CN101671692A

CN101671692A - Method for constructing GDP-mannose system synthesized by coupling and fermenting gene engineering strains

Info

Publication number: CN101671692A
Application number: CN200910070580A
Authority: CN
Inventors: 李玉; 孔董俊; 王春霞; 路福平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2009-09-25
Filing date: 2009-09-25
Publication date: 2010-03-17

Abstract

The invention relates to a method for constructing a GDP-mannose system synthesized by coupling and fermenting gene engineering strains. The method comprises the following steps: metabolic engineeringstains are constructed by using the genetic engineering technology to realize efficient expression of various enzymes during the metabolic pathway to further achieve the purpose of a great amount ofsynthesized GDP-mannose. The method breaks the bottleneck that a great amount of fucosylation oligosaccharide cannot be obtained more easily, provides an effective way for producing the human milk oligosaccharide at a large scale, provides the raw materials for the production of the natural oligosaccharide medicine, complies with the current development trend of the biomedical field, not only hashigher basic theory research value, but also has tremendous economic and social benefits, and has a broad market development prospect.

Description

Utilize the construction process of the synthetic GDP-seminose system of coupling and fermenting gene engineering strains

Technical field

What the present invention relates to is the constructing technology of engineering strain in the genetically engineered field, especially a kind of construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains.

Background technology

WHO finds that to 36 investigation that derive from 14 countries in back 6 months of birth, the dysentery incidence of breast feeding babies than the on average low 4-5 of not breast fed baby doubly.

Why breast milk can effectively reduce the morbidity degree and the death toll of ewborn infant dysentery, mainly contain panimmunity regulatory factors such as anti-infective, anti-inflammatory, mainly comprise secretor type antibody (sIgA), oligosaccharides, lactoferrin, white corpuscle, cytokine etc. owing to it.Because it is fully grown that the immunity system of ewborn infant was failed before 2 one full year of life, these factors can be by disturbing the adhesion of pathogenic bacteria on enteron aisle, the adjusting body reaches the opposing diseases in infants to Inflammatory response, stimulation intestinal tract and the immune maturation of pathogenic bacteria purpose in the breast milk.

The content of human milk oligosaccharides is the third-largest material in people Ruzhong after lactose, lipid.Studies show that, breast-feeding or the baby who feeds with the breast milk substitute that is added with human milk oligosaccharides, the unwanted bacteria number in its ight soil reduces greatly; Simultaneously, compare with common breast milk substitute feeding infant, the mortality ratio that causes because of infectation of bacteria also obviously reduces.This shows, human milk oligosaccharides has significantly anti-infective and some important non-specific immunity function, some researchs have proved that also human milk oligosaccharides can promote the propagation of bifidus bacillus in baby's digestive tube, and clostridium and enterococcal quantity are significantly reduced.

The anti-microbial pathogen adhesion function of human milk oligosaccharides has caused people's extensive concern, and many pharmaceuticals also begin to carry out exploitation and the clinical study with the anti-adhesive medicine of human milk oligosaccharides and analogue thereof, and still, a large amount of acquisitions of human milk oligosaccharides also are not easy.At present, function and preparation research about human milk oligosaccharides are not effectively carried out yet in China, China mainly concentrates on the exploitation of function oligosaccharides class material and adopts chemical processes such as acid, alkali, enzyme, oxidation to degrade to natural phant, microbial polysaccharide to prepare low-molecular-weight oligosaccharides, as oligochitosan, Nutriflora P etc., this series products is successfully listing at present.Though also in constantly carrying out, only be to be prepared with chemical synthesis process, and great majority work only is in conceptual phase to the exploitation of oligosaccharides medicine.Also there are research unit or enterprise to produce some oligosaccharides, but only are to utilize the interior normal enzyme of microbe system to produce by microbial fermentation processes, and to also not carrying out by the research of genetic engineering technique exploitation oligosaccharides.Along with the continuous parsing of increasing Glycosylase and gene thereof and constantly illustrating of microorganism metabolism controlling route, making up metabolic engineering bacteria production structure oligosaccharides specific, definite functions becomes possibility.But how obtaining a large amount of cheap glycosyl donors also is vital for the preparation of oligosaccharides.At present, no matter chemical method or enzyme process synthesis of oligose, its required glycosyl is expensive for equal body.

