCAACTAATCAAGGTAAGGAATTT TTGAAGAGACTATTTTCATTGACCGAATCAGAGTTTTATAGAGGATTTTTACTAGGAAATGAGAATTTTTGGAAATT TTTAAGAAAAGTTACAGCAATGAAAGAGCAGAGCGAGAGCATTTTTGAATATTTAAATGAATCGATCAAGACAGACA GCAATATTTTGACAAATGAGAACTTCATGTGGGTCCTAGGACTATTAGATGAAATTTCATCAATGGGTGCCGTTGGA AATCACTGGGAAATAGAATACAAGAAATTGACAGAAAGTGGTCATAAAATTGATAAGGAGAATCCATACAAGAAATC GATCGAATTATCATTGAAATCCATTCAACTAACATCACACTTGCTGGAAGATAATAACGATCTGCGTAAAAACGAGA TATTCGCTATTATTCAAGCTTTGGCACATCAATGCATCAATCCGTGTAAGCAGATAAGTGAATTTGCAGTGGTAACG CTAGAGCAGACGCTCATCAATAAAATCGAAATTCCAACTAATGAGATGGAATCGGTAGAAGAATTAATTGAGGGCGG ATTACTACCGTTGCTAAATTCGAGTGAAACACAGGAAGACCAGAAAATCCTCATTTCATCCATATTAACAATAATTT CAAATGTTTATTTGCATTATTTGAAACTAGGGAAGACAAGCAACGAAACGTTTTTGAAAATTTTGAGTATTTTCAAT AAATTTGTAGAGGACTCAGATATTGAAAAAAAGCTACAGCAATTAATACTTGATAAGAAGAGTATTGAGAAGGGCAA CGGTTCATCATCTCATGGATCTGCACATGAACAAACACCAGAGTCAAACGACGTTGAAATTGAGGCTACTGCGCCAA TTGATGACAATACAGACGATGATAACAAACCGAAGTTATCTGATGTAGAAAAGGATTAAAGATGCTAAGAGATAGTG ATGATATTTCATAAATAATGTAATTCTATATATGTTAATTACCTTTTTTGCGAGGCATATTTATGGTGAAGGATAAG TTTTGACCATCAAAGAAGGTTAATGTGGCTGTGGTTTCAGGGTCCATAAAGCTTTTCAATTCATCATTTTTTTTTTA TTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGG AGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAA CTGCACAGAACAAAAACCTGCAGGAAACGAAGGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTG GCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTT CGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGC GCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGC GCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTT CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGC CTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCT GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAGATCCGTCGAGTTCAAGAGAAAAAAAAA GAAAAAGCAAAAAGAAAAAAGGAAAGCGCGCCTCGTTCAGAATGACACGTATAGAATGATGCATTACCTTGTCATCT TCAGTATCATACTGTTCGTATACATACTTACTGACATTCATAGGTATACATATATACACATGTATATATATCGTATG CTGCAGCTTTAAATAATCGGTGTCACTACATAAGAACACCTTTGGTGGAGGGAACATCGTTGGTACCATTGGGCGAG GTGGCTTCTCTTATGGCAACCGCAAGAGCCTTGAACGCACTCTCACTACGGTGATGATCATTCTTGCCTCGCAGACA ATCAACGTGGAGGGTAATTCTGCTAGCCTCTGCAAAGCTTTCAAGAAAATGCGGGATCATCTCGCAAGAGAGATCTC CTACTTTCTCCCTTTGCAAACCAAGTTCGACAACTGCGTACGGCCTGTTCGAAAGATCTACCACCGCTCTGGAAAGT GCCTCATCCAAAGGCGCAAATCCTGATCCAAACCTTTTTACTCCACGCGCCAGTAGGGCCTCTTTAAAAGCTTGACC GAGAGCAATCCCGCAGTCTTCAGTGGTGTGATGGTCGTCTATGTGTAAGTCACCAATGCACTCAACGATTAGCGACC AGCCGGAATGCTTGGCCAGAGCATGTATCATATGGTCCAGAAACCCTATACCTGTGTGGACGTTAATCACTTGCGAT TGTGTGGCCTGTTCTGCTACTGCTTCTGCCTCTTTTTCTGGGAAGATCGAGTGCTCTATCGCTAGGGGACCACCCTT TAAAGAGATCGCAATCTGAATCTTGGTTTCATTTGTAATACGCTTTACTAGGGCTTTCTGCTCTGTCATCTTTGCCT TCGTTTATCTTGCCTGCTCATTTTTTAGTATATTCTTCGAAGAAATCACATTACTTTATATAATGTATAATTCATTA TGTGATAATGCCAATCGCTAAGAAAAAAAAAGAGTCATCCGCTAGGGGAAAAAAAAAAATGAAAATCATTACCGAGG CATAAAAAAATATAGAGTGTACTAGAGGAGGCCAAGAGTAATAGAAAAAGAAAATTGCGGGAAAGGACTGTGTTATG ACTTCCCTGACTAATGCCGTGTTCAAACGATACCTGGCAGTGACTCCTAGCGCTCACCAAGCTCTTAAAACGGGAAT TTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACGCGCTGA CGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGAGCAAATTAAAGCCTTCGAGCGTCC CAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAAAATTT GAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACT CCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCGAC AAAGGAAAAGGGGCCTGTTTATATCCTAATATCGTTAGAGTTCTGTCCTTGGAAGACGTTTGGCAACAACTGATTAA TAAAAGATGCTGGAGTAGTAGTGTCGTCCCTAGGAAAGAAATAGAAGAACAAGAAAGTGACAAAGTAGCATGCTAAG CAGTAATAAATCCAGTGTGACTCAGTCTTCCTTGTTTGCTCAGGTGCATATTTCTTCTGAGAGTCCTGTAACTTCCT AGGCAAAGGGTTTTGTCCGAATGACTTACAGACCTGGTCAGAAGTTAATTTAGATCCTGCCAAGACTATTGGGACTC CTGTACCAGGGTGAGTTGATGCACCGACGAAAAATAAATTGTCATATCTGTTAGTAGAGTCCTTTGTTGAAGGTCTG AACCACAAAACCTGAAAGACATCATGTGATAAACCTAATATTGATCCCCTCCACAAGTTGAACTTAGATTGCCAGAC TGATGGGTCGTTGACCTCCTCGTGTTCTATCAAGTTAGCGAAGTTGTTGACTCCCAACCTCCTCTCTATGACCTCCA AGACCATCTTTCTAGCCCTGTTGACCAACTCTGGGTAGTTCTCCTCTGCTGAGTTACCTGTCTTAGACTTCATGTGA CCAATTGGAACTAAAACTATAATTGAGTCCTTATTTGGAGGTGCTGCAGACTCGTCTATCCTTGAAGGAACGTTGAC GTAGAAAGATGCTTCAGAAGGCAAACCGAAATCGTTAAAAATCTCGTCGAATGACTCCTTGTAAGCCTCAGCCAAGA AAATGTTGTGGACGTCCAATTGAGGGACCTTAGTAGACATTGACCAGTAAAAAGAAATTGATGAAGATGTTAATTTC TTAGATGCCAAAGTCTTCTTTGTCCAGTTGCAAGGTGGCAACAAGTGGTGGTAAGCGTAGACCAAGTCAGCGTTGCA GACGACAGCGTCTGCCTCAATGACTTCTCCAGACTCCAAAGTGACACCAGTGACCCTCTTGTCTTTATCGACAGTGT TAATCTTAGCGACAGGAGATTGGTACCTGAACTCAGCTCCGTACTTCTTAGAAGCTATAGACTCCAACTTTTGGACG ACCATGTTGAAACCACCCCTTGGGTACCAAATTCCCTCTGCGAACTCTGTATATTGCAACAATGAGTAGACTGCAGG TGCGTCGTAAGGTGACATACCCATGTACATTGTTTGAAAAGTAAAAGCCATCCTCATCTTTTTTGTTTGGAAGTATT TTGATGCTCTGTCGTATATCTTACCGAATAAGTGCAACCTAAAAATCTCTGGGACGTACTGTAACCTTATTAAGTCC CATATAGTTTCGAAGTTTCTCTTTATAGCAATGAATGTACCCTGCTCGTAGTGGACGTGTGTCTCTTTCATGAAATC TAAGAACCTACCGAATCCTAAAGGTCCCTCAATCCTGTCCAACTCACCCTTCATCTTTGTTAAGTCTGATGACAATT GGACAGCGTCACCGTCGTCGAAATGGACTTTGTAATTGTTGTCACATCTTAATAAGTCCAAGTGGTCTCCTATCCTC TCGTCTAAGTCAGCGAAAGCGTCCTCAAACAACTTAGGCATCAAGTACAATGAAGGTCCCTGGTCGAACCTGTGACC GTCGTGGTGAATGAAAGAGCACCTTCCTCCAGAGAAGTCGTTCTTCTCGACGACAGTGACTCTGAAACCCTCCCTTG CTAACCTTGCTGCTGTTGCAGTACCTCCTATTCCAGCACCTATGACGACAATGTGCTTCTTCTGATCAGACATTTAT ATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACC ACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAA ACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGC GGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGT GCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCA GTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAG CCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCAT AACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAA AATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGTCACAACCACCATTATTGGACCACGC TACACAAACTATGGCAAACGGTTCTAAATCTTTCGCTACTGCTGCTAAATTATTCGACCCAGCAACAAGAAGATCTG TATTGATGTTGTACACCTGGTGTAGACATTGCGATGACGTTATAGATGACCAAACTCACGGTTTTGCTTCAGAAGCT GCAGCCGAAGAAGAAGCTACACAAAGATTGGCAAGATTAAGAACTTTGACATTAGCTGCATTCGAAGGTGCCGAAAT GCAAGATCCAGCTTTTGCCGCTTTCCAAGAAGTTGCATTAACCCATGGTATTACTCCTAGAATGGCTTTGGATCACT TAGACGGTTTTGCAATGGATGTCGCCCAAACAAGATACGTAACCTTCGAAGACACTTTAAGATATTGTTACCATGTC GCCGGTGTTGTCGGTTTGATGATGGCTAGAGTAATGGGTGTTAGAGATGAAAGAGTTTTAGATAGAGCATGTGACTT GGGTTTAGCCTTCCAATTGACAAACATAGCTAGAGATATAATAGATGACGCAGCCATAGACAGATGCTATTTGCCAG CTGAATGGTTACAAGATGCAGGTTTGACTCCTGAAAATTACGCTGCAAGAGAAAACAGAGCCGCTTTAGCCAGAGTT GCTGAAAGATTGATAGATGCAGCCGAACCATATTACATCTCTTCACAAGCTGGTTTGCATGATTTGCCACCTAGATG CGCATGGGCCATTGCTACCGCAAGATCTGTTTACAGAGAAATCGGTATTAAAGTCAAGGCTGCAGGTGGTTCCGCAT GGGATAGAAGACAACACACTTCTAAAGGTGAAAAGATCGCTATGTTGATGGCCGCTCCTGGTCAAGTTATTAGAGCA AAGACCACCAGAGTCACCCCAAGACCAGCCGGTTTATGGCAAAGACCTGTTTAAATTGAATTGAATTGAAATCGATA GATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTA TTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAATTACGCTCCTC TTTTAATGCCTTTATGCAGTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAAC TCGAAAATTCTGCGTTCGTTGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTA TTACCCGGGAATCTCGGTCGTAATGATTTCTATAATGACGAAAAAAAAAAAATTGGAAAGAAAAAGCTTCATGGCCT TTATAAAAAGGAACTATCCAATACCTCGCCAGAACCAAGTAACAGTATTTTACGGGGCACAAATCAAGAACAATAAG ACAGGACTGTAAAGATGGACGCATTGAACTCCAAAGAACAACAAGAGTTCCAAAAAGTAGTGGAACAAAAGCAAATG AAGGATTTCATGCGTTTGTACTCTAATCTGGTAGAAAGATGTTTCACAGACTGTGTCAATGACTTCACAACATCAAA GCTAACCAATAAGGAACAAACATGCATCATGAAGTGCTCAGAAAAGTTCTTGAAGCATAGCGAACGTGTAGGGCAGC GTTTCCAAGAACAAAACGCTGCCTTGGGACAAGGCTTGGGCCGATAAGGTGTACTGGCGTATATATATCTAATTATG TATCTCTGGTGTAGCCCATTTTTAGCATGTAAATATAAAGAGAAACCATATCTAATCTAACCAAATCCAAACAAAAT TCAATAGTTACTATCGCTTTTTTCTTTCTGTATCGCAAATAAGTGAAAATTAAAAAAGAAAGATTAAATTGGAAGTT GGATATGGGCTGGAACAGCAGCAGTAATCGGTATCGGGTTCGCCACTAATGACGTCCTACGATTGCACTCAACAGAC CTTGACGCTCACGCCGTAGCGGGCGACAAGTCAAACGGAACAACCGTTGCCGTTCCCATCGGAGTCCGACCTAGGCC GAACTCCGTGAATTTCTGATAACAACGGTCGGTAAAGACTGGTTCCCCAGTATATTTCTTCTCTCAGGAGCAGGGGC CAATGCCAAAAGCGACATTAACCCGGAGGACAAGGCTCCACTGTGTTCCACCGAATTTCCCACCTGATAATATCTGA TAACCCGCCCATAGGTGGGGATCCTTCTGTAAACAGGTTTCTTAATCGTAGGAATTACCACTGTTCCACTGCCAATC GCAGCTCCCAGAGTTTCGTTCCCAGCCGCGAGCACCACAGCGTACCATGTGCGCCACGAGGCCTCAAACGTAAAACA ATCGAAACGAAAAGAAACAGACTATAGGGGAGTATAGAGACAGCCGGCCAATAAGAAGAGGAAAAAGAAATACTAGC GTTTATCAATGTGGGTCGTTAACATATCCTGTTGACAATGATTACAGGTTAAAAGGTAGCGTAAGTGAATATTAACT ATGGATATTCTTATACTTAGATAAGAGACATATAAAACACACGATGATTGATTGATTTTTATGACCAATATATGTAA TTCGTAATTCAGATAGTTTTTATACTTTTAATGTGTGCCGAACCATATTACTAGTATATGTAACTACCATGTTATGT TCAATTGGCAGATCTTTAACTCGGCTTTAGTTATCCAAGTTACTTGCAATATTTCCTTCTGCGAGAGTACATTTGCC CTTAAACG (SEQ ID NO:2).
