CN113502297B

CN113502297B - Recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, construction method and application thereof

Info

Publication number: CN113502297B
Application number: CN202110654277.6A
Authority: CN
Inventors: 段烨红; 梁书利; 林影
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2023-08-18
Anticipated expiration: 2041-06-11
Also published as: CN113502297A

Abstract

The invention discloses recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, a construction method and application thereof, wherein the recombinant pichia pastoris is obtained by expressing fucose-1-guanosine phosphate transferase and fucose kinase in a genome of Pichia pastoris GS115. The strain capable of synthesizing the guanosine diphosphate fucose is obtained through modification, so that the guanosine diphosphate fucose can be efficiently synthesized. The construction method of the recombinant pichia pastoris is simple, convenient to use and has good application prospect.

Description

Recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, and a construction method and application thereof.

Background

Guanylate diphosphate fucose (GDP-L-Fuc) is a nucleotide form of fucose (L-fucse), which provides fucosyl receptors for fucosylation reactions, and plays an important role in tissue growth, angiogenesis, fertilized cell adhesion, inflammation, and tumor metastasis of living organisms, and the like. Meanwhile, oligosaccharides containing fucosyl groups are a major component of breast milk, whose functions are involved in a wide range of biological processes, such as prevention of pathogen infection, improvement of immune system response, reduction of inflammatory response, and the like. The fucosylated oligosaccharides are synthesized under the catalysis of fucosyltransferase with guanyldiphosphate fucose (GDP-L-fucose) as a fucosyl donor. Because of the great demand created by the wide range of roles of fucosyl oligosaccharides, many pharmaceutical companies have attempted to efficiently synthesize sufficient guanosine diphosphate fucose by chemical and biological methods. In chemical synthesis, guanosine diphosphate fucose is synthesized starting from L-fucopyranosyl tetraacetic acid, but chemical synthesis is accompanied by a number of protection and deprotection steps, which complexity makes chemical synthesis impractical for industrial applications. Biosynthesis is based on the discovery in the organism of two metabolic pathways for the synthesis of GDP-L-fuse: salvage pathways and de novo pathways, both of which occur in the cytoplasm. Guanosine diphosphate fucose biosynthesis is shown in figure 1.

The salvage pathway is found in the metabolic pathway of humans, where exogenous fucose is transferred into the cell, and ATP is phosphorylated (EC2.7.1.52) by the consumption of fucose kinase to form fucose-1-phosphate (Fuc-1-P). Fuc-1-P binds Guanosine Triphosphate (GTP) to GDP-L-fucose under the catalysis of fucose-1-phosphate guanyl transferase (EC2.7.7.30). From the head synthesis pathway, which was first found in bacteria and then found to be present in plants, mammals and invertebrates, GDP-fucose was synthesized catalytically from GDP-mannose by guanosine diphosphate mannose dehydratase (GMD, ec 4.2.1.47) and guanosine diphosphate mannose epimerase (WCAG, EC1.1.1.271).

Pichia pastoris is widely used as an excellent host for expression of foreign proteins, and its product is FDA certified as "generally regarded as safe" (GRAS) safety level.

Therefore, how to use pichia pastoris to synthesize guanosine diphosphate fucose efficiently by biological methods is still a problem to be solved in the art.

Disclosure of Invention

The invention aims to provide a construction method and application of recombinant pichia pastoris for synthesizing guanosine diphosphate fucose, and the constructed recombinant pichia pastoris can efficiently synthesize guanosine diphosphate fucose.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a vector comprising an expression sequence for a fucose-1-phosphate guanosine transferase and/or an expression sequence for a fucose kinase.

In some embodiments of the invention, the amino acid sequence of the fucose-1-phosphogguanosine transferase is shown in SEQ ID NO.1, and the amino acid sequence of the fucose kinase is shown in SEQ ID NO. 2.

In some embodiments of the invention, the fucose-1-phosphate guanylate transferase is derived from the FPGT gene of a wild boar (Sus scrofa) and the fucose kinase gene is derived from the FCSK gene of the wild boar (Sus scrofa).

In some embodiments of the invention, the expression sequence of the fucose-1-phosphogguanosine transferase is shown in SEQ ID NO.3 and the expression sequence of the fucose kinase is shown in SEQ ID NO. 4.

In some embodiments of the invention, the sequence of the vector is shown in SEQ ID NO. 5.

In a second aspect of the invention, there is provided a recombinant cell comprising the expression vector of the first aspect of the invention, wherein the cell is a non-plant cell.

In some embodiments of the invention, the cell is pichia pastoris.