Summary of the invention

The purpose of this invention is to provide a kind of construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains, this construction process utilizes the E.coli expression system, to being that the relevant enzyme of the synthetic GDP-seminose of substrate efficiently expresses with the seminose, utilize gene recombination technology to make up the recombinant escherichia coli strain that contains these three kinds of enzymes, carry out the method for the synthetic GDP-seminose of coupled fermentation effect, the engineering strain that this method makes up can increase the copy number of three enzyme genes, improve the expression amount of enzyme, thereby can act on the synthetic GDP-seminose of substrate seminose.

The objective of the invention is to be achieved through the following technical solutions:

A kind of construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains, construction process may further comprise the steps:

(1) gene amplification: the sequences Design primer according to hexokinase gene glk, mannose-phosphate mutase gene manB in the e. coli k-12 genome and mannose-1-phosphate guanosine acyltransferase gene manC amplifies three enzyme gene orders in the intestinal bacteria;

(2) construction of recombinant plasmid: the method for cutting connection with enzyme links to each other above-mentioned three kinds of goal gene respectively with carrier pET-22b, obtain carrying the recombinant expression vector of enzyme gene, and carries out the double digestion checking;

(3) structure of engineering strain: the recombinant expression vector that above-mentioned checking is correct is transformed among the host strain BL21 (DE3), obtains containing three kinds of engineering strains of recombinant plasmid;

(4) the synthetic GDP-seminose of mixed fermentation: the mixed fermentation of three engineering strains makes the product of engineering strain act on the synthetic GDP-seminose of seminose by changing the cell permeability.

And three kinds of engineering strains of described step (3) are respectively: BL21 (DE3)/pET-22b-glk, BL21 (DE3)/pET-22b-manB and BL21 (DE3)/pET-22b-manC.

And the moiety of described step (4) mixed fermentive culture medium is g/L:BL21 (DE3)/pET-22b-gL weight in wet base 25; BL21 (DE3)/pET-22b-manB weight in wet base 15; BL21 (DE3)/pET-22b-manC weight in wet base 25; Seminose 30; Phytic acid 5; KH ₂PO ₄25; MgSO ₄.7H ₂O 5; ATP 5; Nymeen S-215 4.

Positively effect of the present invention:

1, the present invention is based on the industrialization intestinal bacteria, adopt the genetically engineered recombinant technology to make up BL21 (DE3)/pET-22b-glk, BL21 (DE3)/pET-22b-manB and BL21 (DE3)/three kinds of engineering strains of pET-22b-manC, the utilization fermentation engineering carries out mixed fermentation with three strain engineering bacterias, thereby act on the synthetic GDP-seminose of seminose, for the suitability for industrialized production of oligosaccharides provides a feasible way, yield height not only, and can save industrial materials, have obvious social benefits.

2, the present invention is a purpose to produce the GDP-seminose, with the seminose is starting point, utilize genetic engineering technique to make up the metabolic engineering bacterial strain, various enzymes efficiently expresses in the realization pathways metabolism, and then reach a large amount of synthetic of GDP-seminose, provide valid approach for further producing human milk oligosaccharides in batches, break a large amount of synthetic fucosylation oligosaccharides and obtain difficult bottleneck in a large number; Especially the continuous deterioration problem of resistance problem that causes for the common abuse of current microbiotic adopts gentle methods of treatment such as anti-adhesive therapy etc. to treat, and is exactly wherein one of desirable selection and utilize human milk oligosaccharides opposing pathogenic bacterial infection.The present invention provides raw material for natural oligosaccharides medicine production, for the realization of this target provides possibility, meet current bio-pharmaceutical field development trend, not only have stronger fundamental research and be worth, and having huge economic benefit and social benefit, the prospect of marketing is wide.

