CN107815461A

CN107815461A - Rumen Fungi xylose isomerase gene and its application

Info

Publication number: CN107815461A
Application number: CN201711063478.9A
Authority: CN
Inventors: 王智; 顿宝庆; 李桂英; 路明
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2017-11-02
Filing date: 2017-11-02
Publication date: 2018-03-20
Anticipated expiration: 2037-11-02
Also published as: CN107815461B

Abstract

The present invention relates to a kind of new xylose isomerase gene from Rumen Fungi, its nucleotide sequence is as shown in SEQ ID NO.2.The present invention imports the recombinant plasmid comprising the gene in host cell, obtains a kind of new Yeast engineering bacteria.It is verified by experiments, after the gene is expressed in saccharomyces cerevisiae, the saccharomyces cerevisiae for not possessing conversion xylose originally for the ability of xylulose obtains the conversion capability.The engineering bacteria can prepare ethanol and other tunnings by the culture medium containing xylose that ferments.

Description

Rumen Fungi xylose isomerase gene and its application

Technical field：

The present invention relates to a kind of xylose isomerase gene, and in particular to xylose isomerase gene from Rumen Fungi and its The function that xylose is xylulose is converted, the invention further relates to the recombinant plasmid containing the gene and engineered strain, and with the engineering Bacterium prepares the application of ethanol and other tunnings by the culture medium containing xylose that ferments.

Background technology：

It is both economically and environmentally beneficial mode that lignocellulose is produced into ethanol in a manner of industrialization.Because from plant Lignocellulosic material is the renewable resource that can largely obtain.But many yeast for being capable of fermentative production of ethanol are (as made Brewer yeast) carbon source can not be used as using xylose.Accordingly, it is desirable to provide the ferment of alcohol fermentation can be carried out as carbon source using xylose It is female.

Saccharomyces cerevisiae has the complete enzyme system that xylulose is metabolized, if it is possible to which the xylose in the lignocellulosic of plant is different Structure, generate product, the xylose such as xylulose, saccharomyces cerevisiae can metabolism xylulose generation ethanol and can serve as carbon source progress Alcohol fermentation.If can be effectively ethanol by wood-sugar fermentation, the yield of lignocellulose ethanol fermentation can be made original On the basis of increase by 25%, cellulose ethanol production cost will reduce by 20% or so.Determined so xylose fermentation for producing ethanol turns into One of economically viable key factor of Production of Alcohol from Lignocellulose.

Xylose isomerase (D-Xylose isomerase, XI, EC 5.3.1.5) needed for xylose isomerase is catalyzed five carbon Sugared D- xyloses are D- xyluloses, are widely present in bacterium, small part fungi.It is complete that there is saccharomyces cerevisiae xylulose to be metabolized Whole enzyme system, xylulose enter pentose phosphate pathway, fermentation generation ethanol.If it find that it is new can in saccharomyces cerevisiae high efficient expression Xylose isomerase, xylose isomerase can just be melted into xylulose by saccharomyces cerevisiae, reach using xylose and to carry out second as carbon source The purpose of alcohol fermentation.

The xylose isomerase gene of prior art comes from microorganism more.Once someone distinguished a variety of sources of clonal expression Xylose isomerase gene, such as Escherichia coli, Bacillus subtilis, Thormotoga species, Thermus species etc..But there was only a few so far and the mostly xylose isomerase gene of thermophilic bacteria is in second Activity expression is obtained in alcohol production tradition strain Saccharomyces cerevisiae, and generally due to work at a temperature of 30 DEG C or so of alcohol fermentation Property it is too low and as xylose metabolism approach rate-limiting step.

The content of the invention

The purpose of the present invention is that new xylose isomerase gene is filtered out from fungi, and by the channel genes make wine ferment It is female, so as to get Yeast Genetics engineering bacteria obtain ability that xylose is xylulose.

