CN107475269B - acyl-CoA thioesterase gene of candida tropicalis and application thereof - Google Patents
acyl-CoA thioesterase gene of candida tropicalis and application thereof Download PDFInfo
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- 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
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- C12P7/00—Preparation of oxygen-containing organic compounds
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- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
Abstract
The invention relates to an acyl-CoA thioesterase gene of candida tropicalis and application thereof. The nucleotide sequence of the acyl-CoA thioesterase gene ctaCA of the candida tropicalis is shown in SEQ ID No. 1. The amino acid sequence of the acyl-CoA thioesterase protein CtAcA is shown in SEQ ID NO. 2. The invention discovers for the first time that acyl-CoA thioesterase gene ctaCA in candida tropicalis is a key gene in the conversion process of long-chain dibasic acid, the expression of the acyl-CoA thioesterase gene ctaCA can promote the conversion of the long-chain dibasic acid-CoA to free long-chain dibasic acid, and the invention lays a foundation for realizing the synthesis of the long-chain dibasic acid by a new way by taking oil as a raw material.
Description
Technical Field
The invention relates to an acyl-CoA thioesterase gene of candida tropicalis and application thereof, belonging to the technical field of genetic engineering.
Background
The long-chain dicarboxylic acid generally refers to a straight-chain aliphatic dicarboxylic acid having a carbon chain containing 12 or more carbon atoms and having a carboxyl group at each of the α and ω positions. The product has high industrial application value, and the long-chain dibasic acid is an important chemical intermediate for synthesizing special nylon, high-grade musk, adhesive, hot melt adhesive, medicine, pesticide and the like. At present, 2 methods are mainly used for producing long-chain dicarboxylic acid at home and abroad: chemical and fermentation processes. The chemical synthesis of long-chain dicarboxylic acid has complex process, harsh reaction conditions and high cost, and only a few countries such as the United states, Germany and the like utilize the chemical synthesis to produce the long-chain dicarboxylic acid at present. Compared with a chemical synthesis method, the microbial fermentation method is replacing the chemical synthesis method to produce the long-chain dicarboxylic acid by virtue of the characteristics of strong reaction specificity, wide raw material source, low cost, mild reaction conditions and the like. Therefore, many researchers turn the target to microbial fermentation with wide development prospect and great industrial value. The microbial fermentation method is characterized in that normal alkane is used as a raw material, and the oxidation performance of Candida tropicalis is utilized to oxidize methyl groups at two ends of the normal alkane at normal temperature and normal pressure to generate dibasic acid with the corresponding chain length of matrix alkane. At present, the industrialization of producing the long carbon chain dibasic acid by fermenting alkane serving as a substrate is realized in China, and the eleven-tetradecanedioic acid prepared by a biological method is put on the market. Such as chinese patent document CN1570124A (application No. 2004100182557), chinese patent document CN1844404A (application No. CN200610038331X), chinese patent document CN101225411A (application No. 2007101958427), chinese patent document CN102115769A (application No. 2009102565907), chinese patent document CN102115768A (application No. 2009102565890), chinese patent document CN102115766A (application No. 2009102565871), chinese patent document CN102115765A (application No. 2009102565867), chinese patent document CN102061316A (application No. 2010101603101), and chinese patent document CN103805642A (application No. 2012104397995).
At present, the technology for producing long-chain dicarboxylic acid by a microbial fermentation method, particularly the breeding aspect of microorganisms, is mature day by day, for example, Chinese patent document CN105400796A (application No. 201511003830) discloses a long-chain fatty acid transporter gene pxa1p of candida tropicalis positioned on a peroxidase membrane, and the synthesis of the gene is blocked by genetic engineering to realize the improvement of the yield of the long-chain dicarboxylic acid. Chinese patent document CN103992959A (application No. 2014101755564) improves the yield of long-chain dibasic acid of candida tropicalis by adding one copy of CYP monooxygenase gene, Chinese patent document CN102839133A (application No. CN201110168672X) screens a mutant strain of pox4 gene, fao gene and CYP52A18 gene by strain mutation breeding, and the mutant strain has high conversion performance on substances such as alkane, fatty acid and the like with different carbon chain lengths.