Description of drawings:

Fig. 1 is an amplification electrophorogram of the present invention, wherein: 1, hexokinase glk; 2, mannose-phosphate mutase gene manB; 3, mannose-1-phosphate guanosine acyltransferase gene manC;

Fig. 2 is pET-glk of the present invention, pET-manB, the structure iron of the recombinant expression plasmid of pET-manC, the wherein structure of a, recombinant plasmid pET-glk; The structure of b, recombinant plasmid pET-manB; The structure of c, recombinant plasmid pET-manC;

Fig. 3 utilizes the structure schema of the synthetic GDP-seminose system of engineering strain mixed fermentation for the present invention.

Embodiment

Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.

The construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains involved in the present invention, be to utilize escherichia expression system BL21 (DE3)/pET-22b, to catalysis is that the relevant enzyme (hexokinase gene (glk), mannose-phosphate mutase gene (manB) and mannose-1-phosphate guanosine acyltransferase gene (manC)) of the synthetic GDP-seminose of substrate efficiently expresses with the seminose, make up three reorganization large intestine bacterial strains that comprise these three kinds of enzymes respectively, carry out the efficient synthetic of GDP-seminose by the metabolic regulation means.Three used enzyme sources of the present invention are E.coli-K12, among the present invention recombinant vectors host cell be e. coli bl21 (DE3).

The recombinant expression vector of three important enzyme dna sequences in the metabolic process; Recombinant expression vector is except that the dna sequence dna that comprises codase, also has the required controlling elements of this gene of expression, these carriers carry complete open-reading frames, promptly contain a promotor and a terminator, one section multiple clone site is arranged between promotor and terminator, these multiple clone site include some single restriction enzyme sites respectively, select for use restriction enzyme that carrier is cut into wire, and the enzyme gene is connected on the selected carrier, thereby be built into a recombinant vectors that between promotor and terminator, contains goal gene with dna ligase.

The construction process step is:

1, the amplification of hexokinase gene (glk), mannose-phosphate mutase gene (manB) and mannose-1-phosphate guanosine acyltransferase gene (manC)

Extract the genome of intestinal bacteria E.coli-K12, design following primer:

Pglk1:5 '-GGAATTC CATATGATGACAAAGTATGCATTAGTCGGT-3 ' restriction enzyme site is Nde I;

Pglk2:5 '-CGC GGATCCTTACAGAATGTGACCTAAGGTCTG-3 ' restriction enzyme site is BamH I;

PmanB1:5 '-GGAATTC CATATGATGAAAAAATTAACCTGCTTT-3 ' restriction enzyme site is Nde I;

PmanB2:5 '-CCC AAGCTTTTACTCGTTCAGCAACGTC-3 ' restriction enzyme site is HindIII;

PmanC1:5 '-CATG CCATGGATATGACAAAGTATGCATTAGTCGGT-3 ' restriction enzyme site is Nco I;

Pmanc2:5 '-CCC AAGCTTTTACAGAATGTGACCTAAGGTCTG-3 ' restriction enzyme site is HindIII.

With intestinal bacteria E.coli-K12 chromosomal DNA is that template is carried out pcr amplification, in the following order, in sterilization EP pipe, mix,

A. adopt the pcr amplification system of 50 μ L:

B. the PCR condition that is used for amplifying target genes:

The amplified production of gained is carried out the agarose gel electrophoresis detection, detect amplified production 0.9kb (glk), 1.3kb (manB), 1.4kb (manC), the result as shown in Figure 1, can see at about 0.9kb, 1.3kb, 1.4kb locate to occur a specific band, its size fits like a glove with the goal gene size, is connected on the pET-22b carrier, obtain pET-glk, pET-manB, pET-manC, with its order-checking as can be known (entrust Beijing precious biological) increase the hexokinase gene, the dna sequence dna of mannose-phosphate mutase gene and mannose-1-phosphate guanosine acyltransferase gene is as the back table.