The technical problems to be solved by the invention are as follows：

A kind of new xylose isomerase gene is filtered out from fungi；

Recombinant vector comprising above-mentioned xylose isomerase and its gene is provided；

The recombinant bacterial strain for including above-mentioned xylose isomerase gene is provided；

The above-mentioned recombinant bacterial strain of gained is applied to conversion xylose generation xylulose, and then generates ethanol.

The present inventor obtains a kind of new xylose isomerase from natural Rumen Fungi, is named as XI, and obtained one Kind encodes the xylose isomerase gene xylA of the enzyme, it is demonstrated experimentally that the gene has the function by xylose into xylulose, fits Used together in the energy, wine brewing and food industry.

Although the present invention is initially the invention completed by screening the fungi, the result of the present invention is to have obtained gene Sequence.Other people are independent of this fungi, as long as learning that sequence is obtained with the xylose isomerase of the present invention.

The nucleotide sequence of the xylose isomerase gene is as shown in SEQ ID NO.2.The xylose of the xylA gene codes Isomerase XI amino acid sequence is as shown in SEQ ID NO.1.

Between above-mentioned xylose isomerase gene xylA is inserted into the suitable restriction enzyme site of expression vector by the present invention, Make its nucleotide sequence is exercisable to be connected with expression regulation sequence, obtain including xylose isomerase gene xylA restructuring matter Grain.The expression vector is preferably saccharomyces cerevisiae multicopy constitutive expression carrier pYES-FBA, obtains recombinant plasmid pYES- FBA-XI。

As the most preferred embodiment of the present invention, between the suitable restriction enzyme site of expression vector It is between the Spe I on plasmid pYES2 and the restriction enzyme sites of Xba I, the nucleotide sequence is located under FBA1 promoters Swim and regulated and controled by it, obtain expression of recombinant yeast plasmid pYES-FBA-XI.

Above-mentioned expression of recombinant yeast plasmid pYES-FBA-XI conversions are entered host cell by the present invention, and obtaining can be efficient Express the recombinant bacterial strain of above-mentioned xylose isomerase.

The host cell is brewing yeast cell, Pichia pastoris or many types of inferior yeast cells, preferred host cell For brewing yeast cell (Saccharomyces cerevisiae), it is furthermore preferred that being to convert expression of recombinant yeast plasmid to make Brewer yeast T1308-U, obtain recombinant bacterial strain B1 (detailed in Example 4).

Present invention also offers the recombinant bacterial strain for including above-mentioned xylose isomerase gene, preferably recombinant bacterial strain B1.Restructuring Bacterial strain B1 can the above-mentioned xylose isomerase of high efficient expression, the enzyme can be used for fermentation the material containing xylose, by xylose Cheng Mu Ketose, and then it is metabolized generation ethanol and other tunnings.

Present invention also offers a kind of application of gained xylose isomerase, comprise the following steps：

1st, with claim recombinant plasmid transformed host cell, recombinant bacterial strain B1 (detailed in Example 4) is obtained；

2nd, recombinant bacterial strain B1, expression restructuring xylose isomerase are cultivated.

Wherein, xylose isomerase of the invention has following application：

1st, energy industry：

Can be D- xyluloses by a large amount of existing D- xyloses in lignincellulose hydrolysis thing, and D- xyluloses Again can be by traditional alcohol production bacterial strain such as Saccharomyces cerevisiae transformant into valuable fuel.

2nd, food industry

Xylose isomerase can be catalyzed D-Glucose in vitro and be converted into D-Fructose, therefore " the glucose isomerase that is otherwise known as Enzyme ".This latter activity can be used for producing fructose syrup in the industry, can be used as food additives.Fructose syrup is one to finish The product of sucrose can be substituted entirely, and food and beverage industry are can be widely used in as sucrose.

Embodiment

Plasmid, the bacterial strain lifted in following examples are only intended to be described in further detail the present invention, not to this The substantive content of invention is any limitation as.In fact, with present invention discover that gene and method, those skilled in the art can obtain Other a variety of genetic engineering bacterial strains having by xylose for xylulose ability, do not depart from the spirit and think Road.

