CN101899454B - 2-D-arbaitol dehydrogenase gene, recombinant protein thereof, escherichia coli containing gene and application thereof - Google Patents
2-D-arbaitol dehydrogenase gene, recombinant protein thereof, escherichia coli containing gene and application thereof Download PDFInfo
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Abstract
The invention discloses a new 2-D-arbaitol dehydrogenase gene, a recombinant protein thereof, escherichia coli containing the gene and an application thereof, wherein the base sequence of the 2-D-arbaitol dehydrogenase encoding gene is as shown in SEQ ID No. 1; the recombinant protein encoded by the gene is 2-D-arbaitol dehydrogenase and has the amino acid sequence as shown in SEQ ID No.2; the escherichia coli is to lead escherichia coli BL21 (DE3) to contain the 2-D-arbaitol dehydrogenase encoding gene, and the 2-D-arbaitol dehydrogenase encoding gene comprises the base sequence as shown in the SEQ ID No. 1; and the escherichia coli can be synthesized into D-arbaitol by taking ribulose or glucose as a substrate after induction. The invention firstly discloses a method for introducing the2-D-arbaitol dehydrogenase encoding gene into the escherichia coli BL21 (DE3) for forming a genetic engineering strain, thereby successfully constructing a metabolic pathway of synthesizing the D-arbaitol by taking the glucose as the substrate.
Description
Technical field
The present invention relates to 2-D-arabitol dehydrogenase gene and the recombinant protein thereof of candiyeast H2 (Candida sp.H2); More particularly; Relate to a kind of gene that from candiyeast H2 (Candida sp.H2), is separated to; The engineering bacteria that utilizes gene engineering method to make up, and use engineering bacteria to produce the D-arabitol.
Background technology
D-arabitol (D-arabitol) is that a kind of white crystal, pleasantly sweet, specific rotation are 138 ° pentasaccharides alcohol.Ooze one of important products of mikrobe as anti-height, it extensively is present in nature, is mainly used in industries such as food, chemical industry, medicine.At present, the purposes of the tool market potential of D-arabitol is as the synthetic Xylitol of raw material.
People start from mid-term in 20th century to the research of accumulation D-arabitol bacterial strain, and external report is many, and domestic research is fewer.It is reported that most anti-height ooze bacterial strain can both synthesize the D-arabitol, but the bacterial strain of high yield D-arabitol not a lot; Also there is the part superior strain to be obtained, mainly concentrates on fungi by screening.Current, mainly there are shortcomings such as yield is low, fermentation period is long, and by product is many in the method for the synthetic D-arabitol of microbial fermentation D-glucose.Be directed to these shortcomings, utilize genetically engineered to transform or rebuild engineering bacteria and demonstrate wide prospect.
At present, the research for 2-D-arabitol desaturase and gene thereof is very few.From results reported: most gene length is 843-849 base, and primary structure is a 281-283 amino acid, and it belongs to the short-chain dehydrogenase enzyme family analysis revealed through aminoacid sequence; The method that obtains mainly is through making up genomic library and λ-cDNA library or passing through the purifying original protein.But do not see that the 2-D-arabitol dehydrogenase gene that passes through to be obtained makes up the report that genetic engineering bacterium is studied.
Before this, the applicant has obtained a strain D-arabitol superior strain candiyeast H2 (Candida sp.H2) through seed selection.This candida bacterial strain H2 on August 28th, 2009 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, its preserving number is CGMCC No.3255.Gene and recombinant protein research thereof to the 2-D-arabitol desaturase among the candiyeast H2 (Candida sp.H2) have very big realistic meaning.
Summary of the invention
The purpose of this invention is to provide a kind of new 2-D-arabitol dehydrogenase gene and recombinant protein and the intestinal bacteria and the application thereof that contain this gene.
For realizing above-mentioned purpose, the present invention is cloned into a 2-D-arabitol dehydrogenase gene through PCR from candiyeast H2 (Candida sp.H2); Utilize the gene engineering method construction recombination plasmid and change over to and make up genetic engineering bacterium in the escherichia expression system; For adopting engineering bacteria is that substrate production D-arabitol provides a feasible approach with glucose.
The present invention realizes that the technical scheme that its purpose is taked is: the base sequence of this 2-D-arabitol dehydrogenase coding genes is shown in SEQ ID NO:1.