However, it is still a current research focus to develop strains with higher ability to produce long-chain dicarboxylic acids and methods for producing them.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an acyl-CoA thioesterase gene of candida tropicalis and application thereof.
The technical scheme of the invention is as follows:
an acyl-CoA thioesterase gene ctacA for regulating and controlling candida tropicalis, the nucleotide sequence is shown in SEQ ID NO. 1.
The gene ctacA for regulating acyl-CoA thiolase of Candida tropicalis is derived from Candida tropicalis, and the expression of the gene ctacA can promote the conversion of long-chain dibasic acid-CoA to free long-chain dibasic acid.
An acyl-CoA thiolase protein CtAcA, the amino acid sequence of which is shown in SEQ ID NO. 2.
A recombinant expression vector comprises an acyl-CoA thioesterase gene ctacA with a nucleotide sequence shown as SEQ ID NO. 1.
A recombinant cell comprising the above recombinant expression vector or expressing the above acyl-CoA thioesterase gene ctacA.
The acyl-CoA thioesterase gene ctaCA is applied to preparation of long-chain dibasic acid by modifying candida tropicalis.
According to a preferred embodiment of the present invention, the steps of the application are as follows:
constructing multi-copy recombinant candida of acyl-CoA thioesterase gene ctaCA or replacing a promoter to realize the over-expression of the acyl-CoA thioesterase gene ctaCA.
The over-expression of the acyl-CoA thioesterase gene ctaCA is realized by constructing multi-copy recombinant candida of the acyl-CoA thioesterase gene ctaCA or replacing a promoter, so that the conversion rate of long-chain dibasic acid-CoA in cells of candida tropicalis to free long-chain dibasic acid can be increased, the conversion of the long-chain dibasic acid of a product is increased, the internal consumption is reduced, and the yield and the output of the long-chain dibasic acid of the candida tropicalis are further improved.
Advantageous effects
The invention discovers for the first time that acyl-CoA thioesterase gene ctaCA in candida tropicalis is a key gene in the conversion process of long-chain dibasic acid, the expression of the acyl-CoA thioesterase gene ctaCA can promote the conversion of the long-chain dibasic acid-CoA to free long-chain dibasic acid, and the invention lays a foundation for realizing the synthesis of the long-chain dibasic acid by a new way by taking oil as a raw material.
Drawings
FIG. 1, growth curves of Candida tropicalis original strain and Candida tropicalis mutant strain;
FIG. 2 is a bar graph of the fermentation results of long-chain dibasic acids of Candida tropicalis original bacteria and Candida tropicalis mutant bacteria;
Detailed Description
The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
The source of the biological material is as follows:
plasmid pPIC9K was purchased from Baozoite, Inc.;
candida tropicalis (Candida tropicalis) was purchased from the China center for Industrial microbial cultures Collection (CICC); the number of the strain is CICC 1798;
example 1 verification of the Functions of the ctaCA Gene of Candida tropicalis
1. The construction method of the candida tropicalis genetic engineering recombinant strain comprises the following steps:
(1) extracting genome DNA of Candida tropicalis (Candida tropicalis) thalli, and carrying out PCR amplification by taking the genome DNA as a template to obtain a homology arm ctaCA1 with the length of 523bp, wherein the PCR primer sequence is as follows:
CtacA F1:GGAATTCCCACTATTTTGGCAGAGTT；
CtacA R1:CTGGCAAACTTTCTTCGTCATGATACCTGCT；
wherein, the underlined is the EcoR I restriction site;
the PCR amplification system is 50 mu l:
2 × HiFi-PCR master 25 μ l, concentration 10 μmol/L primer CtacA F12.5. mu.l of primer CtacA R with a concentration of 10. mu. mol/L12.5. mu.l, template 2.5. mu.l, in ddH2O, complementing 50 mu l;
the PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 57 ℃ for 30sec, extension at 72 ℃ for 1.5min, 30 cycles; extending at 72 deg.C for 10min, and storing at-20 deg.C;
