CN110499259A - A kind of solution ester Ye Shi yeast YW100-1 and its application - Google Patents
A kind of solution ester Ye Shi yeast YW100-1 and its application Download PDFInfo
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- CN110499259A CN110499259A CN201910661781.1A CN201910661781A CN110499259A CN 110499259 A CN110499259 A CN 110499259A CN 201910661781 A CN201910661781 A CN 201910661781A CN 110499259 A CN110499259 A CN 110499259A
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- shi yeast
- glycerol
- solution ester
- ester
- gene
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- 150000002148 esters Chemical class 0.000 title claims abstract description 93
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- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 82
- 229940107700 pyruvic acid Drugs 0.000 claims abstract description 41
- 101001009678 Homo sapiens Glycerol-3-phosphate dehydrogenase, mitochondrial Proteins 0.000 claims abstract description 23
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Classifications
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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Abstract
The invention discloses a kind of solution ester Ye Shi yeast YW100-1 and its applications; the solution ester Ye Shi yeast YW100-1 is to be overexpressed glycerokinase GUT1 in glycerol metabolism in solution ester Ye Shi yeast; glycerokinase GUT2; glycerol dehydrogenase enzyme GCY1; dihydroxyacetone kinase DAK1; dihydroxyacetone kinase DAK2 and NADH kinases POS5, while knocking out what diacylglycerol acyltransferase gene DGA2 and glycerol-3-phosphate gene GPD2 in solution ester Ye Shi yeast was obtained.When bacterial strain of the present invention is using glycerol as carbon source, output of pyruvic acid increases 66.2%, reaches 121.2g/L, provides strain excellent for pyruvic acid industrialized production.
Description
(1) technical field
The present invention relates to a kind of construction method for solving ester Ye Shi Yeast engineering bacteria and applications, belong to technical field of bioengineering.
(2) background technique
Pyruvic acid is also important one of organic acid as the intermediate in microbial metabolism approach, in biochemical industry, system
There is extensive effect in medicine, food and scientific domain.In medical industry, pyruvic acid is a kind of important medicine intermediate,
It can be used for synthesizing levodopa, anti-inflammatory analgesic quinophan, anti-tubercular drug isoniazid calcium pyruvinate, Anthelvet drug etc., in addition,
Pyruvates (such as CALCIUM PYRUVIC, Potassium pyruvate, acetone creatine acid salt etc.) are widely used in slimming health product medicine class.In food
Industry, pyruvic acid have effects that natural anti-corrosion and fresh-keeping as food additives.In chemical industry, ethyl pyruvate conduct
Skin-whitening agents can inhibit the formation of the melanin in skin, have good skin whitening efficacy.In addition, pyruvic acid is made
For the precursor of the bio-fuel of a new generation, domestic and international market demand is increased rapidly, has wide application value.
The method of Pyruvate production mainly has chemical synthesis, enzyme transforming process, microbe fermentation method three categories at present.Chemistry
Synthetic method mainly using tartaric acid as raw material, chemical synthesis pyruvic acid, but this method pollution weight, it is at high cost, industrialized production by
Limitation is arrived.Enzyme transforming process is mainly the dehydrogenase system utilized in microorganism, converts pyruvic acid for lactic acid, but this method turns
Rate is low, at high cost, and there is presently no realize industrialized production.Microbe fermentation method mainly utilizes microorganism, with glucose
Or glycerol etc. is inexpensively carbon source, passes through microbial fermentation, directly production pyruvic acid.The bacterial strain that microbe fermentation method uses at present
Have saccharomyces cerevisiae (Saccharomyces cerevisiae), Escherichia coli (Escherichia coli), smooth ball intends ferment
(Torulopsis glabrata), solution ester Ye Shi yeast (Yarrowia lipolytica) etc..Turn with chemical synthesis and enzyme
The advantages that change method is compared, and is had and is polluted small, high conversion rate, at low cost, and industrialized main method at present.
Although solving ester Ye Shi yeast has preferable Pyruvate production ability, turning for glycerol synthesis pyruvic acid is utilized
Rate and synthesis rate are still to be improved, and degradation of pyruvate speed is fast to be led to not largely accumulate.It solves in ester Ye Shi yeast
There are two glycerol catabolic pathways, one is glycerol-3-phosphate approach, in cytoplasm glycerol glycerokinase (by
GUT1 coding) under the action of phosphoric acid turn to glycerol-3-phosphate, then shuttle enter mitochondria, in glycerol-3-phosphate dehydrogenase
It is phosphoric acid dihydroxyacetone (DHA) that glycerol-3-phosphate is aoxidized under the action of (being encoded by GUT2), and subsequent phosphoric acid dihydroxyacetone (DHA) is transported again
It returns cytoplasm and enters glycolysis metabolism approach.Another is dihydroxyacetone (DHA) (DHA) approach, the glycerol dehydrogenase in this approach
Glycerol is oxidized to DHA by (being encoded by GCY1), then phosphorus under the action of dihydroxyacetone kinase (being encoded by DAK1 and DAK2)
Acidification is that phosphoric acid dihydroxyacetone (DHA) enters glycolysis metabolism approach.Ester Ye Shi yeast is solved as a kind of high Lipid-producing Yeast, two acyls
Base glycerol acyltransferase (being encoded by DGA2) can use the important component trigalloyl of phosphoric acid dihydroxyacetone (DHA) Synthetic Oil
Glycerol.The synthesis of glycerol first through dihydroxyacetone phosphate, is converted into glycerol 3-phosphate, after through glycerol-3-phosphate (by
GPD1, GPD2 coding), synthetic glycerine.Some researches show that, GPD2 knock out meeting so that NADH intracellular accumulation, to break
The growth of NADH/NADPH balance influence thallus, and Pos5 can use ATP and turn to NADH phosphoric acid as a kind of NADH kinases
NADPH。
In consideration of it, the present invention will strengthen point of glycerol by enhancing glycerol-3-phosphate approach and dihydroxyacetone (DHA) approach
Solution metabolic pathway accelerates the decomposition of glycerol, while weakening the synthesis way of glycerol, to realize pyruvic acid in the cell a large amount of
Accumulation.
(3) summary of the invention
It is an object of the present invention to provide a kind of solution ester Ye Shi Yeast engineering bacterias of high yield pyruvic acid, and its building and application, are
The industrialized production of pyruvic acid provides excellent species.
The technical solution adopted by the present invention is that:
The present invention provides a kind of solution ester Ye Shi yeast (Yarrowia lipolytica) YW100-1 of high yield pyruvic acid, institute
Stating solution ester Ye Shi yeast YW100-1 is to be overexpressed glycerokinase GUT1 in glycerol metabolism, glycerokinase in solution ester Ye Shi yeast
GUT2, glycerol dehydrogenase enzyme GCY1, dihydroxyacetone kinase DAK1 and dihydroxyacetone kinase DAK2, while knocking out solution ester Ye Shi
Diacylglycerol acyltransferase gene DGA2 enhances the catabolism of glycerol in yeast, knocks out glycerol-3-phosphate base
The anabolism of glycerol is reduced because of GPD2 and overexpression NADH kinases POS5, a plant height of acquisition produces the solution ester of pyruvic acid
Family name's Yeast engineering bacteria.
Further, the solution ester Ye Shi yeast YW100-1 is constructed as follows:
(1) by from GUT1 gene, GUT2 gene and the promoter pEXP1 segment of solution ester Ye Shi yeast, pass through weight
Folded extend obtains GUT1-pEXP1-GUT2, is connected on carrier JMP113, obtains carrier E14；
(2) it by the carrier E14 of building Not I digestion, is transferred in wild type solution ester Ye Shi yeast AS2.1405, obtains
Solution ester Ye Shi Yeast engineering bacteria ZS102 after conversion；
(3) will from solution ester Ye Shi yeast GCY1, DAK1, DAK2 gene, promoter pTEF, pEXP1, pGPD, with
And 5 ' the ends and 3 ' ends of the homology arm KU70 for integration, obtain 5 ' KU70-URA3-pTEF-DAK1 respectively by overlap-extension PCR,
Tri- large fragments of DAK1-pEXP1-DAK2-pGPD, pGPD-GYC1-3 ' KU70, are transferred in ZS102, the solution after being converted
Ester Ye Shi Yeast engineering bacteria ZS104；
(4) by 5 ' the end GPD2 from the POS5 gene and pTEF of solution ester Ye Shi yeast, and for knocking out GPD2
With 3 ' end GPD2, bis- segments of 5 ' GPD2-URA3-pTEF-POS5 and POS5-3 ' GPD2 are obtained by overlap-extension PCR, are transferred to
Solution ester Ye Shi Yeast engineering bacteria ZS106 in ZS104, after obtaining homologous recombination；
(5) by the 5 ' ends for being used to knock out and 3 ' end DGA2,5 ' DGA2-URA3-DGA2 segments is obtained by overlap-extension PCR, are turned
Enter into ZS106, obtains solution ester Ye Shi yeast YW100-1.
Further, glycerokinase GUT1 gene nucleotide series are glycerokinase GUT2 gene core shown in SEQ ID NO.1
Nucleotide sequence be SEQ ID NO.2 shown in, dihydroxyacetone kinase DAK1 gene nucleotide series be SEQ ID NO.3 shown in,
Dihydroxyacetone kinase DAK2 gene nucleotide series are glycerol dehydrogenase GCY1 gene nucleotide sequence shown in SEQ ID NO.4
It is classified as shown in SEQ ID NO.5, NADH kinases POS5 gene nucleotide series are diacylglycerol acyl shown in SEQ ID NO.6
5 ' DGA2 gene nucleotide series of based transferase are 3 ' DGA2 gene of diacylglycerol acyltransferase shown in SEQ ID NO.7
Nucleotides sequence is classified as shown in SEQ ID NO.8, and promoter pEXP1 nucleotides sequence is classified as shown in SEQ ID NO.9,5 ' KU70 nucleosides
Acid sequence is shown in SEQ ID NO.10, and Loxp-URA3-Loxp nucleotides sequence is classified as shown in SEQ ID NO.11, promoter
PTEF nucleotides sequence is classified as shown in SEQ ID NO.12, and promoter pGPD nucleotides sequence is classified as shown in SEQ ID NO.13, and 3 '
KU70 nucleotides sequence is classified as shown in SEQ ID NO.14, and 5 ' GPD2 gene nucleotide series of glycerol-3-phosphate are SEQ ID
Shown in NO.15,3 ' GPD2 gene nucleotide series are shown in SEQ ID NO.16.