2, construction of recombinant plasmid

(1) preparation of expression vectors

Carry the e. coli jm109 bacterial strain (available from precious biotech firm) of plasmid pET-22b at the LB inoculation of medium of penbritin (50 μ g/mL), spend the night in 37 ℃ of shaking culture.1.5mL bacterium liquid is changed in the Eppendorf tube, the 12000r/min clock, centrifugal 30s collects thalline, abandons supernatant, controls dried raffinate.Precipitation is resuspended in the solution 1 of 100 μ L precoolings, and (10mmol EDTA PH8.0), mixes for 50mmol sucrose, 25mmolTris.(0.2mol NaoH 1%SDS) covers the tight mouth of pipe, shakes up gently, places that 1-2min is limpid to liquid on ice to add the solution 2 of the new configuration of 200 μ L.Solution 3 (the 3mol acetate first that adds 150 μ L precoolings, PH4.8) rotate centrifuge tube gently, solution 3 is mixed in the heavy-gravity bacterial lysate, ice bath 3-5min, 12000r/min, centrifugal 5min, supernatant is transferred in another pipe, the 12000r/min clock, centrifugal 5min moves on to supernatant in another centrifuge tube again.The dehydrated alcohol that adds 2-2.5 times of volume, mixing, ice bath (or-20 ℃) is placed 30min.12000r/min, centrifugal 5min collects the plasmid DNA precipitation.Precipitate 2-3 time with 70% washing with alcohol, discard raffinate, air drying 10-20min is with the distilled water dissolution precipitation of 20 μ L.The pET-22b that is obtained promptly can be used as the carrier that connects amylase gene.

(2) structure of hexokinase gene (glk), mannose-phosphate mutase gene (manB) and mannose-1-phosphate guanosine acyltransferase gene (manC) expression vector

1. to carrier pET-22b and glk, manB, manC PCR product carries out double digestion, then electrophoresis, cut glue and reclaim plasmid and PCR product after enzyme is cut, all select for use 50 μ L enzymes to cut system:

a.

b.

c.

Above-mentioned endonuclease bamhi adopts DNA purification kit (TaKaBa company) to carry out purifying, and the linear purifying pET-22b and glk, manB, the manC that are obtained promptly can be used to construction recombination plasmid.

2. carrier and goal gene is connected

All select 10 μ L linked systems for use:

Gained connects mixture and adopts DNA purification kit (TaKaBa company) to carry out purifying, and the purifying after product is used for electrotransformation transformed into escherichia coli BL21 (DE3).

3, the structure of engineering strain

The competent cell for preparing e. coli bl21 (DE3) as follows: connect intestinal bacteria-BL21 (DE3) slant strains and be inoculated in the 5mL LB substratum, 37 ℃ of shaking culture 2-3h make cell reach logarithmic phase (OD ₆₀₀=0.6), triangular flask transferred to place 20min on ice, 3000r/min, 4 ℃ of centrifugal 15min, collecting cell, with 300 μ L, 10% glycerine suspension cell, 3000r/min, 4 ℃ of centrifugal 15min, this process repeats once, at last with cell suspension in 300 μ L, 10% glycerine, divide the centrifuge tube that installs to precooling by each part 40 μ L, place-70 ℃ of preservations then.

During use competent cell is placed thawing on ice, simultaneously electricity is transformed cup and be also placed in cooled on ice.In the competence of a pipe 40 μ L, add the above-mentioned connection product of 5 μ L, add electricity behind the mixing and transform in the cup, touch liquid to guarantee that bacterium and DNA suspension are positioned at electricity and transform the cup bottom.Open electric conversion instrument, adjust to the Ecl shelves, promptly aim at intestinal bacteria and transform one grade that is provided with, dry water of condensation and fog that electricity transforms the cup outside, put in the electric conversion instrument, by the shelves of above-mentioned setting, the electricity that starts pair cell transforms.After transforming end, fast as far as possible taking-up electricity revolving cup adds 1mL SOC nutrient solution, changes over to behind the mixing in the 1.5mL centrifuge tube, cultivates 1h in 37 ℃.Be applied on the LA flat board that contains penbritin (100 μ g/mL) by each dull and stereotyped 100 μ L, be inverted overnight incubation (16-20h) for 37 ℃.The single bacterium colony of picking from the flat board is inoculated in the liquid LB substratum, cultivates 12-18h in 37 ℃, extracts plasmid DNA then in a small amount, carries out the double digestion evaluation with corresponding restriction enzyme.The recombinant plasmid structure is seen Fig. 2.