Do not make the experimental methods of molecular biology illustrated, equal reference in following examples《Molecular Cloning:A Laboratory guide》 Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description.

Test material and reagent

1st, bacterial strain and carrier：Saccharomyces cerevisiae expression pYES2 is purchased from Invitrogen companies, Wine brewing yeast strain T1308-U and bacillus coli DH 5 alpha are preserved by place laboratory.

2nd, enzyme and other biochemical reagents：Restriction endonuclease and ligase are purchased from NEB companies, and other reagents are not as illustrated All it is domestic reagent (can be commercially available from common biochemical Reagent Company).

3rd, culture medium：

1. Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, l%NaCl, pH7.0)

2. yeast culture medium YPD (1% yeast extract, 2% peptone, 2% glucose, flat board add 2% agar)

3. Selective agar medium SC (0.67%YNB, 2% glucose, flat board add 2% agar)

The extraction of the Rumen Fungi genome DNA of embodiment 1

1. taking Rumen Fungi bacterial strain about 0.5g, pulverized rapidly in liquid nitrogen；

2. adding 4ml extract solutions, quick oscillation mixes；

3. add isometric 4ml chloroform：Isoamyl alcohol (24：1), vortex 3-5min；

4. 10000rpm, 4 DEG C, 5min；

5. the chloroform of supernatant volume：Isoamyl alcohol (24：1) extract again once (10000rpm, 4 DEG C of centrifugation 5min)；

6. taking supernatant, -20 DEG C of pre- cold isopropanols of 2/3 times of volume or the absolute ethyl alcohol precipitation of 2.5 times of volumes are added, is mixed It is even, stand about 30min；

7. choosing flocculent deposit with capillary glass rod, rinsed for several times with 75% ethanol, then rinsed 1 time with absolute ethyl alcohol repeatedly, Drying, is resuspended in 500 μ l TE；

8. 1 μ l RNaseA (10mg/ml) are added, 37 DEG C of processing 1h；

9. use phenol：Chloroform：Isoamyl alcohol (25：24：And chloroform 1)：Isoamyl alcohol (24：1) each extracting 1 time (10000rpm, 4 DEG C Centrifuge 5min)；

10. supernatant is taken, the absolute ethyl alcohol of 3M NaAc, the 2.5V volumes of 1/10V volumes, -70 DEG C of precipitation more than 30min. 12000rpm, 4 DEG C of centrifugation 10min, abandons supernatant, and precipitation is rinsed with 75% ethanol, is air-dried, and is dissolved in 200 μ l pH8.0 ddH₂O In, -20 DEG C save backup.

The Rumen Fungi xylose isomerase gene xylA of embodiment 2 clone

The Rumen Fungi STb gene extracted using embodiment 1 enters performing PCR amplification as template.Amplification is served after obtaining product recovery Hai Shenggong biotech firms are sequenced, and analyze sequencing sequence by comparing, it was demonstrated that the sequence of acquisition includes xylose isomerase full-length gene Sequence, overall length 1284bp, sequence as shown in SEQ ID NO.2,

The structure of the xylose isomerase gene expression vector plasmid of embodiment 3

Primer, including EcoR I and Xba I are designed with 5 ' and 3 ' terminal sequences of the gene of coding Rumen Fungi xylose isomerase Site.With EcoR I and the digestion PCR primers of Xba I.End-product is cloned into as caused by pYES2 on carrier.In the carrier, GAL1 promoters on pYES2 are replaced with FBA1 promoters to ensure the constitutive expression of xylose isomerase, so as to eliminate culture To the demand of galactolipin in base.FBA1 promoters clone to obtain from saccharomyces cerevisiae genome.The promoter be digested into Nhe I- The fragments of EcoR I.The PCR primer of the encoding gene of FBA1 promoters and xylose isomerase is connected to is cut with EcoR I and Xba I On the pYES2 cut, the recombinant plasmid pYES-FBA-XI containing xylose isomerase gene is finally given.