The coded recombinant protein recombinant protein of 2-D-arabitol dehydrogenase coding genes of the present invention is a 2-D-arabitol desaturase, has the aminoacid sequence shown in SEQ ID NO:2.
Further, the recombinant vectors of recombinant protein according to the invention is the pET30-a expression vector that contains 2-D-arabitol dehydrogenase coding genes, and the base sequence of said 2-D-arabitol dehydrogenase coding genes is shown in SEQ ID NO:1.
Intestinal bacteria provided by the invention are in e. coli bl21 (DE3), to contain 2-D-arabitol dehydrogenase coding genes, and this 2-D-arabitol dehydrogenase coding genes has the base sequence shown in SEQ ID NO:1.
Further, the recombinant vectors in the intestinal bacteria according to the invention is the pET30-a expression vector that contains 2-D-arabitol dehydrogenase coding genes, and the base sequence of said 2-D-arabitol dehydrogenase coding genes is shown in SEQ ID NO:1.
The application that intestinal bacteria of the present invention are used to prepare the D-arabitol is that these intestinal bacteria are the synthetic D-arabitol of substrate through inducing the back with ribulose or glucose.
Compared with prior art, the invention has the beneficial effects as follows: the 2-D-arabitol dehydrogenase coding genes that obtains is separated in (1) from candiyeast H2 (Candida sp.H2) be a new gene; (2) the present invention disclose first 2-D-arabitol dehydrogenase coding genes is imported e. coli bl21 (DE3) thus form a genetic engineering bacterium successfully to make up one be the metabolic pathway of the synthetic D-arabitol of substrate with glucose, its transformation efficiency is 17%.
The preservation information of biological material specimens:
The biological material specimens of preservation: candiyeast H2 (Candida sp.H2);
Depositary institution: (be called for short: CGMCC) at China Committee for Culture Collection of Microorganisms common micro-organisms center;
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica (postcode: 100101);
Preservation date: on August 28th, 2009;
Preservation registration number: CGMCC No.3255.
Description of drawings
Fig. 1 is the electrophoresis result of the PCR product of the 2-D-arabitol dehydrogenase gene among the candiyeast H2 (Candida sp.H2), wherein, and M:DNA Mark; 1: the purpose band; 2: contrast.
Fig. 2 is the SDS-PAGE electrophorogram of the 2-D-arabitol desaturase among the candiyeast H2 (Candida sp.H2), wherein, and M: protein Mark; 1: e. coli bl21 (DE3) cell pyrolysis liquid; 2: through IPTG inductive e. coli bl21 (DE3) cell pyrolysis liquid; 3: e. coli bl21 (DE3) cell pyrolysis liquid that contains 2-D-arabitol dehydrogenase gene; 4: e. coli bl21 (DE3) cell pyrolysis liquid that contains 2-D-arabitol dehydrogenase gene through the IPTG inductive.
Embodiment
Further specify the present invention below.Below enforcement only be for illustrative purposes, and and unrestricted protection scope of the present invention.