In some preferred embodiments of the invention, the cell is pichia pastoris GS115.

In a third aspect of the present invention, there is provided a method for constructing a recombinant cell according to the second aspect of the present invention, comprising the steps of: transferring the vector of the first aspect of the invention into a cell.

In some embodiments of the invention, the construction method is specifically to electrotransform competent cells of pichia pastoris GS115 with the vector according to the first aspect of the invention.

In a fourth aspect of the invention there is provided the use of a vector according to the first aspect of the invention or a recombinant cell according to the second aspect of the invention.

In some preferred embodiments of the invention, the use is in particular in the preparation of guanosine diphosphate fucose.

In a fifth aspect of the invention there is provided a method of preparing guanosine diphosphate fucose produced by the recombinant cell of the second aspect of the invention.

In some embodiments of the invention, the fermentation conditions are to introduce the recombinant cell seed solution into a fermentation medium at an inoculum size with an OD600 value of 0.5-1.5, and to culture at 200-300 rpm for 100-140 h at 25-35 ℃.

In some preferred embodiments of the invention, the fermentation conditions are such that the recombinant cell seed fluid is inoculated into the fermentation medium in an inoculated amount having an OD of 1 and incubated at 30 ℃ for 120h at 250 rpm.

The invention also provides guanosine diphosphate fucose prepared by the method of the fifth aspect of the invention.

The beneficial effects of the invention are as follows:

the present invention provides a vector comprising an expression sequence of a fucose-1-phosphate guanylate transferase and/or an expression sequence of a fucose kinase. And provides a recombinant cell which is obtained by expressing fucose-1-phosphate guanosine transferase and a fucose kinase gene on the basis of Pichia pastoris GS115, and a recombinant strain capable of synthesizing guanosine diphosphate fucose is obtained by modification. The construction method of the recombinant pichia pastoris is simple, convenient to use and has good application prospect.

Drawings

FIG. 1 is a schematic representation of guanosine diphosphate fucose biosynthesis.

FIG. 2 is a schematic diagram of the synthesis of guanosine diphosphate fucose plasmids according to example 1 of the present invention.

FIG. 3 is a diagram of PCR-verified agarose gel electrophoresis of yeast colonies transformed with the plasmids expressing the FPGT and FCSK genes in example 1 of the present invention.

FIG. 4 is a high performance liquid chromatogram of example 2 of the present invention.

Fig. 5 is a mass spectrum diagram in example 2 of the present invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

High Performance Liquid Chromatography (HPLC) detection method: agilent, UV detector, C18 column (250 x 4.6mm,5 μm), mobile phase a:20mM triethylamine acetate buffer (TEAA) pH6.0, mobile phase B: acetonitrile flow rate 0.35mL/min, column temperature 40℃and sample injection volume 10. Mu.L.

EXAMPLE 1 construction of recombinant Pichia pastoris

According to the sequences of fucose-1-phosphogguanyltransferase (FPGT) and Fucoskinase (FCSK) of wild boar [ susscrofa (pig) ] published on NCBI, wherein the fucose-1-phosphogguanyltransferase Gene is shown as Gene ID on NCBI: 100513664, the amino acid sequence of which is shown in SEQ ID NO. 1; gene ID of fucose kinase Gene at NCBI: 100625448 the sequence of which is shown in SEQ ID NO.2, both genes are optimized according to the codon preference of Pichia pastoris. The optimized base sequence of the fucose-1-guanosine phosphate transferase is shown as SEQ ID NO.3, and the base sequence of the fucose kinase is shown as SEQ ID NO. 4; the recombinant plasmid with the sequence shown as SEQ ID NO.5 is constructed through enzyme digestion connection. The schematic diagram of the plasmid is shown in FIG. 2.

Wherein:

SEQ ID NO.5：

in SEQ ID No. 5'_"is the gene FPGT region" - "is the FCSK region," … "is the promoter region.

Electrotransformation of competent cells of recombinant Pichia pastoris GS115 with the constructed recombinant plasmid, wherein the addition amount of the recombinant plasmid is 800-1500 ng, and the electrotransformation conditions are as follows: the voltage is 2.5kV, the electric shock reagent is 5.6ms, the recovery time is 1.5 hours at 30 ℃, YPD Z plates with the final concentration of 100mg/L bleomycin resistance are coated, the culture is carried out for 96 hours at 30 ℃, and a plurality of single colonies are selected.