Solution ester Ye Shi yeast (Yarrowia lipolytica) YW100-1 of the present invention, is preserved in Chinese Typical Representative culture
Object collection, the deposit date is on March 18th, 2019, deposit number was CCTCC NO:M 2019168, address are as follows: Wuhan, China
Wuhan University, postcode 430072.
The present invention also provides a kind of solution ester Ye Shi yeast YW100-1 to prepare the application in pyruvic acid in fermentation glycerol,
The application is one of following method: (1) shake flask fermentation: solution ester Ye Shi yeast YW100-1 is inoculated in YPD culture medium, in 30
DEG C, 200rpm is cultivated for 24 hours, obtains seed liquor；With initial cell concentration OD600=0.05 is inoculated in YNG culture medium, in 30 DEG C,
200rpm fermented and cultured is to OD600When for 4.0-5.0 (preferably 4.5), the fermentation liquid containing pyruvic acid is obtained；YPD Liquid Culture matrix
Amount composition: 1% yeast extract, 2% glucose, 2% peptone, solvent are distilled water, and pH value is natural；YNG culture medium quality
Composition: 0.67%YNB (containing ammonium sulfate, without the yeast basic nitrogen source of amino acid), 50g/L glycerol, solvent is distilled water, is used
Na2HPO4Citrate buffer solution adjusts pH to 4.0；
(2) ferment tank: solution ester Ye Shi yeast YW100-1 is seeded in the fermentor containing fermentation medium, in 30-
32 DEG C, pH value 4-4.5, ferment under the conditions of dissolved oxygen amount 40-50%, obtain the fermentation liquid containing pyruvic acid, isolate and purify, obtain
Pyruvic acid；The fermentation medium composition are as follows: 60g/L glycerol, 10g/L (NH4)2SO4、1.4g/L MgSO4·7H2O、2g/L
KH2PO4、0.8g/L CaCl2, 0.5g/L NaCl, 1 μ g/L vitamin B1, solvent is distilled water, and pH value is natural.
Further, when the ferment tank, glycerol is added using batch mode, and 60g/L is added for the first time, works as glycerol depletion
After add glycerol, add 40g/L every time, preferably add 2 times.
Further, before the ferment tank, solution ester Ye Shi yeast YW100-1 first carries out seed and expands culture, then will kind
Sub- liquid is seeded to fermentation medium, the seed culture with the inoculum concentration of volumetric concentration 10% are as follows: solution ester Ye Shi yeast YW100-1
It is seeded to seed culture medium, 30 DEG C of shaking flask culture 18h obtain seed liquor；The seed culture medium: 2g/L glycerol, 0.4g/L pancreas
Peptone, yeast extract 0.2g/L, 0.24g/L KH2PO4、1.7g/L K2HPO4·3H2O, solvent are distilled water, and pH value is certainly
So.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
Present invention solution ester Ye Shi yeast YW100-1 obtained (deposit number is CCTCC NO:M 2019168) and bacterium germination out
Strain solution ester Ye Shi yeast AS2.1405 is compared, and when using glycerol as carbon source, output of pyruvic acid increases 66.2%, reaches
121.2g/L provides strain excellent for pyruvic acid industrialized production.
(4) Detailed description of the invention
Fig. 1 is the building route map for solving ester Ye Shi yeast gene engineering bacteria.
Fig. 2 output of pyruvic acid of bacterium germination AS2.1405 and glycerol residual quantity ratio for shake flask fermentation recombinant bacterium YW100-1 and out
Compared with.
Fig. 3 is 20L ferment tank culture recombinant bacterium YW100-1 and the output of pyruvic acid of bacterium germination AS2.1405 compares out.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Method in the following example is unless otherwise specified known method.
YPD fluid nutrient medium quality composition: 1% yeast extract, 2% glucose, 2% peptone, solvent are distilled water,
PH value is natural.
YNG culture medium quality composition: 0.67%YNB (containing ammonium sulfate, without the yeast basic nitrogen source of amino acid), 50g/L
Glycerol, solvent are distilled water, use Na2HPO4Citrate buffer solution adjusts pH to 4.0.
YPD solid medium is the addition 2g/L agar in YPD fluid nutrient medium.
LB culture medium quality composition: 1% peptone, 0.5% yeast extract, 0.5% sodium chloride, solvent are distilled water,
PH value is natural.
Embodiment 1: the building of the solution ester Ye Shi yeast gene engineering bacteria of high yield pyruvic acid
Process is as shown in Figure 1, the construction step of solution ester Ye Shi yeast YW100-1 is as follows:
1, the building of E14 plasmid
The amplification of GUT1: it by design primer YW238 and YW239, (is purchased from wild type solution ester Ye Shi yeast AS2.1405
Guangdong Culture Collection, number are GIM 2.187) genome be template, amplify GUT1, size is about 1.5kb,
Nucleotides sequence is classified as shown in SEQ ID NO.1.
The amplification of promoter pEXP1: by design primer YW405 and YW240, with wild type solution ester Ye Shi yeast
AS2.1405 genome is template, amplifies pEXP1, size is about 1kb, and nucleotides sequence is classified as shown in SEQ ID NO.9.
The amplification of GUT2: by design primer YW241 and YW242, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies GUT2, size is about 2kb, and nucleotides sequence is classified as shown in SEQ ID NO.2.
The above GUT1, pEXP1, the PCR amplification condition of GUT2 be 98 DEG C initial denaturation 3 minutes；98 DEG C of denaturation are moved back for 10 seconds, 58 DEG C
Extend within fire 10 seconds, 72 DEG C with 2 minutes (30 circulations), 72 DEG C extend 10 minutes again.
Overlap-extension PCR constructs GUT1-pEXP1-GUT2 segment: overlap-extension PCR is using 2 wheel PCR amplifications.The first round: amplification body
System is 25 μ l (2 × PrimeSTAR Max archaeal dna polymerase (be purchased from Beijing Bao doctor object Co., Ltd) 12.5 μ l, according to 1:
3:1 molar ratio mixing GUT1, pEXP1 and GUT2 segment, add distilled water to 25 μ l), 95 DEG C initial denaturation 3 minutes, 98 DEG C change
Property 10 seconds, 58 DEG C are annealed 15 seconds, and 72 DEG C extend 2 minutes (15 circulation), and 72 DEG C extend 10 minutes again.Second wheel: amplification body
System is 50 μ l (2 × PrimeSTAR Max archaeal dna polymerase, 25 μ l, the PCR product that the 2 μ l first steps expand, 1 μ l YW238+
1 μ l YW243 and add distilled water to 50 μ l), 95 DEG C initial denaturation 3 minutes, 98 DEG C be denaturalized 10 seconds, 56 DEG C anneal 15 seconds, 72 DEG C
Extend 2 minutes (30 circulations), 72 DEG C extend 10 minutes again.
The GUT1-pEXP1-GUT2 segment of above-mentioned acquisition (is had using plastic recovery kit purchased from Beijing Bao doctor's object
Limit company) after recycling, using one-step cloning kit (be purchased from Nanjing Vazyme Biotechnology Co., Ltd.), it is cloned into JMP113
Carrier (Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM.New disruption
cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia
Lipolytica.J Microbiol Methods.2003.55 (3): 727-37.) on.Clone's system: 25 × CE of μ l
MultiS buffer, 3 μ l GUT1-pEXP1-GUT2 segments, JMP113,1 μ l Exnase TM after 2 μ l BamH I linearisation
MultiS enzyme, 2 μ l distilled water.37 DEG C of reaction half an hour are placed in and 10 μ l are taken to be added to 100 μ l bacillus coli DH 5s after five minutes on ice
Ice bath 30 minutes again in α competent cell.42 DEG C heat shock 30 seconds, be immediately placed on 2 minutes on ice.1mL LB culture medium is added,
200rpm, 37 DEG C are incubated for 1 hour.Bacterium solution is collected by centrifugation to be coated on the LB plate containing 50 μ g/mL kanamycins, 37 DEG C of trainings overnight
It supports and is identified after growing transformant with primer YW244+YW245, PCR screens 4 positive single colonies, is inoculated in LB culture medium
In cultivated, extract positive colony plasmid and carry out sequence verification, sequencing result shows that carrier E14 is constructed successfully.
1 list of primers of table
2, the building of recombination solution ester Ye Shi Yeast engineering bacteria ZS102
With Not I digested plasmid E14, gel extraction pTEF-GUT1-pEXP1-GUT2 segment, electricity consumption shifting method is transferred to open country
In raw type solution ester Ye Shi yeast AS2.1405 competent cell, recombination solution ester Ye Shi Yeast engineering bacteria ZS102, specific steps are obtained
Are as follows:
Be inoculated with wild type solution ester Ye Shi yeast AS2.1405 in YPD fluid nutrient medium, 30 DEG C are incubated overnight, switching in
Make initial OD in the fresh YPD fluid nutrient medium of 50mL600It is 0.1,30 DEG C of cultures 4-6 hours to OD600Reach 1.0.Centrifugation is gone
Fall supernatant, thallus is outstanding with buffer (0.6M sorbierite, 10mM Tris-HCl, 25mM DTT, 150mM LiAc, pH 7.5)
Static 1 hour after floating.It is centrifuged again, three times with 1M sorbierite.After removing supernatant, with 150mL 1M sorbierite suspension cell, i.e.,
For wild type solution ester Ye Shi yeast AS2.1405 competent cell.Take 80 μ l competent cells and 200ng pTEF-GUT1-
The mixing of pEXP1-GUT2 segment carries out electric turn under the conditions of 1.5kV (E=12.4kV/cm), 200 Ω, 25 μ F, and electricity is rapid after turning
The sorbitol aqueous solution of 1mL 1M is added, plate is applied and is inverted culture 2-3 days, transformant primer in YND solid medium
YW253+YW254 carries out PCR identification.