Δ gal1 is constructed again, and Δ gal7, Δ gal10:pGAL10-tHMG1 knock out box, i.e. knockout box segment 3, knock out box piece Section 3 has the nucleotide sequence as shown in SEQ ID NO:3.Design upstream and downstream primer PCR and expand each segment, make its mutually it Between have the overlapping fragments of 60-80bp, then by way of homologous recombination by the recombination of all segments together, pass through linearization for enzyme restriction Δ gal1 is obtained, Δ gal7, Δ gal10::pGAL10-tHMG1 knock out segment, and the structure for knocking out box segment 3 is as shown in Figure 3.Benefit The segment is integrated into yeast J1011-C-2 base by Li-acetate method yeast conversion respectively with the homologous recombination machinery of yeast itself Because in group, integration site GAL1, GAL7 and GAL10 use SD-Trp solid panel (synthetic yeast nitrogen source YNB after conversion 6.7g/L, glucose 20g/L lack the kilnitamin powder 1.3g/L of tryptophan, 2% agar powder) it is screened, what is obtained turns Beggar extracts Yeast genome progress PCR verifying by dividing after pure culture, and the Strain Designation of good authentication is J1011-C-3.
TTCACCGATTCTGAGCGAATCACAGGTGAGAAATTTGGATTCGAAATAAACCTAAAAAAACTATCCAATAAGGCTTC CATAGGCTTCGTATTTCCCGACCATTCCAATTGGAAAAATTGAGCGCTGTCTTCCATGATCTTAGATAAAGCCTTAA TACTTGGCTCATTTCCATTTGAGGTTAGCTCTAGCAACTGGTAAAGCATTTGAAGACCAGTTGGATCATCTCTATGC TGCCTATAGTAAGTGGAAAATAAAGTAGTAATAACGATACCTGTCACGTTCGAAACGAGATTATTGTTCGAATTATA AAGGCCATGTATAATGTTTGATTTGACGTATCCTAAGTCGTGGCTATTGCTCTTAATTAAATTGTTTCCCCCTAGCA TTGAATTTTTCAACAGCATACCGGCGGTAGCTCTATTGTTCTGAAGATCCTGTAGGGAGTAGTGTTGCTTCAATACA TCATCAGATTCGCCTTCAATTAAAATATAACACAAATAATTGAGAAATTCAGGTTGCAGCTGGAAGTTCTCCATAGC TTCCATTGCGTTATTACGAATCTCTGGATTTGGTGACATACAGTTCTGTAAAAGAGTTGCTAGTTGCAACACATAGT CTTCGGCGGGCTTCCATGTCGATGCCATCTTTATTCACTTAACTACTGCTAACAATTCTGGAAACCAAAGACTGCGG AATATTCTGATATGTATTACTACTATTCGCTGCTCTTCTGCATAATTAATACTGAAAAGTTTTTCATACTTTTAAAC ATAACCTTTTTTTAAGCAAAACTCTATGACCCGGATTAGAAAACTACGAAAAGAGGGTAATAACATAGGTGCAGGAT TTCCATCGATAACGACGCCGACAATGAGCCTTGCTGCAACATCCAATTAGGACTAATAACTATCGTAGGAATTTCTA CGTAATAAACTTCAACAGAGCCTAAAATTTGAAAATAAATAATCTAGAGGGGAAACTTAAAGAAATTCTATTCTTGT CAATAAAGTGGAAATCTGTCAGATGTCACAGTTTCTTTATTTGTGACACATATTTTCAACATAAATTCAGGCATTAG TGCTGTAAGCACAAAAAGTTGTGGCGATATGAATATTCCAGATTTTACTTACAAGCTGCATTGTAGTCTTACAATTC TTTTTTTCTTTTTTTTTTTTTATGGAAAGGACCACTCTTACATAACTAGAATAGCATTAAGAATCAGATTTACAGAT AAAGATGACATTATTTTATATATATATTGTCACTCCGTTCAAGTCGACAACCAATAAAAAATTTAAAAAAAGCCAGG CAGTTAATAGAAAAAATATGATATGAATGAATATTCCACTTTCTTTTCACAGCTTGTCTGTAAGCGGATGCCGGGAG CAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGA TTGTACTGAGAGTGCACCATAAACGACATTACTATATATATAATATAGGAAGCATTTAATAGACAGCATCGTAATAT ATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTAC GTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCC AAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCT GTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGC TCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAA GGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAAT CAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTG GCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCA GTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGAT TGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGAC GCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATG GTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTT AAGTATTGTTTGTGCACTTGCCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAA TTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAA ATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCC ACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCAT CACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGA GCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGC AAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATT CGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG GGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGA GCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACTACACGGTCCAATGGATAAACATTTTTTATCAACACTA TGATATATAAATATAATAGTTTTTCGTATATATATTCCTTTTTTTGGTCAATTTTTGAAATTTTCGTAGAAAAGGGA GAGACAAAACACATTATATCAATGAAAACGTACAAAAAGTAGATAAAGTCAGTGCTTAAACACGTCTTTTCCTTAAA AATACTTTATTATTTTTATTTTATTGAGAGGGTGGTTTAAAAATAGAAATAGAGAGAGAGGTACATACATAAACATA CGCGCACAAAAGCAGAGATTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGATTTATATCAGTGGCGTCCA AATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATATGACTTTGAACCAAATGGCCGGCTGCTAGG GCAGCACATAAGGATAATTCACCTGCCAAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGG AGCGGTAGCATGCGGGCCTCTTACACCTAATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGA TGGTACCTACTTCGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAG TTGGAACTTTCAACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAATTAGCTGCATGTGC GTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAATTCTTAGCAATGTTCAACTCAACCAATGCGGAAA CATCACTTTTTAACACTTTTCTGACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACGACCTTCG ATCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTTCCCAGCC ATACTCTTCTACCATTTGCTTTAATGAGTATTCGACACCTTTAGAAATCATATTCATACCCATTGCGTCACCAGTAG TTGTTCTAAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATCTTGATGTA GAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAGGCACCAGATCTTTTCAA AGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAAAACAGTTGTTGCACCACCGCCAGCATTGA TTGCCTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCA TCGATAACCAAGGGGCCTATAACACCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATAC GCGGTCGTAGTCATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGAAAGAGCCT TCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGAATA ACCAAGGCAGCGACCTCTTTGTTCTTCAATTGTTTTGTATTTCCACTACTTAATAATGCTTCTAATTCTTCTAAAGG ACGTATTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATCTTCCTCACTAGATGATGAAGGTCCTGATGAGCTCG ATTGCGCAGATGATAAACTTTTGACTTTCGATCCAGAAATGACTGTTTTATTGGTTAAAACCATTTATATTGAATTT TCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATAC ATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCT TTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAG CCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGC CGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGG CCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTC TGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTT AACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAA TATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAGCGCAATGATTGAATAGTCAAAGATTTTTTTTTTTTAA TTTTTTTTTTTTCATAGAACTTTTTATTTAAATAAATCACGTCTATATATGTATCAGTATAACGTAAAAAAAAAAAC ACCGTCAGTTAAACAAAACATAAATAAAAAAAAAAAGAAGTGTCAAATCAAGTGTCAAATGTATACTTCTTTTTTTT ACTTTGTTCAGAACAACTTCTCATTTTTTTCTACTCATAACTTTAGCATCACAAAGTACACAATAATAACGAGTAGT AACACTTTTATAGTTCATACATGCTTCAACTACTTAATAAATGATTGTATGATAATGTTTTCAATGTAAGAGATTTC GATTATCCACAAACTTTGAAACACAGGGACACAATTCTTGATATGCTTTCAACCGCTGCGTTTTGGATACCTATTCT TGACATAATATGACTACCATTTTGTTATTGTACGTGGGGCAGTTGACGTCTTATCATATGTCAAAGTCATTTGCGAA GTTCTTGGCAAGTTGCCAACTGACGAGATGCAGTAAAAAGAGATTGCCGTCTTGAAACTTTTTGTCCTTTTTTTTTT CCGGGGACTCTACGAGAACCCTTTGTCCTACTGATTAATTTTGTACTGAATTTGGACAATTCAGATTTTAGTAGACA AGCGCGAGGAGGAAAAGAAATGACAGAAAAATTCCGATGGACAAGAAGATAGGAAAAAAAAAAAGCTTTCACCGATT TCCTAGACCGGAAAAAAGTCGTATGACATCAGAATGAAAAATTTTCAAGTTAGACAAGGACAAAATCAGGACAAATT GTAAAGATATGATAAATCATTTGATTCAGCGCCAATTTGCCCTTTTCCATTTTCCATTAAATCTCTGTTCTCTCTTA CTTATATGATGATTAGGTATCATCTGTATAAAACTCCTTTCTTAATTTCACTCTAAAGCATACCCCATAGAGAAGAT CTTTCGGTTCGAAGACATTCCTACGCATAATAAGAATAGGAGGGAATAATGCCAGACAATCTATCATTACATTTAAG CGGCTCTTCAAAAAGATTGAACTCTCGCCAACTTATGGAATCTTCCAATGAGACCTTTGCGCCAAATAATGTGGATT TGGAAAAAGAGTATAAGTCATCTCAGAGTAATATAACTACCGAAGTTTATGAGGCATCGAGCTTTGAAGAAAAAGTA AGCTCAGAAAAACCTCAATACAGCTCATTCTGGAAGAAAATCTATTATGAATATGTGGTCGTTGACAAATCAATCTT GGGTGTTTCTATTCTGGATTCATTTATGTACAACCAGGACTTGAAGCCCGTCGAAAAAGAAAGGCGGGTTTGGTCCT GGTACAATTATTGTTACTTCTGGCTTGCTGAATGTTTCAATATCAACACTTGGCAAATTGCAGCTACAGGTCTACAA CTGGGTCTAAATTGGTGGCAGTGTTGGATAACAATTTGGATTGGGTACGGTTTCGTTGGTGCTTTTGTTGTTTTGGC CTCTAGAGTTGGATCTGCTTATCATTTGTCATTCCCTATATCATCTAGAGCATCATTCGGTATTTTCTTCTCTTTAT GGCCTGTTATTAATAGAGTCGTCATGGCCATCGTTTGGTATAGTGTCCAAGCTTATATTGCGGCAACTCCCGTATCA TTAA (SEQ ID NO:3).
In following experiment, inventor is further transformed J1011-C-3 bacterial strain, described in detail below:
The selection of embodiment 2 promoter and terminator
In order to be induced with galactolipin, need to knock out the gene GAL1, GAL7, GAL10 of metabolism galactolipin.Therefore special The background strain for levying promoter is to knock out gene GAL1,7,10.Galactolipin price is higher, is not particularly suited for industrialized production, because This is knocking out gene GAL1, GAL80 is knocked out on the basis of 7,10, to reach by the control to glucose amount to gene expression Regulation.Obtained starting sub-information is used to construct the expression of control carotenogenesis related gene.
As a result shown in, in Δ GAL1/7/10/80 bacterial strain and Δ GAL1/7/10 bacterial strain whether inducible promoter or It is that constitutive promoter is almost the same in stationary phase intensity.The i.e. inducible base of galactolipin is not added as it can be seen that knocking out GAL80 and can play Because of expression, induction intensity is suitable with galactolipin induction intensity.
Yeast belongs to eucaryote, is overexpressed gene in vivo at it and needs to select suitable promoter and terminator, passes through Repeated multiple times experiment, inventor have selected PGAL1, PGAL7, PGAL10, PGAL1-10, PGAL10-1And PHXT1Starting as expressing gene Son.Wherein, PGAL1With the nucleotide sequence as shown in SEQ ID NO:4, PGAL7With the nucleosides as shown in SEQ ID NO:5 Acid sequence, PGAL10With the nucleotide sequence as shown in SEQ ID NO:6, PGAL1-10With the core as shown in SEQ ID NO:7 Nucleotide sequence, PGAL10-1With the nucleotide sequence as shown in SEQ ID NO:8, PHXT1With the core as shown in SEQ ID NO:9 Nucleotide sequence.
AGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCG GTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTAT GGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGG ATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACA GATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCA AATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATA(SEQ ID NO:4).
GACGGTAGCAACAAGAATATAGCACGAGCCGCGAAGTTCATTTCGTTACTTTTGATATCGCTCACAACTATTGCGAA GCGCTTCAGTGAAAAAATCATAAGGAAAAGTTGTAAATATTATTGGTAGTATTCGTTTGGTAAAGTAGAGGGGGTAA TTTTTCCCCTTTATTTTGTTCATACATTCTTAAATTGCTTTGCCTCTCCTTTTGGAAAGCTATACTTCGGAGCACTG TTGAGCGAAGGCTCATTAGATATATTTTCTGTCATTTTCCTTAACCCAAAAATAAGGGAAAGGGTCCAAAAAGCGCT CGGACAACTGTTGACCGTGATCCGAAGGACTGGCTATACAGTGTTCACAAAATAGCCAAGCTGAAAATAATGTGTAG CTATGTTCAGTTAGTTTGGCTAGCAAAGATATAAAAGCAGGTCGGAAATATTTATGGGCATTATTATGCAGAGCATC AACATGATAAAAAAAAACAGTTGAATATTCCCTCAAAA (SEQ ID NO:5).
TACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATG GGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGA TTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAA TATAA (SEQ ID NO:6).
TATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATGACATTTGAAT AAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGTTA ATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATTATCCTAT GGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAA AGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTG AAGACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAAT ATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTT ACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAAC TTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAA (SEQ ID NO:7).
TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAA TACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAA AAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCC GAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAG ATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATT GGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTT TTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCT GCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAAC AAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATA (SEQ ID NO:8).
