CN101560475A - Uracil auxotroph Hansenula yeast, construction method thereof and application thereof - Google Patents

Abstract

The invention discloses uracil auxotroph Hansenula yeast gene engineering host strain Hansenula yeast YH-11 CGMCC NO.2976 with stable heredity, and simultaneously provides a construction method for the Hansenula yeast. The construction method comprises the following steps: 1) constructing recombinant plasmid p18HURA containing orotidine-5-phosphate decarboxylase gene (HURA3) of the Hansenula yeast; and 2) constructing recombinant plasmid pHURK for the HURA3 with disrupted gene; and converting the plasmid pHURK of step 2) into the Hansenula yeast by using a yeast whole cell conversion method, and obtaining the uracil auxotroph strain through screening. The invention further discloses application of the constructed uracil auxotroph Hansenula yeast gene engineering host strain Hansenula yeast in producing foreign protein.

Description

Uracil auxotroph Hansenula yeast and construction process thereof and application

Technical field

The present invention relates to a kind of debaryomyces hansenii bacterium and construction process and its thereof application in producing foreign protein of recombinating.The present invention is uracil auxotroph Hansenula yeast and construction process and application in particular.

Background technology

Utilize genetic engineering technique to obtain have important biomolecule to learn the important goal that active protein is modern biotechnology.In protein expression in vitro matter, importantly when making great efforts to improve expression amount, keep the space structure of product consistent with native protein to greatest extent with biologic activity.People have set up intestinal bacteria, baculovirus, mammalian cell and yeast four big exogenous gene expression systems and have efficiently expressed external with the protein that adapts to various sources for this reason.Yeast expression system wherein is the strong instrument that the research eukaryotic protein is expressed and analyzed, have and transcribe the post-treatment rhetorical function, being suitable for stably express has the exogenous protein of function. compare with mammalian expression system with insect expression system, yeast expression system is easy and simple to handle, with low cost, can ferment on a large scale, be optimal reorganization eukaryotic protein production preparation instrument.

In the time of decades, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) has obtained using widely as first eukaryotic gene expression system in the past.But also there are many weak points in this expression system in application process, and the secretion level of, expressing protein not high as the exogenous gene expression level is low and the polypeptide chain glycosylation is excessive, bacterial strain instability etc.And other yeast strain, especially methyl alcohol nutritional type yeast then demonstrates bigger advantage on expression of exogenous gene: recombinant bacterial strain genetic stability height, the expression amount of foreign protein and secernment efficiency are than the high 10-100 of yeast saccharomyces cerevisiae doubly, and therefore the phenomenon that does not have excessive glycosylation is suitable for large scale fermentation and produces target protein.Debaryomyces hansenii wherein (Hansenula) also has its distinctive advantage except that having above-mentioned advantage: (1) can utilize strong promoters such as methanol oxidase gene (MOX) promotor and formaldehyde dehydrogenase gene (FMD) promotor to start expression of exogenous gene efficiently; (2) the frequency height of non-homogeneous reorganization, thereby recombinant plasmid can be incorporated on the karyomit(e) by high copy, easily obtains the transformant that high copy is integrated; (3) foreign protein of Biao Daing is stored in the peroxysome usually, can make its degraded of avoiding the intracellular protein enzyme, and has reduced the toxic action of pair cell; (4) can carry out high density fermentation with the substratum of cheapness, further improve the expression of exogenous gene level; (5) high temperature resistant, optimum growth temperature is 37 ℃-43 ℃, and growth velocity is fast, and the incubation time of large scale fermentation is short, pollution rate is low.Therefore yeast expression system is subjected to increasing the attention and utilization.

Summary of the invention

The purpose of this invention is to provide the more stable uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium of a kind of heredity.Uracil auxotroph Hansenula Yeast gene engineering host bacterium provided by the invention is vitamin B13 glycosides-ruined debaryomyces hansenii bacterium of 5-phosphate decarboxylase gene (HURA3).

Uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium (Hansenula) the YH-11CGMCC N0.2976 that the invention discloses, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on March 24th, 2009, preserving number is YH-11CGMCC NO.2976, classification called after: unusual pichia spp, Pichia anomala.Bacterium colony is convex, oyster white, glossy, neat in edge, and the cell ovalize, the optimum growth temperature of this bacterial strain is 37 ℃, pH is 5.5.

The present invention further discloses the method that makes up uracil auxotroph Hansenula yeast, carry out according to the following steps:

1) structure contains the recombinant plasmid p18HURA of vitamin B13 glycosides-5-phosphate decarboxylase gene (HURA3) of debaryomyces hansenii bacterium;

2) make up the ruined recombinant plasmid pHURK of HURA3 gene;

With step 2) plasmid pHURK transform the debaryomyces hansenii bacterium with yeast intact cell conversion method, obtain the uracil auxotrophy bacterial strain through screening.

Being described in detail as follows of concrete construction process:

1) recombinant vectors of structure vitamin B13 glycosides-screened marker gene deactivation of 5-phosphate decarboxylase gene; Recombinant vectors can be pHURK.

2) recombinant vectors in the step 1) is imported in the wild-type debaryomyces hansenii bacterium, be preferably debaryomyces hansenii bacterium (Hansenula).Screening obtains vitamin B13 glycosides-ruined debaryomyces hansenii bacterium of 5-phosphate decarboxylase gene (HURA3).Wherein, described selection markers gene can be G418 resistant gene, neomycin resistance gene or cupric ion resistant gene.

Described G418 resistant gene can be KanMX.Derive from pFA6a-KanMX.

The carrier that sets out that is used for making up the recombinant vectors of described vitamin B13 glycosides-screened marker gene deactivation of 5-phosphate decarboxylase gene (HURA3) can be plasmid vector, filobactivirus carrier or the yeast shuttle vectors in genetically engineered field, be preferably pUC18, pUC19, pUC118, pUC119 etc., especially be preferably pUC18 or pUC19.

Described KanMX can derive from pFA6a-KanMX.

Described wild-type debaryomyces hansenii bacterium is preferably debaryomyces hansenii bacterium (Hansenula).

The recombinant vectors of described vitamin B13 glycosides-screened marker gene deactivation of 5-phosphate decarboxylase gene can be pHURK.

Described reorganization debaryomyces hansenii bacterium is debaryomyces hansenii bacterium (Hansenula).

Foreign protein of the present invention is amylase or phytase.

The present invention has made up the uracil auxotroph Hansenula yeast pearl, particularly debaryomyces hansenii bacterium (Hansenula) YH-11CGMCC NO.2976 of inheritance stability by genetic engineering means.5 bases have been inserted in the HURA3 gene inside of the debaryomyces hansenii mutant strain of the HURA3 gene function disappearance that the present invention obtains, phase shift mutation takes place after causing the 10th codon, make cell can not produce activated vitamin B13 glycosides-5-phosphate decarboxylase, with uracil auxotroph Hansenula yeast mutant (Hansenula) the called after YH-11 of this HURA3 gene function disappearance.The at present domestic and international used all advantages of inferior yeast of this bacterial strain ratio:

(1) mitotic division genetic stability height, reverse mutation rate is low.

(2) have autonomously replicating sequence (HARS), the frequency height of reorganization, recombinant plasmid can be incorporated on the karyomit(e) by high copy, easily obtains the transformant that high copy is integrated.

(3) can utilize strong promoters such as methanol oxidase (MOX) promotor and formaldehyde dehydrogenase gene (FMD) promotor, start expression of exogenous gene efficiently.

(4) proteic expression is carried out in peroxysome usually, can make its degraded of avoiding intracellular protease, and has reduced the toxic action of pair cell.

(5) can utilize cheap chemosynthesis substratum to carry out the cell high density fermentation, not need to add expensive natural constituents (as yeast powder, peptone), further reduce production cost and improved the expression of exogenous gene level.

(6) high temperature resistant, optimum growth temperature is 37-430C, and growth velocity is fast, and the incubation time of large scale fermentation is short.

(7) be the sole carbon source and the energy with glycerine or methyl alcohol in the fermentation, can utilize the microorganism of glycerine few, the microorganism that can utilize methyl alcohol still less, fermentation microbiological contamination rate is low.