3, the building of ester Ye Shi Yeast engineering bacteria ZS104 is solved
The amplification of 5 ' KU70: by design primer YW600 and YW601, with wild type solution ester Ye Shi yeast AS2.1405 base
Because group is template, the 5 ' upstreams of KU70 are amplified, size is about 1.2kb, and nucleotides sequence is classified as shown in SEQ ID NO.10.
The amplification of Loxp-URA3-Loxp: by design primer YW602 and YW603, using plasmid JMP113 as template, amplification
Loxp-URA3-Loxp out, size are about 1.8kb, and nucleotides sequence is classified as shown in SEQ ID NO.11.
The amplification of pTEF: by design primer YW604 and YW605, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter pTEF, size is about 0.4kb, and nucleotides sequence is classified as shown in SEQ ID NO.12.
The amplification of DAK1: by design primer YW606 and YW607, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter DAK1, size is about 2.2kb, and nucleotides sequence is classified as shown in SEQ ID NO.3.
The amplification of pEXP1: by design primer YW608 and YW609, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter pEXP1, size is about 1kb, and nucleotides sequence is classified as shown in SEQ ID NO.9.
The amplification of DAK2: by design primer YW610 and YW611, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter DAK2, size is about 2.2kb, and nucleotides sequence is classified as shown in SEQ ID NO.4.
The amplification of pGPD: by design primer YW612 and YW613, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter pGPD, size is about 0.94kb, and nucleotides sequence is classified as shown in SEQ ID NO.13.
The amplification of GYC1: by design primer YW614 and YW615, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter GYC1, size is about 1.5kb, and nucleotides sequence is classified as shown in SEQ ID NO.5.
The amplification of 3 ' KU70: by design primer YW616 and YW617, with wild type solution ester Ye Shi yeast AS2.1405 base
Because group is template, the 3 ' downstreams of KU70 are amplified, size is about 1kb, and nucleotides sequence is classified as shown in SEQ ID NO.14.
The PCR of above 5 ' KU70, Loxp-URA3-Loxp, pTEF, DAK1, pEXP1, DAK2, pGPD, GYC1,3 ' KU70
Amplification condition is the same as shown in step 1.
The DNA fragmentation of above-mentioned acquisition is assembled by three 5 ' KU70-URA3-pTEF-DAK1 of large fragment using overlap-extension PCR,
DAK1-pEXP1-DAK2-pGPD, pGPD-GYC1-3 ' KU70.
Overlap-extension PCR constructs 5 ' KU70-URA3-pTEF-DAK1 segments.The first round: according to 1:3:3:1 molar ratio mixing 5 '
KU70, URA3, pTEF, DAK1 segment, amplification condition are same as above.Second wheel: the PCR product expanded using the first round as template,
It adds 1 μ l YW600+1 μ l YW607 and carries out PCR amplification.
Overlap-extension PCR constructs DAK1-pEXP1-DAK2-pGPD segment.The first round: it is mixed according to 1:3:3:1 molar ratio
DAK1, pEXP1, DAK2, pGPD segment, amplification condition are same as above.Second wheel: the PCR product expanded using the first round as template,
It adds 1 μ l YW618+1 μ l YW613 and carries out PCR amplification.
Overlap-extension PCR constructs pGPD-GYC1-3 ' KU70 segment.The first round: mixing pGPD, GYC1 according to 1:3:1 molar ratio,
3 ' KU70 segments, amplification condition are same as above.Second wheel: the PCR product expanded using the first round adds 1 μ l YW612+ as template
1 μ l YW617 carries out PCR amplification.
5 ' the KU70-URA3-pTEF-DAK1 that above-mentioned overlap-extension PCR is obtained, DAK1-pEXP1-DAK2-pGPD, pGPD-
After three large fragment glue recycling of GYC1-3 ' KU70, to be transferred to solution ester Ye Shi yeast recombination work after each segment 50ng mixing
In journey bacterium ZS102, transformant uses primer YW619+YW620 respectively, and YW621+YW622, YW226+YW227 carry out PCR identification.
2 list of primers of table
4, the building of ester Ye Shi Yeast engineering bacteria ZS106 is solved
The amplification of 5 ' GPD2: by design primer YW626 and YW627, with wild type solution ester Ye Shi yeast AS2.1405 base
Because group is template, 5 ' GPD2 are amplified, size is about 1kb, and nucleotides sequence is classified as shown in SEQ ID NO.15.
The amplification of Loxp-URA3-Loxp: by design primer YW628 and YW603, using plasmid JMP113 as template, amplification
Loxp-URA3-Loxp out, size are about 1.8kb, and nucleotides sequence is classified as shown in SEQ ID NO.11.
The amplification of pTEF: by design primer YW604 and YW629, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter pTEF, size is about 0.4kb, and nucleotides sequence is classified as shown in SEQ ID NO.12.
The amplification of POS5: by design primer YW630 and YW631, with wild type solution ester Ye Shi yeast AS2.1405 gene
Group is template, amplifies promoter POS5, size is about 1.2kb, and nucleotides sequence is classified as shown in SEQ ID NO.6.
The amplification of 3 ' GPD2: by design primer YW632 and YW633, with wild type solution ester Ye Shi yeast AS2.1405 base
Because group is template, 3 ' GPD2 are amplified, size is about 1.2kb, and nucleotides sequence is classified as shown in SEQ ID NO.16.
Shown in the PCR amplification conditional synchronization rapid 1 of above 5 ' GPD2, Loxp-URA3-Loxp, pTEF, POS5,3 ' GPD2.
The DNA fragmentation of above-mentioned acquisition is assembled by two 5 ' GPD2-URA3-pTEF-POS5 of large fragment using overlap-extension PCR,
POS5-3’GPD2。
Overlap-extension PCR constructs 5 ' GPD2-URA3-pTEF-POS5 segments.The first round: according to 1:3:3:1 molar ratio mixing 5 '
GPD2, URA3, pTEF, POS5 segment, amplification condition are same as above.Second wheel: the PCR product expanded using the first round as template,
It adds 1 μ l YW626+1 μ l YW631 and carries out PCR amplification.
Overlap-extension PCR constructs POS5-3 ' GPD2 segment.The first round: according to 1:1 molar ratio mix POS5,3 ' GUT2 segments,
Amplification condition is same as above.Second wheel: the PCR product expanded using the first round as template, addition 1 μ l YW630+1 μ l YW633 into
Row PCR amplification.
Two large fragment glue of the 5 ' GPD2-URA3-pTEF-POS5 that above-mentioned overlap-extension PCR is obtained, POS5-3 ' GPD2 return
After receipts, to be transferred in solution ester Ye Shi yeast recombination engineering ZS104 after each segment 50ng mixing, transformant uses primer respectively
YW634+YW635, YW636+YW637 carry out PCR identification.
3 list of primers of table
5, the building of ester Ye Shi Yeast engineering bacteria YW100-1 is solved
The amplification of 5 ' DGA2: by design primer YW646 and YW647, with wild type solution ester Ye Shi yeast AS2.1405 base
Because group is template, 5 ' DGA2 are amplified, size is about 1kb, and nucleotides sequence is classified as shown in SEQ ID NO.7.
The amplification of Loxp-URA3-Loxp: by design primer YW648 and YW649, using plasmid JMP113 as template, amplification
Loxp-URA3-Loxp out, size are about 1.8kb, and nucleotides sequence is classified as shown in SEQ ID NO.11.
The amplification of 3 ' DGA2: by design primer YW650 and YW651, with wild type solution ester Ye Shi yeast AS2.1405 base
Because group is template, 3 ' DGA2 are amplified, size is about 1kb, and nucleotides sequence is classified as shown in SEQ ID NO.8.
Shown in the PCR amplification conditional synchronization rapid 1 of above 5 ' DGA2, Loxp-URA3-Loxp, 3 ' DGA2.
The DNA fragmentation of above-mentioned acquisition is assembled by 5 ' DGA2-URA3-3 ' DGA2 of large fragment using overlap-extension PCR.It will be above-mentioned
After 5 ' DGA2-URA3-3 ' the DGA2 segment glue recycling that overlap-extension PCR obtains, it is transferred to solution ester Ye Shi yeast recombination engineering
In ZS106, transformant carries out PCR identification with primer YW652+YW653.
4 list of primers of table
The GUT1-pEXP1-GUT2 segment that overlap-extension PCR obtains is connected with the plasmid JMP113 plasmid that BamH I is linearized
One-step cloning method is crossed, E14 is constructed, it is thin to be transferred to solution ester Ye Shi yeast AS2.1405 competence for subsequent Not I digestion E14 plasmid
In born of the same parents, ZS102 is constructed.5 ' KU70-URA3-pTEF-DAK1, the DAK1-pEXP1-DAK2-pGPD that overlap-extension PCR is obtained,
PGPD-GYC1-3 ' KU70 large fragment is transferred in solution ester Ye Shi yeast ZS102 competent cell, constructs ZS104.Overlapping is prolonged
5 ' GPD2-URA3-pTEF-POS5 of acquisition are stretched, POS5-3 ' GPD2 large fragment is transferred to solution ester Ye Shi yeast ZS104 competence
In cell, ZS106 is constructed.5 ' DGA2-URA3-3 ' the DGA2 large fragments that overlap-extension PCR obtains are transferred to solution ester Ye Shi yeast
In ZS106 competent cell, building recombination engineering YW100-1 (solution ester Ye Shi yeast YW100-1), March 18 in 2019
It is preserved in China typical culture collection center day, deposit number is CCTCC NO:M 2019168, and address Wuhan, China Wuhan is big
It learns, postcode 430072.