4, the synthetic GDP-seminose of the mixed fermentation of three strain engineering strains

BL21 (DE3)/pET-22b-glk, BL21 (the DE3)/pET-22b-manB and BL21 (the DE3)/pET-22b-manC engineering strain that build are cultivated in the LB substratum respectively, when the thalli growth amount reaches OD ₆₀₀=0.4～0.6 o'clock preparation bacterium as the synthetic GDP-seminose of mixed fermentation.

The synthetic GDP-seminose of mixed fermentation carries out in the LB substratum that shakes 30mL in the bottle of 250mL, and its moiety is (g/L): BL21 (DE3)/pET-22b-glk, 25 (thalline weight is weight in wet base); BL21 (DE3)/pET-22b-manB, 15; BL21 (DE3)/pET-22b-manC, 25; Seminose, 30; Phytic acid, 5; KH ₂PO ₄, 25; MgSO ₄.7H ₂O, 5; ATP, 5; Nymeen S-215,4 (tensio-active agent, the perviousness of increase bacterium surface); Reaction system maintains 7.2 with the NaOH of 4N with the pH value, is reflected at 32 ℃, carries out 22h under the condition of 900r/min.

Reaction mixture is centrifugal under 4 ℃, 4000r/min, and 15min removes thalline.Getting supernatant 100 μ L mixes with (1-benzene-3 methyl-5 pyrazolone) PMP/ methanol solution (015mol/L) of 100 μ L, 70 ℃ are reacted 30min down, take out postcooling to room temperature, add the 1.5mL pure water, vacuum-drying, repeat 2 times, dried matter is fully removed organic phase after the vibration with 1mL pure water and the dissolving of 1mL chloroform, repeats 3 times, behind the speed high speed centrifugation 2min of aqueous phase solution with 13000r/min, carry out HPLC and analyze.Chromatographic condition is: chromatographic column is Waters Symmetry C18 (3.9mm * 150mm * 5 μ m).Mobile phase A is 90%0.1mol/L ammonium acetate (pH=5.0)+10% acetonitrile, and Mobile phase B is 75%0.1mol/L ammonium acetate (pH=5.0)+25% acetonitrile.Adopt gradient elution: 45%B moving phase 55min, 100%B moving phase, flow velocity is 1mL/min, and column temperature is 30 ℃, and sample size 10 μ L detect wavelength 254nm.

The output that external standard method is calculated the GDP-seminose is 10mg/L, the regression equation of GDP-seminose:

y＝0.001+12.7x(R＝0.9998)

The mixed fermentation of three engineering strains makes the product of engineering strain act on the synthetic GDP-seminose of seminose by changing the cell permeability.

SEQUENCE?LISTING

＜110〉University Of Science and Technology Of Tianjin

＜120〉utilize coupling and fermenting gene engineering strains to synthesize the construction process of GDP-seminose system