(8) expression amount of foreign protein and secernment efficiency are higher about 10 times than yeast saccharomyces cerevisiae, and do not have the excessive glycosylation phenomenon, therefore are suitable for large scale fermentation and produce target protein.

Description of drawings:

Fig. 1 is the structure synoptic diagram of recombinant plasmid p18HURA;

Fig. 2 is that uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium makes up synoptic diagram;

Fig. 3 is the physical map of recombinant plasmid pMOX-Amy;

Fig. 4 is the physical map of recombinant plasmid pMOX α-Amy;

Fig. 5 is the physical map of recombinant plasmid pMOX α-appA.

Embodiment

For simple and purpose clearly, hereinafter appropriate omission the description of known technology, in order to avoid those unnecessary details influences are to the description of the technical program.The present invention is described further below in conjunction with example.Wherein among the embodiment used restriction enzyme all available from TaKaRa company.

Embodiment 1

The structure of debaryomyces hansenii bacterium (Hansenula)

One, it is as follows to make up the building process of recombinant plasmid p18HURA recombinant plasmid p18HURA of the vitamin B13 glycosides-5-phosphate decarboxylase gene (HURA3) contain the debaryomyces hansenii bacterium:

1, the acquisition of the vitamin B13 of debaryomyces hansenii bacterium glycosides-5-phosphate decarboxylase gene (HURA3)

Primer sequence according to the nucleotide sequence design of the HURA3 of the debaryomyces hansenii bacterium of having reported is as follows:

Primer 1:5 '-CCA GGATCCTCAACATTTCCCTGAATAAT-3 ' (sequence 1 in the sequence table) (line part base is a BamH I recognition site)

Primer 2: 5 '-CGA GAATTCTCACTAGTATTC CCGCGACT-3 ' (sequence 2 in the sequence table) (line part base is an EcoR I recognition site)

Total DNA with debaryomyces hansenii bacterium (Hansenula) JCM3621 (ATCC34438) is a template, under the guiding of primer 1 and primer 2, carry out PCR reaction amplification HURA3, the PCR reaction system is: template DNA 0.6 μ g, primer 10.5 μ mol/L, primer 2 0.5 μ mol/L, dNTP 200 μ mol/L, 10 * PCR damping fluid, 5 μ L, pfu archaeal dna polymerase (Promega company) 1 unit is mended to 50 μ L with deionized water, adds 20 μ l paraffin oils behind the mixing; With homemade PCR instrument TC-96AE the PCR reaction conditions being set is: 94 ℃ 5 minutes, circulate 1 time; 94 ℃ 40 seconds, 70 ℃ 1 minute, 50 ℃ 1 minute, 29 circulations; 94 ℃ 40 seconds, 70 ℃ 15 minutes, 50 ℃ 1 minute, circulate 1 time.

The PCR product reclaimed and purifying after, carry out agarose gel electrophoresis and detect, the result shows the dna fragmentation that has obtained big or small about 0.9kb, with expect big or small consistent, with its called after HURA3.

2, with the PCR product of step 1 after BamHI and EcoRI enzyme are cut, with the plasmid vector pUC18 (TaKaRa company) that cuts through same enzyme enzyme at T ₄Under the dna ligase effect, 16 ℃ of reactions connected in 10-16 hour, obtained recombinant plasmid, with its called after p18HURA.Recombinant plasmid p18HURA is carried out enzyme with BamHI and EcoRI restriction enzyme cuts evaluation, the result show pcr amplification to the HURA3 fragment have identical restriction enzyme mapping with HURA3 fragment among the GenBank; Adopt two deoxidation cessation method to measure the nucleotide sequence of the HURA3 that inserts recombinant plasmid p18HURA, the result shows that HURA3 and the sequence homology of the HURA3 that has reported are 100%, illustrates that amplification has obtained the encoding sequence of debaryomyces hansenii bacterium vitamin B13 glycosides-5-phosphate decarboxylase gene.