Embodiment 2: the comparison of the shake flask fermentation of wild type AS2.1405 and recombination engineering YW100-1
By wild type solution ester Ye Shi yeast strain AS2.1405 and recombination engineering YW100-1, it is inoculated in YPD liquid respectively
In culture medium, in 30 DEG C in 250mL triangular flask, 200rpm is cultivated for 24 hours, obtains seed liquor；With initial cell concentration OD600=
0.05 is inoculated in respectively in YNG culture medium, and every kind of bacterial strain does three groups of parallel analysis, and in 30 DEG C, 200rpm fermented and cultured 16h is extremely
OD600When about 4.5, the concentration of 10mL fermentation liquid high effective liquid chromatography for measuring pyruvic acid and glycerol is taken, as a result sees Fig. 2.
The measurement of concentrations of pyruvate: taking 10mL fermentation liquid, and 6000rpm is centrifuged 5min, collects supernatant, is measured with C18 column
The content of pyruvic acid.Mobile phase: 0.1% phosphoric acid；Flow velocity: 1ml min-1；Injector temperature: 28 DEG C；Sample volume: 10 μ l.
Shown in Fig. 2, wild-type strain ferments 16h in YNG culture medium, and the yield of pyruvic acid is about 3.5g/L, and glycerol is residual
Allowance is about 10.2g/L.The yield of the pyruvic acid of engineering bacteria YW100-1 is about 5.95g/L, and glycerol residual quantity is about 7.4g/L.
It follows that engineering bacteria YW100-1 fermentation production of acetone acid yield is apparently higher than wild bacterium germination out.
Embodiment 3: wild type AS2.1405 and engineering bacteria YW100-1 produces the comparison of pyruvic acid in 20L ferment tank
Seed culture medium: 2g/L glycerol, 0.4g/L tryptone, yeast extract 0.2g/L, 0.24g/L KH2PO4、
1.7g/L K2HPO4·3H2O, solvent are distilled water, and pH value is natural.
Fermentation medium composition: 60g/L glycerol, 10g/L (NH4)2SO4、1.4g/L MgSO4·7H2O、2g/L KH2PO4、
0.8g/L CaCl2, 0.5g/L NaCl, 1 μ g/L vitamin B1, solvent is distilled water, and pH value is natural.
Inoculation wild type solution ester Ye Shi yeast strain AS2.1405 and engineering bacteria YW100-1 is in YPD fluid nutrient medium respectively
In, 30 DEG C are incubated overnight, and subsequent 200ml is transferred in 3L seed culture medium, 30 DEG C of shaking flask culture 18h, then with volumetric concentration
10% inoculum concentration is inoculated in the 20L fermentor equipped with 12L fermentation medium, fermented and cultured 1h.During entire fermented and cultured,
It is flowed with 20%NaOH plus adjusts pH value to 4.0, dissolved oxygen amount is maintained at 40% or so, and fermentation temperature control is 30 DEG C.According to fermentation
Glycerol residual concentration in tank respectively added 40g/L glycerol at 50,64 hours respectively, and glycerol 1440g is added altogether.
The process that the glycerol fermentation of two plants of bacterium produces pyruvic acid is as shown in Figure 3.Wild-type strain fermentation 120h, output of pyruvic acid
Reach maximum value 72.9g/L, pyruvic acid/glycerol conversion yield is about 0.52g/g.Engineering bacteria YW100-1 fermentation 120h, pyruvic acid produce
Amount reaches maximum value 121.2g/L, and pyruvic acid/glycerol conversion yield reaches 0.865g/g.Compared with bacterium germination out, engineering bacteria YW100-1
Fermentation output of pyruvic acid improve 66.2%, and be apparently higher than other genetic engineering bacteriums of reported maximum output.By
This is as it can be seen that solution ester Ye Shi yeast recombinant strain YW100-1 can provide strain excellent for pyruvic acid industrialized production.
Sequence table
<110>Zhejiang Polytechnical University
<120>a kind of solution ester Ye Shi yeast YW100-1 and its application
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1512
<212> DNA
<213>unknown (Unknown)
<400> 1
atgtcttcct acgtaggagc tctcgaccag ggtaccacct ccacccgttt cattctcttt 60
tcgcctgacg gcaagcccgt ggcatcccac cagatcgaat tcacccagat ctacccccac 120
cccggatggg tggagcacga ccccgaggag ctcgtgagct cgtgtctgga gtgcatgtcg 180
tcggtggcca aggaaatgcg aacccagggc atcaaggtgg ccgacgtgaa ggcgatcgga 240
atcaccaacc agcgagaaac caccgtgctt tgggacattg agaccggcca gcccctgtac 300
aacgccattg tgtggtccga cgcccgaacc ggcgacaccg tcaagaagct cgaggcccag 360
cccggcgctg acgaaatccc caagctctgt ggcctgcccc tgtccaccta ctttgccgga 420
gtcaaggtcc gatggatcct ggataacgtc aaggaggccc gagagtgcta cgatcgaggc 480
aagctggcct tctccaccat cgactcgtgg ctgctctaca acctcacggg cggcctcaac 540
ggcggcgccc acatcaccga cacctccaac gcctcccggt ccatgttcat gaacattgag 600
accctcaagt acgacgagaa gctcatcaag ttctttggcg tcgagaagct cattctcccc 660
aagattgtct cgtccgccga ggtctacggc cgaatcggaa ccggcccctt cgccaacatc 720
cccctggccg gctgtctcgg tgaccagtcc gccgccctcg tcggccagaa ggcctttgag 780
cccggccagg ccaagaacac atatggaacc ggctgcttcc tgctctacaa cgccggcgag 840
aagcccatca tctccaacaa cggcctgctg accaccgtcg gctaccactt caagggccag 900
aagcccgtct acgctctgga gggctccatc tccgtcgccg gctcgtgcat caagtggctg 960
cgagacaaca ttggtctcat tgagtcttcc gagcagattg gagagcttgc ctcccaggtc 1020
gacgactctg ccggcgtggt gtttgtcacc gctctgtccg gcctgtttgc cccctactgg 1080
cgaaccgacg cccgaggcac cattctgggt ctcactcagt tcaccaccaa ggcccacatt 1140
tgccgagccg ccctggaggc tacttgtttc cagacccggg ccattctcga cgccatggcc 1200
aaggactctg gtaagccctt caccaagctg cgagtcgacg gaggaatgac caactcggac 1260
attgctatgc agatccaggc cgacattctt ggcattgagg tcgagcgacc cgccatgcga 1320
gagaccaccg ctctgggtgc cgccattgct gccggctttg ccgttggcgt gtggaagtcc 1380
attgaggatc ttaaggacat caacaccgag ggcatgaccg agtttgcttc caagaccaac 1440
gaggaggagc gggccgccat gatgaagcag tggaaccggg gcattgagcg agctgttggc 1500
tggcttgagt aa 1512
<210> 2
<211> 1839
<212> DNA
<213>unknown (Unknown)
<400> 2
atgttcagaa ccattcgaaa acccgcgtgg gctgctgccg ccgccgtggc agccgctggc 60
gctggagccg tcgccctgtc tgtgcctgcc caggcccagg aggagctcca caagaagcac 120
aaattcacag tgccccccgt ggccgccgag cccccctctc gagccgccca gctcgagaag 180
atgaagaccg aggagtttga tctcgtcgtt gttggtggag gagctaccgg atccggtatc 240
gccctcgacg ctgtcacacg aggcctcaag gttgctctgg tcgagcgaga cgatttctcc 300
tgcggaacct cgtcccgatc caccaagctc atccacggag gtgtccgata cctcgagaag 360
gctgtgtgga acctcgacta caaccagtac gagctggtca aggaggccct gcacgagcga 420
aaggtcttcc tcgacattgc tccccacctc acctttgctc tgcccatcat gatccccgtc 480
tacacctggt ggcagcttcc ctacttctgg atgggtgtca agtgctacga tctgcttgcc 540
ggccgacaga acctcgagtc ctcttacatg ctctcccgat cccgtgctct cgatgccttc 600
cccatgcttt ccgatgacaa gctcaagggc gccattgtct actatgatgg ctcccagaac 660
gactctcgaa tgaacgtttc tcttattatg actgctgttg agaagggtgc caccatcctg 720
aaccattgcg aggtcaccga gctcaccaag ggcgccaatg gccagctcaa cggtgttgtt 780
gccaaggata ctgacggaaa cgctggatcc ttcaacatca aggccaagtg tgtcgttaat 840
gctactggac ccttcactga ctctctgcga cagatggacg acaagaacac caaggagatc 900
tgtgctcctt cctccggtgt tcacatcatt ctccccggtt actactcccc caagaagatg 960
ggactccttg accccgctac ttctgacggc cgagttatct tcttcctccc ctggcaggga 1020
aacacccttg ccggtactac tgaccagcct accaagatca ctgctaaccc tatcccctcc 1080
gaggaggaca ttgacttcat tctcaacgag gtccgacact acgttgaggg caaggttgat 1140