Successful integration of fucose-1-phosphate guanosine transferase (FPGT) and fucose kinase (FCSK) into the yeast genome was confirmed by bleomycin resistance plate screening, colony PCR validation. The agarose gel electrophoresis identification results are shown in FIG. 3, wherein the bands 1, 2, 3, 4 and 5 are all colony samples, and M is Marker. Colony PCR can be seen to verify that there is a special band, demonstrating successful integration of fucose-1-phosphate guanosine transferase and fucose kinase in the Pichia pastoris GS115 genome. And confirming that the fucose-1-guanosine phosphate transferase and the fucose kinase which are derived from the wild boar are successfully expressed, and obtaining the recombinant pichia pastoris GS115.

EXAMPLE 2 fermentative production of guanosine diphosphate fucose

The recombinant pichia pastoris in the example 1 is prepared into seed liquid, and the formula of the seed liquid culture medium is as follows: glycerol 10g/L, tryptone 10g/L, yeast powder 5g/L, naCl g/L; the seed liquid preparation method comprises the following steps: single colonies on fresh plates were picked and cultured in seed medium for 24h.

Inoculating the seed solution into a fermentation medium according to an inoculum size with an OD value of 1, wherein the fermentation medium comprises the following formula: 20g/L peptone, 10g/L yeast powder and 1.34% YNB;10%10 XpH6.0 phosphate buffer, at 30 ℃ under 250rpm conditions for 120 hours, every 24 hours to feed 1% carbon source, carbon source is methanol, 48 hours after fermentation, every 24 hours to add 1g/L fucose to the culture medium to the end of fermentation.

After the fermentation was completed, guanosine diphosphate fucose was measured in the fermentation cells by high performance liquid chromatography, the results are shown in FIG. 4, and further confirmed by mass spectrometry, and the content of guanosine diphosphate fucose is 289.8mg/L, as shown in FIG. 5.

The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

<110> university of North China

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gttcctgatg gttcttacaa ctacatgact aactccgctt cagaattttt gcattccttg 1020

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gttttgcaag gtcatcattt gcaattgcat ggtgctccat ccagagcttt tactttggtt 1320