<130>2009-09-18

<160>9

<170>PATENTIN?VERSION?3.3

<210>1

<211>37

<212>DNA

<213>PGLK1

<400>1

GGAATTCCAT?ATGATGACAA?AGTATGCATT?AGTCGGT 37

<210>2

<211>33

<212>DNA

<213>PGLK2

<400>2

CGCGGATCCT?TACAGAATGT?GACCTAAGGT?CTG 33

<210>3

<211>34

<212>DNA

<213>PMANB1

<400>3

GGAATTCCAT?ATGATGAAAA?AATTAACCTG?CTTT 34

<210>4

<211>28

<212>DNA

<213>PMANB2

<400>4

CCCAAGCTTT?TACTCGTTCA?GCAACGTC 28

<210>5

<211>36

<212>DNA

<213>PMANC1

<400>5

CATGCCATGG?ATATGACAAA?GTATGCATTA?GTCGGT 36

<210>6

<211>33

<212>DNA

<213>PMANC2

<400>6

CCCAAGCTTT?TACAGAATGT?GACCTAAGGT?CTG 33

<210>7

<211>966

<212>DNA

＜213〉hexokinase (GLK)

<400>7

ATGACAAAGT?ATGCATTAGT?CGGTGATGTG?GGCGGCACCA?ACGCACGTCT?TGCTCTGTGT 60

GATATTGCCA?GTGGTGAAAT?CTCGCAGGCT?AAGACCTATT?CAGGGCTTGA?TTACCCCAGC 120

CTCGAAGCGG?TCATTCGCGT?TTATCTTGAA?GAACATAAGG?TCGAGGTGAA?AGACGGCTGT 180

ATTGCCATCG?CTTGCCCAAT?TACCGGTGAC?TGGGTGGCGA?TGACCAACCA?TACCTGGGCG 240

TTCTCAATTG?CCGAAATGAA?AAAGAATCTC?GGTTTTAGCC?ATCTGGAAAT?TATTAACGAT 300

TTTACCGCTG?TATCGATGGC?GATCCCGATG?CTGAAAAAAG?AGCATCTGAT?TCAGTTTGGT 360

GGCGCAGAAC?CGGTCGAAGG?TAAGCCTATT?GCGGTTTACG?GTGCCGGAAC?GGGGCTTGGG 420

GTTGCGCATC?TGGTCCATGT?CGATAAGCGT?TGGGTAAGCT?TGCCAGGCGA?AGGCGGTCAC 480

GTTGATTTTG?CGCCGAATAG?TGAAGAAGAG?GCCATTATCC?TCGAAATATT?GCGTGCGGAA 540

ATTGGTCATG?TTTCGGCGGA?GCGCGTGCTT?TCTGGCCCTG?GGCTGGTGAA?TTTGTATCGC 600

GCAATTGTGA?AAGCTGACAA?CCGCCTGCCA?GAAAATCTCA?AGCCAAAAGA?TATTACCGAA 660

CGCGCGCTGG?CTGACAGCTG?CACCGATTGC?CGCCGCGCAT?TGTCGCTGTT?TTGCGTCATT 720

ATGGGCCGTT?TTGGCGGCAA?TCTGGCGCTC?AATCTCGGGA?CATTTGGCGG?CGTGTTTATT 780

GCGGGCGGTA?TCGTGCCGCG?CTTCCTTGAG?TTCTTCAAAG?CCTCCGGTTT?CCGTGCCGCA 840

TTTGAAGATA?AAGGGCGCTT?TAAAGAATAT?GTCCATGATA?TTCCGGTGTA?TCTCATCGTC 900

CATGACAATC?CGGGCCTTCT?CGGTTCCGGT?GCACATTTAC?GCCAGACCTT?AGGTCACATT 960

CTGTAA 966

<210>8

<211>1371

<212>DNA

＜213〉mannose-phosphate mutase (MANB)