GATTTTACGTATATCAACTAGTTGACGATTATGATATCTTTATAGATTTTAAGGTGGGGAAAGAACATGAGACCCCA GATGGAATTGATTATGGGGACATTGTTGCCTTTATATATAATTTCAATATACTAATTCAAATGATTAAAAACGTGAG GGGGACACGCAACTTCGGGTGTTAAGAAATATTTTGCTACATTAGATAATGGTGGAGTTTCCTGGCTTGTCGGATAA AAGCCATCAAATGTCGCAGCAGCTCATGTTTACGTTTGCTGTCTTCTGCCCACGTCATATGAGTGGTATTCTTCTAT CAGCACTTGATGAATATTCTTTTTCTCATATATCTGAAAGACAAAAGATCGGCACGGCAATGCCCTGCAGCATTTCT TCCTAGTTTTTCCGAATTTCCATTACGTATTGGATCTTGTGCGCATATTTGTCAGTCCTTCACGGAAAAAAAAAAAG AGCACTGGGTCACTTCGGAAAAACTTTTGACTCAATGCAACAGTGTCATAATCCTTTGCGCTGTCTCTTTGAAGAAA AATCAGGAGTGCAAGATATCGATTAATTCCTTGGAAGTTATGATGGTTAGTCTTAGTTTAACTCTCTTGAAGAAGGG TTTTTTCAGTTGGTCAACACTCTTTAGAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAGAGAATTCTTCATGTAATTT ACCATGATTCTACGTTTTTGCAAGCAAAAATGAAGATAATCCGAGCGCATGCGCAAGTAGTCCCTGCCATGCCGCTT CGGAAAAACTTTCGAAACCAATACTCCTTACATCCGCCTTGAATTTAGGACCTACAATTGTTGTCTCTTCTGCTTGA ACCAATAAGCCCTAGAAACCTTACACCCTAATTTGCACAAGAAAACTACGAAAAAATTTTTTCCTCTTTTTTCCGCA GCTCCAATCTCGCCCTGCTATTTTTTTGGTTCGGAGAAAAATAGTCGGTCGATATGAATTGAAGTTTCATTGTGGCC ACTCGTTTAGGAATGGAATAACCTCCGCGAATGGAAAAAAAGAACTCGGAATATCAAAAATTCCGGCTCAAAGGAAA AATTTGCTTCAGGAGGGGGGAATTATATTCCAGATGAGACCTGCA (SEQ ID NO:9).
Select arbitrary terminator (such as TADH2, TGAL10, TCYC1, TGPM1And TPGK1Deng) terminator as expressing gene, The combination collocation of these elements can be realized the overexpression of multiple genes.
The building of 3 second generation engineered strain of embodiment
In the present embodiment, inventor adjusts PaCrtE on J1011-C-3 bacterial strain based on J1011-C-3 bacterial strain (Pantoea ananatis), PagCrtB (Pantoea Agglomerans) and BtCrtI (Blakeslea trispora) this The copy number of three genes obtains second generation bacterial strain.
Wherein the concrete property of second generation engineered strain is as shown in table 1.According to the suitable promoter of design alternative and termination Son expands each segment with upstream and downstream primer PCR, and making it between each other has the overlapping fragments of 60-80bp, then passes through homologous recombination Mode by all segments recombinate together, each respective segments are obtained by linearization for enzyme restriction, utilize the homologous heavy of yeast itself The segment is integrated on yeast J1011-C-3 genome by group mechanism respectively by Li-acetate method yeast conversion, is used after conversion Screening resistance solid panel is screened, and obtained transformant extracts Yeast genome progress PCR verifying by dividing after pure culture, at See Table 1 for details for the bacterial strain of function verifying.
Table 1: second generation engineered strain
Bacterial strain Characteristic and knockout box
J1011-C-5 J1011-C-3 derivative strain；BtCrtI. is overexpressed in original basis
J1011-C-6 J1011-C-3 derivative strain；BtCrtI and PagCrtB is overexpressed in original basis
J1011-C-7 J1011-C-3 derivative strain；BtCrtI and PagCrtB and PaCrtE is overexpressed in original basis
J1011-C-33 J1011-C-3 derivative strain；PagCrtB and PaCrtE is overexpressed in original basis
J1011-C-34 J1011-C-3 derivative strain；PaCrtE is overexpressed in original basis
J1011-C-35 J1011-C-3 derivative strain；BtCrtI and PaCrtE is overexpressed in original basis
J1011-C-36 J1011-C-3 derivative strain；PagCrtB. is overexpressed in original basis
Wherein, BtCrtI has the nucleotide sequence as shown in SEQ ID NO:10, and PagCrtB has such as SEQ ID NO: Nucleotide sequence shown in 11, PaCrtE have the nucleotide sequence as shown in SEQ ID NO:12.
TTATATCCTAATATCGTTAGAGTTCTGTCCTTGGAAGACGTTTGGCAACAACTGATTAATAAAAGATGCTGGAGTAG TAGTGTCGTCCCTAGGAAAGAAATAGAAGAACAAGAAAGTGACAAAGTAGCATGCTAAGCAGTAATAAATCCAGTGT GACTCAGTCTTCCTTGTTTGCTCAGGTGCATATTTCTTCTGAGAGTCCTGTAACTTCCTAGGCAAAGGGTTTTGTCC GAATGACTTACAGACCTGGTCAGAAGTTAATTTAGATCCTGCCAAGACTATTGGGACTCCTGTACCAGGGTGAGTTG ATGCACCGACGAAAAATAAATTGTCATATCTGTTAGTAGAGTCCTTTGTTGAAGGTCTGAACCACAAAACCTGAAAG ACATCATGTGATAAACCTAATATTGATCCCCTCCACAAGTTGAACTTAGATTGCCAGACTGATGGGTCGTTGACCTC CTCGTGTTCTATCAAGTTAGCGAAGTTGTTGACTCCCAACCTCCTCTCTATGACCTCCAAGACCATCTTTCTAGCCC TGTTGACCAACTCTGGGTAGTTCTCCTCTGCTGAGTTACCTGTCTTAGACTTCATGTGACCAATTGGAACTAAAACT ATAATTGAGTCCTTATTTGGAGGTGCTGCAGACTCGTCTATCCTTGAAGGAACGTTGACGTAGAAAGATGCTTCAGA AGGCAAACCGAAATCGTTAAAAATCTCGTCGAATGACTCCTTGTAAGCCTCAGCCAAGAAAATGTTGTGGACGTCCA ATTGAGGGACCTTAGTAGACATTGACCAGTAAAAAGAAATTGATGAAGATGTTAATTTCTTAGATGCCAAAGTCTTC TTTGTCCAGTTGCAAGGTGGCAACAAGTGGTGGTAAGCGTAGACCAAGTCAGCGTTGCAGACGACAGCGTCTGCCTC AATGACTTCTCCAGACTCCAAAGTGACACCAGTGACCCTCTTGTCTTTATCGACAGTGTTAATCTTAGCGACAGGAG ATTGGTACCTGAACTCAGCTCCGTACTTCTTAGAAGCTATAGACTCCAACTTTTGGACGACCATGTTGAAACCACCC CTTGGGTACCAAATTCCCTCTGCGAACTCTGTATATTGCAACAATGAGTAGACTGCAGGTGCGTCGTAAGGTGACAT ACCCATGTACATTGTTTGAAAAGTAAAAGCCATCCTCATCTTTTTTGTTTGGAAGTATTTTGATGCTCTGTCGTATA TCTTACCGAATAAGTGCAACCTAAAAATCTCTGGGACGTACTGTAACCTTATTAAGTCCCATATAGTTTCGAAGTTT CTCTTTATAGCAATGAATGTACCCTGCTCGTAGTGGACGTGTGTCTCTTTCATGAAATCTAAGAACCTACCGAATCC TAAAGGTCCCTCAATCCTGTCCAACTCACCCTTCATCTTTGTTAAGTCTGATGACAATTGGACAGCGTCACCGTCGT CGAAATGGACTTTGTAATTGTTGTCACATCTTAATAAGTCCAAGTGGTCTCCTATCCTCTCGTCTAAGTCAGCGAAA GCGTCCTCAAACAACTTAGGCATCAAGTACAATGAAGGTCCCTGGTCGAACCTGTGACCGTCGTGGTGAATGAAAGA GCACCTTCCTCCAGAGAAGTCGTTCTTCTCGACGACAGTGACTCTGAAACCCTCCCTTGCTAACCTTGCTGCTGTTG CAGTACCTCCTATTCCAGCACCTATGACGACAATGTGCTTCTTCTGATCAGACAT (SEQ ID NO:10).
ATGTCACAACCACCATTATTGGACCACGCTACACAAACTATGGCAAACGGTTCTAAATCTTTCGCTACTGCTGCTAA ATTATTCGACCCAGCAACAAGAAGATCTGTATTGATGTTGTACACCTGGTGTAGACATTGCGATGACGTTATAGATG ACCAAACTCACGGTTTTGCTTCAGAAGCTGCAGCCGAAGAAGAAGCTACACAAAGATTGGCAAGATTAAGAACTTTG ACATTAGCTGCATTCGAAGGTGCCGAAATGCAAGATCCAGCTTTTGCCGCTTTCCAAGAAGTTGCATTAACCCATGG TATTACTCCTAGAATGGCTTTGGATCACTTAGACGGTTTTGCAATGGATGTCGCCCAAACAAGATACGTAACCTTCG AAGACACTTTAAGATATTGTTACCATGTCGCCGGTGTTGTCGGTTTGATGATGGCTAGAGTAATGGGTGTTAGAGAT GAAAGAGTTTTAGATAGAGCATGTGACTTGGGTTTAGCCTTCCAATTGACAAACATAGCTAGAGATATAATAGATGA CGCAGCCATAGACAGATGCTATTTGCCAGCTGAATGGTTACAAGATGCAGGTTTGACTCCTGAAAATTACGCTGCAA GAGAAAACAGAGCCGCTTTAGCCAGAGTTGCTGAAAGATTGATAGATGCAGCCGAACCATATTACATCTCTTCACAA GCTGGTTTGCATGATTTGCCACCTAGATGCGCATGGGCCATTGCTACCGCAAGATCTGTTTACAGAGAAATCGGTAT TAAAGTCAAGGCTGCAGGTGGTTCCGCATGGGATAGAAGACAACACACTTCTAAAGGTGAAAAGATCGCTATGTTGA TGGCCGCTCCTGGTCAAGTTATTAGAGCAAAGACCACCAGAGTCACCCCAAGACCAGCCGGTTTATGGCAAAGACCT GTTTAA (SEQ ID NO:11).
ATGACTGTTTGTGCTAAGAAACATGTTCATTTGACAAGAGATGCTGCAGAACAATTGTTGGCAGATATCGATAGAAG ATTGGATCAATTGTTACCAGTTGAAGGTGAAAGAGATGTTGTTGGTGCTGCAATGAGAGAAGGTGCTTTGGCACCAG GTAAAAGAATCAGACCAATGTTGTTGTTGTTGACTGCTAGAGATTTGGGTTGTGCAGTTTCTCATGATGGTTTGTTA GATTTGGCTTGTGCAGTTGAAATGGTTCATGCTGCATCATTGATCTTGGATGATATGCCATGTATGGATGATGCTAA ATTGAGAAGAGGTAGACCAACTATTCATTCTCATTATGGTGAACATGTTGCTATTTTAGCTGCAGTTGCTTTGTTAT CAAAAGCATTTGGTGTTATTGCTGATGCAGATGGTTTGACACCATTGGCTAAAAATAGAGCAGTTTCTGAATTGTCA AACGCTATCGGTATGCAAGGTTTGGTTCAAGGTCAATTCAAAGATTTGTCTGAAGGTGACAAACCAAGATCAGCTGA AGCAATTTTGATGACTAACCATTTCAAGACTTCTACATTATTTTGTGCTTCTATGCAAATGGCATCAATTGTTGCTA ATGCATCTTCAGAAGCTAGAGATTGTTTGCATAGATTTTCATTGGATTTGGGTCAAGCATTTCAATTGTTAGATGAT TTGACTGATGGTATGACTGATACTGGTAAAGATTCTAATCAAGATGCTGGTAAATCAACATTGGTTAATTTGTTAGG TCCAAGAGCTGTTGAAGAAAGATTGAGACAACATTTGCAATTAGCATCTGAACATTTGTCAGCTGCATGTCAACATG GTCATGCTACACAACATTTCATCCAAGCATGGTTCGATAAGAAATTAGCTGCAGTT TCTTAA (SEQ ID NO:12).
Further, inventor is by experimental verification, as a result as shown in figure 5, under the conditions of shaking flask, J1011-C-5, and 6,7 There is the performance of high yield lycopene more than J1011-C-3 with J1011-C-33~36.
The building of the engineered strain of 4 cryptiogene of embodiment
In the present embodiment, inventor describes in detail knocks out the acquisition of other gene on the basis of above-mentioned engineering bacteria The experimentation of cryptiogene engineered strain.
In saccharomyces cerevisiae, there are many potential genes to have significantly after knockout to Cellular Accumulation lycopene compound It influences, therefore constructs a series of background strains for increasing yield of lycopene.Construction method is in the gene for needing to inactivate Being that label building is corresponding with hygromycin gene knocks out box segment, knocks out box segment and sees Fig. 4.The segment is passed through into lithium acetate Method yeast conversion is integrated into respectively in above-mentioned engineering bacteria, is finally screened to obtain doubtful band with the plate containing hygromycin resistance Resistant bacterial strain, is verified with PCR, and obtained forth generation engineered strain is shown in Table 2.Wherein, the inactivation in box segment is knocked out Gene order can be downloaded from NCBI, so as to design to obtain the knockout frame of Fig. 6.
Table 2: silencing engineered strain characteristic
Further, inventor is by experimental verification, and J1011-C-9,10,11,13,15,19 all have than J1011-C-3 The more performance of high yield lycopene.
Embodiment 5 is overexpressed the building of the engineered strain of gene
In the present embodiment, inventor describes in detail is overexpressed relevant experimentation on above-mentioned engineered strain.
The expression cassette for selecting related gene in suitable promoter and terminator building table 3, is selected on the basis of embodiment 4 Suitable gene is selected as insertion point, each segment is expanded by design upstream and downstream primer PCR, it is made to have 60- between each other The overlapping fragments of 80bp, then recombinated all segments together by way of homologous recombination, it is by linearization for enzyme restriction The genetic fragment that cryptiogene can be overexpressed gene again can be achieved.The segment is passed through using the homologous recombination machinery of yeast itself Li-acetate method yeast conversion is integrated into respectively on above-mentioned engineered strain genome, is screened, is obtained using screening flat board after conversion To transformant extract Yeast genome by dividing after pure culture and carry out PCR verifying, see Table 3 for details for the bacterial strain of good authentication.
Further, inventor has high yield tomato red by experimental verification, J1011-C-4 and J1011-C-20~32 The performance of element.