gtgcgacgag aggacgttct ggccgcctgg tccggaatcc gaccccttgt ccgggacccc 1200
cacgccaaga acaccgagtc tcttgtccga aaccatctca tcacctactc cgagtctggt 1260
cttgtcacca ttgctggcgg aaagtggacc acttaccgac agatggctga ggagactgtc 1320
gatgcctgca ttgccaagtt cggtctcaag cctgaaatct ccgccaaggc cgtcacccga 1380
gacgtcaagc tcatcggtgc taaggactgg actcctctca cttacattga tctgatccag 1440
caggaggacc ttgaccccga ggttgctaag cacctttctg agaactacgg atctcgagct 1500
ttcaccgttg cttctcttgc tgagatgccc acccccgaac ccggtgtgat cccccagtct 1560
actctcacaa agggtaagcg aatcctgtac ccctacccct acctcgatgc cgagtgcaag 1620
tactctatga agtacgagta tgccaccacc gccatcgact tccttgctcg acgaactcgt 1680
cttgctttcc ttaacgccgc tgccgcctac gaggctctcc ctgaggtcat tgagatcatg 1740
gccaaggagc tccagtggga cgaggctcga aaggagcagg aattcaacac cggtgtcgag 1800
tacctctact ccatgggcct tacccccaag gacaaataa 1839
<210> 3
<211> 1014
<212> DNA
<213>unknown (Unknown)
<400> 3
atgttccggt cagtatataa acgggggcga atcctgtcca aaatccccat caaacaacac 60
atccagatca cccacaaaca cacaatgact tctctcgact ttactctcaa caacggcaag 120
accatccctg ccatcggtct tggaacctgg aagtccacca ccgaggaggt ggctggcgcc 180
gtcgagtgcg ccctcaccga gggaggctac cgacacattg acaccgcctt caactaccga 240
aacgaagacg ccgtcggact cggaatcaag cgagccatgg agaagggtgt caagcgagaa 300
gacatcttcg tcaccaccaa gatctgggtc acctaccacg accgagtcga ggagaacctc 360
gacatgtctc tggagcgact gggtcttgac tacgtcgaca tgctcctcat ccactggccc 420
gttcccctca accctaacgg taacgacccc gtctaccccc tgcgacccga tggctctcga 480
gacattgacg agtccggctc ccagcccaag acctggaagc agatggaggc tgttctgaag 540
accggcaaga ccaagtctat cggtgtctcc aacttctcca tcccttacct cgaggagctg 600
ctcaaggagg ccgaggttgt ccccgccgtc aaccaggtcg agctccaccc tctgctgccc 660
cagctcgagc tcatggaatt ctgcaagaag aacaacattg tcatgaccgc cttctctccc 720
tttggctctg tcggtggccc tctgctcaag aacgagctcg tcgtctctct ggccgacaag 780
tacaacacct ctcccggagg aatcctcacc tcctaccaca ttggtaacgg caccgtggtc 840
atccccaagt ctgtcaccaa ctctcgaatc gtcgagaacg gaaagtccgc cgtcaccctg 900
tcccaggagg acctcaaggc tctgaacgac ctccacaaga ccgagggtat ccaccgaacc 960
tccaagccca agtggggtgt tgacctcgga ttccccgact ttgacttctg ctaa 1014
<210> 4
<211> 1719
<212> DNA
<213>unknown (Unknown)
<400> 4
atgaccacta aacagttcca attcgactcg gatccgctca attctgccct tgccgccacc 60
gcggaggcct caggcctcgc ttacctcccc aagagcaagg tcatctacta ccctctgacc 120
aacgacaagg tgacgttgat ttcaggtgga ggagctggcc acgagcctgc tcagaccggg 180
tttgtgggtc ccggactgct ggatgcggcc gtgtcgggcc agatctttgc ctcaccttcc 240
accaaacaga tcattgccgg agtcaatgcc gtcaagtcgc aacggggctc catcattatc 300
gtcatgaact acactggcga tgtgatccac tttggaatgg ccgccgagca gctgcggtcc 360
cgatatgact accacgccga actggtgtcc attggcgacg acatttccgt caacaagaag 420
gccggacgac gaggtctggc aggaaccgtt cttgttcaca agatcgcagg ccatcttgcc 480
cgagatggct gggacgtcgg agtgcttgct gaagctctgc gaaccaccgc cgccaacctg 540
gccaccgtgg ctgcgtctct ggaacactgc actgtacctg gcagaaagtt cgagaccgaa 600
ctggcggccg atgagatgga gattggcatg ggtatccaca acgagcccgg tgtcaagacc 660
atcaagattg gcaaggttga gtctctgctg gacgaattgg tcgacaagtt cgagccctcc 720
aagcaggact ttgtgccctt caacaagggc gacgaggtgg tgctgctggt caattccctc 780
ggaggagtct cttctctgga actccacgcc attgccaaca ttgcccagac aaagttcgag 840
aaggtgctgg gcgtcaagac cgtgcgactt attgttggca acttcatggc tgccttcaac 900
ggtcctggct tctctttgac tctgctcaac gtcaccacga ccgccaagaa gggcaacttt 960
gacgttctgg gagccctgga cgctcccgtg tccaccgccg cctggccctc tctgcagcag 1020
aaggacaagc ctgccaacgg cggtgtccag gaggagaagg agaccgactc ggacaagcct 1080
gctgagccta ctggaatcaa ggccgacgga aagctgttca aggccatgat tgagagtgct 1140
gttgacgatc tcaagaagga ggagccccag attaccaaat acgacactat tgctggcgat 1200
ggagactgtg gagagactct gttggctgga ggcgacggta ttctggacgc tatcaagaac 1260
aagaagattg accttgatga tgccgctgga gtggctgata tttctcacat cgtcgagaac 1320
tccatgggag gcacctcggg aggtctctac tccatcttct tctccggtct cgtggtcggt 1380
atcaaggaga ccaaggccaa ggagctgtct gtcgatgtgt ttgccaaggc atgtgagact 1440
gctctggaga ctctttctaa gtacacccag gcccgagtcg gcgaccgaac cctcatggac 1500
gcacttgttc cctttgtaga gaccctcagc aagaccaagg acttcgccaa ggccgtagag 1560
gctgctcgga agggcgccga cgagacttcc aagctgcctg ccaattttgg ccgtgcctcg 1620
tatgtgaacg aggagggatt ggagaacatt cctgaccctg gagctcttgg actggccgtc 1680
attttcgaag gtcttctcaa ggcctgggag aagaagtag 1719
<210> 5
<211> 1692
<212> DNA
<213>unknown (Unknown)
<400> 5
atgaaacact tcgccaaaac gaatctcgtc aatctctacc tcgaatctct cctggctagc 60
aacccgcaac tgggtctagt ggaagatcag agaatcattt attacaagaa gaaaaagtcg 120
gacaaggtcc gagttatttc gggcggcgga tcgggccatg agcccagctg gagcggtctt 180
gtgggctcgg gactactaga tgctgctgtc tgtggagaca tttttgcatc tccttcggcc 240
agacaggtta tggccggtat cagagcctcg gaacccgaca gtggcgttat tctggcaatc 300
acaaactaca cgggcgacaa gctgcacttt ggactggccc aagagaagtt ccaggccgag 360
tctggaggca tgcaggtggc agtcatccct gtgactgatg atgtcgctct cggacgaacc 420
cggtcctcaa aggtcggaag acggggactg gcgggaaacc tgcttgttct caagtccatg 480
ggagcctgtg ctgaggctgg aggttccttt gatcacgtct ccaatgtagg acgggcagtg 540
aacgacggac tggtcacggt gggatgttct ctggaccact gcagtgttcc tggtcgaaca 600
gacgtggact ttcatatccc tcatgacaag gctgtacttg gaatgggtat tcacaacgaa 660
cgaggacttg ttgaggtcga cattcccgaa cggcctgaag atctcatcaa acagatgttg 720
actcttttgc tagaccccaa cgacaaggag cgagcctttg tgtccttcaa ggagaaggac 780
gaggttattc tgctggtcaa caactttggt gggctgtcta atctcgagaa tggagcccta 840
actcaagtgg ccctgtctgt tctggagcag gattataaca ttgttccctg tcgagtcctg 900
tctggagcct ttgagacgtc gctagacggc ccaggctttt caatcactct ttacaaccct 960
tcatactctg caactctcgt tgaaaaatta tctagcaaac agcttctaga gctcatcgat 1020
gctccaactg atgctcctgc ttggccaagg gtcggtgtca acgagcccaa gaaacagaag 1080
gtgctctcca agcaggagga gctagcggcc aaggactgcg aagagtcgcc ttatgacgag 1140
cttgtttcgc gtatttgcaa acatgtcatt tcaatcgagc cttctctcac cacctgggat 1200
actgtaatgg gtgatggaga ctgcggtatg gcagccaaag acgcggcact tcacattcaa 1260
aaggagtgga attcccgcaa gcagtcttct ttaaagggaa ctcttaatct cctctcgtcc 1320
tgcctggatg acatgggtgg ctctctggga gccattctgg gcatctttgt tagtgctctc 1380
atctacaacc tgcaaaagga aggagttgaa caggctccaa aggcggttgg attggcttca 1440
aaatctctcc agacacacac acaggctcgc aagggtgacc gaacggtcat ggactctctg 1500
attcccttct gtgaagtcta cgcctcgtct ggaagtcttc aacatgcagc caaagccgct 1560
caggagggag cagaaagcac aaagaccctc aaagctcagt atggacgagc cagttatgtt 1620
tcaaagaccg cagatgttcc cgatcccgga gcctggctgt ttgccgcagt tgttgaccag 1680
ctttccaagt ag 1692
<210> 6
<211> 1581
<212> DNA
<213>unknown (Unknown)
<400> 6
atggaagtcc gacgacgaaa aatcgacgtg ctcaaggccc agaaaaacgg ctacgaatcg 60
ggcccaccat ctcgacaatc gtcgcagccc tcctcaagag catcgtccag aacccgcaac 120
aaacactcct cgtccaccct gtcgctcagc ggactgacca tgaaagtcca gaagaaacct 180
gcgggacccc cggcgaactc caaaacgcca ttcctacaca tcaagcccgt gcacacgtgc 240
tgctccacat caatgctttc gcgcgattat gacggctcca accccagctt caagggcttc 300
aaaaacatcg gcatgatcat tctcattgtg ggaaatctac ggctcgcatt cgaaaactac 360
ctcaaatacg gcatttccaa cccgttcttc gaccccaaaa ttactccttc cgagtggcag 420
ctctcaggct tgctcatagt cgtggcctac gcacatatcc tcatggccta cgctattgag 480
agcgctgcca agctgctgtt cctctctagc aaacaccact acatggccgt ggggcttctg 540
cataccatga acactttgtc gtccatctcg ttgctgtcct acgtcgtcta ctactacctg 600
cccaaccccg tggcaggcac aatagtcgag tttgtggccg ttattctgtc tctcaaactc 660