Embodiment 1:
Three base sequences that derive from the 2-D-arabitol dehydrogenase gene of white candiyeast (candida albicans) (GeneBank No.L16227.1), candida tropicalis (candida tropicalis) (GeneBank No.U00675.1) and candida dubliniensis (candida dubliniensis) (GeneBank No.XM002420767.1) are carried out multiple ratio to obtaining conserved sequence [5 '
1ATGGA-TCCTCATCATACTGGTCATACGACA
31------
813CTTGGT-GT-GATGG-GGTTACGAATGTTGGTAA
8493 ']; Design a pair of primer HF [5 '-ATGGANTCCGCATCATACTGGTCATACG-3 '] and HR [5 '-TTACCAACATTCGTAACCNCCATCNAC-3 '] with conserved sequence then and carry out the pcr gene amplification, wherein the condition of PCR is 94 ℃ of 45S, 52 ℃ of 30S, 72 ℃ of 1min totally 35 circulations.Get the above-mentioned PCR product of 40 μ l and carry out the agarose gel electrophoresis evaluation, as shown in Figure 1, find between 800-900bp, to have a specific amplification band.Reclaim test kit to the recovery of tapping rubber of above-mentioned PCR product through the quick glue of dna fragmentation.The PCR product that reclaims is connected with the T carrier; The back transforms the entering bacillus coli DH 5 alpha and increases; The picking bacterial plaque checks order, and the result of order-checking is [SEQ ID NO:1 is the nucleotide sequence of candiyeast H2 (Candidasp.H2) 2-D-arabitol dehydrogenase gene] shown in SEQ ID NO.1.The aminoacid sequence that utilizes the coded recombinant protein of the base sequence of SEQ ID NO.1 is [shown in SEQ ID NO.2; This sequence is the aminoacid sequence of candiyeast H2 (Candida sp.H2) 2-D-arabitol desaturase] compare with the aminoacid sequence of the D-arabitol desaturase of having reported, the result is following:
1 MESAS-YWSYDNIVPSFRLDGRLAILTGGSGGLAAVVARALLAQGADIALIDVNLERTQQ 59
2 MSTDSQYWAYDNIVPSFRLDGRLAILTGGSGGLAAVVARALLAKGADVALVDMNLERTQQ 60
3 -MDSSSYWSYDNIVPSFRLDGKLVIITGGSGGLSAVVSRALLAKGADIALIDMNLERTQQ 59
4 -MES-AYWSYENIVPSFRLDGKLVILTGGSGGLAAVVSRALLAKGADIALVDMNLERTQQ 58
5 -MDS-AYWSYDNIVPSFRLDGKLVILTGGSGGLAAVVSRALLAKGADVALVDMNLERTQQ 58
6 -----MDYSYANVVPNFRLDGRLAIITGGSGGLAAVISRALLAQGADVALIDMNLERTKS 55
7 -----MDYSYDNVVPNFRLDGRLAIVTGGSGGLASVISRALLAQGADVALIDMNLERTNV 55
8 -----MDYSYDNVVPSFRLDGRLAILTGGSGGLAAVVSRALLAQGAQIALLDMNLERTKA 55
9 ----MSEYSYENVVPNFRLDGRLAILTGGSGGMSHVVSRALVAQGADVALVDINLERTKQ 56
::* *:**.*****:*.*:******:: *::***:*:**::**:*:*****:
1 AAKEILLWGEEQMKGKHESPIGQVSAWSCNIGDAESVELTFKAINEHHGKISDLLINSAG 119
2 AAKDVLIWGEEQMKGKNESPIGQVSAWACNIGDAESVELTFKAINEHHAKIADLLVNSAG 120
3 AARDVLQWGEEQMKGKHESPIGQVSAWSCNIGDAEAVELTFKAINEHHGKVASVLINTAG 119
4 AARDVLQWGEEQMKGKYESPIGQVSAWSCNIGDAEAVDMTFKAINEHHGKISSVLVNTAG 118
5 AARDVLQWGEEQMKGKYESPIGQVSAWSCNIGDAEAVDLTFKAINEHHGKISSVLVNTAG 118
6 AAKEVLGWGEETLKGEHASAIGQVSAWSCNIGDAEAVDATFSSINEHHGKIADLLINTAG 115
7 AAKELLKWGEDTLKGAHESAIGQVSAWSCDIGDAGSVEQTFVAINEKHGKIADLLINSAG 115
8 AAKELETWGQESLKGAHESPVGAVSAWSCNIGDFEQVEECFKNINEHHDMVADLLINTAG 115