ggtagattgg attcctggga aagacaaggt gctggtactt acttgaatat gtcctggtct 1380

aagttttttc aaaagactgg tattagagat tgggatttgt gggaccctga tactcctcca 1440

actgaaagat gtttgttgtc cgctagattg tttccagttt tgcatccatt gagagctttg 1500

ggaccacaag atatgttgtg gatgttagat cctcaagaag atggaggtaa ggctttgaga 1560

gcttggagag attcatggag attgtcatgg gaacaattgc aaccatgttt ggatagagct 1620

gctactttgg cttctagaag agatttgttt tttagacaag ctttgcataa ggctcgccat 1680

gttttggaag ctagacaaga tttgtccttg agaccattga ttagagctgc tgttagagaa 1740

ggatgtccag gtccattgat ggctactttg gatcaagttg ctgctggagc tggtgatcca 1800

ggagttgctg ctagagcttt ggcttgtgtt gctgatattt tgggatgtat ggctgaaggt 1860

agaggtggtt tgagatcagg acctgctgct aacccagaat gggttagacc attttcttac 1920

ttggaatgtg gtgatttggc tggaggagtt gaagctttgg ctcaagaaag agaaaagtgg 1980

ttgtctagac ctgctttgtt ggttagagct gctagacatt acgaaggtgc tggacaaatt 2040

ttgattagac aagctgttat gtccgctcaa cattttgttt ctactgaacc agttgaattg 2100

ccagctccag gtcaatgggt tgttgctgaa tgtccagcta gagttgattt ttcaggagga 2160

tggtccgata ctccaccatt ggcttacgaa catggaggtg ctgttttggg attggctgtt 2220

agagttgatg gtagaagacc tattggtgct agagctagac gtattccaga accagaattg 2280

tggttggctg ttggacctca acatgataag atggctatga agattgtttg tagatccttg 2340

gatgatttgc aagattactg tcaacctcat gctccaggag ctttgttgaa ggctgctttt 2400

atttgtgctg gtattttgtc cgttcattca gaattgtcct tgtccgaaca attgttgtgt 2460

acttttggag gaggatttga attgcaaact tggtccgaat tgccacatgg ttcaggattg 2520

ggtacttctt ctattttggc tggtgctgct ttggctgctt tgcaaagagt tgctggtaga 2580

gctgttggta ctgaagcttt gattcatgct gttttgcatt tggaacaagt tttgactact 2640

ggaggaggat ggcaagatca agttggagga ttgatgccag gtattaaggt tggaagatcc 2700

cgtgcgcaat tgccattgaa ggttgaagtt gaagaaatta ctgttcctgc tggttttgtt 2760

caaaagttga acgatcattt gttgttggtt tacactggta agactagatt ggctagaaac 2820

ttgttgcaag atgttttgag atcctggtac gctagattgc caccagttgt tcaaaacgct 2880

cataacttgg ttcaacaaac tgaagaatgt gctgaagctt ttagacaagg ttccttgcca 2940

agattgggtc aatgtttgac ttcttactgg gaacaaaaga agttgatggc tccaggatgt 3000

gaaccattgg ctgttagaca tatgatggat gctttggctc ctcatgttca tggtcaatcc 3060

ttggctggtg ctggtggagg aggttttttg tacttgttga ctaaggaacc tagacaaaag 3120

gaagctttgg aagctgtttt ggctaagact gaaggattgg gtaactactc agttcatttg 3180

gttgaagttg atactcaagg attgtccttg caattgttgg gaactgaaac ttctactgga 3240

tgttcctttt aa 3252

<210> 5

<211> 13774

<212> DNA

<213> artificial sequence

<400> 5

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180

cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420

taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 480

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagccggtg 540

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 600

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 660

tcatgagatc agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca 720

tctctgaaat atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc 780

cggggtaaaa cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct 840

tccaccgccc gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc 900

cccttgcagc aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg 960

acctagcagc ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg 1020

catgtcatga gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca 1080

attttggttt ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca 1140

attgaacaac tatatggctg ctgcttcagc tccattggga gtttccttgc aagaagctac 1200

tcaaagaaga ttgagaagat tttccgaatt gagaggtaag ccagttgctg ctggtgaatt 1260

ttgggatatt gttgctatta ctgctgctga tgaaaagcaa gaattggctt acaagcaaca 1320

attgtccgaa aagttgaaga agaaggaatt gccattggga gttcaatact tggtttttgt 1380

tgatccagct ggtgctaaga ttggtaacgg aggtagcact ttgtgtgctt tgagatgttt 1440

ggaaaagttg tacggtgatc aatggaactc ctttactatt ttgttgattc atagcggggg 1500

atactcccaa agattgccta acgcttcagc tttgggtaag atttttactg ctttgccatt 1560

tggttcacct atttaccaaa tgttggaatt gaagttggct atgtacattg attttccatc 1620

acatatgaac cctggtattt tggttacttg tgctgatgat attgaattgt actccattgg 1680