gcctcatacg ccctcactaa ctcggatctc cgaaaagccg caattcatgc ccagaagctc 720
gacaagacgc aagacgataa cgaaaaggaa tccacctcgt cttcctcttc ttcagatgac 780
gcagagactt tggcagacat tgacgtcatt cctgcatact acgcacagct gccctacccc 840
cagaatgtga cgctgtcgaa cctgctgtac ttctggtttg ctcccacact ggtctaccag 900
cccgtgtacc ccaagacgga gcgtattcga cccaagcacg tgatccgaaa cctgtttgag 960
ctcgtctctc tgtgcatgct tattcagttt ctcatcttcc agtacgccta ccccatcatg 1020
cagtcgtgtc tggctctgtt cttccagccc aagctcgatt atgccaacat ctccgagcgc 1080
ctcatgaagt tggcctccgt gtctatgatg gtctggctca ttggattcta cgctttcttc 1140
cagaacggtc tcaatcttat tgccgagctc acctgttttg gaaacagaac cttctaccag 1200
cagtggtgga attcccgctc cattggccag tactggactc tatggaacaa gccagtcaac 1260
cagtacttta gacaccacgt ctacgtgcct cttctcgctc ggggcatgtc gcggttcaat 1320
gcgtcggtgg tggttttctt tttctccgcc gtcatccatg aactgcttgt cggcatcccc 1380
actcacaaca tcatcggagc cgccttcttc ggcatgatgt cgcaggtgcc tctgatcatg 1440
gctactgaga accttcagca tattaactcc tctctgggcc ccttccttgg caactgtgca 1500
ttctggttca cctttttcct gggacaaccc acttgtgcat tcctttatta tctggcttac 1560
aactacaagc agaaccagta g 1581
<210> 7
<211> 1838
<212> DNA
<213>unknown (Unknown)
<400> 7
atgttcagaa ccattcgaaa acccgcgtgg gctgctgccg ccgccgtggc agccgctggc 60
gctggagccg tcgccctgtc tgtgcctgcc caggcccagg aggagctcca caagaagcac 120
aaattcacag tgccccccgt ggccgccgag cccccctctc gagccgccca gctcgagaag 180
atgaagaccg aggagtttga tctcgtcgtt gttggtggag gagctaccgg atccggtatc 240
gccctcgacg ctgtcacacg aggcctcagg ttgctctggt cgagcgagac gatttctcct 300
gcggaacctc gtcccgatcc accaagctca tccacggagg tgtccgatac ctcgagaagg 360
ctgtgtggaa cctcgactac aaccagtacg agctggtcaa ggaggccctg cacgagcgaa 420
aggtcttcct cgacattgct ccccacctca cctttgctct gcccatcatg atccccgtct 480
acacctggtg gcagcttccc tacttctgga tgggtgtcaa gtgctacgat ctgcttgccg 540
gccgacagaa cctcgagtcc tcttacatgc tctcccgatc ccgtgctctc gatgccttcc 600
ccatgctttc cgatgacaag ctcaagggcg ccattgtcta ctatgatggc tcccagaacg 660
actctcgaat gaacgtttct cttattatga ctgctgttga gaagggtgcc accatcctga 720
accattgcga ggtcaccgag ctcaccaagg gcgccaatgg ccagctcaac ggtgttgttg 780
ccaaggatac tgacggaaac gctggatcct tcaacatcaa ggccaagtgt gtcgttaatg 840
ctactggacc cttcactgac tctctgcgac agatggacga caagaacacc aaggagatct 900
gtgctccttc ctccggtgtt cacatcattc tccccggtta ctactccccc aagaagatgg 960
gactccttga ccccgctact tctgacggcc gagttatctt cttcctcccc tggcagggaa 1020
acacccttgc cggtactact gaccagccta ccaagatcac tgctaaccct atcccctccg 1080
aggaggacat tgacttcatt ctcaacgagg tccgacacta cgttgagggc aaggttgatg 1140
tgcgacgaga ggacgttctg gccgcctggt ccggaatccg accccttgtc cgggaccccc 1200
acgccaagaa caccgagtct cttgtccgaa accatctcat cacctactcc gagtctggtc 1260
ttgtcaccat tgctggcgga aagtggacca cttaccgaca gatggctgag gagactgtcg 1320
atgcctgcat tgccaagttc ggtctcaagc ctgaaatctc cgccaaggcc gtcacccgag 1380
acgtcaagct catcggtgct aaggactgga ctcctctcac ttacattgat ctgatccagc 1440
aggaggacct tgaccccgag gttgctaagc acctttctga gaactacgga tctcgagctt 1500
tcaccgttgc ttctcttgct gagatgccca cccccgaacc cggtgtgatc ccccagtcta 1560
ctctcacaaa gggtaagcga atcctgtacc cctaccccta cctcgatgcc gagtgcaagt 1620
actctatgaa gtacgagtat gccaccaccg ccatcgactt ccttgctcga cgaactcgtc 1680
ttgctttcct taacgccgct gccgcctacg aggctctccc tgaggtcatt gagatcatgg 1740
ccaaggagct ccagtgggac gaggctcgaa aggagcagga attcaacacc ggtgtcgagt 1800
acctctactc catgggcctt acccccaagg acaaataa 1838
<210> 8
<211> 1200
<212> DNA
<213>unknown (Unknown)
<400> 8
atgcgactac tcatccgccg aaccggtata acacggcccc acagcgtgca agcgcgccga 60
tccacatgga ttcggcttct ctcgaccgag atattgcatg cagaactgct tcccgaccgc 120
cagtcgcccc actacgtcca ggagtcgacc tctctgtcat ctctggtgtg ggacaagcct 180
ctggaaaacg ttctgatcgt caaaaaaccc tgggaccaca atgtgcgcga gtcgctcatc 240
cagatggcat ctcacatcca gcgccggtac ccccgagtca acattctggt ggaggaacat 300
gtggccgacg aggtccagaa gcagattgga gccgcaggcg tgaccgccat ccacacgggg 360
ccaggagagg tgctgagaaa caagacggat ctgctcgtga ctctgggagg cgacggaact 420
attctacatg ccacctccat gtttgcttcc ggagaagtgc cgccggtgct gtccttttcg 480
ctggggactc tgggtttcct gctgccgttt gatttcaagg acttcaaaac tgcattcgac 540
atggtgtact cgtcgcaggc ctcggtggtc aaccgcgccc gcctagcatg tcagaaaatg 600
tccattcgca aggaaatcac ccacttgccc tcccaatcgc acattgaaca caactcaacc 660
catgtctacg gcaatcccga cgactacaat cttagcccac taacctacgc catgaacgac 720
atcaacatcc accgtggagc tgagccgcat ctcaccaagc tcgacatcca cgttgacggc 780
gagttcatca cccgagccat tgctgacggt gtcaccatcg ccacacccac gggctccacg 840
gcctactcgc tgtcgtctgg cggctccatt gtgcatcccc gagtcgcctg cattctgctg 900
acccccatct gtccgcgatc gctgtcattc cggcctctca ttttcccagc cacctccaaa 960
atatgcatca ccgcctcgtc cgaatctcga ggtagaggcg ccgagctgtc tgtcgacgga 1020
atcgccaagg gtctggttcg acccagcgac aagattctgg tcgaaagcga aaccggccac 1080
aactcgggca tctggtgcgt ggccaagaca gacagagact gggtcagtgg cctcaacggg 1140
ttactgggct tcaatagcag ttttggcaag ggcggggagg cgtcaggcga tgttgcttag 1200
<210> 9
<211> 1000
<212> DNA
<213>unknown (Unknown)
<400> 9
ggagtttggc gcccgttttt tcgagcccca cacgtttcgg tgagtatgag cggcggcaga 60
ttcgagcgtt tccggtttcc gcggctggac gagagcccat gatgggggct cccaccacca 120
gcaatcaggg ccctgattac acacccacct gtaatgtcat gctgttcatc gtggttaatg 180
ctgctgtgtg ctgtgtgtgt gtgttgtttg gcgctcattg ttgcgttatg cagcgtacac 240
cacaatattg gaagcttatt agcctttcta ttttttcgtt tgcaaggctt aacaacattg 300
ctgtggagag ggatggggat atggaggccg ctggagggag tcggagaggc gttttggagc 360
ggcttggcct ggcgcccagc tcgcgaaacg cacctaggac cctttggcac gccgaaatgt 420
gccacttttc agtctagtaa cgccttacct acgtcattcc atgcatgcat gtttgcgcct 480
tttttccctt gcccttgatc gccacacagt acagtgcact gtacagtgga ggttttgggg 540
gggtcttaga tgggagctaa aagcggccta gcggtacact agtgggattg tatggagtgg 600
catggagcct aggtggagcc tgacaggacg cacgaccggc tagcccgtga cagacgatgg 660
gtggctcctg ttgtccaccg cgtacaaatg tttgggccaa agtcttgtca gccttgcttg 720
cgaacctaat tcccaatttt gtcacttcgc acccccattg atcgagccct aacccctgcc 780
catcaggcaa tccaattaag ctcgcattgt ctgccttgtt tagtttggct cctgcccgtt 840
tcggcgtcca cttgcacaaa cacaaacaag cattatatat aaggctcgtc tctccctccc 900
aaccacactc acttttttgc ccgtcttccc ttgctaacac aaaagtcaag aacacaaaca 960
accaccccaa cccccttaca cacaagacat atctacagca 1000
<210> 10
<211> 1193
<212> DNA
<213>unknown (Unknown)
<400> 10
caatgaatta agtctccgtg ttaccatctt agatatgaac actaaatatg ccaagttctc 60
tttcccctac atgattcata gcacttgccc aaagacgagg agactttctt cttacgacat 120
atacatcaat agaccattca tggcaagaaa gaagtactcg aacataggcc cataaagtac 180
gagtacagta cactacacta cactacttgt accattctac ccggggtctg ccggcttgta 240
cacaccgaca gcactcgtac tctcccacga atgctccggc tgccgacatc aacacgatct 300
caaaagcgca tactgagctt cctttcctag ctcttccttc cttcaactcg ataaatacat 360
tggatatata catgtgtggc gactgtcgac ttgatgttta gagtgtccag atccgcaaga 420
tcggctcgca cttgtgttgt gttgtttcaa atcagcctgt cgttttgtgt cgtttgagat 480
cattctgtct cactcttagg ctcgcttaga accgacaacg gagaatccgg gctcggtttt 540
tcggtcggcc ttgatctggg ccttggactt gtactggtcg gccatctcca cgttgaccag 600
ctccttgacc ttgtagagct gaccggcgat accaggagac accttgtagt acttctggga 660
gccgaccttg cccagaccga gggtcttgag cacgtcacgt gttctccacg gcattcgcag 720
gatagatcgg acctgtgtga ctttgtagaa catggcgttt caggtggttg cgtgagtgtg 780