9 AAKGVLSWAEATLKGDKASPVGMVSAWSCNIGDAESVATTFAEINEAHGKIADLLINTAG 116
**: : *.: :** *.:* ****:*:*** * * *** * ::.:*:*:**
1 YAENFPAEEYPAKNAESIVKVNGLGAFYVSQSFARPLIAANKKGSIILIGSMSGTIVNDP 179
2 YAENFPAEEYPAANAEAIMKVNGLGAFYVSQSFARALIANNKPGSIILIGSMSGTIVNDP 180
3 YAENFPAEEYPAKNAENIMKVNGLGSFYVSQAFARPLIQNNMTGSIILIGSMSGTIVNDP 179
4 YAENFPAEEYPAKNAENLMKVNGLGSFYVSQAFARPLIQNNMTGSIILIGSMSGTIVNDP 178
5 YAENFPAEEYPAKNAENLMKVNGLGSFYVSQAFARPLIQNNMTGSIILIGSMSGTIVNDP 178
6 YCENFPAETYPATNAESIMKVNGLGSFYVSQSFARPLIQNNLRGSIILIGSMSGTIVNDP 175
7 YCENFPAEEYPARNAEGIMKVNGLGAFYVSQSFARPLIQNNLRGSIILIGSMSGTIVNDP 175
8 YCENFPAEEYPSANAEGILKVNGLGAFYVSQAFARPLISSNKKGSIILVGSMSGTIVNDP 175
9 YCENFPAEDYPALNAEGIMRVNGLGSFYVAQAFAKPLIQNNLRGSIILIGSMSGTIVNDP 176
*.****** **: *** :::*****:***:*:**:.** * *****:***********
1 QPQCMYNMSKAGVIHLVRSLACEWAKYNIRVNTLSPGYILTPLTRNVIAGHADMKAEWES 239
2 QPQCMYNMSKAGVIHLCRSLAVEWAKYNIRVNTLSPGYILTPLTRNVISGHADMKAEWES 240
3 QPQCMYNMSKAGVIHLARSLACEWAKYNIRVNTLSPGYILTPLTRNVISGHTEMKTEWES 239
4 QPQCMYNMSKAGVIHLARSLACEWAKYNIRVNTLSPGYILTPLTRNVISGHTEMKAEWES 238
5 QPQCMYNMSKAGVIHLARSLACEWAKYNIRVNTLSPGYILTPLTRNVISGHTEMKTEWES 238
6 QPQCMYNMSKAGVIHLVRSLACEWAKYNIRVNTLSPGYILTPLTRNVISGHTEMKEAWES 235
7 QPQAMYNMSKAGVIHLVRSLACEWAKYNIRVNTLSPGYILTPLTKNVIAGHSEMKDAWES 235
8 QPQCMYNMSKAGVIHLTRSLACEWAKFNIRVNTLSPGYILTPLTRNVISGHSDMKEAWES 235
9 QPQCMYNMSKAGVIHLVRSLACEWAKYNIRVNSLSPGYILTPLTKNVISGHAEMKEAWES 236
***.************ **** ****:*****:***********:***:**::** ***
1 KIPMKRMAEPKEFVGSILYLASDSASSYTTGHNLVVDGGYECW 282
2 KIPMKRMAEPKEFVGSILYLASDSASSYTTGHNLVVDGGYECW 283
3 KIPMKRMAEPKEFVGSILYLASDSASSYTTGHNLVVDGGYECW 282
4 KIPMKRMAEPKEFVGSILYLASESASSYTTGHNLVVDGGYECW 281
5 KIPMKRMAEPKEFVGSILYLASESASSYTTGHNLVVDGGYECW 281
6 KIPMKRMAEPKEFVGSILYLASETASSYTTGHNLVVDGGYECW 278
7 KIPMKRMAEPKEFVGSILYLASESASSYTTGHNLVVDGGYECW 278
8 KVPMKRMAEPKEFVGSILYLASESASSYTTGHNLVVDGGYECW 278
9 KIPMKRMADPKEFVGSILYLASETASSYTTGHNLVVDGGYECW 279
*:******:*************::*******************
Wherein, 1: candiyeast H2 (Candida sp.H2); 2:Lodderomyces elongisporus yeast NRRLYB-4239 (Lodderomyces elongisporus NRRLYB-4239) (GenBank accession No.XP_001523855.1); 3: candida tropicalis MYA-3404 (Candida tropicalis MYA-3404) (GenBank accession No.XP_002548729.1); 4: candida dubliniensis CD 36 (Candida dubliniensis CD 36) (GenBank accession No.XP_002420812.1); 5: white candiyeast (Candida albicans) (GenBank accession No.P43066.1); 6: pichia stipitis CBS6054 (Pichia stipitis CBS6054) (GenBank accessionNo.XP_001385035.1); 7: Pichia guilliermondii ATCC 6260 (Pichia guilliermondiiATCC6260) (GenBank accession No.XP_001486239.1); 8: the inferior Dbaly yeast CBS 767 of the Chinese (Debaryomyces hansenii CBS767) (GenBank accession No.XP_456858.1); 9: Candida lusitaniae ATCC 42720 (Clavispora lusitaniae ATCC42720) (GenBank accession No.XP_002619221.1)." * ": identical; ": ": highly similar; ". ": part is similar; Space: different or jagged fully.