Table 3: it is overexpressed the engineered strain of gene
Wherein, INO2 gene has the nucleotide sequence as shown in SEQ ID NO:13, and gapN gene has such as SEQ ID Nucleotide sequence shown in NO:14, PYC2 gene have the nucleotide sequence as shown in SEQ ID NO:15, SMAE1 gene tool Just like nucleotide sequence shown in SEQ ID NO:16, MDH2 gene has the nucleotide sequence as shown in SEQ ID NO:17, POS5 gene has the nucleotide sequence as shown in SEQ ID NO:18, and pntA gene has the core as shown in SEQ ID NO:19 Nucleotide sequence, pntB gene have the nucleotide sequence as shown in SEQ ID NO:20, and ADH2 gene has such as SEQ ID NO: Nucleotide sequence shown in 21, ACS6 gene have the nucleotide sequence as shown in SEQ ID NO:22, and ALD6 gene has such as Nucleotide sequence shown in SEQ ID NO:23, EUTE gene have the nucleotide sequence as shown in SEQ ID NO:24, ERG12 Gene has the nucleotide sequence as shown in SEQ ID NO:25, and IDI1 gene has the nucleotide as shown in SEQ ID NO:26 Sequence, ERG10 gene have the nucleotide sequence as shown in SEQ ID NO:27, and MVD1 gene has such as SEQ ID NO:28 institute The nucleotide sequence shown, ERG13 gene have the nucleotide sequence as shown in SEQ ID NO:29, and tHMG1 gene has such as SEQ Nucleotide sequence shown in ID NO:30, ERG8 gene have the nucleotide sequence as shown in SEQ ID NO:31, yap1 gene With the nucleotide sequence as shown in SEQ ID NO:32.Spt15-5 gene has the nucleotide as shown in SEQ ID NO:51 Sequence.Taf25-3 gene has the nucleotide sequence as shown in SEQ ID NO:52.
ATGTCCCAAGCAACTGGGAACGAATTACTGGGTATCCTAGATCTGGATAACGATATAGACTTTGAAACTGCTTACCA AATGCTCAGCAGTAACTTCGACGACCAAATGTCTGCGCACATACATGAAAACACGTTTAGTGCAACTTCCCCTCCTC TGTTAACACACGAGCTCGGCATAATTCCTAACGTGGCAACCGTGCAACCCTCTCACGTAGAAACTATACCTGCCGAT AACCAAACTCATCATGCTCCTTTGCATACTCATGCTCACTATCTAAATCACAACCCTCATCAACCAAGCATGGGTTT TGATCAAACGCTTGGTCTCAAGTTGTCTCCTTCCAGTTCGGGGTTGTTGAGCACGAATGAATCGAATGCCATTGAAC AGTTTTTAGACAATCTAATATCACAGGATATGATGTCTTCCAACGCTTCCATGAACTCCGATTCACATCTACATATA AGATCACCAAAAAAGCAGCATAGGTATACCGAATTAAATCAAAGATATCCTGAAACACATCCACACAGTAACACAGG GGAGTTACCCACAAACACAGCAGATGTGCCAACTGAGTTCACCACGAGGGAAGGACCTCATCAGCCTATCGGCAATG ACCACTACAACCCGCCACCGTTTTCAGTACCTGAGATACGAATCCCAGACTCTGATATTCCAGCCAATATCGAGGAC GACCCTGTGAAGGTACGGAAATGGAAACACGTTCAAATGGAGAAGATACGAAGAATAAACACCAAAGAAGCCTTTGA AAGGCTCATTAAATCAGTAAGGACCCCACCAAAGGAAAACGGGAAAAGAATTCCCAAGCATATTCTTTTAACTTGTG TAATGAACGATATCAAGTCCATTAGAAGCGCAAATGAAGCACTACAGCACATACTGGATGATTCCTGA(SEQ ID NO:13).
ATGACTAAGCAATACAAGAACTACGTTAACGGTGAATGGAAGTTGTCTGAAAACGAAATTAAAATCTATGAACCTGC TTCAGGTGCAGAATTAGGTTCCGTTCCAGCTATGAGTACAGAAGAAGTAGATTACGTTTACGCTTCTGCTAAGAAAG CCCAACCTGCTTGGAGAGCATTGTCATATATCGAAAGAGCTGCATACTTACATAAGGTTGCTGATATATTGATGAGA GACAAGGAAAAGATTGGTGCAATCTTATCTAAAGAAGTAGCCAAGGGTTATAAATCCGCTGTTAGTGAAGTTGTCAG AACCGCTGAAATTATAAACTACGCCGCTGAAGAAGGTTTAAGAATGGAAGGTGAAGTTTTGGAAGGTGGTTCTTTCG AAGCAGCCTCTAAAAAGAAAATTGCAGTAGTTAGAAGAGAACCTGTCGGTTTAGTATTGGCCATTTCCCCTTTCAAT TATCCAGTAAACTTAGCTGGTAGTAAGATCGCCCCAGCTTTGATTGCTGGTAATGTTATAGCCTTTAAACCACCTAC ACAAGGTTCTATTTCAGGTTTGTTATTGGCAGAAGCCTTCGCTGAAGCAGGTTTGCCTGCTGGTGTTTTTAACACAA TTACCGGTAGAGGTTCCGAAATCGGTGACTACATTGTCGAACACCAAGCTGTAAACTTCATCAACTTCACTGGTTCT ACAGGTATAGGTGAAAGAATCGGTAAAATGGCTGGTATGAGACCAATTATGTTAGAATTGGGTGGTAAAGATTCTGC AATAGTTTTAGAAGATGCCGACTTAGAATTGACCGCTAAGAATATCATTGCCGGTGCTTTTGGTTATTCCGGTCAAA GATGTACTGCAGTCAAAAGAGTTTTAGTCATGGAAAGTGTTGCCGATGAATTGGTCGAAAAGATAAGAGAAAAGGTT TTGGCATTGACTATCGGTAATCCTGAAGATGACGCCGACATCACTCCATTGATCGATACAAAGTCAGCTGACTACGT TGAAGGTTTGATTAATGATGCAAACGACAAGGGTGCAACCGCCTTGACTGAAATTAAAAGAGAGGGTAACTTAATCT GCCCAATCTTGTTCGATAAGGTTACTACAGACATGAGATTAGCTTGGGAAGAACCATTTGGTCCTGTCTTGCCAATA ATCAGAGTTACATCTGTCGAAGAAGCTATCGAAATATCTAATAAGTCAGAATATGGTTTGCAAGCATCAATCTTTAC AAACGATTTCCCTAGAGCCTTTGGTATTGCTGAACAATTGGAAGTAGGTACCGTTCATATCAACAACAAGACACAAA GAGGTACCGATAATTTTCCATTCTTGGGTGCTAAGAAATCTGGTGCAGGTATCCAAGGTGTCAAATACTCCATTGAA GCTATGACCACTGTAAAGAGTGTCGTATTTGATATTAAATAA (SEQ ID NO:14).
ATGTATCCAAGGGTCTATGAAGATTTCCAAAAGATCAGAGAAACATACGGTGATTTATCAGTTCTACCAACCAAAAA TTTCCTAGCACCAGCAGAACCTGATGAAGAAATCGAAGTCACCATCGAACAAGGTAAGACTTTGATTATCAAATTGC AAGCTGTTGGTGACTTAAATAAGAAAACTGGGCAAAGAGAAGTGTATTTTGAATTGAACGGTGAATTAAGAAAGATC AGAGTTGCAGACAAGTCACAAAACATACAATCTGTTGCTAAACCAAAGGCTGATGTCCACGATACTCACCAAATCGG TGCACCAATGGCTGGTGTTATCATAGAAGTTAAAGTACATAAAGGGTCTTTGGTGAAAAAGGGCGAATCGATTGCTG TTTTGAGTGCCATGAAAATGGAAATGGTTGTCTCTTCACCAGCAGATGGTCAAGTTAAAGATGTTTTCATTAGGGAT GGTGAAAGTGTTGACGCATCAGATTTGTTGGTTGTCCTAGAAGAAGAAACCCTACCCCCATCCCAAAAAAAGTAA (SEQ ID NO:15).
ATGTGGCCTATTCAGCAATCGCGTTTATATTCTTCTAACACTAGATCGCATAAAGCTACCACAACAAGAGAAAATAC TTTCCAAAAGCCATACAGCGACGAGGAGGTCACTAAAACACCCGTCGGTTCTCGCGCCAGAAAGATCTTCGAAGCTC CTCACCCACATGCCACTCGTTTGACTGTAGAAGGTGCCATAGAATGTCCCTTGGAGAGCTTTCAACTTTTAAACTCT CCTTTATTTAACAAGGGTTCTGCATTTACACAAGAAGAAAGGGAAGCGTTTAATTTAGAAGCATTGCTACCACCACA AGTGAACACTTTGGACGAACAACTGGAAAGAAGCTACAAGCAGTTATGCTATTTGAAGACGCCCTTGGCCAAAAACG ACTTCATGACGTCTTTGAGAGTACAGAACAAAGTCCTATATTTTGCATTAATAAGGAAACATATCAAGGAATTAGTT CCTATCATTTACACCCCAACCGAAGGTGATGCTATTGCTGCCTATTCCCACAGGTTCAGAAAGCCAGAAGGTGTGTT TTTAGACATTACCGAACCTGATTCCATCGAACGTAGATTGGCTACATACGGTGGAGACAAAGATGTAGACTACATCG TTGTGTCGGATTCGGAAGGTATTCTGGGAATTGGTGACCAAGGTATCGGTGGTGTACGTATTGCTATCTCCAAATTG GCATTGATGACGCTGTGCGGTGGTATTCATCCCGGCCGTGTGCTACCTGTGTGTTTGGACGTCGGTACTAACAACAA GAAACTAGCCCGTGACGAATTGTACATGGGTAACAAGTTCTCCAGAATCAGGGGTAAGCAATATGACGACTTCTTGG AAAAATTCATCAAGGCCGTTAAGAAAGTGTATCCAAGCGCCGTTCTGCATTTCGAAGATTTCGGTGTTAAGAACGCT AGAAGATTGCTAGAAAAGTACAGGTACGAATTGCCATCATTCAACGATGACATTCAGGGCACCGGTGCCGTCGTGAT GGCCTCGTTGATTGCTGCTTTGAAACATACCAACAGAGACTTGAAAGACACCAGAGTGCTTATTTACGGTGCCGGGT CTGCGGGCCTCGGTATCGCAGACCAAATTGTGAATCATATGGTCACGCACGGCGTTGACAAGGAAGAAGCGCGCAAG AAAATCTTCTTGATGGACAGACGTGGGTTAATTCTACAATCTTACGAGGCTAACTCCACTCCCGCCCAACACGTATA CGCTAAGAGTGATGCGGAATGGGCTGGTATCAACACCCGCTCTTTACATGATGTGGTGGAGAACGTCAAACCAACGT GTTTGGTTGGCTGCTCCACACAAGCAGGCGCATTCACTCAAGATGTCGTAGAAGAAATGCACAAGCACAATCCTAGA CCGATCATTTTCCCATTATCCAACCCTACTAGACTACACGAAGCCGTTCCTGCCGATTTAATGAAGTGGACCAACAA CAACGCTCTTGTAGCTACCGGATCTCCTTTCCCACCTGTTGACGGTTACCGTATCTCGGAGAACAACAATTGTTACT CTTTCCCAGGTATCGGTTTAGGTGCCGTACTATCGCGTGCCACCACTATCACAGACAAGATGATCTCCGCTGCAGTG GACCAACTAGCCGAATTGTCGCCACTAAGAGAGGGCGACTCGAGACCTGGGTTGCTACCCGGCCTGGACACCATCAC CAACACCTCTGCGCGTCTAGCTACCGCTGTGATCTTGCAAGCACTCGAGGAGGGAACCGCCCGTATCGAGCAAGAAC AAGTACCGGGAGGAGCTCCCGGCGAAACTGTCAAGGTTCCTCGTGACTTTGACGAATGTTTACAGTGGGTCAAAGCC CAAATGTGGGAGCCTGTGTACAGACCTATGATCAAGGTCCAACATGACCCATCGGTGCACACCAACCAATTGTAG (SEQ ID NO:16).
ATGCCTCACTCAGTTACACCATCCATAGAACAAGATTCGTTAAAAATTGCCATTTTAGGTGCTGCCGGTGGTATCGG GCAGTCGTTATCGCTGCTTTTGAAAGCTCAGTTGCAATACCAGTTAAAGGAGAGCAACCGGAGCGTTACCCACATTC ATCTGGCTCTTTACGATGTCAACCAAGAAGCCATCAACGGTGTTACCGCCGACTTGTCTCATATAGACACCCCCATT TCCGTGTCGAGCCACTCTCCTGCAGGTGGCATTGAGAACTGTTTGCATAACGCTTCTATTGTTGTCATTCCTGCAGG TGTTCCAAGAAAACCTGGCATGACTCGTGATGACTTATTTAACGTGAATGCTGGTATCATTAGCCAGCTCGGTGATT CTATTGCAGAATGTTGTGATCTTTCCAAGGTCTTCGTTCTTGTCATTTCCAACCCTGTTAATTCTTTAGTCCCAGTG ATGGTTTCTAACATTCTTAAGAACCATCCTCAGTCTAGAAATTCCGGCATTGAAAGAAGGATCATGGGTGTCACCAA GCTCGACATTGTCAGAGCGTCCACTTTTCTACGTGAGATAAACATTGAGTCAGGGCTAACTCCTCGTGTTAACTCCA TGCCTGACGTCCCTGTAATTGGCGGGCATTCTGGCGAGACTATTATTCCGTTGTTTTCACAGTCAAACTTCCTATCG AGATTAAATGAGGATCAATTGAAATATTTAATACATAGAGTCCAATACGGTGGTGATGAAGTGGTCAAGGCCAAGAA CGGTAAAGGTAGTGCTACCTTATCGATGGCCCATGCCGGTTATAAGTGTGTTGTCCAATTTGTTTCTTTGTTATTGG GTAACATTGAGCAGATCCATGGAACCTACTATGTGCCATTAAAAGATGCGAACAACTTCCCCATTGCTCCTGGGGCA GATCAATTATTGCCTCTGGTGGACGGTGCAGACTACTTTGCCATACCATTAACTATTACTACAAAGGGTGTTTCCTA TGTGGATTATGACATCGTTAATAGGATGAACGACATGGAACGCAACCAAATGTTGCCAATTTGCGTCTCCCAGTTAA AGAAAAATATCGATAAGGGCTTGGAATTCGTTGCATCGAGATCTGCATCATCTTAA (SEQ ID NO:17).