taaaatcgtg tctttcagaa gttacaaatt tcaccgcatt tagagtttat gcagatgggc 840
ggtgtgtggt tgggagttcg atttccgtgc gtgcatttga tcttgatgaa ttggatttgt 900
acatgaggaa gagcacgtca agcaccgcct actgcaaact cgtgaatatt gagattattg 960
aggaaattca aggaaaattc agatcagatt tgagagcaaa gtccaacaat actacacaat 1020
ccctttcctg tattcttcca ccatcgtcat cgtcgtctgt cttctcttca gctttttaat 1080
ttcactcccc acaaacccaa atttagctgc atcattcatc aacctccaat tataactata 1140
catcgcgaca cgaacacgaa acacgaacca cgaaccgccg ctttttgaaa atg 1193
<210> 11
<211> 1726
<212> DNA
<213>unknown (Unknown)
<400> 11
gcactttgct agatagagtc gagaattacc ctgttatccc tagataactt cgtatagcat 60
acattatacg aagttattct gaattccgag aaacacaaca acatgcccca ttggacagac 120
catgcggata cacaggttgt gcagtaccat acatactcga tcagacaggt cgtctgacca 180
tcatacaagc tgaacagcgc tccatacttg cacgctctct atatacacag ttaaattaca 240
tatccatagt ctaacctcta acagttaatc ttctggtaag cctcccagcc agccttctgg 300
tatcgcttgg cctcctcaat aggatctcgg ttctggccgt acagacctcg gccgacaatt 360
atgatatccg ttccggtaga catgacatcc tcaacagttc ggtactgctg tccgagagcg 420
tctcccttgt cgtcaagacc caccccgggg gtcagaataa gccagtcctc agagtcgccc 480
ttaggtcggt tctgggcaat gaagccaacc acaaactcgg ggtcggatcg ggcaagctca 540
atggtctgct tggagtactc gccagtggcc agagagccct tgcaagacag ctcggccagc 600
atgagcagac ctctggccag cttctcgttg ggagagggga ctaggaactc cttgtactgg 660
gagttctcgt agtcagagac gtcctccttc ttctgttcag agacagtttc ctcggcacca 720
gctcgcaggc cagcaatgat tccggttccg ggtacaccgt gggcgttggt gatatcggac 780
cactcggcga ttcggtgaca ccggtactgg tgcttgacag tgttgccaat atctgcgaac 840
tttctgtcct cgaacaggaa gaaaccgtgc ttaagagcaa gttccttgag ggggagcaca 900
gtgccggcgt aggtgaagtc gtcaatgatg tcgatatggg tcttgatcat gcacacataa 960
ggtccgacct tatcggcaag ctcaatgagc tccttggtgg tggtaacatc cagagaagca 1020
cacaggttgg ttttcttggc tgccacgagc ttgagcactc gagcggcaaa ggcggacttg 1080
tggacgttag ctcgagcttc gtaggagggc attttggtgg tgaagaggag actgaaataa 1140
atttagtctg cagaactttt tatcggaacc ttatctgggg cagtgaagta tatgttatgg 1200
taatagttac gagttagttg aacttataga tagactggac tatacggcta tcggtccaaa 1260
ttagaaagaa cgtcaatggc tctctgggcg gaattcgtat aacttcgtat agcaggagtt 1320
atccgaagcg ataattaccc tgttatccct agaatcgata gagaccgggt tggcggcgca 1380
tttgtgtccc aaaaaacagc cccaattgcc ccaattgacc ccaaattgac ccagtagcgg 1440
acccaacccc ggcgagagcc cccttcaccc cacatatcaa acctcccccg gttcccacac 1500
ttgccgttaa gggcgtaggg tactgcagtc tggaatctac gcttgttcag actttgtact 1560
agtttctttg tctggccatc cgggtaaccc atgccggacg caaaatagac tactgaaaat 1620
ttttttgctt tgtggttggg actttagcca agggtataaa agaccaccgt ccccgaatta 1680
cctttcctct tcttttctct ctctccttgt caactcacac ccgaag 1726
<210> 12
<211> 444
<212> DNA
<213>unknown (Unknown)
<400> 12
gcaaaggagg ggctggtgag ggcgtctgga agtcgaccag agaccgggtt ggcggcgcat 60
ttgtgtccca aaaaacagcc ccaattgccc caattgaccc caaattgacc cagtagcggg 120
cccaaccccg gcgagagccc ccttctcccc acatatcaaa cctcccccgg ttcccacact 180
tgccgttaag ggcgtagggt actgcagtct ggaatctacg cttgttcaga ctttgtacta 240
gtttctttgt ctggccatcc gggtaaccca tgccggacgc aaaatagact actgaaaatt 300
tttttgcttt gtggttggga ctttagccaa gggtataaaa gaccaccgtc cccgaattac 360
ctttcctctt cttttctctc tctccttgtc aactcacacc cgaaatcgtt aagcatttcc 420
ttctgagtat aagaatcatt caaa 444
<210> 13
<211> 941
<212> DNA
<213>unknown (Unknown)
<400> 13
ataaaccgga cgcagtagga tgtcctgcac gggtcttttt gtggggtgtg gagaaagggg 60
tgcttggaga tggaagccgg tagaaccggg ctgcttgggg ggatttgggg ccgctgggct 120
ccaaagaggg gtaggcattt cgttggggtt acgtaattgc ggcatttggg tcctgcgcgc 180
atgtcccatt ggtcagaatt agtccggata ggagacttat cagccaatca cagcgccgga 240
tccacctgta ggttgggttg ggtgggagca cccctccaca gagtagagtc aaacagcagc 300
agcaacatga tagttggggg tgtgcgtgtt aaaggaaaaa aaaagaagct tgggttatat 360
tcccgctcta tttagaggtt gcgggataga cgccgacgga gggcaatggc gccatggaac 420
cttgcggata tcgatacgcc gcggcggact gcgtccgaac cagctccagc agcgtttttt 480
ccgggccatt gagccgactg cgaccccgcc aacgtgtctt ggcccacgca ctcatgtcat 540
gttggtgttg ggaggccact ttttaagtag cacaaggcac ctagctcgca gcaaggtgtc 600
cgaaccaaag aagcggctgc agtggtgcaa acggggcgga aacggcggga aaaagccacg 660
ggggcacgaa ttgaggcacg ccctcgaatt tgagacgagt cacggcccca ttcgcccgcg 720
caatggctcg ccaacgcccg gtcttttgca ccacatcagg ttaccccaag ccaaaccttt 780
gtgttaaaaa gcttaacata ttataccgaa cgtaggtttg ggcgggcttg ctccgtctgt 840
ccaaggcaac atttatataa gggtctgcat cgccggctca attgaatctt ttttcttctt 900
ctcttctcta tattcattct tgaattaaac acacatcaac a 941
<210> 14
<211> 1219
<212> DNA
<213>unknown (Unknown)
<400> 14
ctagggaggc acatctaaac gaataacgaa tattaatgat accatcatat ctcagaacat 60
gtatgactgc tgcttccaaa cgatatgagg atgagtcctc tttcagatta agatagagta 120
caaatatatt atctatatac tggtgtctgt gcgatgtcgt atgagcggtg aatcatgtga 180
ctgtcacgtg gtttggccca agttacaccg tagctacgcc tttcttgacc gtctccatgg 240
tcttctgggc gggttgacag tttccactgg atgagcgtcc gcctcctgtt cctgtcgttg 300
tccctgcagc tcagcctcaa tcttctgacc gagctcggag tccagggaaa tgccaacagg 360
ttgtccaagc aacatcatgg tttggtgggc agccgtgatc tcatcgtcgt tggataccat 420
tcggtacttg gcctcaatct gcacaaagta gcggtaccac tggtttcgag caaaccgctc 480
caattgagcc tctccgtcga gagagagagt aggtgattgc tccaacttgc ggccaaaatg 540
aagttctcga ctcacctttt tgaagcggtt cttcttgccc atcttggtgg cgaaagtagt 600
ggctagtggt ggatgacttt gtataatgta ccgatgaaga gggttgtatt tgctcagtaa 660
gaagtagcga gtgaaatcag atgacttaac gagagcaaag ggcaatggaa tacctgctgc 720
ctgattaaca acagcttctg tgtcgtttct ctcttgtgaa tgagtgtgtt gctagaggta 780
ggttggcact ccaatgttac gacacacaat agtctataga gcactacaaa gggctatatc 840
gtcaactgct ctattgtagc tacagtacag tacataccat caagtgaaca atggaccacc 900
aaactcggca ctaagccaat agaacctttg cggcctcctt tatcacgttt ctatatacct 960
tgtccattta tgtgccaccc tttagtcttg gtcgttcact tcagctcaac ttcagccatg 1020
atagcaagat gatctgaagg atacatgtca atgcgaggct gaccactggg ctcgggcccc 1080
atatcctcct caaggggcat cttcaacaga ctcttgacct ggacctcatc gctgttgttg 1140
gacgaaacga aaatgtagtc caaaagaccc ctccaggcgt gggcccagtt actgaagcga 1200
ggctccttct tgtgcttgc 1219
<210> 15
<211> 1034
<212> DNA
<213>unknown (Unknown)
<400> 15
agcggtttgt tttctatggc atgttgttga cgcatgctgc caacggctat tcaacggtga 60
caacggatga tgctgtcaca tgacgccatt ttttatgttg tatccaacag cacggtacta 120
aaacaggcca tttgtaaagg cctcactcag ctcacacacg ctcaacggtc acgataaggt 180
cgcactagag gcgttagttg gtttcaagaa tagtggttat tggtcttggg atacgggttg 240
gacaatatac aaatgggctc gcgtacactt atacagtcct accattctgt cgccctctga 300
ttctccgcca catcagccac gccgcaacgt ctcctcctca tccccctcct gctcttccac 360
tcgcaaaacg tccaaactca attgtgtcaa aattggaggt tctcttcgtt tgagcctacc 420
attttcaatt ttttagttgc gacagcggcc cggtcagagg ttcacaacaa ggtctagaga 480
cactttgtca tggggccgag aaggaccata aaaaccaaac gatggtcacg tcaggtcaat 540
tactgaccag tctcacatcc gacccctcgc gtgctcgacc ggaggatttc tctgcactcg 600
tccttgcata cctcggctag cgggatttat tcaccaatca cacagccgag agtttttccg 660
gacccttcat ccaacagctt agagttgcat gagtcagtag caacgtagac tttgagcctt 720
tgtgacagat gtccaagtgc agcacgttgt aggaaaataa ggtgaaggat tggccaatgt 780
gaacagaggc gacaagagtc cgtctggagg gcttgttgta gtcaattgcc cgcgcaattg 840
attgacctca tcgtttctgc cggaaccccc ccacaagccc ggataaatag acacgcccca 900
caagccgttc gtctggtctg ctcacagcac acttccattt aaaattcaaa caaagcgcac 960
caccgcaaag catacttaac ccactcaatg tagacgtcgc ggaacttctc tttcctaccc 1020