We can find out from the right result of above-mentioned multiple ratio; The aminoacid sequence of the recombinant protein of the new 2-D-arabitol dehydrogenase gene coding that the present invention obtained is up to 91% with the similarity of reporting sequence, explains that the 2-D-arabitol dehydrogenase gene that the present invention clone obtains is a new gene.
Embodiment 2:
With 2-D-arabitol dehydrogenase gene (shown in SEQ ID NO.1) be stencil design a pair of expression primer HEF [5 '-
CATATGGAATCCGCATCATACTGGTC-3 '] (annotate: the line part is the restriction enzyme site of NdeI) and HER [5 '-
AAGCTTACCAACATTCGTAACCTCCAT-3 '] (annotate: the line part be the restriction enzyme site of Hind III) carry out pcr gene and clone, and wherein the condition of PCR is 94 ℃ of 45S, 50 ℃ of 30S, 72 ℃ of 1min totally 30 circulations.Above-mentioned PCR product is connected with the T carrier, transforms and get into bacillus coli DH 5 alpha, the picking bacterial plaque checks order.Sequencing result and 2-D-arabitol dehydrogenase gene (shown in SEQ IDNO.1) are compared, and the result shows that the 2-D-arabitol dehydrogenase gene (shown in SEQ ID NO.1) that successfully will contain NdeI and HindIII restriction enzyme site inserts the T carrier.Utilize NdeI and HindIII that the T carrier and the pET-30a expression vector (containing NdeI and HindIII restriction enzyme site) of the 2-D-arabitol dehydrogenase gene (shown in SEQ IDNO.1) that contains NdeI and HindIII restriction enzyme site are carried out double digestion then; The back is cut product with the T4 ligase enzyme to enzyme and is connected; The back transforms and gets into e. coli bl21 (DE3), and the picking bacterial plaque checks order.Sequencing result and 2-D-arabitol dehydrogenase gene (shown in SEQID NO.1) are compared; The result shows sequence 100% pairing, explains that pET30-a recombinant vectors that contains 2-D-arabitol dehydrogenase coding genes and the e. coli bl21 (DE3) that contains this carrier are successfully made up.In addition; Connecing the e. coli bl21 (DE3) that contains 2-D-arabitol dehydrogenase gene contains in the LB substratum of kantlex at 37 ℃ in 200ml; Cultivate under the condition of 250rpm and reached 0.5-1.0 up to the OD value in 2-3 hour; The IPTG that the back adds 0.02mmol induced 10 hours; The result is as shown in Figure 2, the 4th swimming lane (being added with the above-mentioned back cell pyrolysis liquid sample of inducing) about 31KDa, have one clearly all the other swimming lanes of band do not have protein band, explain that the coded recombinant protein of 2-D-arabitol dehydrogenase gene is successfully efficiently expressed after IPTG induces.
Embodiment 3:
Connecing the e. coli bl21 (DE3) that contains 2-D-arabitol dehydrogenase gene contains in the LB substratum of kantlex at 37 ℃ in 200ml; Cultivate under the condition of 250rpm and reached 0.5-1.0 up to OD value in 2-3 hour, the IPTG of back adding 0.02mmol induced 10 hours; Back under the 5000rpm condition centrifugal 15 minutes; Twice of sodium chloride solution washing precipitation with 0.9%; Under the 5000rpm condition centrifugal 15 minutes again, be placed on 35 ℃ with the ribulose solution of the 50g/L of 200ml suspension cell again, cultivated 3 days under the condition of 200rpm.With above-mentioned solution under the 12000rpm condition centrifugal 20 minutes, stay supernatant to utilize performance liquid chromatography-evaporative light scattering detection logotype method (HPLC-ELSD) detection by quantitative solution composition.The detected result of HPLC-ELSD shows, contains 40g/L D-arabitol in the solution, and transformation efficiency is 80%; And original e. coli bl21 (DE3) can not transform ribulose generation D-arabitol in reaction system identical with the BL21 that contains 2-D-arabitol dehydrogenase gene (DE3) and reaction conditions; Explain that the expressed endonuclease capable catalysis ribulose of 2-D-arabitol dehydrogenase gene forms the D-arabitol.