(2) extracting pPIC9K plasmid, using the plasmid as a template, carrying out PCR amplification to obtain Kan fragment with length of 1523bp, wherein the PCR primer sequence is as follows:
Kan F2:TCTTGGGGTTGAGGCCGTTGAGCA；
Kan R2:ATTGTGTGAATTCAGTGAGTCAGTCATCAGG；
wherein, the underlined is the EcoR I restriction site;
the PCR amplification system is 50 mu l:
2 × HiFi-PCR master 25 μ L, primer Kan-F with concentration of 10 μmol/L22.5. mu.l of primer Kan-R with a concentration of 10. mu. mol/L22.5. mu.l, template 2.5. mu.l, in ddH2O, complementing 50 mu l;
the PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 3.5min, 30 cycles; extending at 72 deg.C for 10min, and storing at-20 deg.C;
(3) performing overlapping PCR on the CtacA1 fragment prepared in the step (1) and the kan fragment prepared in the step (2) to prepare a CtacA1-kan fragment with the length of 2046 bp; the primary amplification system of the overlapping PCR is 25 mu l:
CtacA1 fragment 4. mu.l; 4 μ l of kan fragment; 2 × HiFi-PCR master 12.5 μ l; ddH2O 4.5μl；
The primary amplification procedure for the overlapping PCR is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 57 ℃ for 30sec, extension at 72 ℃ for 1.5min, 5 cycles; extending for 2min at 72 ℃;
the complementary amplification system of the overlapping PCR is 25 μ l:
upstream primer CtacA F 12 mu l of the solution; downstreamPrimer Kan R2 2μl；2×HiFi-PCR master 12.5μl；ddH2O 8.5μl；
The complementary amplification procedure for overlapping PCR is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 5min, 30 cycles; extending at 72 deg.C for 10min, and storing at-20 deg.C;
2. candida tropicalis competence was prepared by the following steps:
(i) inoculating Candida tropicalis (Candida tropicalis) into 250ml triangular flask containing 50ml thallus proliferation medium, culturing at 30 deg.C and 200rpm/min overnight in shaking table;
the thallus multiplication culture medium comprises the following components per liter:
2g of glucose, 2g of peptone and 1g of yeast extract powder, wherein the pH value is natural;
(ii) spreading the overnight-cultured bacterial liquid on a solid YPD culture medium, and culturing at 30 ℃ for 1-2 days to obtain a Candida tropicalis (Candida tropicalis) single colony; picking single colony by using an inoculating loop, putting the single colony into 50ml of thallus multiplication culture medium, culturing for 12h at 30 ℃ and 200rpm/min, transferring, and culturing for 10 h;
the YPD solid culture medium comprises the following components per liter:
2g of glucose, 2g of peptone, 1g of yeast extract powder and 2g of agar, wherein the pH value is natural;
(iii) taking 1.5ml of bacterial liquid into an Ep tube, centrifuging at 3000rpm/min for 1min, collecting thalli, and blowing and beating suspended cells by using 1.5ml of precooled sterile water;
(iv) centrifuging at 3000rpm/min for 1min, discarding the supernatant, and suspending the cells with 1ml of pre-cooled sterile water;
(v) centrifuging at 3000rpm/min for 1min, discarding the supernatant, and suspending the cells with 1ml of 1mol/L precooled sorbitol;
(vi) centrifuging at 3000rpm/min for 1min, discarding supernatant, and suspending with 80 μ L precooled sorbitol to obtain Candida tropicalis electrotransformation competence; and (4) storing the prepared competent cells at-80 ℃ for later use.
3. Transformation of the CtacA1-kan fragment into Candida tropicalis cells
(i) The CtacA1-kan fragment is cut by restriction enzyme EcoR I, the cutting system is as follows, the total system is 40 mu L:
(ii) concentrating and purifying the enzyme digestion product
(1) Adding 1/10 volumes of 3M sodium acetate and 2.5 times volume of anhydrous ethanol, and placing in a refrigerator at-20 deg.C for 20 min;
(2)12000r/min, centrifuging for 5min to obtain precipitate;
(3)300 μ L of ethanol with 70% volume percentage resuspend the pellet;
(4) centrifuging at 12000r/min for 5min, removing ethanol, and air drying at 37 deg.C for 30 min;
(5) adding 15-18 mu L ddH2O resuspend DNA and store at-20 ℃.