The preparation of competent escherichia coli cell and the conversion of plasmid

1st, the preparation (calcium chloride transformation) of competent cell

1. it is inoculated into the LB Liquid Cultures containing 5ml from picking single bacterium colony on Escherichia coli (E.coli) DH5 α flat boards of activation In the test tube of base, 37 DEG C of shaken cultivation 2.5h to 3h make the OD of bacterium solution₆₀₀Value reaches 0.4 to 0.6, cooled on ice culture to 0 ℃；

2. culture is poured into sterile 1.5ml centrifuge tube；

3. 4 DEG C, 4000rpm centrifugations 10min；

4. abandoning supernatant, thalline is collected；

5. the CaCl of the 0.1M with the 0.5ml of precooling₂Thalline is resuspended, centrifugation, abandons supernatant；

6. the CaCl of the 0.1M with the 0.5ml of precooling₂Thalline is resuspended, ice bath 15min, centrifugation, abandons supernatant；

7. add the 0.1M of 200 μ l precooling CaCl₂Thalline is resuspended, ice bath is placed.

2nd, plasmid transformed competence colibacillus cell

1. taking 0.5 μ l plasmids to be added in a pipe competent cell, gently rotate to mix content, place 30min on ice；

2. centrifuge tube is placed in into heat shock 90s in 42 DEG C of water-baths, centrifuge tube is not rocked；

3. being rapidly placed in centrifuge tube on ice, 2min is cooled down；

4. 800 μ l LB fluid nutrient mediums are added, 37 DEG C, 200rpm shaking table cultures 45min；

5. 50 μ l nutrient solution is taken to be coated on the LB solid plates containing ampicillin (50 μ g/ml), 37 DEG C of cultures 12 To 16 hours, conversion bacterium colony is checked；

6. picking individual colonies, being inoculated into the test tube of the LB fluid nutrient mediums containing 5ml, 37 DEG C of shaken cultivations extract plasmid enzyme Cut and be verified as correct transformant, proof is then sequenced and contains xylose isomerase gene.

The structure of the engineered strain of embodiment 4

Recombinant plasmid pYES-FBA-XI containing xylose isomerase gene is transferred to not through electroporated method and had xylose For in the saccharomyces cerevisiae T1308-U of xylulose ability, conversion is screened on the SC culture medium flat plates using 2% glucose as carbon source Son.The cell not being converted can not grow on these flat boards.It is different that PCR assay certificate B1 transformants contain band Rumen Fungi xylose The plasmid of structure enzyme gene.

The preparation of Saccharomyces cerevisiae competent cell and the conversion of plasmid：

1st, the preparation (electroporated method) of competent yeast cells

1. in the 50ml centrifuge tubes of the YPD containing 5ml, saccharomyces cerevisiae is cultivated, 30 DEG C overnight；

2. taking 0.1-0.5ml overnight cultures, the 2L shaking flasks of the fresh culture containing 500ml, overnight growth to OD are inoculated with₆₀₀ =1.3-1.5；

3. at 4 DEG C, 1500g centrifugations 5min collects cell, with the aqua sterilisa suspension cell of 500ml precoolings；

4. as above centrifuge, with the aqua sterilisa suspension cell of 250ml precoolings；

5. as above centrifuge, with the 1M sorbierite suspension cells of 20ml precoolings；

6. as above centrifuge, with the 1M sorbierite suspension cells of 1ml precoolings, to final volume about 1.5ml；

Pay attention to：The Electrocompetent cells of 80 μ l equivalent can be frozen, but transformation efficiency can decline a lot.

2nd, the electroporated competent cell of plasmid：

1. taking the 80 above-mentioned cells of μ l to linearize DNA (being dissolved in 5-10 μ lTE) with 5-20 μ g to mix, the 0.2cm electricity of precooling is transferred to In revolving cup；

2. 5min is placed on ice；

3. shocked by electricity according to the saccharomyces cerevisiae parameter that institute's use device is recommended；

4. adding the 1M sorbierites of 1ml precoolings immediately into cup, content is transferred in sterile centrifugation tube；

5. being divided into 200-600 μ l equal portions, it is applied on SC flat boards；

Contain band Rumen Fungi xylose isomerase to generation, PCR assay certificate transformants B1 is cloned 6. being incubated flat board at 30 DEG C

The plasmid pYES-FBA-XI of enzyme gene.