Embodiment 4:
Connecing the e. coli bl21 (DE3) that contains 2-D-arabitol dehydrogenase gene contains in the LB substratum of kantlex at 37 ℃ in 200ml; Cultivate under the condition of 250rpm and reached 0.5-1.0 up to OD value in 2-3 hour, the IPTG of back adding 0.02mmol induced 10 hours; Back under the 5000rpm condition centrifugal 15 minutes; Twice of sodium chloride solution washing precipitation with 0.9%; Under the 5000rpm condition centrifugal 15 minutes again, be placed on 35 ℃ with the ribulose solution of the 80g/L of 200ml suspension cell again, cultivated 6 days under the condition of 200rpm.With above-mentioned solution under the 12000rpm condition centrifugal 20 minutes, stay supernatant to utilize performance liquid chromatography-evaporative light scattering detection logotype method (HPLC-ELSD) detection by quantitative solution composition.The detected result of HPLC-ELSD shows, contains 13.33g/L D-arabitol in the solution, and transformation efficiency is 17%; And original e. coli bl21 (DE3) can not generate the D-arabitol by transforming glucose in reaction system identical with the BL21 that contains 2-D-arabitol dehydrogenase gene (DE3) and reaction conditions; Explanation imports the synthetic route of successfully having built a D-arabitol behind the BL21 (DE3) with 2-D-arabitol dehydrogenase gene, for adopting engineering bacteria to produce the D-arabitol a feasible new way is provided.
SEQUENCE LISTING
< 110>Hangzhou Baojing Biology Chemical Co., Ltd
< 120>a kind of 2-D-arabitol dehydrogenase gene and recombinant protein thereof and the intestinal bacteria and the application thereof that contain this gene
<160>2
<170>PatentIn version 3.1
<210>1
<211>849
<212>DNA
< 213>candiyeast H2 (Candida sp.H2)
<400>1
atggaatccg catcatactg gtcatacgac aatatcgtcc catcattcag actagatggc 60
agattagcca tcttgactgg tggttcaggt ggtttagccg ctgttgtcgc tagagcctta 120
cttgcacaag gtgccgatat tgcccttatt gatgtcaatt tggaaagaac acaacaagcc 180
gctaaggaaa ttttactttg gggtgaagaa caaatgaagg gcaaacacga atctccaatt 240
ggtcaagttt ctgcctggtc ttgtaacatc ggagacgctg aatcagtcga attgacattt 300
aaagctatta acgaacatca cggtaagatt tcagacttgt tgattaattc tgcgggttat 360
gctgaaaact tcccagctga agaatatcct gccaagaatg ctgaatccat agtgaaggtt 420
aatggattgg gtgcatttta cgtttcacaa tcatttgcaa gacctttaat tgctgccaac 480
aagaaaggct ctattattct tattggatca atgtcgggta ccattgtcaa tgatcctcaa 540
ccacaatgta tgtacaacat gtcaaaggct ggtgttatac atttggttcg gtcattggct 600
tgtgaatggg ctaaatataa catcagggtc aacactttga gtcctggtta tattttgact 660
ccattgacta gaaatgtcat tgctggtcat gctgatatga aggctgaatg ggaaagcaaa 720
attccaatga aacgtatggc tgaaccaaag gaattcgttg gatctatttt gtatttggct 780
tctgattccg cctcatctta tactactggg cataacttgg tcgtagatgg aggttacgaa 840
tgttggtaa 849
<210>2
<211>282
<212>PRT
< 213>candiyeast H2 (Candida sp.H2)
<400>2
Met Glu Ser Ala Ser Tyr Trp Ser Tyr Asp Asn Ile Val Pro Ser Phe
1 5 10 15
Arg Leu Asp Gly Arg Leu Ala Ile Leu Thr Gly Gly Ser Gly Gly Leu
20 25 30
Ala Ala Val Val Ala Arg Ala Leu Leu Ala Gln Gly Ala Asp Ile Ala
35 40 45
Leu Ile Asp Val Asn Leu Glu Arg Thr Gln Gln Ala Ala Lys Glu Ile
50 55 60
Leu Leu Trp Gly Glu Glu Gln Met Lys Gly Lys His Glu Ser Pro Ile
65 70 75 80
Gly Gln Val Ser Ala Trp Ser Cys Asn Ile Gly Asp Ala Glu Ser Val
85 90 95
Glu Leu Thr Phe Lys Ala Ile Asn Glu His His Gly Lys Ile Ser Asp