Two, the structure concrete steps of the ruined recombinant plasmid pHURK of HURA3 gene are as follows:

1, carrier pFA6a-KanMX (the Wach A that contains G418 resistant gene (KanMX), Brachat A, PoehlmannR, Philippsen P. (1994) New heterologous modules for classical or PCR-based genedisruptions in Saccharomyces cerevisiaea.Yeast, 10:1793-1808);

2, use the recombinant plasmid p18HURA of restriction enzyme EcoRV and SacI double digestion embodiment 1, plasmid vector pFA6a-KanMX with Restriction enzyme Sma I and SacI double digestion step 1, above-mentioned enzyme is cut product reclaim the p18HURA linear fragment of about 3.6Kb and G418 resistant gene (KanMX) fragment of 1.48Kb through the low melting-point agarose gel electrophoresis respectively, and the dna fragmentation that reclaims is dissolved in TE damping fluid (10mM Tris-HCl respectively, 1mM EDTA, pH8.0) ,-20 ℃ preservation is standby;

3, with the p18HURA linear fragment and G418 resistant gene (KanMX) the fragment T of step 2 ₄Dna ligase connects, it is destroyed to obtain in the p18HURA carrier HURA3, it is the recombinant plasmid that the HURA3 gene function is blocked, with its called after pHURK, the ligation system is: 3 μ l deionized waters, 1 μ l 10 * connection damping fluid, 1 μ l concentration is the linearizing p18HURA of 0.05pmol/ μ l, 4 μ l concentration are KanMX fragment and the 1 μ l T of 0.02pmol/ μ l ₄Dna ligase; Condition of contact is: 22 ℃, and incubation 10-16 hour.To connect product transformed into escherichia coli competent cell again, with blue hickie method screening positive clone, the 37 ℃ of cultivations in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) of the single colony inoculation of picking white were used the alkaline lysis method of extracting plasmid after 12-24 hour, obtained recombinant plasmid pHURK.

Three, the acquisition of debaryomyces hansenii bacterium (Hansenula)

Transform the preparation of solution:

0.1M Lithium Acetate solution (LiAc-TE): Lithium Acetate is dissolved in TE damping fluid (pH8.0), and making its final concentration is 0.1M;

40%PEG-4000 Lithium Acetate solution: 20 gram PEG4000 are dissolved among the 0.1M LiAc-TE, are settled to 50ml;

Calf thymus DNA: the 4mg calf thymus DNA is dissolved in 1ml TE damping fluid (pH8.0), 4 ℃ 12-24 hour, aspirate mixing repeatedly, place 100 ℃ of boiling water to boil 10 minutes after, rapidly ice bath carries out packing after the cooling ,-20 ℃ of preservations are standby.

The plasmid pHURK of step 2 is transformed the wild-type debaryomyces hansenii with yeast intact cell conversion method, obtain uracil auxotrophy (because of the HURA3 gene function is blocked) bacterial strain through screening, concrete steps are as follows:

1, the wild-type debaryomyces hansenii being inoculated in the YEPD slant medium activates, be inoculated in the 5ml YEPD liquid nutrient medium with transfering loop picking one ring thalline, 37 ℃ of 150rpm shaking table shaking culture, get the somatic cells suspension that 1.5ml is in logarithmic phase, 5000rpm collected thalline in centrifugal 1 minute, after of the thalline cleaning of 0.5ml 0.1M Lithium Acetate solution with collection, again somatic cells is resuspended in the 100 μ l 0.1M Lithium Acetate solution, obtain the somatic cells suspension; Add 5-10 μ g plasmid pHURK and 10 μ g calf thymus DNAs in the somatic cells suspension, 37 ℃ are incubated 10 minutes, add 700 μ l40%PEG-4000 Lithium Acetate solution again, mixing, and 37 ℃ are incubated 1.5 hours; Add methyl-sulphoxide in the transformation system, making its final concentration is 10% (v/v), 45 ℃ of water-bath thermal shocks 5 minutes; Add 1ml liquid YEPD substratum again, 37 ℃ of insulations 30 minutes, centrifugal collection somatic cells is resuspended in the somatic cells of collecting in the liquid YEPD substratum that 2ml contains 1mg/ml G418, cultivates 12 hours for 37 ℃;