agaatccgaa tttattagat ttgataagcc aggatttact gctttggctc atccatcatc 1740

cttgactgtt ggtactactc atggagtttt tgttttggaa ccatttaaca gattggaata 1800

cagagatttg gaatacagat gttgtcatag atttttgcat aagccatcta ttgaaatgat 1860

gtacaagttt gatgctgttt gtagacctgg taacttttcc caacaagatt ttggtggagg 1920

tgatactcca tccttgaagc tagatccaga atacgtttac actgattccg ttttttacat 1980

ggatcataag actgctaaga agttgttggc tttttacgaa aagattgata ctttgaactg 2040

tgaaattgat gcttacggtg attttttgca agctttggga ccaggagcta ctgttgaata 2100

cactagaaac acttctaacg ttactaagga agaatccgaa ttggttgata tgagacaaag 2160

aatttttcat ttgttgaagg gtactccatt gaacgttatt gttttgaaca actctaagtt 2220

ttaccatatt ggtactacta aggaatactt gtttcatttt acttctgatt cttccttgaa 2280

gtccgaattg ggattgcaat caattgcttt ttctattttt ccagctattc cagaatactc 2340

aggtaacaag tcatgtatta ttcaatctat tttggattct agatgttcct tggctccagg 2400

atcagttgtt gaatactcta gattgggacc tgatgtttcc gttggtgaaa actgtattat 2460

ttcaggttca catattatta ctagagctat tttgccagct tactcctttg tttgttcctt 2520

gtccttgaag attaacggac atattaagta ctcaactatg gcttgtggag ttcaagataa 2580

cttgaagaag aacgttaaga ctttgtccga tgttaagttg ttgcaatttt ttggagtttc 2640

cttgttgtcc tgtttggatg tttggaactt ggaagttact gaagaattgt tttcaggtaa 2700

caagacttgt ttgtccttgt ggaacgctag aatttttcca gtttgttctt ccttgtccga 2760

ttccgttatt gcttccttga agatgttgaa cgctgttcaa tctaagtcag ttttttcttt 2820

gaacaactac aagttgttgt caattgaaga aatgttggtt tacaaggatg ttgaagatat 2880

gattacttac agagaacaaa tttttttgga aattgctttg aacagaaagc aatccgtttt 2940

ggaaacttct taagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca 3000

tcatcatcat catcattgag ttttagcctt agacatgact gttcctcagt tcaagttggg 3060

cacttacgag aagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg 3120

cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga 3180

ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag 3240

ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg 3300

tatttcccac tcctcttcag agtacagaag attaagtgag aacgcgtaga tcttttttgt 3360

agaaatgtct tggtgtcctc gtccaatcag gtagccatct ctgaaatatc tggctccgtt 3420

gcaactccga acgacctgct ggcaacgtaa aattctccgg ggtaaaactt aaatgtggag 3480

taatggaacc agaaacgtct cttcccttct ctctccttcc accgcccgtt accgtcccta 3540

ggaaatttta ctctgctgga gagcttcttc tacggccccc ttgcagcaat gctcttccca 3600

gcattacgtt gcgggtaaaa cggaggtcgt gtacccgacc tagcagccca gggatggaaa 3660

agtcccggcc gtcgctggca ataatagcgg gcggacgcat gtcatgagat tattggaaac 3720

caccagaatc gaatataaaa ggcgaacacc tttcccaatt ttggtttctc ctgacccaaa 3780

gactttaaat ttaatttatt tgtccctatt tcaatcaatt gaacaactat ttcgaaacga 3840

ggaattcatg gaacaaccta agggagttga ttggactgtt attattttga cttgtcaata 3900

caaggattcc gttgaagttt ttcaaaagga attggaaatt agacaaaaga gagaacaaat 3960

tccagcttct actttgttgt tggctgttga agatcctgaa gttcatgttg gtagtggagg 4020

agctactttg aacgctttgt tggttgctgc tgaacatttg tccgctagag ctggatttac 4080

tgttgttact tccgatgttt tgcattccgc ttggattttg attttgcata tgggtagaga 4140

ttttccattt gatgattgtg gtagagcttt tacttgtttg ccagttgaaa acccacaagc 4200

tccagttgaa gctgttgttt gtaacttgga ttgtttgttg gatattatgt cacatagatt 4260

gggaccagga tcacctccag gagtttgggt ttgttctact gatatgttgt tgtcagttcc 4320

acctaaccca ggtattaact gggatggatt tagaggtgct agagttattg ctttgccagg 4380

aagcactgct tacgctagaa accatggagt ttacttgact gattcccaag gatttgtttt 4440

ggatatttac taccaaggaa ctgaagctga aattcaaaga tgtgctagac ctgatggtca 4500

agttccattg gtttccggta ttgttttttt ttccgttgaa actgctgaac atttgttggc 4560

tactcatgtt tccccaccat tggatgcttg tacttacatg ggattggatt caggtgctca 4620

accagttcaa ttgtcattgt tttttgatat tttgttgtgt atggctagaa acgttagaag 4680

agaagatttt ttggttggta gaccaccaga aatgggtaga ggtgatatgg aaactgaagg 4740

ttacttgaga ggtgctagag ctgaattgtg gagagaattg agagatcaac cattgacttt 4800

ggcttacgtt cctgatggtt cttacaacta catgactaac tccgcttcag aatttttgca 4860

ttccttgact tttccaggtg cttccggtgc tcaagttgtt cattcccaag ttgaagaaca 4920

acaattgttg ggtgctggtt cttccgttgt ttcctgtttg ttggaaggac cagttcaatt 4980

gggaccaggt tccgttttgc aacattgtca tttgagagga cctattcata ttggtactgg 5040