The measure of the engineered strain xylose isomerase enzyme activity of embodiment 5

1st, experimental subjects

Experimental strain：The saccharomyces cerevisiae recombinant bacterial strain B1 of pYES-FBA-XI containing carrier (detailed in Example 4)；

Control strain：The Wine brewing yeast strain of the carrier containing pYES-FBA.

2nd, experimental method

Grown in the weighing apparatus culture using the mixture of glucose/xylose as sole carbon source, determine control strain respectively and contain Carrier pYES-FBA-XI saccharomyces cerevisiae recombinant strains B1 xylose isomerase enzyme activity.The measure of enzyme activity is carried out as follows：

Supernatant after appropriate brewing yeast cell is broken (passes through OD₅₉₅It is consistent that measure is adjusted to total protein concentration) 100mM Tris-HCl pH7.0 buffer solutions, 10mMMgCl₂, 2U SDH (sorbitol dehydrogenase, Roche companies) and 0.15mM NADH are (also Prototype cigarette amino acid adenine-dinucleotide, Roche companies), mix, 500mM xylose starts reaction, 30 DEG C, detect under 340nm Amount (doing time sweep in 340nm) oxidized NADH.One enzyme-activity unit (U) of xylose isomerase (XI) is defined as every point Clock converts 1 μm of ol substrate.

3rd, specific operation process

1. picking single bacterium colony is seeded in 20ml SC nutrient solutions respectively, 30 DEG C of 200rpm shake training 48h；

2. 1600g centrifuges 5min, supernatant, precipitation 10mM pH7.5 potassium-phosphate buffer are abandoned (EDTA containing 2mM) is washed twice；

3. it is resuspended in 100mM pH 7.5 potassium-phosphate buffer (2mM MgCl₂And 1mM dithiothreitol)；

4. ultrasonication, 4 DEG C of 36000g centrifuge 20min, and supernatant is used as enzyme activity analysis and total protein measure.

4th, measurement result

Control strain does not measure xylose isomerase enzyme activity；

The saccharomyces cerevisiae recombinant bacterial strains of pYES-FBA-XI containing carrier B1 xylose isomerase enzyme activity determination is the total eggs of 0.95U/mg In vain.

5th, conclusion

After the xylose isomerase of Rumen Fungi xylose isomerase gene coding is expressed in saccharomyces cerevisiae, host cell is assigned By the ability that xylose is xylulose.

The measure of the engineered strain xylose utilization of embodiment 6

1st, experimental subjects

Experimental strain：The saccharomyces cerevisiae recombinant bacterial strain B1 (detailed in Example 4) of the pYES-FBA-XI containing carrier；

2nd, experimental method

In the weighing apparatus that the mixture (initial concentration is respectively 20.0g/L and 10.0g/L) with glucose/xylose is sole carbon source Change culture growth, 30 DEG C of 200rpm shake training 48h, high performance liquid chromatography (HPLC) measure supernatant glucose and xylose content, pushed away Calculate sugared utilization ratio.

3rd, result

Test result is shown：Cultivated by 48h, the glucose of recombinant bacterial strain P culture mediums has almost been fully utilized, xylose profit Reach 59.41% with rate；The glucose of control strain culture medium has almost been fully utilized, and xylose utilization rate only reaches 2.19%.

4th, conclusion

The xylose utilization rate of Rumen Fungi xylose isomerase gene engineered strain is much higher than control strain.