(iii) Electric conversion
Measuring the concentration of the ctacA1-kan fragment by using a nucleic acid ultramicro spectrophotometer (BioFuture MD2000), performing electrotransformation after the concentration reaches 500 mu G/ml, wherein the electrotransformation condition is 1500V and 5ms, then culturing in a recovery solution containing 1mol/L sorbitol, recovering the obtained cells, coating 100 mu L of the recovered cells on a YPD solid culture medium containing 1mg/mLG418 (geneticin), culturing at 30 ℃ for 3 days, and screening a transformant with G418 resistance;
the resuscitation solution is 1mol/L sorbitol;
the YPD solid culture medium comprises the following components per liter:
2g of glucose, 2g of peptone, 1g of yeast extract powder and 2g of agar, and the pH value is natural.
4. Culture and identification of positive recombinant bacteria
Inoculating the transformant obtained by screening into YPD liquid culture medium containing G418 resistance, culturing overnight, sucking 1mL of bacterial liquid, extracting genome DNA by using kit provided by Shanghai bioengineering Co., Ltd, taking the obtained genome DNA as template, CtacAF F1And Kan R2PCR amplification was performed for the primers. Agarose gel electrophoresis demonstrated the transformation of the exogenous fragment ctacA1-kan into the genome.
The YPD liquid culture medium comprises the following components per liter:
2g of glucose, 2g of peptone and 1g of yeast extract powder, and the pH value is natural.
The method for verifying the influence of the knockout of the ctaCA gene on the oil absorption rate of cells by utilizing the fermentation of the candida tropicalis genetic engineering recombinant bacteria comprises the following steps:
respectively inoculating candida tropicalis primordium and the recombinant strain seed liquid into YPD liquid culture media, and culturing for 20 hours at the temperature of 30 ℃; OD was measured every two hours600And obtaining the growth curves of the original candida tropicalis bacteria and the recombinant candida tropicalis bacteria, wherein the results are shown in figure 1.
The fermentation medium comprises the following components:
20g of peptone, 10g of yeast powder, 20g of glucose and water, and the pH value is 7.0.
OD according to FIG. 1600The growth rate of the recombined candida tropicalis is similar to that of candida tropicalis protobacteria, and the knockout of the gene does not influence the metabolism of a glucose carbon source of the candida tropicalis.
Example 2 Candida tropicalis ctaCA Gene multicopy Strain construction
On the basis of obtaining the full-length gene of the ctaCA, a specific primer is designed to clone a target gene, and a recombination reaction system is configured by a vector and the target gene through a seamless cloning technology to carry out recombination reaction. Transformed into DH5 alpha competence and positive clones were selected. After the sequencing is correct, the extracted plasmid is electrically transferred into the competence of the candida tropicalis. Fermentation verifies the influence of increasing the copy number of the ctaCA gene on the oil absorption rate of cells. The technical core is that a vector is linearized by utilizing a homologous recombination principle, and a terminal sequence of the linearized vector is introduced into the 5 ' end of an insert PCR primer, so that the 5 ' and 3 ' extreme ends of a PCR product respectively have sequences (15 bp-20 bp) consistent with the two terminals of the linearized vector. After the PCR product with the carrier terminal sequences at the two ends and the linearized carrier are mixed according to a certain proportion, the conversion can be carried out only by reacting for 30min under the catalysis of seamless exchange enzyme, the directional cloning is completed, and the positive rate can reach more than 95%. The method comprises the following specific steps:
(i) extracting genome DNA of Candida tropicalis, performing PCR amplification by using the genome DNA as a template to obtain a ctaCA gene with the length of 3325bp, wherein the PCR primer sequence is as follows:
CtacA F2:ctcactatagggagagcggccgcTTCTTCATAATAATGCTAACTT；
CtacA R2:catccggaagatctggcggccgcATTATAATAATTTGATTTTC；
wherein the Not I restriction site is underlined;
the PCR amplification system is 50 mu l:
the PCR amplification procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15sec, annealing at 51 ℃ for 15sec, extension at 72 ℃ for 2min, 30 cycles; extending at 72 deg.C for 5min, and storing at-20 deg.C;
(ii) the plasmid vector was digested with restriction enzyme Not I in the following manner, for a total of 50. mu.L:
the vector is a pZERO-Blunt cloning vector which is constructed in a laboratory and has a G418 resistance label;
(iii) purifying the enzyme digestion product by using a SanPrep column type PCR product purification reagent box column, dephosphorizing the column purification product, configuring a recombination system for recombination reaction, converting and coating the reaction product, and selecting a single colony to identify a positive clone by adopting a colony PCR method; go to Shanghai Boshang sequencing.