ATGTTTGTCAGGGTTAAATTGAATAAACCAGTAAAATGGTATAGGTTCTATAGTACGTTGGATTCACATTCCCTAAA GTTACAGAGCGGCTCGAAGTTTGTAAAAATAAAGCCAGTAAATAACTTGAGGAGTAGTTCATCAGCAGATTTCGTGT CCCCACCAAATTCCAAATTACAATCTTTAATCTGGCAGAACCCTTTACAAAATGTTTATATAACTAAAAAACCATGG ACTCCATCCACAAGAGAAGCGATGGTTGAATTCATAACTCATTTACATGAGTCATACCCCGAGGTGAACGTCATTGT TCAACCCGATGTGGCAGAAGAAATTTCCCAGGATTTCAAATCTCCTTTGGAGAATGATCCCAACCGACCTCATATAC TTTATACTGGTCCTGAACAAGATATCGTAAACAGAACAGACTTATTGGTGACATTGGGAGGTGATGGGACTATTTTA CACGGCGTATCAATGTTCGGAAATACGCAAGTTCCTCCGGTTTTAGCATTTGCTCTGGGCACTCTGGGCTTTCTATT ACCGTTTGATTTTAAGGAGCATAAAAAGGTCTTTCAGGAAGTAATCAGCTCTAGAGCCAAATGTTTGCATAGAACAC GGCTAGAATGTCATTTGAAAAAAAAGGATAGCAACTCATCTATTGTGACCCATGCTATGAATGACATATTCTTACAT AGGGGTAATTCCCCTCATCTCACTAACCTGGACATTTTCATTGATGGGGAATTTTTGACAAGAACGACAGCAGATGG TGTTGCATTGGCCACTCCAACGGGTTCCACAGCATATTCATTATCAGCAGGTGGATCTATTGTTTCCCCATTAGTCC CTGCTATTTTAATGACACCAATTTGTCCTCGCTCTTTGTCATTCCGACCACTGATTTTGCCTCATTCATCCCACATT AGGATAAAGATAGGTTCCAAATTGAACCAAAAACCAGTCAACAGTGTGGTAAAACTTTCTGTTGATGGTATTCCTCA ACAGGATTTAGATGTTGGTGATGAAATTTATGTTATAAATGAGGTCGGCACTATATACATAGATGGTACTCAGCTTC CGACGACAAGAAAAACTGAAAATGACTTTAATAATTCAAAAAAGCCTAAAAGGTCAGGGATTTATTGTGTCGCCAAG ACCGAGAATGACTGGATTAGAGGAATCAATGAACTTTTAGGATTCAATTCTAGCTTTAGGCTGACCAAGAGACAGAC TGATAATGATTAA (SEQ ID NO:18).
ATGCGAATTGGCATACCAAGAGAACGGTTAACCAATGAAACCCGTGTTGCAGCAACGCCAAAAACAGTGGAACAGCT GCTGAAACTGGGTTTTACCGTCGCGGTAGAGAGCGGCGCGGGTCAACTGGCAAGTTTTGACGATAAAGCGTTTGTGC AAGCGGGCGCTGAAATTGTAGAAGGGAATAGCGTCTGGCAGTCAGAGATCATTCTGAAGGTCAATGCGCCGTTAGAT GATGAAATTGCGTTACTGAATCCTGGGACAACGCTGGTGAGTTTTATCTGGCCTGCGCAGAATCCGGAATTAATGCA AAAACTTGCGGAACGTAACGTGACCGTGATGGCGATGGACTCTGTGCCGCGTATCTCACGCGCACAATCGCTGGACG CACTAAGCTCGATGGCGAACATCGCCGGTTATCGCGCCATTGTTGAAGCGGCACATGAATTTGGGCGCTTCTTTACC GGGCAAATTACTGCGGCCGGGAAAGTGCCACCGGCAAAAGTGATGGTGATTGGTGCGGGTGTTGCAGGTCTGGCCGC CATTGGCGCAGCAAACAGTCTCGGCGCGATTGTGCGTGCATTCGACACCCGCCCGGAAGTGAAAGAACAAGTTCAAA GTATGGGCGCGGAATTCCTCGAGCTGGATTTTAAAGAGGAAGCTGGCAGCGGCGATGGCTATGCCAAAGTGATGTCG GACGCGTTCATCAAAGCGGAAATGGAACTCTTTGCCGCCCAGGCAAAAGAGGTCGATATCATTGTCACCACCGCGCT TATTCCAGGCAAACCAGCGCCGAAGCTAATTACCCGTGAAATGGTTGACTCCATGAAGGCGGGCAGTGTGATTGTCG ACCTGGCAGCCCAAAACGGCGGCAACTGTGAATACACCGTGCCGGGTGAAATCTTCACTACGGAAAATGGTGTCAAA GTGATTGGTTATACCGATCTTCCGGGCCGTCTGCCGACGCAATCCTCACAGCTTTACGGCACAAACCTCGTTAATCT GCTGAAACTGTTGTGCAAAGAGAAAGACGGCAATATCACTGTTGATTTTGATGATGTGGTGATTCGCGGCGTGACCG TGATCCGTGCGGGCGAAATTACCTGGCCGGCACCGCCGATTCAGGTATCAGCTCAGCCGCAGGCGGCACAAAAAGCG GCACCGGAAGTGAAAACTGAGGAAAAATGTACCTGCTCACCGTGGCGTAAATACGCGTTGATGGCGCTGGCAATCAT TCTTTTTGGCTGGATGGCAAGCGTTGCGCCGAAAGAATTCCTTGGGCACTTCACCGTTTTCGCGCTGGCCTGCGTTG TCGGTTATTACGTGGTGTGGAATGTATCGCACGCGCTGCATACACCGTTGATGTCGGTCACCAACGCGATTTCAGGG ATTATTGTTGTCGGAGCACTGTTGCAGATTGGCCAGGGCGGCTGGGTTAGCTTCCTTAGTTTTATCGCGGTGCTTAT AGCCAGCATTAATATTTTCGGTGGCTTCACCGTGACTCAGCGCATGCTGAAAATGTTCCGCAAAAATTAA(SEQ ID NO:19)
ATGTCTGGAGGATTAGTTACAGCTGCATACATTGTTGCCGCGATCCTGTTTATCTTCAGTCTGGCCGGTCTTTCGAA ACATGAAACGTCTCGCCAGGGTAACAACTTCGGTATCGCCGGGATGGCGATTGCGTTAATCGCAACCATTTTTGGAC CGGATACGGGTAATGTTGGCTGGATCTTGCTGGCGATGGTCATTGGTGGGGCAATTGGTATCCGTCTGGCGAAGAAA GTTGAAATGACCGAAATGCCAGAACTGGTGGCGATCCTGCATAGCTTCGTGGGTCTGGCGGCAGTGCTGGTTGGCTT TAACAGCTATCTGCATCATGACGCGGGAATGGCACCGATTCTGGTCAATATTCACCTGACGGAAGTGTTCCTCGGTA TCTTCATCGGGGCGGTAACGTTCACGGGTTCGGTGGTGGCGTTCGGCAAACTGTGTGGCAAGATTTCGTCTAAACCA TTGATGCTGCCAAACCGTCACAAAATGAACCTGGCGGCTCTGGTCGTTTCCTTCCTGCTGCTGATTGTATTTGTTCG CACGGACAGCGTCGGCCTGCAAGTGCTGGCATTGCTGATAATGACCGCAATTGCGCTGGTATTCGGCTGGCATTTAG TCGCCTCCATCGGTGGTGCAGATATGCCAGTGGTGGTGTCGATGCTGAACTCGTACTCCGGCTGGGCGGCTGCGGCT GCGGGCTTTATGCTCAGCAACGACCTGCTGATTGTGACCGGTGCGCTGGTCGGTTCTTCGGGGGCTATCCTTTCTTA CATTATGTGTAAGGCGATGAACCGTTCCTTTATCAGCGTTATTGCGGGTGGTTTCGGCACCGACGGCTCTTCTACTG GCGATGATCAGGAAGTGGGTGAGCACCGCGAAATCACCGCAGAAGAGACAGCGGAACTGCTGAAAAACTCCCATTCA GTGATCATTACTCCGGGGTACGGCATGGCAGTCGCGCAGGCGCAATATCCTGTCGCTGAAATTACTGAGAAATTGCG CGCTCGTGGTATTAATGTGCGTTTCGGTATCCACCCGGTCGCGGGGCGTTTGCCTGGACATATGAACGTATTGCTGG CTGAAGCAAAAGTACCGTATGACATCGTGCTGGAAATGGACGAGATCAATGATGACTTTGCTGATACCGATACCGTA CTGGTGATTGGTGCTAACGATACGGTTAACCCGGCGGCGCAGGATGATCCGAAGAGTCCGATTGCTGGTATGCCTGT GCTGGAAGTGTGGAAAGCGCAGAACGTGATTGTCTTTAAACGTTCGATGAACACTGGCTATGCTGGTGTGCAAAACC CGCTGTTCTTCAAGGAAAACACCCACATGCTGTTTGGTGACGCCAAAGCCAGCGTGGATGCAATCCTGAAAGCTCTG TAA (SEQ ID NO:20)
ATGTCTATTCCAGAAACTCAAAAAGCCATTATCTTCTACGAATCCAACGGCAAGTTGGAGCATAAGGATATCCCAGT TCCAAAGCCAAAGCCCAACGAATTGTTAATCAACGTCAAGTACTCTGGTGTCTGCCACACCGATTTGCACGCTTGGC ATGGTGACTGGCCATTGCCAACTAAGTTACCATTAGTTGGTGGTCACGAAGGTGCCGGTGTCGTTGTCGGCATGGGT GAAAACGTTAAGGGCTGGAAGATCGGTGACTACGCCGGTATCAAATGGTTGAACGGTTCTTGTATGGCCTGTGAATA CTGTGAATTGGGTAACGAATCCAACTGTCCTCACGCTGACTTGTCTGGTTACACCCACGACGGTTCTTTCCAAGAAT ACGCTACCGCTGACGCTGTTCAAGCCGCTCACATTCCTCAAGGTACTGACTTGGCTGAAGTCGCGCCAATCTTGTGT GCTGGTATCACCGTATACAAGGCTTTGAAGTCTGCCAACTTGAGAGCAGGCCACTGGGCGGCCATTTCTGGTGCTGC TGGTGGTCTAGGTTCTTTGGCTGTTCAATATGCTAAGGCGATGGGTTACAGAGTCTTAGGTATTGATGGTGGTCCAG GAAAGGAAGAATTGTTTACCTCGCTCGGTGGTGAAGTATTCATCGACTTCACCAAAGAGAAGGACATTGTTAGCGCA GTCGTTAAGGCTACCAACGGCGGTGCCCACGGTATCATCAATGTTTCCGTTTCCGAAGCCGCTATCGAAGCTTCTAC CAGATACTGTAGGGCGAACGGTACTGTTGTCTTGGTTGGTTTGCCAGCCGGTGCAAAGTGCTCCTCTGATGTCTTCA ACCACGTTGTCAAGTCTATCTCCATTGTCGGCTCTTACGTGGGGAACAGAGCTGATACCAGAGAAGCCTTAGATTTC TTTGCCAGAGGTCTAGTCAAGTCTCCAATAAAGGTAGTTGGCTTATCCAGTTTACCAGAAATTTACGAAAAGATGGA GAAGGGCCAAATTGCTGGTAGATACGTTGTTGACACTTCTAAATAA (SEQ ID NO:21).
ATGTCTCAGACTCACAAGCATGCTATTCCTGCTAACATAGCTGACAGATGCTTAATAAATCCAGAACAGTATGAAAC TAAATACAAGCAATCTATAAACGATCCTGACACTTTCTGGGGAGAGCAGGGTAAGATATTGGACTGGATTACACCAT ACCAAAAGGTCAAGAACACTTCATTTGCTCCTGGTAATGTTTCTATTAAATGGTACGAGGACGGTACATTAAACTTA GCAGCAAACTGCTTGGACAGGCACTTGCAAGAGAACGGAGACAGAACTGCAATAATTTGGGAGGGTGACGACGCATC TCAATCTAAGCACATATCATACAGAGAGTTACACAGAGACGTTTGCAGATTCGCTAACACATTGTTGGATTTGGGAA TAAAGAAAGGTGACGTTGTTGCAATTTATATGCCTATGGTTCCTGAGGCTGCTGTCGCTATGTTGGCTTGCGCAAGG ATAGGAGCTGTCCATTCTGTCATTTTTGGTGGTTTTTCACCTGAGGCAATAGCAGGTAGGATAATTGATTCATCTTC AAGGTTGGTCATAACTGCAGATGAGGGAGTCAGGGCAGGAAGATCTATTCCATTGAAGAAGAACGTTGACGATGCAT TGAAGAACCCAAACGTTACTTCTGTCGAGCACGTCATTGTCTTAAAAAGAACTGGAAACGACATAGACTGGCAAGAG GGTAGGGACTTATGGTGGAGGGACTTGATTGAGAAGGCTTCACCTGAACACCAGCCAGAGGCAATGAATGCAGAAGA CCCATTGTTTATTTTATATACATCTGGATCTACAGGAAAGCCAAAGGGAGTCTTGCATACTACAGGTGGTTACTTGG TCTATGCAGCAACAACTTTTAAGTATGTCTTCGACTACCACCCTGGTGACATTTACTGGTGCACAGCTGACGTTGGA TGGGTCACAGGTCACTCTTACTTGTTGTACGGACCTTTGGCATGCGGTGCTACTACATTGATGTTCGAGGGAGTCCC TAACTGGCCTACACCTGCTAGGATGTGCCAGGTTGTCGACAAGCACCAGGTCAACATACTGTACACAGCACCAACAG CTATTAGGGCATTGATGGCAGAGGGTGACAAGGCAATTGAGGGAACAGACAGGTCTTCTTTGAGAATATTGGGATCT GTCGGTGAGCCAATAAACCCAGAGGCATGGGAGTGGTACTGGAAGAAGATAGGAAAGGAGAAATGTCCAGTCGTCGA CACATGGTGGCAGACAGAGACAGGTGGATTTATGATAACTCCTTTGCCAGGAGCTATAGAGTTGAAGGCTGGTTCAG CAACAAGGCCATTCTTCGGAGTCCAGCCTGCATTGGTCGACAACGAGGGTCATCCACAGGAAGGTGCTACAGAGGGT AACTTAGTCATTACTGACTCTTGGCCTGGTCAGGCTAGGACATTGTTCGGTGACCATGAGAGGTTCGAGCAGACTTA CTTTTCTACATTTAAAAATATGTACTTTTCTGGAGACGGTGCTAGAAGGGATGAAGATGGTTACTATTGGATAACTG GTAGGGTCGATGATGTCTTAAACGTCTCAGGACACAGGTTGGGAACTGCTGAAATTGAATCAGCATTGGTCGCTCAC CCTAAGATAGCTGAGGCAGCTGTCGTCGGAATTCCTCACGCAATTAAGGGACAGGCAATTTACGCATACGTCACTTT GAACCACGGTGAAGAGCCATCACCAGAGTTGTACGCAGAGGTTAGGAACTGGGTCAGGAAGGAGATTGGACCTTTGG CAACTCCAGACGTCTTGCACTGGACAGACTCATTGCCTAAGACAAGGTCTGGAAAAATAATGAGGAGAATATTGAGG AAAATTGCTGCTGGAGATACTTCAAACTTGGGTGACACTTCTACATTGGCTGACCCAGGTGTTGTCGAGAAACCATT GGAGGAAAAACAGGCAATAGCTATGCCATCTTAA (SEQID NO:22).