atgttttgtt tcgggcttgg atgctgctca atccgaagct ttgcattcct tggaattgca 5100

tgatttggtt ttgcaaggtc atcatttgca attgcatggt gctccatcca gagcttttac 5160

tttggttggt agattggatt cctgggaaag acaaggtgct ggtacttact tgaatatgtc 5220

ctggtctaag ttttttcaaa agactggtat tagagattgg gatttgtggg accctgatac 5280

tcctccaact gaaagatgtt tgttgtccgc tagattgttt ccagttttgc atccattgag 5340

agctttggga ccacaagata tgttgtggat gttagatcct caagaagatg gaggtaaggc 5400

tttgagagct tggagagatt catggagatt gtcatgggaa caattgcaac catgtttgga 5460

tagagctgct actttggctt ctagaagaga tttgtttttt agacaagctt tgcataaggc 5520

tcgccatgtt ttggaagcta gacaagattt gtccttgaga ccattgatta gagctgctgt 5580

tagagaagga tgtccaggtc cattgatggc tactttggat caagttgctg ctggagctgg 5640

tgatccagga gttgctgcta gagctttggc ttgtgttgct gatattttgg gatgtatggc 5700

tgaaggtaga ggtggtttga gatcaggacc tgctgctaac ccagaatggg ttagaccatt 5760

ttcttacttg gaatgtggtg atttggctgg aggagttgaa gctttggctc aagaaagaga 5820

aaagtggttg tctagacctg ctttgttggt tagagctgct agacattacg aaggtgctgg 5880

acaaattttg attagacaag ctgttatgtc cgctcaacat tttgtttcta ctgaaccagt 5940

tgaattgcca gctccaggtc aatgggttgt tgctgaatgt ccagctagag ttgatttttc 6000

aggaggatgg tccgatactc caccattggc ttacgaacat ggaggtgctg ttttgggatt 6060

ggctgttaga gttgatggta gaagacctat tggtgctaga gctagacgta ttccagaacc 6120

agaattgtgg ttggctgttg gacctcaaca tgataagatg gctatgaaga ttgtttgtag 6180

atccttggat gatttgcaag attactgtca acctcatgct ccaggagctt tgttgaaggc 6240

tgcttttatt tgtgctggta ttttgtccgt tcattcagaa ttgtccttgt ccgaacaatt 6300

gttgtgtact tttggaggag gatttgaatt gcaaacttgg tccgaattgc cacatggttc 6360

aggattgggt acttcttcta ttttggctgg tgctgctttg gctgctttgc aaagagttgc 6420

tggtagagct gttggtactg aagctttgat tcatgctgtt ttgcatttgg aacaagtttt 6480

gactactgga ggaggatggc aagatcaagt tggaggattg atgccaggta ttaaggttgg 6540

aagatcccgt gcgcaattgc cattgaaggt tgaagttgaa gaaattactg ttcctgctgg 6600

ttttgttcaa aagttgaacg atcatttgtt gttggtttac actggtaaga ctagattggc 6660

tagaaacttg ttgcaagatg ttttgagatc ctggtacgct agattgccac cagttgttca 6720

aaacgctcat aacttggttc aacaaactga agaatgtgct gaagctttta gacaaggttc 6780

cttgccaaga ttgggtcaat gtttgacttc ttactgggaa caaaagaagt tgatggctcc 6840

aggatgtgaa ccattggctg ttagacatat gatggatgct ttggctcctc atgttcatgg 6900

tcaatccttg gctggtgctg gtggaggagg ttttttgtac ttgttgacta aggaacctag 6960

acaaaaggaa gctttggaag ctgttttggc taagactgaa ggattgggta actactcagt 7020

tcatttggtt gaagttgata ctcaaggatt gtccttgcaa ttgttgggaa ctgaaacttc 7080

tactggatgt tccttttaag cggccgccag cttgggcccg aacaaaaact catctcagaa 7140

gaggatctga atagcgccgt cgaccatcat catcatcatc attgagtttt agccttagac 7200

atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa 7260

tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt 7320

ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc 7380

ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa 7440

tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta 7500

agtgagacct tcgtttgtgc ggatccatga catttccctt gctacctgca tacgcaagtg 7560

ttgcagagtt tgataattcc ttgagtttgg taggaaaagc cgtgtttccc tatgctgctg 7620

accagctgca caacctgatc aagttcactc aatcgactga gcttcaagtt aatgtgcaag 7680

ttgagtcatc cgttacagag gaccaatttg aggagctgat cgacaacttg ctcaagttgt 7740

acaataatgg tatcaatgaa gtgattttgg acctagattt ggcagaaaga gttgtccaaa 7800

ggatgatccc aggcgctagg gttatctata ggaccctggt tgataaagtt gcatccttgc 7860

ccgctaatgc tagtatcgct gtgccttttt cttctccact gggcgatttg aaaagtttca 7920

ctaatggcgg tagtagaact gtttatgctt tttctgagac cgcaaagttg gtagatgtga 7980

cttccactgt tgcttctggt ataatcccca ttattgatgc tcggcaattg actactgaat 8040

acgaactttc tgaagatgtc aaaaagttcc ctgtcagtga aattttgttg gcgtctttga 8100

ctactgaccg ccccgatggt ctattcacta ctttggtggc tgactcttct aattactcgt 8160

tgggcctggt gtactcgtcc aaaaagtcta ttccggaggc tataaggaca caaactggag 8220

tctaccaatc tcgtcgtcac ggtttgtggt ataaaggtgc tacatctgga gcaactcaaa 8280

agttgctggg tatcgaattg gattgtgatg gagactgctt gaaatttgtg gttgaacaaa 8340

caggtgttgg tttctgtcac ttggaacgca cttcctgttt tggccaatca aagggtctta 8400