100 105 110
Leu Leu Ile Asn Ser Ala Gly Tyr Ala Glu Asn Phe Pro Ala Glu Glu
115 120 125
Tyr Pro Ala Lys Asn Ala Glu Ser Ile Val Lys Val Asn Gly Leu Gly
130 135 140
Ala Phe Tyr Val Ser Gln Ser Phe Ala Arg Pro Leu Ile Ala Ala Asn
145 150 155 160
Lys Lys Gly Ser Ile Ile Leu Ile Gly Ser Met Ser Gly Thr Ile Val
165 170 175
Asn Asp Pro Gln Pro Gln Cys Met Tyr Asn Met Ser Lys Ala Gly Val
180 185 190
Ile His Leu Val Arg Ser Leu Ala Cys Glu Trp Ala Lys Tyr Asn Ile
195 200 205
Arg Val Asn Thr Leu Ser Pro Gly Tyr Ile Leu Thr Pro Leu Thr Arg
210 215 220
Asn Val Ile Ala Gly His Ala Asp Met Lys Ala Glu Trp Glu Ser Lys
225 230 235 240
Ile Pro Met Lys Arg Met Ala Glu Pro Lys Glu Phe Val Gly Ser Ile
245 250 255
Leu Tyr Leu Ala Ser Asp Ser Ala Ser Ser Tyr Thr Thr Gly His Asn
260 265 270
Leu Val Val Asp Gly Gly Tyr Glu Cys Trp
275 280
Claims (6)
1. 2-D-arabitol dehydrogenase coding genes, it is characterized in that: its base sequence is shown in SEQ ID NO:1.
2. the recombinant protein of the coded by said gene of a claim 1, it is characterized in that: this recombinant protein is a 2-D-arabitol desaturase, and its aminoacid sequence is shown in SEQ ID NO:2.
3. recombinant protein according to claim 2; It is characterized in that: the recombinant vectors of this recombinant protein is the pET30-a expression vector that contains 2-D-arabitol dehydrogenase coding genes, and the base sequence of said 2-D-arabitol dehydrogenase coding genes is shown in SEQ ID NO:1.
4. intestinal bacteria, it is characterized in that: in e. coli bl21 (DE3), contain 2-D-arabitol dehydrogenase coding genes, the base sequence of this 2-D-arabitol dehydrogenase coding genes is shown in SEQ ID NO:1.
5. intestinal bacteria according to claim 4; It is characterized in that: the recombinant vectors in these intestinal bacteria is the pET30-a expression vector that contains 2-D-arabitol dehydrogenase coding genes, and the base sequence of said 2-D-arabitol dehydrogenase coding genes is shown in SEQ ID NO:1.
6. claim 4 or 5 the intestinal bacteria application that is used to prepare the D-arabitol, it is characterized in that: these intestinal bacteria are the synthetic D-arabitol of substrate through inducing the back with ribulose or glucose.
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| WO2023220546A1 (en) * | 2022-05-09 | 2023-11-16 | Cargill, Incorporated | Genetically modified yeast and fermentation processes for the production of arabitol |
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| CN101624574A (en) * | 2009-08-06 | 2010-01-13 | 南京林业大学 | Yeast engineering bacteria for expressing high-stability xylanase, construction method and application thereof |
| CN101638640A (en) * | 2009-09-07 | 2010-02-03 | 北京利德曼生化股份有限公司 | Glucose-6-phosphoric acid dehydrogenase and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof |
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| CN101624574A (en) * | 2009-08-06 | 2010-01-13 | 南京林业大学 | Yeast engineering bacteria for expressing high-stability xylanase, construction method and application thereof |
| CN101638640A (en) * | 2009-09-07 | 2010-02-03 | 北京利德曼生化股份有限公司 | Glucose-6-phosphoric acid dehydrogenase and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof |
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