The dephosphorylation system comprises the following components:
the recombination system is as follows:
the PCR primer sequences are as follows:
CtacA F2:ctcactatagggagagcggccgcATAGAAGAGTTATTAAAATG；
CtacA R2:catccggaagatctggcggccgcATACCACACAGAGAGAATACAT；
(iv) after confirming that the sequence information is correct, the corresponding plasmid was extracted and electrotransferred into Candida tropicalis competence, the procedure was as described in example 1- (iii), and the culture and identification of the positive recombinant bacteria were as described in example 1.
The method for verifying the influence of increasing the copy number of the ctaCA gene on the yield of the dibasic acid by utilizing the fermentation of the candida tropicalis genetic engineering recombinant bacteria comprises the following steps:
respectively inoculating multi-copy recombinant candida tropicalis, candida tropicalis original bacteria and candida tropicalis genetic engineering recombinant bacteria into YPD liquid culture media, and culturing for 14 hours at the temperature of 30 ℃; respectively inoculating 10ml of multi-copy recombinant bacterium liquid, 10ml of original bacterium liquid and 10ml of recombinant bacterium liquid into 100ml of fermentation culture medium, respectively adding 5ml of grease after culturing for 12 hours, and entering an acid production period; in the acid production period, the pH is adjusted to 7.5 every 12 hours or 24 hours, and the acid production period lasts for 4-5 days.
The fermentation medium comprises the following components:
glucose 64g/L, (NH)4)2SO41g/L yeast extract 2g/L, VB1 0.1g/L、NaCl 2g/L、KH2PO4 4g/L、Na2HPO4·12H2O10.08 g/L and urea 2g/L, Mg2SO4·7H2O6.15 g/L, water preparation, pH 7.0;
after the fermentation is finished, the yield of the dibasic acid is measured by an acid-base titration method, and the result is shown in fig. 2.
According to the yield of the long-chain dibasic acid shown in FIG. 2, the yield of the long-chain dibasic acid (DCA) of the recombined Candida tropicalis is greatly reduced compared with that of the Candida tropicalis original bacteria, the yield of the long-chain dibasic acid of the multi-copy recombined Candida tropicalis is improved by 58% compared with that of the Candida tropicalis original bacteria, and the thallus does not show poor growth due to the increase of the copy number. Therefore, the capability of yeast transformation to produce long-chain dibasic acid is enhanced after the copy number of the ctaCA gene is increased, and the ctaCA gene is a key gene for candida tropicalis long-chain dibasic acid transformation.