gagccatgga agccaccttg tgggatcgta agagcaatgc tccagaaggt tcttatacca 8460

aacggttatt tgacgacgaa gttttgttga acgctaaaat tagggaggaa gctgatgaac 8520

ttgcagaagc taaatccaag gaagatatag cctgggaatg tgctgactta ttttattttg 8580

cattagttag atgtgccaag tacggtgtga cgttggacga ggtggagaga aacctggata 8640

tgaagtccct aaaggtcact agaaggaaag gagatgccaa gccaggatac accaaggaac 8700

aacctaaaga agaatccaaa cctaaagaag tcccttctga aggtcgtatt gaattgtgca 8760

aaattgacgt ttctaaggcc tcctcacaag aaattgaaga tgcccttcgt cgtcctatcc 8820

agaaaacgga acagattatg gaattagtca aaccaattgt cgacaatgtt cgtcaaaatg 8880

gtgacaaagc ccttttagaa ctaactgcca agtttgatgg agtcgctttg aagacacctg 8940

tgttagaagc tcctttccca gaggaactta tgcaattgcc agataacgtt aagagagcca 9000

ttgatctctc tatagataac gtcaggaaat tccatgaagc tcaactaacg gagacgttgc 9060

aagttgagac ttgccctggt gtagtctgct ctcgttttgc aagacctatt gagaaagttg 9120

gcctctatat tcctggtgga accgcaattc tgccttccac ttccctgatg ctgggtgttc 9180

ctgccaaagt tgctggttgc aaagaaattg tttttgcatc tccacctaag aaggatggta 9240

cccttacccc agaagtcatc tacgttgccc acaaggttgg tgctaagtgt atcgtgctag 9300

caggaggcgc ccaggcagta gctgctatgg cttacggaac agaaactgtt cctaagtgtg 9360

acaaaatatt tggtccagga aaccagttcg ttactgctgc caagatgatg gttcaaaatg 9420

acacatcagc cctgtgtagt attgacatgc ctgctgggcc ttctgaagtt ctagttattg 9480

ctgataaata cgctgatcca gatttcgttg cctcagacct tctgtctcaa gctgaacatg 9540

gtattgattc ccaggtgatt ctgttggctg tcgatatgac agacaaggag cttgccagaa 9600

ttgaagatgc tgttcacaac caagctgtgc agttgccaag ggttgaaatt gtacgcaagt 9660

gtattgcaca ctctacaacc ctatcggttg caacctacga gcaggctttg gaaatgtcca 9720

atcagtacgc tcctgaacac ttgatcctgc aaatcgagaa tgcttcttct tatgttgatc 9780

aagtacaaca cgctggatct gtgtttgttg gtgcctactc tccagagagt tgtggagatt 9840

actcctccgg taccaaccac actttgccaa cgtacggata tgcccgtcaa tacagcggag 9900

ttaacactgc aaccttccag aagttcatca cttcacaaga cgtaactcct gagggactga 9960

aacatattgg ccaagcagtg atggatctgg ctgctgttga aggtctagat gctcaccgca 10020

atgctgttaa ggttcgtatg gagaaactgg gacttattta aggatcctac cgttcgtata 10080

gcatacatta tacgaagtta taacatccaa agacgaaagg ttgaatgaaa cctttttgcc 10140

atccgacatc cacaggtcca ttctcacaca taagtgccaa acgcaacagg aggggataca 10200

ctagcagcag accgttgcaa acgcaggacc tccactcctc ttctcctcaa cacccacttt 10260

tgccatcgaa aaaccagccc agttattggg cttgattgga gctcgctcat tccaattcct 10320

tctattaggc tactaacacc atgactttat tagcctgtct atcctggccc ccctggcgag 10380

gttcatgttt gtttatttcc gaatgcaaca agctccgcat tacacccgaa catcactcca 10440

gatgagggct ttctgagtgt ggggtcaaat agtttcatgt tccccaaatg gcccaaaact 10500

gacagtttaa acgctgtctt ggaacctaat atgacaaaag cgtgatctca tccaagatga 10560

actaagtttg gttcgttgaa atgctaacgg ccagttggtc aaaaagaaac ttccaaaagt 10620

cggcataccg tttgtcttgt ttggtattga ttgacgaatg ctcaaaaata atctcattaa 10680

tgcttagcgc agtctctcta tcgcttctga accccggtgc acctgtgccg aaacgcaaat 10740

ggggaaacac ccgctttttg gatgattatg cattgtctcc acattgtatg cttccaagat 10800

tctggtggga atactgctga tagcctaacg ttcatgatca aaatttaact gttctaaccc 10860

ctacttgaca gcaatatata aacagaagga agctgccctg tcttaaacct ttttttttat 10920

catcattatt agcttacttt cataattgcg actggttcca attgacaagc ttttgatttt 10980

aacgactttt aacgacaact tgagaagatc aaaaaacaac taattattcg aaacggaatt 11040

gtgagcggat aacaaaggat gtccaattta ctgaccgtac accaaaattt gcctgcatta 11100

ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt cagggatcgc 11160

caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc gtgggcggca 11220

tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt tcgcgattat 11280

cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca tttgggccag 11340

ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca 11400

ctggttatgc ggcgcatccg aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct 11460

ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc 11520

tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct gttacgtata 11580

gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg gagaatgtta 11640

atccatattg gcagaacgaa aacgctggtt agcaccgcag gtgtagagaa ggcacttagc 11700