ATGACTAAGCTACACTTTGACACTGCTGAACCAGTCAAGATCACACTTCCAAATGGTTTGACATACGAGCAACCAAC CGGTCTATTCATTAACAACAAGTTTATGAAAGCTCAAGACGGTAAGACCTATCCCGTCGAAGATCCTTCCACTGAAA ACACCGTTTGTGAGGTCTCTTCTGCCACCACTGAAGATGTTGAATATGCTATCGAATGTGCCGACCGTGCTTTCCAC GACACTGAATGGGCTACCCAAGACCCAAGAGAAAGAGGCCGTCTACTAAGTAAGTTGGCTGACGAATTGGAAAGCCA AATTGACTTGGTTTCTTCCATTGAAGCTTTGGACAATGGTAAAACTTTGGCCTTAGCCCGTGGGGATGTTACCATTG CAATCAACTGTCTAAGAGATGCTGCTGCCTATGCCGACAAAGTCAACGGTAGAACAATCAACACCGGTGACGGCTAC ATGAACTTCACCACCTTAGAGCCAATCGGTGTCTGTGGTCAAATTATTCCATGGAACTTTCCAATAATGATGTTGGC TTGGAAGATCGCCCCAGCATTGGCCATGGGTAACGTCTGTATCTTGAAACCCGCTGCTGTCACACCTTTAAATGCCC TATACTTTGCTTCTTTATGTAAGAAGGTTGGTATTCCAGCTGGTGTCGTCAACATCGTTCCAGGTCCTGGTAGAACT GTTGGTGCTGCTTTGACCAACGACCCAAGAATCAGAAAGCTGGCTTTTACCGGTTCTACAGAAGTCGGTAAGAGTGT TGCTGTCGACTCTTCTGAATCTAACTTGAAGAAAATCACTTTGGAACTAGGTGGTAAGTCCGCCCATTTGGTCTTTG ACGATGCTAACATTAAGAAGACTTTACCAAATCTAGTAAACGGTATTTTCAAGAACGCTGGTCAAATTTGTTCCTCT GGTTCTAGAATTTACGTTCAAGAAGGTATTTACGACGAACTATTGGCTGCTTTCAAGGCTTACTTGGAAACCGAAAT CAAAGTTGGTAATCCATTTGACAAGGCTAACTTCCAAGGTGCTATCACTAACCGTCAACAATTCGACACAATTATGA ACTACATCGATATCGGTAAGAAAGAAGGCGCCAAGATCTTAACTGGTGGCGAAAAAGTTGGTGACAAGGGTTACTTC ATCAGACCAACCGTTTTCTACGATGTTAATGAAGACATGAGAATTGTTAAGGAAGAAATTTTTGGACCAGTTGTCAC TGTCGCAAAGTTCAAGACTTTAGAAGAAGGTGTCGAAATGGCTAACAGCTCTGAATTCGGTCTAGGTTCTGGTATCG AAACAGAATCTTTGAGCACAGGTTTGAAGGTGGCCAAGATGTTGAAGGCCGGTACCGTCTGGATCAACACATACAAC GATTTTGACTCCAGAGTTCCATTCGGTGGTGTTAAGCAATCTGGTTACGGTAGAGAAATGGGTGAAGAAGTCTACCA TGCATACACTGAAGTAAAAGCTGTCAGAATTAAGTTGTAA (SEQID NO:23).
ATGAATCAACAGGATATTGAACAGGTGGTGAAAGCGGTACTGCTGAAAATGCAAAGCAGTGACACGCCGTCCGCCGC CGTTCATGAGATGGGCGTTTTCGCGTCCCTGGATGACGCCGTTGCGGCAGCCAAAGTCGCCCAGCAAGGGTTAAAAA GCGTGGCAATGCGCCAGTTAGCCATTGCTGCCATTCGTGAAGCAGGCGAAAAACACGCCAGAGATTTAGCGGAACTT GCCGTCAGTGAAACCGGCATGGGGCGCGTTGAAGATAAATTTGCAAAAAACGTCGCTCAGGCGCGCGGCACACCAGG CGTTGAGTGCCTCTCTCCGCAAGTGCTGACTGGCGACAACGGCCTGACCCTAATTGAAAACGCACCCTGGGGCGTGG TGGCTTCGGTGACGCCTTCCACTAACCCGGCGGCAACCGTAATTAACAACGCCATCAGCCTGATTGCCGCGGGCAAC AGCGTCATTTTTGCCCCGCATCCGGCGGCGAAAAAAGTCTCCCAGCGGGCGATTACGCTGCTCAACCAGGCGATTGT TGCCGCAGGTGGGCCGGAAAACTTACTGGTTACTGTGGCAAATCCGGATATCGAAACCGCGCAACGCTTGTTCAAGT TTCCGGGTATCGGCCTGCTGGTGGTAACCGGCGGCGAAGCGGTAGTAGAAGCGGCGCGTAAACACACCAATAAACGT CTGATTGCCGCAGGCGCTGGCAACCCGCCGGTAGTGGTGGATGAAACCGCCGACCTCGCCCGTGCCGCTCAGTCCAT CGTCAAAGGCGCTTCTTTCGATAACAACATCATTTGTGCCGACGAAAAGGTACTGATTGTTGTTGATAGCGTAGCCG ATGAACTGATGCGTCTGATGGAAGGCCAGCACGCGGTGAAACTGACCGCAGAACAGGCGCAGCAGCTGCAACCGGTG TTGCTGAAAAATATCGACGAGCGCGGAAAAGGCACCGTCAGCCGTGACTGGGTTGGTCGCGACGCAGGCAAAATCGC GGCGGCAATCGGCCTTAAAGTTCCGCAAGAAACGCGCCTGCTGTTTGTGGAAACCACCGCAGAACATCCGTTTGCCG TGACTGAACTGATGATGCCGGTGTTGCCCGTCGTGCGCGTCGCCAACGTGGCGGATGCCATTGCGCTAGCGGTGAAA CTGGAAGGCGGTTGCCACCACACGGCGGCAATGCACTCGCGCAACATCGAAAACATGAACCAGATGGCGAATGCTAT TGATACCAGCATTTTCGTTAAGAACGGACCGTGCATTGCCGGGCTGGGGCTGGGCGGGGAAGGCTGGACCACCATGA CCATCACCACGCCAACCGGTGAAGGGGTAACCAGCGCGCGTACGTTTGTCCGTCTGCGTCGCTGTGTATTAGTCGAT GCGTTTCGCATTGTTTAA (SEQ ID NO:24).
ATGTCATTACCGTTCTTAACTTCTGCACCGGGAAAGGTTATTATTTTTGGTGAACACTCTGCTGTGTACAACAAGCC TGCCGTCGCTGCTAGTGTGTCTGCGTTGAGAACCTACCTGCTAATAAGCGAGTCATCTGCACCAGATACTATTGAAT TGGACTTCCCGGACATTAGCTTTAATCATAAGTGGTCCATCAATGATTTCAATGCCATCACCGAGGATCAAGTAAAC TCCCAAAAATTGGCCAAGGCTCAACAAGCCACCGATGGCTTGTCTCAGGAACTCGTTAGTCTTTTGGATCCGTTGTT AGCTCAACTATCCGAATCCTTCCACTACCATGCAGCGTTTTGTTTCCTGTATATGTTTGTTTGCCTATGCCCCCATG CCAAGAATATTAAGTTTTCTTTAAAGTCTACTTTACCCATCGGTGCTGGGTTGGGCTCAAGCGCCTCTATTTCTGTA TCACTGGCCTTAGCTATGGCCTACTTGGGGGGGTTAATAGGATCTAATGACTTGGAAAAGCTGTCAGAAAACGATAA GCATATAGTGAATCAATGGGCCTTCATAGGTGAAAAGTGTATTCACGGTACCCCTTCAGGAATAGATAACGCTGTGG CCACTTATGGTAATGCCCTGCTATTTGAAAAAGACTCACATAATGGAACAATAAATACAAACAATTTTAAGTTCTTA GATGATTTCCCAGCCATTCCAATGATCCTAACCTATACTAGAATTCCAAGGTCTACAAAAGATCTTGTTGCTCGCGT TCGTGTGTTGGTCACCGAGAAATTTCCTGAAGTTATGAAGCCAATTCTAGATGCCATGGGTGAATGTGCCCTACAAG GCTTAGAGATCATGACAAAGTTAAGTAAATGTAAAGGCACCGATGACGAGGCCGTAGAAACTAATAATGAACTGTAT GAACAACTATTGGAATTGATAAGAATAAATCATGGACTGCTTGTCTCAATCGGTGTTTCTCATCCTGGATTAGAACT TATTAAAAATCTGAGCGATGATTTGAGAATTGGCTCCACAAAACTTACCGGTGCTGGTGGCGGCGGTTGCTCTTTGA CTTTGTTACGAAGAGACATTACTCAAGAGCAAATTGACAGTTTCAAAAAGAAATTGCAAGATGATTTTAGTTACGAG ACATTTGAAACAGACTTGGGTGGGACTGGCTGCTGTTTGTTAAGCGCAAAAAATTTGAATAAAGATCCTAAAATCAA ATCCCTAGTATTCCAATTATTTGAAAATAAAACTACCACAAAGCAACAAATTGACGATCTATTATTGCCAGGAAACA CGAATTTACCATGGACTTCATAA (SEQ ID NO:25).
ATGACTGCCGACAACAATAGTATGCCCCATGGTGCAGTATCTAGTTACGCCAAATTAGTGCAAAACCAAACACCTGA AGACATTTTGGAAGAGTTTCCTGAAATTATTCCATTACAACAAAGACCTAATACCCGATCTAGTGAGACGTCAAATG ACGAAAGCGGAGAAACATGTTTTTCTGGTCATGATGAGGAGCAAATTAAGTTAATGAATGAAAATTGTATTGTTTTG GATTGGGACGATAATGCTATTGGTGCCGGTACCAAGAAAGTTTGTCATTTAATGGAAAATATTGAAAAGGGTTTACT ACATCGTGCATTCTCCGTCTTTATTTTCAATGAACAAGGTGAATTACTTTTACAACAAAGAGCCACTGAAAAAATAA CTTTCCCTGATCTTTGGACTAACACATGCTGCTCTCATCCACTATGTATTGATGACGAATTAGGTTTGAAGGGTAAG CTAGACGATAAGATTAAGGGCGCTATTACTGCGGCGGTGAGAAAACTAGATCATGAATTAGGTATTCCAGAAGATGA AACTAAGACAAGGGGTAAGTTTCACTTTTTAAACAGAATCCATTACATGGCACCAAGCAATGAACCATGGGGTGAAC ATGAAATTGATTACATCCTATTTTATAAGATCAACGCTAAAGAAAACTTGACTGTCAACCCAAACGTCAATGAAGTT AGAGACTTCAAATGGGTTTCACCAAATGATTTGAAAACTATGTTTGCTGACCCAAGTTACAAGTTTACGCCTTGGTT TAAGATTATTTGCGAGAATTACTTATTCAACTGGTGGGAGCAATTAGATGACCTTTCTGAAGTGGAAAATGACAGGC AAATTCATAGAATGCTATAA (SEQID NO:26).
ATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTCATTCCAGGGTTCTCTATCCTCCAAGAC AGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGGCGCCTTGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTG ACGAAATTATTTTTGGTAACGTTCTTTCTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTTTGGCTGCCGGT TTGAGTAATCATATCGTTGCAAGCACAGTTAACAAGGTCTGTGCATCCGCTATGAAGGCAATCATTTTGGGTGCTCA ATCCATCAAATGTGGTAATGCTGATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCATACTACATGC CAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGAAAGAGATGGGTTGAACGATGCG TACGATGGTCTAGCCATGGGTGTACACGCAGAAAAGTGTGCCCGTGATTGGGATATTACTAGAGAACAACAAGACAA TTTTGCCATCGAATCCTACCAAAAATCTCAAAAATCTCAAAAGGAAGGTAAATTCGACAATGAAATTGTACCTGTTA CCATTAAGGGATTTAGAGGTAAGCCTGATACTCAAGTCACGAAGGACGAGGAACCTGCTAGATTACACGTTGAAAAA TTGAGATCTGCAAGGACTGTTTTCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATCAACGATGG TGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAGCCTTTGGCTATTATCAAAGGTT GGGGTGAGGCCGCTCATCAACCAGCTGATTTTACATGGGCTCCATCTCTTGCAGTTCCAAAGGCTTTGAAACATGCT GGCATCGAAGACATCAATTCTGTTGATTACTTTGAATTCAATGAAGCCTTTTCGGTTGTCGGTTTGGTGAACACTAA GATTTTGAAGCTAGACCCATCTAAGGTTAATGTATATGGTGGTGCTGTTGCTCTAGGTCACCCATTGGGTTGTTCTG GTGCTAGAGTGGTTGTTACACTGCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTAAT GGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGA (SEQ ID NO:27).
ATGACCGTTTACACAGCATCCGTTACCGCACCCGTCAACATCGCAACCCTTAAGTATTGGGGGAAAAGGGACACGAA GTTGAATCTGCCCACCAATTCGTCCATATCAGTGACTTTATCGCAAGATGACCTCAGAACGTTGACCTCTGCGGCTA CTGCACCTGAGTTTGAACGCGACACTTTGTGGTTAAATGGAGAACCACACAGCATCGACAATGAAAGAACTCAAAAT TGTCTGCGCGACCTACGCCAATTAAGAAAGGAAATGGAATCGAAGGACGCCTCATTGCCCACATTATCTCAATGGAA ACTCCACATTGTCTCCGAAAATAACTTTCCTACAGCAGCTGGTTTAGCTTCCTCCGCTGCTGGCTTTGCTGCATTGG TCTCTGCAATTGCTAAGTTATACCAATTACCACAGTCAACTTCAGAAATATCTAGAATAGCAAGAAAGGGGTCTGGT TCAGCTTGTAGATCGTTGTTTGGCGGATACGTGGCCTGGGAAATGGGAAAAGCTGAAGATGGTCATGATTCCATGGC AGTACAAATCGCAGACAGCTCTGACTGGCCTCAGATGAAAGCTTGTGTCCTAGTTGTCAGCGATATTAAAAAGGATG TGAGTTCCACTCAGGGTATGCAATTGACCGTGGCAACCTCCGAACTATTTAAAGAAAGAATTGAACATGTCGTACCA AAGAGATTTGAAGTCATGCGTAAAGCCATTGTTGAAAAAGATTTCGCCACCTTTGCAAAGGAAACAATGATGGATTC CAACTCTTTCCATGCCACATGTTTGGACTCTTTCCCTCCAATATTCTACATGAATGACACTTCCAAGCGTATCATCA GTTGGTGCCACACCATTAATCAGTTTTACGGAGAAACAATCGTTGCATACACGTTTGATGCAGGTCCAAATGCTGTG TTGTACTACTTAGCTGAAAATGAGTCGAAACTCTTTGCATTTATCTATAAATTGTTTGGCTCTGTTCCTGGATGGGA CAAGAAATTTACTACTGAGCAGCTTGAGGCTTTCAACCATCAATTTGAATCATCTAACTTTACTGCACGTGAATTGG ATCTTGAGTTGCAAAAGGATGTTGCCAGAGTGATTTTAACTCAAGTCGGTTCAGGCCCACAAGAAACAAACGAATCT TTGATTGACGCAAAGACTGGTCTACCAAAGGAATAA (SEQ ID NO:28).