2, with after 1000 times of the bacterium liquid dilutions, get 200 μ L and coat on the YEPD flat board that contains 0.1mg/ml G418, cultivated 3-5 days for 37 ℃, the screening transformant;

3, the uracil auxotrophy bacterial strain in the screening transformant, concrete steps are as follows:

The single bacterium colony mixing in sterilized water that on the YEPD resistant panel, grows in the picking step 2, room temperature left standstill 4 hours, be inoculated in solid YNB (yeast growth substrate basal culture medium) culture plate then respectively, add on the solid YNB culture plate and solid YEPD flat board of 35 μ g/ml uridylics (Ura), cultivated 48 hours for 37 ℃, acquisition can normal growth on solid YNB culture plate that adds 35 μ g/ml uridylics (Ura) and YEPD flat board, and the bacterial strain that on the YNB substratum, can not grow, above-mentioned bacterial strains is streak culture on the YEPD flat board, 50 single bacterium colonies of each bacterial strain picking place sterilized water respectively, after room temperature leaves standstill 4 hours, be inoculated in the YNB culture plate of making carbon source with glucose respectively, add the YNB culture plate of 35 μ g/ml uridylics (Ura) and the YNB that makes carbon source with methyl alcohol, add on the YNB culture plate culture plate of 35 μ g/ml uridylics (Ura), cultivated 48 hours for 37 ℃, the result shows: containing on the YNB substratum of uridylic and can grow, and nonviable bacterial strain is the uridylic deficient strain on the YNB substratum that lacks uridylic, (the methyl alcohol utilization of other phenotype, 37 ℃ of well-growns) all identical with wild-type;

4, the uracil auxotrophy bacterial strain that step 3 is obtained is identified:

1) with plasmid YEp352 (Hill JE, Meyers AM, Koerner TJ, Tzagoloff A.Yeast/E.coli shuttlevectors with multiple unique restriction sites.Yeast, 1993,9:163-167.) transforming above-mentioned uridylic deficient strain, acquisition can be the debaryomyces hansenii bacterium mutant strain of HURA3 gene function disappearance by the bacterial strain of yeast saccharomyces cerevisiae URA3 gene complementation from the above-mentioned uracil auxotrophy bacterial strain of biological function proof;

2) total DNA of the uracil auxotrophy bacterial strain of extraction debaryomyces hansenii wild type strain and step 1) acquisition.With restriction enzyme HindIII and PstI digested plasmid pFA6a-KanMX, reclaim the dna fragmentation of the G418 resistant gene inside of 0.7kb, carrying out Southern hybridization as probe detects, total DNA of wild type strain and uracil auxotrophy bacterial strain does not all produce hybridization signal as a result, show that introducing intracellular KanMX by plasmid pHURK both be not inserted into HURA3 gene inside, was not inserted into chromosomal other positions yet;

3) with step 2) total DNA of the uracil auxotrophy bacterial strain that extracts is template, under the guiding of the primer 1 of step 1 and primer 2, carry out the HURA3 gene in the high-fidelity PCR reaction amplification bacterial strain, the PCR product is carried out nucleotide sequencing with dideoxy method, compare by sequential analysis and homology, 5 bases have been inserted in the HURA3 gene inside that shows the debaryomyces hansenii mutant strain that lacks at the HURA3 gene function that obtains, phase shift mutation takes place after causing the 10th codon, make cell can not produce activated vitamin B13 glycosides-5-phosphate decarboxylase, with uracil auxotroph Hansenula yeast mutant (Hansenula) the called after YH-11 of this HURA3 gene function disappearance, this bacterial strain has been preserved in Tianjin rich safe biological products company limited and has been uracil auxotroph Hansenula Yeast gene engineering host bacterium-debaryomyces hansenii bacterium (Hansenula) YH-11.