SEQUENCE LISTING
<110> university of Qilu Industrial science
<120> acyl-CoA thioesterase gene of Candida tropicalis and application thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1401
<212> DNA
<213> Candida tropicalis
<400> 1
atgaatcctg ctgaggctgc agatgcagca gccactattt tggcagagtt gcgagataag 60
caaatcaatc caaataaagt aacttggata gatgcattaa aagaacgtga aaaattgcgt 120
gccgagggga aaacaataga tagttttagt tatgttgatc caaagaccac agttgtcggt 180
gagaaaacac gaagtgattc attttctttc ttattattac cttttaagga cgataaatgg 240
ctttgcgacg catacataaa tgcttttgga cgacttagag tggcccaatt atttcaagat 300
cttgatgcac ttgccggtag aattgcttat agacattgtt ctcctgctga accagttaat 360
gtcacagcaa gtgtggatag agtatatatg gtgaagaaag tcgatgagat taacaactat 420
aattttgttt tagctggttc tgttacttgg actggtagat cttccatgga gatcacagtg 480
aaaggttacg catttgaaga tgctgttcct gaaatcacta acgaagaaag tttgccagca 540
gagaacgtgt ttttggctgc taatttcaca ttcgttgcaa gaaatccact tacacataaa 600
tcatttgcta taaaccgatt attacctgtc acagaaaaag attggattga ttatagaaga 660
gctgaatccc ataatgctaa aaagaagtta atggctaaga ataaaaagat acttgagcca 720
accgcagagg aatctaaatt gatctacgac atgtggaaat cctcaaaatc tttaaaaaat 780
attgatcgtc aggatgatgg aatagcattt atgaaggaca ccacaatgaa aagtaccatg 840
tttatgcaac ctcaatatag aaacagacat tcttatatga tttttggtgg ttacttatta 900
cgtcaaacat tcgagcttgc ctattgtaca gcagccacct tttcattagc cggaccaaga 960
tttgttagtt tagattcaac tacttttaaa aatccagttc ctgtcggttc agtcttaacc 1020
atggactcct ctatttctta caccgaacac gtacacgatg ggatagaaga gatagactct 1080
gattctcctt ttaatttctc tcttcctgcc actaataagt tgtccaagaa tccagaagca 1140
ttcttgtcag aacctgggac tttgattcaa gtcaaagtag atacttatat tcagcaattg 1200
gaacagtcag aaaagaagcc agccggtacc ttcatatatt ctttctacgt tccaaaggaa 1260
agtgtcagtg tggatggtaa agcttcttat tgtaccgtta tcccacagac ttactctgaa 1320
atgatgacat atgttggtgg tagaagaaga gcacaggaaa ccgctaatta tgtagaaact 1380
ttaccaagta ctaacaacta a 1401
<210> 2
<211> 466
<212> PRT
<213> Candida tropicalis
<400> 2
Met Asn Pro Ala Glu Ala Ala Asp Ala Ala Ala Thr Ile Leu Ala Glu
1 5 10 15
Leu Arg Asp Lys Gln Ile Asn Pro Asn Lys Val Thr Trp Ile Asp Ala
20 25 30
Leu Lys Glu Arg Glu Lys Leu Arg Ala Glu Gly Lys Thr Ile Asp Ser
35 40 45
Phe Ser Tyr Val Asp Pro Lys Thr Thr Val Val Gly Glu Lys Thr Arg
50 55 60
Ser Asp Ser Phe Ser Phe Leu Leu Leu Pro Phe Lys Asp Asp Lys Trp
65 70 75 80
Leu Cys Asp Ala Tyr Ile Asn Ala Phe Gly Arg Leu Arg Val Ala Gln
85 90 95
Leu Phe Gln Asp Leu Asp Ala Leu Ala Gly Arg Ile Ala Tyr Arg His
100 105 110
Cys Ser Pro Ala Glu Pro Val Asn Val Thr Ala Ser Val Asp Arg Val
115 120 125
Tyr Met Val Lys Lys Val Asp Glu Ile Asn Asn Tyr Asn Phe Val Leu
130 135 140
Ala Gly Ser Val Thr Trp Thr Gly Arg Ser Ser Met Glu Ile Thr Val
145 150 155 160
Lys Gly Tyr Ala Phe Glu Asp Ala Val Pro Glu Ile Thr Asn Glu Glu
165 170 175
Ser Leu Pro Ala Glu Asn Val Phe Leu Ala Ala Asn Phe Thr Phe Val
180 185 190
Ala Arg Asn Pro Leu Thr His Lys Ser Phe Ala Ile Asn Arg Leu Leu
195 200 205
Pro Val Thr Glu Lys Asp Trp Ile Asp Tyr Arg Arg Ala Glu Ser His