ATGAAACTCTCAACTAAACTTTGTTGGTGTGGTATTAAAGGAAGACTTAGGCCGCAAAAGCAACAACAATTACACAA TACAAACTTGCAAATGACTGAACTAAAAAAACAAAAGACCGCTGAACAAAAAACCAGACCTCAAAATGTCGGTATTA AAGGTATCCAAATTTACATCCCAACTCAATGTGTCAACCAATCTGAGCTAGAGAAATTTGATGGCGTTTCTCAAGGT AAATACACAATTGGTCTGGGCCAAACCAACATGTCTTTTGTCAATGACAGAGAAGATATCTACTCGATGTCCCTAAC TGTTTTGTCTAAGTTGATCAAGAGTTACAACATCGACACCAACAAAATTGGTAGATTAGAAGTCGGTACTGAAACTC TGATTGACAAGTCCAAGTCTGTCAAGTCTGTCTTGATGCAATTGTTTGGTGAAAACACTGACGTCGAAGGTATTGAC ACGCTTAATGCCTGTTACGGTGGTACCAACGCGTTGTTCAACTCTTTGAACTGGATTGAATCTAACGCATGGGATGG TAGAGACGCCATTGTAGTTTGCGGTGATATTGCCATCTACGATAAGGGTGCCGCAAGACCAACCGGTGGTGCCGGTA CTGTTGCTATGTGGATCGGTCCTGATGCTCCAATTGTATTTGACTCTGTAAGAGCTTCTTACATGGAACACGCCTAC GATTTTTACAAGCCAGATTTCACCAGCGAATATCCTTACGTCGATGGTCATTTTTCATTAACTTGTTACGTCAAGGC TCTTGATCAAGTTTACAAGAGTTATTCCAAGAAGGCTATTTCTAAAGGGTTGGTTAGCGATCCCGCTGGTTCGGATG CTTTGAACGTTTTGAAATATTTCGACTACAACGTTTTCCATGTTCCAACCTGTAAATTGGTCACAAAATCATACGGT AGATTACTATATAACGATTTCAGAGCCAATCCTCAATTGTTCCCAGAAGTTGACGCCGAATTAGCTACTCGCGATTA TGACGAATCTTTAACCGATAAGAACATTGAAAAAACTTTTGTTAATGTTGCTAAGCCATTCCACAAAGAGAGAGTTG CCCAATCTTTGATTGTTCCAACAAACACAGGTAACATGTACACCGCATCTGTTTATGCCGCCTTTGCATCTCTATTA AACTATGTTGGATCTGACGACTTACAAGGCAAGCGTGTTGGTTTATTTTCTTACGGTTCCGGTTTAGCTGCATCTCT ATATTCTTGCAAAATTGTTGGTGACGTCCAACATATTATCAAGGAATTAGATATTACTAACAAATTAGCCAAGAGAA TCACCGAAACTCCAAAGGATTACGAAGCTGCCATCGAATTGAGAGAAAATGCCCATTTGAAGAAGAACTTCAAACCT CAAGGTTCCATTGAGCATTTGCAAAGTGGTGTTTACTACTTGACCAACATCGATGACAAATTTAGAAGATCTTACGA TGTTAAAAAATAA (SEQ ID NO:29).
ATGGTTTTAACCAATAAAACAGTCATTTCTGGATCGAAAGTCAAAAGTTTATCATCTGCGCAATCGAGCTCATCAGG ACCTTCATCATCTAGTGAGGAAGATGATTCCCGCGATATTGAAAGCTTGGATAAGAAAATACGTCCTTTAGAAGAAT TAGAAGCATTATTAAGTAGTGGAAATACAAAACAATTGAAGAACAAAGAGGTCGCTGCCTTGGTTATTCACGGTAAG TTACCTTTGTACGCTTTGGAGAAAAAATTAGGTGATACTACGAGAGCGGTTGCGGTACGTAGGAAGGCTCTTTCAAT TTTGGCAGAAGCTCCTGTATTAGCATCTGATCGTTTACCATATAAAAATTATGACTACGACCGCGTATTTGGCGCTT GTTGTGAAAATGTTATAGGTTACATGCCTTTGCCCGTTGGTGTTATAGGCCCCTTGGTTATCGATGGTACATCTTAT CATATACCAATGGCAACTACAGAGGGTTGTTTGGTAGCTTCTGCCATGCGTGGCTGTAAGGCAATCAATGCTGGCGG TGGTGCAACAACTGTTTTAACTAAGGATGGTATGACAAGAGGCCCAGTAGTCCGTTTCCCAACTTTGAAAAGATCTG GTGCCTGTAAGATATGGTTAGACTCAGAAGAGGGACAAAACGCAATTAAAAAAGCTTTTAACTCTACATCAAGATTT GCACGTCTGCAACATATTCAAACTTGTCTAGCAGGAGATTTACTCTTCATGAGATTTAGAACAACTACTGGTGACGC AATGGGTATGAATATGATTTCTAAAGGTGTCGAATACTCATTAAAGCAAATGGTAGAAGAGTATGGCTGGGAAGATA TGGAGGTTGTCTCCGTTTCTGGTAACTACTGTACCGACAAAAAACCAGCTGCCATCAACTGGATCGAAGGTCGTGGT AAGAGTGTCGTCGCAGAAGCTACTATTCCTGGTGATGTTGTCAGAAAAGTGTTAAAAAGTGATGTTTCCGCATTGGT TGAGTTGAACATTGCTAAGAATTTGGTTGGATCTGCAATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTA ATTTAGTGACAGCTGTTTTCTTGGCATTAGGACAAGATCCTGCACAAAATGTTGAAAGTTCCAACTGTATAACATTG ATGAAAGAAGTGGACGGTGATTTGAGAATTTCCGTATCCATGCCATCCATCGAAGTAGGTACCATCGGTGGTGGTAC TGTTCTAGAACCACAAGGTGCCATGTTGGACTTATTAGGTGTAAGAGGCCCGCATGCTACCGCTCCTGGTACCAACG CACGTCAATTAGCAAGAATAGTTGCCTGTGCCGTCTTGGCAGGTGAATTATCCTTATGTGCTGCCCTAGCAGCCGGC CATTTGGTTCAAAGTCATATGACCCACAACAGGAAACCTGCTGAACCAACAAAACCTAACAATTTGGACGCCACTGA TATAAATCGTTTGAAAGATGGGTCCGTCACCTGCATTAAATCCTAA (SEQ ID NO:30)
ATGTCAGAGTTGAGAGCCTTCAGTGCCCCAGGGAAAGCGTTACTAGCTGGTGGATATTTAGTTTTAGATACAAAATA TGAAGCATTTGTAGTCGGATTATCGGCAAGAATGCATGCTGTAGCCCATCCTTACGGTTCATTGCAAGGGTCTGATA AGTTTGAAGTGCGTGTGAAAAGTAAACAATTTAAAGATGGGGAGTGGCTGTACCATATAAGTCCTAAAAGTGGCTTC ATTCCTGTTTCGATAGGCGGATCTAAGAACCCTTTCATTGAAAAAGTTATCGCTAACGTATTTAGCTACTTTAAACC TAACATGGACGACTACTGCAATAGAAACTTGTTCGTTATTGATATTTTCTCTGATGATGCCTACCATTCTCAGGAGG ATAGCGTTACCGAACATCGTGGCAACAGAAGATTGAGTTTTCATTCGCACAGAATTGAAGAAGTTCCCAAAACAGGG CTGGGCTCCTCGGCAGGTTTAGTCACAGTTTTAACTACAGCTTTGGCCTCCTTTTTTGTATCGGACCTGGAAAATAA TGTAGACAAATATAGAGAAGTTATTCATAATTTAGCACAAGTTGCTCATTGTCAAGCTCAGGGTAAAATTGGAAGCG GGTTTGATGTAGCGGCGGCAGCATATGGATCTATCAGATATAGAAGATTCCCACCCGCATTAATCTCTAATTTGCCA GATATTGGAAGTGCTACTTACGGCAGTAAACTGGCGCATTTGGTTGATGAAGAAGACTGGAATATTACGATTAAAAG TAACCATTTACCTTCGGGATTAACTTTATGGATGGGCGATATTAAGAATGGTTCAGAAACAGTAAAACTGGTCCAGA AGGTAAAAAATTGGTATGATTCGCATATGCCAGAAAGCTTGAAAATATATACAGAACTCGATCATGCAAATTCTAGA TTTATGGATGGACTATCTAAACTAGATCGCTTACACGAGACTCATGACGATTACAGCGATCAGATATTTGAGTCTCT TGAGAGGAATGACTGTACCTGTCAAAAGTATCCTGAAATCACAGAAGTTAGAGATGCAGTTGCCACAATTAGACGTT CCTTTAGAAAAATAACTAAAGAATCTGGTGCCGATATCGAACCTCCCGTACAAACTAGCTTATTGGATGATTGCCAG ACCTTAAAAGGAGTTCTTACTTGCTTAATACCTGGTGCTGGTGGTTATGACGCCATTGCAGTGATTACTAAGCAAGA TGTTGATCTTAGGGCTCAAACCGCTAATGACAAAAGATTTTCTAAGGTTCAATGGCTGGATGTAACTCAGGCTGACT GGGGTGTTAGGAAAGAAAAAGATCCGGAAACTTATCTTGATAAATAG (SEQ ID NO:31).
ATGAGTGTGTCTACCGCCAAGAGGTCGCTGGATGTCGTTTCTCCGGGTTCATTAGCGGAGTTTGAGGGTTCAAAATC TCGTCACGATGAAATAGAAAATGAACATAGACGTACTGGTACACGTGATGGCGAGGATAGCGAGCAACCGAAGAAGA AGGGTAGCAAAACTAGCAAAAAGCAAGATTTGGATCCTGAAACTAAGCAGAAGAGGACTGCCCAAAATCGGGCCGCT CAAAGAGCTTTTAGGGAACGTAAGGAGAGGAAGATGAAGGAATTGGAGAAGAAGGTACAAAGTTTAGAGAGTATTCA GCAGCAAAATGAAGTGGAAGCTACTTTTTTGAGGGACCAGTTAATCACTCTGGTGAATGAGTTAAAAAAATATAGAC CAGAGACAAGAAATGACTCAAAAGTGCTGGAATATTTAGCAAGGCGAGATCCTAATTTGCATTTTTCAAAAAATAAC GTTAACCACAGCAATAGCGAGCCAATTGACACACCCAATGATGACATACAAGAAAATGTTAAACAAAAGATGAATTT CACGTTTCAATATCCGCTTGATAACGACAACGACAACGACAACAGTAAAAATGTGGGGAAACAATTACCTTCACCAA ATGATCCAAGTCATTCGGCTCCTATGCCTATAAATCAGACACAAAAGAAATTAAGTGACGCTACAGATTCCTCCAGC GCTACTTTGGATTCCCTTTCAAATAGTAACGATGTTCTTAATAACACACCAAACTCCTCCACTTCGATGGATTGGTT AGATAATGTAATATATACTAACAGGTTTGTGTCAGGTGATGATGGCAGCAATAGTAAAACTAAGAATTTAGACAGTA ATATGTTTTCTAATGACTTTAATTTTGAAAACCAATTTGATGAACAAGTTTCGGAGTTTTGTTCGAAAATGAACCAG GTATGTGGAACAAGGCAATGTCCCATTCCCAAGAAACCCATCTCGGCTCTTGATAAAGAAGTTTTCGCGTCATCTTC TATACTAAGTTCAAATTCTCCTGCTTTAACAAATACTTGGGAATCACATTCTAATATTACAGATAATACTCCTGCTA ATGTCATTGCTACTGATGCTACTAAATATGAAAATTCCTTCTCCGGTTTTGGCCGACTTGGTTTCGATATGAGTGCC AATCATTACGTCGTGAATGATAATAGCACTGGTAGCACTGATAGCACTGGTAGCACTGGCAATAAGAACAAAAAGAA CAATAATAATAGCGATGATGTACTCCCATTCATATCCGAGTCACCGTTTGATATGAACCAAGTTACTAATTTTTTTA GTCCGGGATCTACCGGCATCGGCAATAATGCTGCCTCTAACACCAATCCCAGCCTACTGCAAAGCAGCAAAGAGGAT ATACCTTTTATCAACGCAAATCTGGCTTTCCCAGACGACAATTCAACTAATATTCAATTACAACCTTTCTCTGAATC TCAATCTCAAAATAAGTTTGACTACGACATGTTTTTTAGAGATTCATCGAAGGAAGGTAACAATTTATTTGGAGAGT TTTTAGAGGATGACGATGATGACAAAAAAGCCGCTAATATGTCAGACGATGAGTCAAGTTTAATCAAGAACCAGTTA ATTAACGAAGAACCAGAGCTTCCGAAACAATATCTACAATCGGTACCAGGAAATGAAAGCGAAATCTCACAAAAAAA TGGCAGTAGTTTACAGAATGCTGACAAAATCAATAATGGCAATGATAACGATAATGATAATGATGTCGTTCCATCTA AGGAAGGCTCTTTACTAAGGTGTTCGGAAATTTGGGATAGAATAACAACACATCCGAAATACTCAGATATTGATGTC GATGGTTTATGTTCCGAGCTAATGGCAAAGGCAAAATGTTCAGAAAGAGGGGTTGTCATCAATGCAGAAGACGTTCA ATTAGCTTTGAATAAGCATATGAACTAA (SEQ ID NO:32).