accaccccaa acaa 1034
<210> 16
<211> 1240
<212> DNA
<213>unknown (Unknown)
<400> 16
tacctctact ccatgggcct tacccccaag gacaaataac tgtatagtaa aagcgtatag 60
ccaataagat aatcacttga atgaaggagc agcaactcgt atgtttagca cttcaacgga 120
ctatttcccc gcagcaaaga gactattgct gagttgttga gtatctgctt tacaataatg 180
gggtatggac acacaaggag gggtcttagt gagaagttag ataggtctag catacatgag 240
atcaatgtgg tcttacctat atcgtttgtt atcatttatc ttggtttgaa ttgataacac 300
gagttgttca ttgaagtgat ggcaccgggt ctcacacgca acagttggcg aacaggtcgt 360
attgttcctt agatacgacg ctcttttgga catgatggga agtgaaacta caattacagt 420
agctacatag cttggctaac tagaccgctt acagaaccag tagtcgtcac aagaccacca 480
cgaacaaagt ccaactaccc cactcccacc actcgtattt acttaccgca gatcacacgc 540
ttcggtgtat ctccgtgggg catcgtgggg cattgttcta agttttccgt atggtgcaca 600
gtcggtacgt gctttgacta accagtagaa gttaggctac tgtagtggag attgagcaat 660
gaaacgatga caggaagacc ccaaaatgcg accacctcaa ctatacacgg cttgttgcta 720
ttgccgcctt gcactccaca cagcaaacat gcacacgata tgcactcaag tcttaaccga 780
atgaaggtaa aagtagcaac caacaagcga gagttactgt atacttacaa gttatacgac 840
agtctcactt atcaccaatt ggcaacttga ccgcacagac aaacacccta caatgacctt 900
cctcaatgtg ctctactacg tgctgctggc tgccatcatg atcggcaccg gctacttcta 960
ctacctgtgg ttcactgaga ccaacgacca aaccgagaag atcatcagag ctgcgcttgg 1020
agtctttgat atcgccatct ggtacattct aggtatctcc acctccttta agatcctcac 1080
ccagatgatt ctcgcctgtt tccttgtggt tctggctggt cttaagatct acatcaaccc 1140
tcgagttgga ggggctcttc tggccggcag tctgctattt gtggctgctg tctggttcgg 1200
attccgccga gacggtcgag aggcccgaga cgatcttaac 1240
Claims (10)
1. a kind of solution ester Ye Shi yeast YW100-1 of high yield pyruvic acid, it is characterised in that the solution ester Ye Shi yeast (Yarrowia
Lipolytica) YW100-1 is to be overexpressed glycerokinase GUT1 in glycerol metabolism, glycerokinase in solution ester Ye Shi yeast
GUT2, glycerol dehydrogenase enzyme GCY1, dihydroxyacetone kinase DAK1, dihydroxyacetone kinase DAK2 and NADH kinases POS5, together
When knock out solution ester Ye Shi yeast in diacylglycerol acyltransferase gene DGA2 and glycerol-3-phosphate gene GPD2 obtain
.
2. solution ester Ye Shi yeast YW100-1 as described in claim 1, it is characterised in that the solution ester Ye Shi yeast YW100-1 is pressed
Following method building:
(1) by from GUT1 gene, GUT2 gene and the promoter pEXP1 segment of solution ester Ye Shi yeast, prolonged by overlapping
It stretches and obtains GUT1-pEXP1-GUT2, be connected on carrier JMP113, obtain carrier E14；
(2) it by the carrier E14 NotI digestion of building, is transferred in wild type solution ester Ye Shi yeast AS2.1405, is converted
Solution ester Ye Shi Yeast engineering bacteria ZS102 afterwards；
It (3) will be from the GCY1 of solution ester Ye Shi yeast, DAK1, DAK2 gene, promoter pTEF, pEXP1, pGPD, Yi Jiyong
In the 5 ' ends and 3 ' ends of the homology arm KU70 of integration, 5 ' KU70-URA3-pTEF-DAK1 are obtained respectively by overlap-extension PCR,
Tri- large fragments of DAK1-pEXP1-DAK2-pGPD, pGPD-GYC1-3 ' KU70, are transferred in ZS102, after obtaining conversion
Solution ester Ye Shi Yeast engineering bacteria ZS104；
(4) by 5 ' the end GPD2 and 3 ' from the POS5 gene and pTEF of solution ester Ye Shi yeast, and for knocking out GPD2
GPD2 is held, bis- segments of 5 ' GPD2-URA3-pTEF-POS5 and POS5-3 ' GPD2 are obtained by overlap-extension PCR, are transferred to ZS104
In, the solution ester Ye Shi Yeast engineering bacteria ZS106 after obtaining homologous recombination；
(5) by the 5 ' ends for being used to knock out and 3 ' end DGA2,5 ' DGA2-URA3-DGA2 segments is obtained by overlap-extension PCR, are transferred to
In ZS106, solution ester Ye Shi yeast YW100-1 is obtained.
3. solution ester Ye Shi yeast YW100-1 as described in claim 1, it is characterised in that the glycerokinase GUT1 gene nucleotide
Sequence is shown in SEQ ID NO.1, and glycerokinase GUT2 gene nucleotide series are dihydroxyacetone (DHA) shown in SEQ ID NO.2
Kinases DAK1 gene nucleotide series are shown in SEQ ID NO.3, and dihydroxyacetone kinase DAK2 gene nucleotide series are SEQ
Shown in ID NO.4, glycerol dehydrogenase GCY1 gene nucleotide series are NADH kinases POS5 gene core shown in SEQ ID NO.5
Nucleotide sequence is shown in SEQ ID NO.6.
4. solving ester Ye Shi yeast (Yarrowia lipolytica) YW100-1 described in a kind of claim 1, it is preserved in Chinese allusion quotation
Type culture collection, the deposit date is on March 18th, 2019, deposit number was CCTCC NO:M 2019168, address are as follows: in
Wuhan Wuhan University, state, postcode 430072.
5. solution ester Ye Shi yeast YW100-1 described in a kind of claim 1 prepares the application in pyruvic acid in fermentation glycerol.
6. application as claimed in claim 5, it is characterised in that when fermenting for shake flask fermentation, the method are as follows: solution ester Ye Shi ferment
Female YW100-1 is inoculated in YPD culture medium, and in 30 DEG C, 200rpm is cultivated for 24 hours, obtains seed liquor；With initial cell concentration OD600
=0.05 is inoculated in YNG culture medium, in 30 DEG C, 200rpm fermented and cultured to OD600When for 4.0-5.0, obtain containing pyruvic acid
Fermentation liquid.
7. application as claimed in claim 5, it is characterised in that when fermenting for ferment tank, the method are as follows: ester will be solved
Family name's yeast YW100-1 is seeded in the fermentor containing fermentation medium, in 30-32 DEG C, pH value 4-4.5, dissolved oxygen amount 40-50% item
It ferments under part, obtains the fermentation liquid containing pyruvic acid, isolate and purify, obtain pyruvic acid；The fermentation medium composition are as follows:
60g/L glycerol, 10g/L (NH4)2SO4、1.4g/L MgSO4·7H2O、2g/L KH2PO4、0.8g/L CaCl2、0.5g/L
NaCl, 1 μ g/L vitamin B1, solvent are distilled water, and pH value is natural.
8. the use as claimed in claim 7, it is characterised in that when the ferment tank, glycerol is added using batch mode,
60g/L is added for the first time, glycerol is added after glycerol depletion, adds 40g/L every time.
9. application as claimed in claim 8, it is characterised in that glycerol is added 2 times.
10. the use as claimed in claim 7, it is characterised in that before the ferment tank, solution ester Ye Shi yeast YW100-1 is first
It carries out seed and expands culture, then seed liquor is seeded to fermentation medium, the seed training with the inoculum concentration of volumetric concentration 10%
It supports are as follows: solution ester Ye Shi yeast YW100-1 is seeded to seed culture medium, and 30 DEG C of shaking flask culture 18h obtain seed liquor；The seed
Culture medium: 2g/L glycerol, 0.4g/L tryptone, yeast extract 0.2g/L, 0.24g/L KH2PO4、1.7g/L K2HPO4·
3H2O, solvent are distilled water, and pH value is natural.
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| CN116286899A (en) * | 2023-05-12 | 2023-06-23 | 昆明理工大学 | NADH kinase gene RkNADHK1 and application thereof |
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