<400>8

ATGAAAAAAT?TAACCTGCTT?TAAAGCCTAT?GATATTCGCG?GGAAATTAGG?CGAAGAACTG 60

AATGAAGATA?TCGCCTGGCG?CATTGGTCGC?GCCTATGGCG?AATTTCTCAA?ACCGAAAACC 120

ATTGTGTTAG?GCGGTGATGT?CCGCCTCACC?AGCGAAACCT?TAAAACTGGC?GCTGGCGAAA 180

GGTTTACAGG?ATGCGGGCGT?TGACGTGCTG?GATATTGGTA?TGTCCGGCAC?CGAAGAGATC 240

TATTTCGCCA?CGTTCCATCT?CGGCGTGGAT?GGCGGCATTG?AAGTTACCGC?CAGCCATAAT 300

CCGATGGATT?ATAACGGCAT?GAAGCTGGTT?CGCGAGGGGG?CTCGCCCGAT?CAGCGGAGAT 360

ACCGGACTGC?GCGACGTCCA?GCGTCTGGCT?GAAGCCAACG?ACTTTCCTCC?CGTCGATGAA 420

ACCAAACGCG?GTCGCTATCA?GCAAATCAAC?CTGCGTGACG?CTTACGTTGA?TCACCTGTTC 480

GGTTATATCA?ATGTCAAAAA?CCTCACGCCG?CTCAAGCTGG?TGATCAACTC?CGGGAACGGC 540

GCAGCGGGTC?CGGTGGTGGA?CGCCATTGAA?GCCCGCTTTA?AAGCCCTCGG?CGCGCCCGTG 600

GAATTAATCA?AAGTGCACAA?CACGCCGGAC?GGCAATTTCC?CCAACGGTAT?TCCTAACCCA 660

CTACTGCCGG?AATGCCGCGA?CGACACCCGC?AATGCGGTCA?TCAAACACGG?CGCGGATATG 720

GGCATTGCTT?TTGATGGCGA?TTTTGACCGC?TGTTTCCTGT?TTGACGAAAA?AGGGCAGTTT 780

ATTGAGGGCT?ACTACATTGT?CGGCCTGTTG?GCAGAAGCAT?TCCTCGAAAA?AAATCCCGGC 840

GCGAAGATCA?TCCACGATCC?ACGTCTCTCC?TGGAACACCG?TTGATGTGGT?GACTGCCGCA 900

GGTGGCACGC?CGGTAATGTC?GAAAACCGGA?CACGCCTTTA?TTAAAGAACG?TATGCGCAAG 960

GAAGACGCCA?TCTATGGTGG?CGAAATGAGC?GCCCACCATT?ACTTCCGTGA?TTTCGCTTAC 1020

TGCGACAGCG?GCATGATCCC?GTGGCTGCTG?GTCGCCGAAC?TGGTGTGCCT?GAAAGATAAA 1080

ACGCTGGGCG?AACTGGTACG?CGACCGGATG?GCGGCGTTTC?CGGCAAGCGG?TGAGATCAAC 1140

AGCAAACTGG?CGCAACCCGT?TGAGGCGATT?AACCGCGTGG?AACAGCATTT?TAGCCGTGAG 1200

GCGCTGGCGG?TGGATCGCAC?CGATGGCATC?AGCATGACCT?TTGCCGACTG?GCGCTTTAAC 1260

CTGCGCACCT?CCAATACCGA?ACCGGTGGTG?CGCCTGAATG?TGGAATCGCG?CGGTGATGTG 1320

CCGCTGATGG?AAGCGCGAAC?GCGAACTCTG?CTGACGTTGC?TGAACGAGTA?A 1371

<210>9

<211>1437

<212>DNA

＜213〉mannose-1-phosphate guanosine acyltransferase (MANC)