ATGGCCGATGAGGAACGTTTAAAGGAGTTTAAAGGGGCAAACAAGATAGTGTTTGATCCAAATACCAGACAAGTATG GGAAAACCAGAATCGAGATGGAACAAAACCAGCAACTACTTTCCAGAGTGAAGAGGTCATAAAAAGAGCTGCCCCAG AATCTGAAAAAGACACCTCCGCCACATCAAGTATTGTTCCAGAGCTATAGAACATTGTGGCAACTGTGACTTTGGGG AGCAGGTTAGATCTGAAAACAGTTGCGCTACATGCCCGTATGCAGAATATAACCCCTAGCGTTTTGCTGCTGTCATC ATGCGTATAGAGAGCCAAAAACTACAGCTTTAATTTTTGCCTCAGGGAAAATGGTTGTTACCGGTGCAAAAAGTGAG GATGACTCAAAGCTGGCCAGAAGAATATATGCAGGAATTATCCAAAAAATCAGGTTTGCAGCTAAATTCACAGACTT CAAAATACAAAATATTGTCGGTTCGTGTGACGTTAATTTCCCTATACGTCTAGAAGGGTTAGCATTCAGTCATGGTA CTTTCTCCTCCTATGAGCCAGAATTGTTTCCTGGTTTGATCTATAGAATGGTGAAGCCGAAAATTGTGTTGTTAATT TTTGTTTCAGGAAAGATTGTTCTTCTGGAGCAAAGCAAAGGGAGGAAATTTACCAAGCTTTTGAAGCTATATACCCA GTTCTAAGTGAATTTAGAAAAATGTGA (SEQ ID NO:51)
ATGGATTTTGAGGAAGATTACGATGCGGAGTTTGATGATAATCAAGAAGGACAATTAGAAACACCTTTTCCATCGGT TGCGGGAGCCGATGATGGGGACAATGATAATGATGACTCTGTCGCAGAAAACATGAAGAAGAAGCAAAAGAGAGAGG CTGTAGTGGATGATGGGAGTGAAAATGCATTTGGTATACCCGAATTTACAAGAAAAGATAAGACTCTGGAGGAGATT TTAGAGATGATGGACAGTACTCCTCCTATCATTCCCGATGCAGTAATAGACTACTATTTAACCAAAAACGGGTTTAA CGTAGCATATGTACAAGTGAAACGACTTTTAGCACTTGCTACTCAGAAATTTGTTAGTGATATAGCTAAGGATGCCT ACGAATATTCCAGGATCAGGTCTTCCGTAGCGGTATCTAATGCTAACAACAGTCAGGCGAGAGCTAGGCAGCTATTG CAAGGACAGCGACAGACTGGCGTGCAGCAGATTTCACAACAACAACATCAACAGAATGAGAAGACTACAGCAAGCAT AGTTGTTCTGACGGTGAACGATCCCAGTAGCGCTGTTGCTGAATACGGGCTCAATATAGGTCGCCCAGACTTTTATC GTTAG (SEQ ID NO:52)
The building of the engineered strain of new generation of embodiment 8
According to Examples 1 to 7, effective transformation is overlapped combination, such as by effective knockout gene The combination of two such as Ypl062W, Yer130C, Yer134c, Exg1 are knocked out, and dual-gene knockout can obtain yield more as the result is shown High bacterial strain.
On this basis, effective transformation is overlapped combination by us, forms the engineered strain of a new generation, and bacterial strain is special Property such as following table 4.
The feature of the engineered strain of new generation of table 4.
Note: " √ " represents the gene and is knocked, and "+" represents the gene overexpression one copy, and " * " represents the gene and cross table It is copied up at least two, "-" represents the gene and do not operated and (be not knocked or be not overexpressed)
Further, inventor is had shown that by research, and engineered strain J1011-C-37~84 of new generation have high yield tomato The performance of red pigment.
The building of the new two generations engineered strain of embodiment 9
On the basis of previous generation engineered strain, we pick 18 dominant strains, construct new two on this basis The engineered strain in generation probes into the influence of gapN and EUTE gene pairs yield of lycopene, strain characteristic such as following table 5.
The feature of the new two generations engineered strain of table 5.
Note: " √ " represents the gene and is over-expressed, and "-" represents the gene and is not over-expressed
Further, inventor is had shown that by research, and engineered strain J1011-C-85~138 of new generation have high yield tomato The performance of red pigment.
10 engineering bacteria shaking flask cultivation and fermentation process of embodiment
In the present embodiment, inventor describes the fermented and cultured mistake of Examples 1 to 9 engineered strain obtained in detail Journey.
Shake flask fermentation uses two-stage seed culture, and the recombinant bacterial strain on plate is chosen the PA containing 5mL YPD culture medium In bottle, 30 degree of shaking tables have shaken primary seed solution, and after being incubated overnight (general 14-18h), thallus grows to logarithmic growth phase, and (OD is in 5-8 Left and right), then bacterial strain is transferred in the 250mL shaking flask containing 50mL YPD culture medium with 1% inoculum concentration, shaking flask culture obtains Secondary seed solution.After about 14-18h, the OD of secondary seed is surveyed600Then value is calculated and is inoculated into containing 200mL fermentation medium In the 500mL shaking flask of YPDG, makes thallus final concentration OD600=0.5, need how many secondary seed solutions, then take out calculating Volume is simultaneously centrifuged, and removes supernatant, then thallus is suspended with corresponding fermentation medium and is added in corresponding 500mL shaking flask, is set Start shake flask fermentation in 30 degree of shaking tables.Cell concentration is measured by sampling in (about 4h) at regular intervals, and will be in shaking flask after 8h Newspaper removes, and starts sampling after about 48h and is stored in the production that -80 degree refrigerators remain measurement lycopene.
11 product of embodiment extraction mode compares
In the present embodiment, the mode that inventor extracts product obtained after fermentation process is screened, Screening process is as described below.
Method one: taking out sample from refrigerator and thaw, and takes 500 μ L fermentation liquids in 15mL centrifuge tube (being pre-chilled on ice), 5000g 4 degree of centrifugation 2min collect thallus, go after supernatant that washing thalline is resuspended with 1mL pure water again, add the HCl of 1mL 3N And boiling water bath 3min carrys out smudge cells, centrifugation is washed with water once after removing HCl.It is added (HPLC grades of acetone of 1mL thereto again Not), 0.2g bead, 1% antioxidant shake 5min, then 4 degree of centrifugation 2min of 5000g, shift supernatant to 50mL's In centrifuge tube；It repeats that said extracted liquid is added again, and shakes, then collect extract liquor, until thallus is without obvious yellow；It mixes The acetone extract liquid being collected into takes out 2mL, and 12000rpm is centrifuged 10 minutes, then takes supernatant 1.2mL to the HPLC sample injection bottle of brown In, carry out HPLC analysis.
Method two: taking out sample from refrigerator and thaw, and takes 500 μ L fermentation liquids in 15mL centrifuge tube (being pre-chilled on ice), 5000g 4 degree of centrifugation 2min collect thallus, go after supernatant to be resuspended with 1mL pure water again and washing thalline.4mL is added thereto again Acetone (HPLC rank), 0.2g bead, 1% antioxidant shakes 5min, then ice-bath ultrasonic 5-10min, then 4 degree of centrifugation 2min of 5000g shift supernatant into the centrifuge tube of 50mL；It repeats that said extracted liquid is added again, and shakes, then Extract liquor is collected, until thallus is without obvious yellow；The acetone extract liquid being collected into is mixed, 2mL is taken out, 12000rpm is centrifuged 10 points Clock, then take supernatant 1.2mL into the HPLC sample injection bottle of brown, carry out HPLC analysis.
Detection method: what the detection of lycopene was carried out using quaternary HPLC, detector is UV detector, lycopene Absorbing wavelength be 474nm, chromatographic column Agilent Zorbax C18 (150mm*4.6mm*5 μm), mobile phase A (acetonitrile: water= 9:1) and Mobile phase B (methanol: isopropanol=3:2) presses following condition analysis: 0-90%B (0-15min), 90%B (15- 30min), 90%-0B (30-35min), flow velocity 1mL/min.
As the result is shown: after product is boiled through hydrochloric acid in method one, lycopene can degrade, and yield detection is unstable, and should The sample certain interval of time that method obtains detects again, or even 10mg/L, the sample that method two obtains can be degraded to from 100mg/L More stable, certain interval of time detects again, only from 100mg/L to 90mg/L.The extraction scheme ratio method one of illustration method two It is more stable more preferable.
12 engineering bacterium fermentation tank cultivation and fermentation method of embodiment compares
In the present embodiment, screening is optimized to the condition of fermentation medium in inventor, and experimentation is as described below:
Strain in the present embodiment uses J1011-C-3, and seed culture medium uses YPD culture medium, and fermentation medium uses 4 kinds of culture mediums that shaking flask optimized further explore the best medium of fermentation tank level, and various culture medium prescriptions are as follows:
1. culture medium No. 2: YPD contains salt culture medium
2.5L batch culture base: 2% peptone, 1% yeast extract, 0.8%KH2PO4With 2% glucose；
1L glucose supplemented medium: 500g/L glucose, 5g/L MgSO4, 3.5g/L K2SO4, 0.28g/L Na2SO4, 10g/L yeast extract.
1L ethyl alcohol supplemented medium: 100% ethyl alcohol.
2. No. 5: YPD rich medium of culture medium
2.5L batch culture base: 60g peptone, 30g yeast extract and 30g glucose (more abundanter than YPD)；
1L supplemented medium 1:500g/L glucose, 15g/L yeast extract
500mL supplemented medium 2:300mL dehydrated alcohol, 100ml glycerol, 100mL pure water.
Upper tank culture uses fed-batch mode, is batch culture first, starts after carbon source glucose glycosyl originally runs out of Feed supplement, feed supplement use two-stage feed profile, respectively glucose feed phase (being used for synthesising biological amount) and ethyl alcohol glycerol feed supplement Stage (is used for Product formation).The specific method is as follows.
115 degree of progress skies go out 30 minutes after fermentor is packed into a small amount of pure water.Prepare the fermentation liquid and benefit of batch culture Feed liquid calibrates the pH electrode of fermentor, clamps each pipeline of fermentor, and 115 degree sterilize 30 minutes.Take out fermentor, connection Air pipe line, ventilated (0.1vvm or so) on a small quantity, is opened simultaneously chilled water unit and is cooled down.50 are down to temperature When spending left and right, revolving speed is opened to 100rpm.Calibrate dissolved oxygen electrode.It is 0% that the state of not connected route, which set oxygen dissolving value, subsequent general Revolving speed rises to 600rpm, while ventilatory capacity is adjusted to 2vvm, connects dissolved oxygen electrode route, set after dissolved oxygen stable the value as 100%.Ventilatory capacity and revolving speed are then reduced to fermentation desirable value (ventilatory capacity 1.5vvm, revolving speed 300rpm-600rpm).First certainly Right pH, the temperature was then adjusted to 30 degree.Start to be inoculated with, initial inoculation OD is adjusted to 0.5, and required seed liquor volume is counted according to formula It calculates.(such as fermentating liquid volume is 2500mL, and seed liquor OD value is n, then being inoculated with seed liquor volume is 2500x0.5/n mL).Start It is 5.5 that culture medium, which controls pH value with 2M NaOH, after fermentation, when upper tank, and initial ventilatory capacity is 1.5vvm, and initial stirring rates are set It is set to 300rpm, dissolved oxygen maintains 30% or more (association stirring rate, 300-600rpm).
Fermenting procedure: start feed supplement glucose, initial feed supplement when concentration of glucose is down to 2g/L or so when batch culture Rate is 10mL/L fermentation liquid/h, to maintain the residual concentration of glucose in fermentation liquid (not be down to 0g/ in 2-3g/L or so L), glucose content of OD600 value of every two hour sampling and measuring and detection, increases when concentration of glucose is lower than 1g/L Add feed rate.When the increase of OD value is slow, (initially entering stationary phase) stops feed supplement glucose, and it is residual to start monitoring ethyl alcohol at this time Allowance, starts feed supplement ethyl alcohol glycerol when concentration of alcohol is down to 5-10g/L, and initial feed rate is 2mL/L fermentation liquid/h.Then Ethyl alcohol of every 4h sample detection and glycerol content, when adjustment feed rate maintains ethyl alcohol and glycerol within the scope of 5-10g/L. Color starts to extract the variation of product detection product after changing, and terminates to ferment when lycopene concentration is not further added by.
Product detection mode is identical as method two in embodiment 9.
As the result is shown: No. 5 yield of culture medium are better than culture medium 2, so the culture medium is best medium, fermentation strategies It is two step fed-batch fermentations of optimization, first mends the long thallus of glucose, mends ethyl alcohol in the stationary phase of growth to improve yield.
The upper tank fermented and cultured of 13 bacterial strain of embodiment
In the present embodiment, inventor is according to best fermented and cultured mode obtained in embodiment 12 to the part work of building Journey bacterial strain is fermented, and the results are shown in Table 6.
Table 6:
As can be seen that the engineered strain constructed according to embodiments of the present invention has the performance of high yield lycopene
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned Embodiment is changed, modifies, replacement and variant.

Claims (9)

1. a kind of microorganism, which is characterized in that be overexpressed PaCrtE, PagCrtB and BtCrtI gene.
2. microorganism according to claim 1, which is characterized in that being further overexpressed includes being selected from tHMG1, INO2, Yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, EUTE, At least one of ERG12, IDI1, ERG10, MVD1, ERG13, ERG8 gene.
3. microorganism according to claim 1, which is characterized in that further silencing includes being selected from GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, At least one of Sfk1, Mef1 gene.
4. microorganism according to claim 1, which is characterized in that the microorganism is saccharomycete.
5. microorganism according to claim 1, which is characterized in that further comprise that can operate regulation ERG9 gene.
6. a kind of method for obtaining lycopene characterized by comprising
The described in any item microorganisms of Claims 1 to 5 are subjected to fermentation process；And
Fermentation process product is subjected to extraction processing, to obtain the lycopene.
7. according to the method described in claim 6, it is characterized in that, the fermentation process is realized in the following way:
The microorganism is subjected to basal fermentation processing and the processing of two-stage fed batch fermentation, the basal fermentation processing is in base It is carried out in this fermentation medium, the two-stage fed batch fermentation processing is by the basis of the basic fermentation medium Successively add what the first supplemented medium and the second supplemented medium were realized,
Wherein, the basic fermentation medium be containing 24g/L peptone, 12g/L yeast extract and 12g/L glucose YPD culture medium；
First supplemented medium is the YPD culture medium containing 500g/L glucose and 15g/L yeast extract；
Second supplemented medium is 75% ethyl alcohol and/or 50% glycerol.
8. according to the method described in claim 6, it is characterized in that, the extraction processing includes:
The fermentation process product is subjected to broken homogenate or enzymatic hydrolysis break process and organic extraction processing.
9. any one of Claims 1 to 5 microorganism is preparing the purposes in lycopene.
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