Sequence table

<110>Institute of Crop Science, Chinese Academy of Agricultural Science

<120>Rumen Fungi xylose isomerase gene and its application

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 425

<212> PRT

<213>Rumen Fungi（Piromyces sp.）

<400> 1

Met Asp Gly His Phe Pro Asn Val Pro Glu Val Lys Tyr Glu Gly Pro

1 5 10 15

Asn Ser Thr Asn Ser Lys Asn Pro Leu Ala Phe His Tyr Tyr Asp Pro

20 25 30

Glu Lys Val Val Met Gly Lys Lys Met Lys Asp Trp Leu Arg Phe Ala

35 40 45

Met Cys Trp Trp His Thr Leu Cys Ala Glu Gly Ala Phe Gly Gly Gly

50 55 60

Thr Lys Ser Phe Pro Trp Asn Glu Gly Thr Asp Ala Ile Glu Ile Ala

65 70 75 80

Ala Lys Lys Lys Ala Asp Ala Gly Phe Glu Ile Met Gln Lys Leu Gly

85 90 95

Ile Glu Tyr Tyr Asp Val Asp Leu Val Ser Glu Gly Asn Ser Ile Glu

100 105 110

Glu Tyr Glu Ala Asn Leu Lys Thr Lys Ala Val Val Ala Tyr Leu Lys

115 120 125

Glu Lys Gln Lys Glu Thr Gly Ile Lys Leu Leu Trp Trp Ser Thr Ala

130 135 140

Asn Val Phe Gly His Pro Arg Tyr Met Asn Gly Ala Ser Thr Asn Pro

145 150 155 160

Asp Phe Asp Val Val Ala Arg Ala Ile Val Gln Ile Lys Asn Ala Met

165 170 175

Asp Ala Gly Ile Gly Lys Asn Tyr Val Phe Trp Gly Gly Arg Glu Gly

180 185 190

Tyr Met Ser Leu Leu Asn Thr Asp Thr Asp Gln Lys Arg Glu Lys Glu

195 200 205

His Met Ala Thr Met Leu Thr Met Ala Arg Asp Tyr Tyr Ala Arg Ser

210 215 220

Lys Gly Phe Lys Gly Thr Phe Leu Ile Glu Pro Lys Pro Met Glu Pro

225 230 235 240

Thr Lys His Gln Tyr Asp Val Asp Thr Glu Thr Val Ile Gly Phe Leu

245 250 255

Lys Thr His Asn Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr

260 265 270

Leu Ala Gly His Thr Phe Glu His Phe Glu His Glu Leu Ala Val Ala

275 280 285

Val Asp Asn Gly Met Leu Gly Ser Ile Asp Ala Asn Gly Asp Tyr Gln

290 295 300

Asn Gly Trp Asp Thr Asp Gln Phe Pro Ile Asp Gln Tyr Glu Leu Val

305 310 315 320

Val Gln Ala Trp Met Glu Ile Ile Arg Asn Gly Gly Leu Gly Thr Gly

325 330 335

Gly Thr Asn Phe Asp Ala Lys Thr Arg Arg Asn Ser Thr Asp Leu Glu

340 345 350

Asp Ile Leu Ile Ala His Ile Ser Thr Arg Arg Asn Ser Thr Asp Leu

355 360 365

Glu Asp Ile Leu Ile Ala His Ile Ser Gly Met Asp Gly Met Asp Ala

370 375 380

Met Ala Arg Ala Leu Glu Arg Ala Ala Lys Leu Leu Glu Glu Ser Pro

385 390 395 400

Thr Thr Ser Leu Glu Glu Leu Glu Gln Tyr Gln Glu Leu Ala Glu Ile

405 410 415

Leu Phe Gln Ala Ala Glu Ala Ile Val

420 425

<210> 2

<211> 1278

<212> DNA

<213>Rumen Fungi（Piromyces sp.）

<400> 2

ATGGACGGTC ACTTCCCCAA CGTCCCCGAG GTCAAGTACG AGGGTCCCAA CTCCACCAAC 60

TCCAAGAACC CCCTCGCCTT CCACTACTAC GACCCCGAGA AGGTCGTCAT GGGTAAGAAG 120

ATGAAGGACT GGCTCCGCTT CGCCATGTGC TGGTGGCACA CCCTCTGCGC CGAGGGTGCC 180