Embodiment 2

Uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium (Hansenula) YH-11CGMCCNO.2976 biomass and bioactive detection:

Detect the biomass of uracil auxotroph Hansenula yeast (Hansenula) YH-11

Wild-type debaryomyces hansenii (Hansenula) and uracil auxotroph Hansenula yeast (Hansenula) YH-11 are inoculated in the YEPD slant medium to be activated, be inoculated in the 2ml YEPD liquid nutrient medium 37 ℃ of shaking culture 16 hours with transfering loop picking one ring thalline from the inclined-plane; Transfer in 10ml YEPD liquid nutrient medium 37 ℃ of shaking table shaking culture 16 hours again by 10% inoculum size; Centrifugal collection somatic cells, with cell with aseptic washing twice, after weighing, with four parts of cell div in par aeq, two parts of cells are one group, two groups of cells are inoculated in respectively in the 50ml YEPD liquid nutrient medium and 50ml replaces with methyl alcohol in the liquid nutrient medium of the glucose among the YEPD, again with two parts of cells in every group respectively at 37 ℃ of shaking table shaking culture 24 hours and 48 hours, centrifugal collecting cell, with aseptic washing once, behind the recentrifuge, the cell precipitation of collecting is weighed, calculate its biomass.Be in the substratum of carbon source with glucose, cultivating 24 hours that the biomass of wild type strain and uracil auxotrophy bacterial strain is respectively 27.2g/L and 26.7g/L, cultivating 48 hours that biomass is respectively 37.8g/L and 37.5g/L; With methyl alcohol is in the substratum of carbon source, and the biomass of wild type strain and uracil auxotrophy bacterial strain is respectively 23.4g/L (24 hours), 31.7g/L (48 hours) and 22.9g/L (24 hours), 31.5g/L (48 hours).So debaryomyces hansenii bacterium (Hansenula) YH-11 and wild-type debaryomyces hansenii bacterium no significant difference on biomass, it is more stable to grow.

Embodiment 3

Uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium (Hansenula) YH-11CGMCCNO.2976 genetic stability is analyzed.

The genetic stability analysis of uracil auxotroph Hansenula yeast (Hansenula) YH-11

Uracil auxotroph Hansenula yeast (Hansenula) YH-11 that the HURA3 gene function of above-mentioned structure is blocked is inoculated in to go down to posterity in the YEPD liquid nutrient medium and cultivates 30 times, get 200 μ l cell after diluting and coat the YEPD flat board, cultivated 48 hours for 37 ℃, 100 single bacterium colonies of picking place the 2ml sterilized water respectively at random, at room temperature hungry 4-6 hour; Getting that bacterium liquid after the hunger is inoculated in respectively with glucose is that the YNB of carbon source is dull and stereotyped and add on the YNB flat board of 35 μ g/ml uridylics and to make the YNB of carbon source with methyl alcohol dull and stereotyped and add on the YNB flat board of 35 μ g/ml uridylics, cultivated 48 hours for 37 ℃, the result shows that single bacterium colony all grows adding on the YNB minimum medium of uridylic, then can not grow fully not adding on the YNB minimum medium of uridylic, and the reverse mutation phenomenon does not appear, debaryomyces hansenii bacterium (Hansenula) YH-11 that proof the present invention makes up is a strain inheritance stability, and has kept the uracil auxotrophy mutant strain of wild-type debaryomyces hansenii bacterium physio-biochemical characteristics.

Embodiment 4

Uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium (Hansenula) YH-11CGMCCNO.2976 uses in producing foreign protein.

Express in the born of the same parents of amylase gene in debaryomyces hansenii bacterium (Hansenula) YH-11