ctgggggtaa ctaaactggt cgagcgatgg atttccgtct ctggtgtagc tgatgatccg 11760

aataactacc tgttttgccg ggtcagaaaa aatggtgttg ccgcgccatc tgccaccagc 11820

cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa ctcatcgatt gatttacggc 11880

gctaaggatg actctggtca gagatacctg gcctggtctg gacacagtgc ccgtgtcgga 11940

gccgcgcgag atatggcccg cgctggagtt tcaataccgg agatcatgca agctggtggc 12000

tggaccaatg taaatattgt catgaactat atccgtaccc tggatagtga aacaggggca 12060

atggtgcgcc tgctggaaga tggcgattag cgaacaaaaa ctcatctcag aagaggatct 12120

gaatagcgcc gtcgaccatc atcatcatca tcattgagtt ttagccttag acatgactgt 12180

tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct aatcaagagg 12240

atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt 12300

aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct 12360

gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga 12420

gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac 12480

cttcgtttgt gcggaacccc cacacaccat agcttcaaaa tgtttctact ccttttttac 12540

tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag 12600

catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt 12660

tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat 12720

ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct ttcagtgacc 12780

tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc 12840

ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata gcaatctaat 12900

ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 12960

aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc 13020

gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg 13080

gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag 13140

gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg 13200

tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg 13260

accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac 13320

tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacggcggcc cacgggtccc 13380

aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg 13440

cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc 13500

tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat 13560

atttcaaatt tttctttttt ttctgtacag ataacttcgt atagcataca ttatacgaac 13620

ggtaacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac 13680

gctcgaaggc tttaatttgc aagctggaga ccaacatgtg agcaaaaggc cagcaaaagg 13740

ccaggaaccg taaaaaggcc gcgttgctgg cgtt 13774

Claims

1. A recombinant pichia pastoris cell, wherein the recombinant cell comprises a vector comprising an expression sequence for a fucose-1-phosphate guanosine transferase and an expression sequence for a fucose kinase; the expression sequence of the fucose-1-guanosine phosphate transferase is shown as SEQ ID NO.3, and the expression sequence of the fucose kinase is shown as SEQ ID NO. 4.

2. The recombinant pichia pastoris cell of claim 1, wherein the amino acid sequence of the fucose-1-phosphogguanyltransferase is shown as SEQ ID No.1 and the amino acid sequence of the fucose kinase is shown as SEQ ID No. 2.

3. The recombinant pichia pastoris cell of claim 1, wherein the vector has the sequence shown in SEQ ID No. 5.

4. The recombinant pichia pastoris cell of claim 1, wherein the cell is pichia pastoris GS115.

5. The method for constructing the recombinant pichia pastoris cell according to any one of claims 1 to 4, comprising the steps of: transferring the vector of any one of claims 1 to 4 into a pichia cell.

6. The use of the recombinant pichia pastoris cell of any one of claims 1 to 4 for the preparation of guanosine diphosphate fucose.

7. A method of preparing guanosine diphosphate fucose produced by fermentation of the recombinant pichia pastoris cell of any one of claims 1 to 4.

8. The method according to claim 7, wherein the fermentation conditions are that the seed solution of the recombinant pichia pastoris cells is inoculated into a fermentation medium with an inoculum size of 0.5 to 1.5 OD, and cultured at 25 to 35 ℃ at 200 to 300rpm for 100 to 140 hours.