TTCGGTGGTG GTACCAAGTC CTTCCCCTGG AACGAGGGTA CCGACGCCAT CGAGATCGCC 240

GCCAAGAAGA AGGCCGACGC CGGTTTCGAG ATCATGCAGA AGCTCGGTAT CGAGTACTAC 300

GACGTCGACC TCGTCTCCGA GGGTAACTCC ATCGAGGAGT ACGAGGCCAA CCTCAAGACC 360

AAGGCCGTCG TCGCCTACCT CAAGGAGAAG CAGAAGGAGA CCGGTATCAA GCTCCTCTGG 420

TGGTCCACCG CCAACGTCTT CGGTCACCCC CGCTACATGA ACGGTGCCTC CACCAACCCC 480

GACTTCGACG TCGTCGCCCG CGCCATCGTC CAGATCAAGA ACGCCATGGA CGCCGGTATC 540

GGTAAGAACT ACGTCTTCTG GGGTGGTCGC GAGGGTTACA TGTCCCTCCT CAACACCGAC 600

ACCGACCAGA AGCGCGAGAA GGAGCACATG GCCACCATGC TCACCATGGC CCGCGACTAC 660

TACGCCCGCT CCAAGGGTTT CAAGGGTACC TTCCTCATCG AGCCCAAGCC CATGGAGCCC 720

ACCAAGCACC AGTACGACGT CGACACCGAG ACCGTTATAG GCTTCCTCAA GACCCATAAC 780

GACTTCAAGA TGAACATCGA GGCCAACCAC GCCACCCTCG CCGGTCACAC CTTCGAGCAC 840

TTCGAGCACG AGCTCGCCGT CGCCGTCGAC AACGGTATGC TCGGTTCCAT CGACGCCAAC 900

GGTGACTACC AGAACGGTTG GGACACCGAC CAGTTCCCCA TCGACCAGTA CGAGCTCGTC 960

GTCCAGGCCT GGATGGAGAT CATCCGCAAC GGTGGTCTCG GTACCGGTGG TACCAACTTC 1020

GACGCCAAGA CCCGCCGCAA CTCCACCGAC CTCGAGGACA TCCTCATCGC CCACATCTCC 1080

ACCCGCCGCA ACTCCACCGA CCTCGAGGAC ATCCTCATCG CCCACATCTC CGGTATGGAC 1140

GGTATGGACG CCATGGCCCG CGCCCTCGAG CGCGCCGCCA AGCTCCTCGA GGAGTCCCCC 1200

ACTACTTCTT TGGAAGAACT CGAGCAATAC CAGGAACTGG CTGAGATTCT GTTCCAAGCT 1260

GCTGAAGCTA TCGTTTAA 1278

Claims

1. a kind of xylose isomerase gene, its nucleotide sequence is as shown in SEQ ID NO.2.

2. the purposes of xylose isomerase gene described in claim 1, it is conversion xylose generation xylulose.

3. a kind of xylose isomerase, its amino acid sequence is as shown in SEQ ID NO.1；Encode the nucleotides sequence of the amino acid sequence Row are as shown in SEQ ID NO.2.

A kind of 4. recombinant plasmid, containing the xylose isomerase gene described in claim 1.

5. the recombinant plasmid described in claim 4, it is xylose isomerase gene described in claim 1 and saccharomyces cerevisiae multicopy table Up to obtained from carrier pYES2 connections.

6. a kind of recombinant bacterial strain, include the gene described in claim 1.

7. a kind of Yeast engineering bacteria, contain the recombinant plasmid described in claim 4 or 5.

8. the Yeast engineering bacteria described in claim 7, its host cell is selected from brewing yeast cell, Pichia pastoris or many types of Inferior yeast cells.

9. the purposes of the Yeast engineering bacteria of claim 7 or 8, it is conversion xylose generation xylulose.

10. the purposes described in claim 9, it is that ethanol and other tunnings are prepared by the material containing xylose that ferments.