1) with carrier YIp5 (Struhl K, Stinchcomb DT, et al.High-frequencytransformation of yeast:Autonomous replication of hybrid DNA molecules.Proc Natl Acad Sci USA, 1979,76:1035-1039) be the carrier that sets out.With restriction enzyme SalI difference digested plasmid YIp5 and pHARS (Roggenkamp R, Hansen H, et al.Transformation ofthe methylotrophic yeast Hansenula autonomous replication and integrationvectors.Mol Gen Genet, 1986,202:302-308), the linear YIp5DNA fragment of 5.6kb and the debaryomyces hansenii autonomously replicating sequence HARS of 0.5kb are reclaimed in the low melting-point agarose gel electrophoresis respectively, and the dna fragmentation that reclaims is dissolved in TE damping fluid (10mM Tris-HCl respectively, 1mM EDTA, pH8.0) in, carry out ligation, the ligation system is: 3 μ l deionized waters, 1 μ l 10 * connection damping fluid, 1 μ l concentration is the linearizing YIp5 of 0.05pmol/ μ l, and 4 μ l concentration are HARS fragment and the 1 μ l T4DNA ligase enzyme of 0.06pmol/ μ l; Condition of contact is: 16 ℃, and incubation 10-16 hour.To connect product transformed into escherichia coli competent cell again, by tetracyclin resistance inactivation screening positive clone, single colony inoculation that picking loses tetracyclin resistance in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivate after 12-24 hour, use the alkaline lysis method of extracting plasmid, obtain recombinant plasmid pIHARS5.Behind EcoRI complete degestion plasmid pIHARS5, to carry out partially digestedly again with SalI, the 6.1DNA fragment is reclaimed in the low melting-point agarose gel electrophoresis, is dissolved in the TE damping fluid; With SalI and MunI digested plasmid pMPT121 (Godecke S, Eckart M, et al.Identification of sequences responsible for transcriptional regulation of thestrongly expressed methanol oxidase-encoding gene in hansenula,), the dna fragmentation (MOXp-MOXt) that 1.8kb contains methanol oxidase gene promoter and terminator sequence and multiple clone site is reclaimed in the low melting-point agarose gel electrophoresis, the dna fragmentation that reclaims is connected, the transformed into escherichia coli competent cell, transformant being inoculated in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivated after 12-24 hour, use the alkaline lysis method of extracting plasmid, obtain recombinant plasmid pHAM1.

According to the nucleotide sequence design primer amy1 of the saccharomycopsis fibuligera alpha-amylase gene (AMY) of report (5 '-CCGAATTCAATATGCAAATTTCAAAAGC-3 ', the line part is the EcoRI recognition site) and amy2 (5 '-CTGAATTCTCATGAACAAATGTCAGAAGC-3 ', the line part is the EcoRI recognition site, is that template is carried out PCR reaction amplification alpha-amylase gene (AMY) with saccharomycopsis fibuligera (China Committee for Culture Collection of Microorganisms common micro-organisms center) chromosomal DNA.With EcoRI respectively enzyme cut the alpha-amylase gene and the plasmid pHAM1 of pcr amplification, the dna fragmentation of 1.5kb and the linear carrier fragment of 7.9kb are reclaimed in the low melting-point agarose gel electrophoresis, above-mentioned fragment is carried out ligation under the effect of T4DNA ligase enzyme, the transformed into escherichia coli competent cell, transformant being inoculated in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivated after 12-24 hour, use the alkaline lysis method of extracting plasmid, obtain recombinant vectors pMOX-Amy, the physical map of this plasmid as shown in Figure 3, this plasmid has the URA3 gene;

2) genetic transformation: recombinant plasmid pMOX-Amy is transformed debaryomyces hansenii bacterium (Hansenula) YH-11, on the yeast minimum medium, by the complementary screening of auxotrophy transformant, can be transformant at the bacterial strain that is the dull and stereotyped growth of YNB of carbon source with glucose;

3) checking transformant: picking debaryomyces hansenii bacterium (Hansenula) YH-11 and transformant list bacterium colony place the 2ml sterilized water, hungry at ambient temperature 4-6 hour, getting bacterium liquid after the hunger is inoculated in respectively with glucose and is the YNB culture plate of carbon source and adds the YNB culture plate of 35 μ g/ml uridylics (Ura) and be the YNB culture plate of carbon source with methyl alcohol and add on the YNB culture plate of 35 μ g/ml uridylics (Ura), cultivated 48 hours for 37 ℃, the result shows that transformant all can normal growth at the YNB culture plate of two kinds of different carbon sources that do not contain uridylic (Ura), has verified transformant from nutritional needs.

4) multiple copied transformant screening: transformant is connected among the minimum medium YNB, continuous passage is cultivated 30 times, be coated with the YEPD flat board after the dilution, extracting the total DNA of single bacterium colony, is that probe carries out Southern hybridization detection with amylase gene AMY, and the result shows the recombinant bacterial strain that has obtained the high copy integration of AMY, with its called after YH11-AMY, the YH11-AMY bacterium of will recombinating is inoculated on the YEPD inclined-plane, cultivates 4 ℃ of preservations 48 hours for 37 ℃.