210 215 220
Asn Ala Lys Lys Lys Leu Met Ala Lys Asn Lys Lys Ile Leu Glu Pro
225 230 235 240
Thr Ala Glu Glu Ser Lys Leu Ile Tyr Asp Met Trp Lys Ser Ser Lys
245 250 255
Ser Leu Lys Asn Ile Asp Arg Gln Asp Asp Gly Ile Ala Phe Met Lys
260 265 270
Asp Thr Thr Met Lys Ser Thr Met Phe Met Gln Pro Gln Tyr Arg Asn
275 280 285
Arg His Ser Tyr Met Ile Phe Gly Gly Tyr Leu Leu Arg Gln Thr Phe
290 295 300
Glu Leu Ala Tyr Cys Thr Ala Ala Thr Phe Ser Leu Ala Gly Pro Arg
305 310 315 320
Phe Val Ser Leu Asp Ser Thr Thr Phe Lys Asn Pro Val Pro Val Gly
325 330 335
Ser Val Leu Thr Met Asp Ser Ser Ile Ser Tyr Thr Glu His Val His
340 345 350
Asp Gly Ile Glu Glu Ile Asp Ser Asp Ser Pro Phe Asn Phe Ser Leu
355 360 365
Pro Ala Thr Asn Lys Leu Ser Lys Asn Pro Glu Ala Phe Leu Ser Glu
370 375 380
Pro Gly Thr Leu Ile Gln Val Lys Val Asp Thr Tyr Ile Gln Gln Leu
385 390 395 400
Glu Gln Ser Glu Lys Lys Pro Ala Gly Thr Phe Ile Tyr Ser Phe Tyr
405 410 415
Val Pro Lys Glu Ser Val Ser Val Asp Gly Lys Ala Ser Tyr Cys Thr
420 425 430
Val Ile Pro Gln Thr Tyr Ser Glu Met Met Thr Tyr Val Gly Gly Arg
435 440 445
Arg Arg Ala Gln Glu Thr Ala Asn Tyr Val Glu Thr Leu Pro Ser Thr
450 455 460
Asn Asn
465
Claims (2)
1. acyl-CoA thioesterase genectacAThe application of the long-chain dicarboxylic acid in preparing the long-chain dicarboxylic acid by modifying candida tropicalis;
the acyl-CoA thioesterase genectacAThe nucleotide sequence is shown as SEQ ID NO. 1;
the long-chain dicarboxylic acid is a straight-chain aliphatic dicarboxylic acid with more than 12 carbon atoms and alpha and omega positions respectively provided with a carboxyl.
2. Use according to claim 1, characterized by the following steps:
construction of acyl-CoA Thiolidase GenectacAThe acyl-CoA thioesterase gene is realized by multi-copy recombination candida or replacement of a promoterctacAIs overexpressed.
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| CN1614004A (en) * | 2004-12-07 | 2005-05-11 | 清华大学 | Constructing method for candida tropicalis gene engineering recombinant bacterium |
| CN102839133A (en) * | 2011-06-21 | 2012-12-26 | 上海凯赛生物技术研发中心有限公司 | Strain producing long chain dibasic acid, and application thereof |
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| CN105189731A (en) * | 2012-12-19 | 2015-12-23 | 沃德金有限公司 | Biological methods for preparing a fatty dicarboxylic acid |
| CN106754979A (en) * | 2016-12-26 | 2017-05-31 | 齐鲁工业大学 | A kind of gene of long-chain fat acid transporter of regulation and control candida tropicalis and its application |
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| MY165893A (en) * | 2011-07-06 | 2018-05-18 | Verdezyne Inc | Biological methods for preparing a fatty dicarboxylic acid |
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| CN105189731A (en) * | 2012-12-19 | 2015-12-23 | 沃德金有限公司 | Biological methods for preparing a fatty dicarboxylic acid |
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