<400>9

ATGGCGCAGT?CGAAACTCTA?TCCAGTTGTG?ATGGCAGGTG?GCTCCGGTAG?CCGCTTATGG 60

CCGCTTTCCC?GCGTACTTTA?TCCCAAGCAG?TTTTTATGCC?TGAAAGGCGA?TCTCACCATG 120

CTGCAAACCA?CCATCTGCCG?CCTGAACGGC?GTGGAGTGCG?AAAGCCCGGT?GGTGATTTGC 180

AATGAGCAGC?ACCGCTTTAT?TGTCGCGGAA?CAGCTGCGTC?AACTGAACAA?ACTTACCGAG 240

AACATTATTC?TCGAACCGGC?AGGGCGAAAC?ACGGCACCTG?CCATTGCGCT?GGCGGCGCTG 300

GCGGCAAAAC?GTCATAGCCC?GGAGAGCGAC?CCGTTAATGC?TGGTATTGGC?GGCGGATCAT 360

GTGATTGCCG?ATGAAGACGC?GTTCCGTGCC?GCCGTGCGTA?ATGCCATGCC?ATATGCCGAA 420

GCGGGCAAGC?TGGTGACCTT?CGGCATTGTG?CCGGATCTAC?CAGAAACCGG?TTATGGCTAT 480

ATTCGTCGCG?GTGAAGTGTC?TGCGGGTGAG?CAGGATATGG?TGGCCTTTGA?AGTGGCGCAG 540

TTTGTCGAAA?AACCGAATCT?GGAAACCGCT?CAGGCCTATG?TGGCAAGCGG?CGAATATTAC 600

TGGAACAGCG?GTATGTTCCT?GTTCCGCGCC?GGACGCTATC?TCGAAGAACT?GAAAAAATAT 660

CGCCCGGATA?TCCTCGATGC?CTGTGAAAAA?GCGATGAGCG?CCGTCGATCC?GGATCTCAAT 720

TTTATTCGCG?TGGATGAAGA?AGCGTTTCTC?GCCTGCCCGG?AAGAGTCGGT?GGATTACGCG 780

GTCATGGAAC?GTACGGCAGA?TGCTGTTGTG?GTGCCGATGG?ATGCGGGCTG?GAGCGATGTT 840

GGCTCCTGGT?CTTCATTATG?GGAGATCAGC?GCCCACACCG?CCGAGGGCAA?CGTTTGCCAC 900

GGCGATGTGA?TTAATCACAA?AACTGAAAAC?AGCTATGTGT?ATGCTGAATC?TGGCCTGGTC 960

ACCACCGTCG?GGGTGAAAGA?TCTGGTAGTG?GTGCAGACCA?AAGATGCGGT?GCTGATTGCC 1020

GACCGTAACG?CGGTACAGGA?TGTGAAAAAA?GTGGTCGAGC?AGATCAAAGC?CGATGGTCGC 1080

CATGAGCATC?GGGTGCATCG?CGAAGTGTAT?CGTCCGTGGG?GCAAATATGA?CTCTATCGAC 1140

GCGGGCGACC?GCTACCAGGT?GAAACGCATC?ACCGTGAAAC?CGGGCGAGGG?CTTGTCGGTA 1200

CAGATGCACC?ATCACCGCGC?GGAACACTGG?GTGGTTGTCG?CGGGAACGGC?AAAAGTCACC 1260

ATTGATGGTG?ATATCAAACT?GCTTGGTGAA?AACGAGTCCA?TTTATATTCC?GCTGGGGGCG 1320

ACGCATTGCC?TGGAAAACCC?GGGGAAAATT?CCGCTCGATT?TAATTGAAGT?GCGCTCCGGC 1380

TCTTATCTCG?AAGAGGATGA?TGTGGTGCGT?TTCGCGGATC?GCTACGGACG?GGTGTAA 1437

Claims

1, a kind of construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains, it is characterized in that: construction process may further comprise the steps:

2, the construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains according to claim 1, it is characterized in that: three kinds of engineering strains of described step (3) are respectively: BL21 (DE3)/pET-22b-glk, BL21 (DE3)/pET-22b-manB and BL21 (DE3)/pET-22b-manC.

3, the construction process that utilizes the synthetic GDP-seminose system of coupling and fermenting gene engineering strains according to claim 1, it is characterized in that: the moiety of described step (4) mixed fermentive culture medium is g/L:BL21 (DE3)/pET-22b-gL weight in wet base 25; BL21 (DE3)/pET-22b-manB weight in wet base 15; BL21 (DE3)/pET-22b-manC weight in wet base 25; Seminose 30; Phytic acid 5; KH ₂PO ₄25; MgSO ₄.7H ₂O 5; ATP 5; Nymeen S-215 4.