5) recombinant bacterial strain is cultivated: after debaryomyces hansenii recombinant bacterial strain YH11-AMY activation, be inoculated in 10 milliliters of YNB liquid nutrient mediums with transfering loop picking one garland cells, the bacterium liquid of 37 ℃ of shaking table shaking culture acquisition in 14-20 hour is as liquid spawn, liquid spawn inserted by 10% inoculum size to be equipped with 100 milliliters be that 2% cane molasses is synthetic medium (the 5.0g potassium primary phosphate of carbon source with total reducing sugars content, the 10g monoammonium phosphate, 4.5g bitter salt, 5.0g ammonium sulfate, 2.3g Repone K, 0.5g sodium-chlor, 0.75g calcium chloride, 0.1g ferric ammonium sulfate, 8mg Salzburg vitriol, 30mg Zinc vitriol, the 40mg manganous sulfate, 0.1g VitB1,0.3mg vitamin H, an amount of molasses, water is settled to 1 liter), 37 ℃ of shaking culture 48 hours;

6) inducing culture: adding methyl alcohol to final concentration in above-mentioned culture is 1%, 37 ℃ of shaking table shaking culture 48 hours, during added methyl alcohol every 8 hours in 0.5% ratio;

7) after cultivation finished, 5000 rev/mins of centrifugal 5 minutes collecting cells were measured cell endo-amylase activity according to a conventional method, showed under these conditions, and the wet cell weight of every liter of nutrient solution is the 80-85 gram, and the amylase of every gram wet cell is 28-32 enzyme activity unit.

The secreting, expressing of amylase gene in debaryomyces hansenii bacterium (Hansenula) YH-11CGMCC No.2976

1) design and synthesize primer amy3 (5 '-CC CTGCAGAATATGCAAATTTCAAAAGC-3 ', the line part is the PstI recognition site), primer amy2 in embodiment 2, with saccharomycopsis fibuligera (China Committee for Culture Collection of Microorganisms common micro-organisms center) chromosomal DNA is template, carries out PCR reaction amplification alpha-amylase gene (AMY).

Design and synthesize primer α P1 (5 '-TAA GAATTCAAAATGAGATTTCCTT-3 ', the line part is the EcoRI recognition site) and α P2 (5 '-CGT CTGCAGCTCAGCTTCAGCCTCTCTT-3 ', the line part is the PstI recognition site), be template with plasmid pMET α B (Invitrogen company), carry out the sequence MF α 1s of coding yeast saccharomyces cerevisiae α-factor signal peptide of the about 0.3kb of PCR reaction amplification.

Cut the plasmid pHAM1 that makes up among the embodiment 2 with restriction enzyme EcoRI enzyme, cut pcr amplification product AMY and MF α 1s in the present embodiment with restriction enzyme EcoRI and PstI enzyme, linear pHAM1 fragment, the alpha-amylase gene AMY of 1.5kb and the α-factor signal peptide sequence MF α 1s of 0.3kb of 7.9kb reclaimed in the low melting-point agarose gel electrophoresis, and above-mentioned three dna fragmentations are at T ₄Carry out ligation under the dna ligase effect, the transformed into escherichia coli competent cell, transformant being inoculated in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivated after 12-24 hour, use the alkaline lysis method of extracting plasmid, obtain recombinant vectors pMOX α-Amy, the physical map of this plasmid as shown in Figure 4, this recombinant vectors has the URA3 gene, and secretory signal sequence is arranged in external source gene coded sequence upstream, can carry out the secreting, expressing of foreign protein;

Step 2)-6) identical with embodiment 2;

7) after cultivation finishes, 5000 rev/mins of centrifugal 5 minutes collecting cells and supernatant liquor, measure the extracellular amylase activity according to a conventional method, show under these conditions, the wet cell weight of every liter of nutrient solution is the 83-87 gram, every liter of total amylase of nutrient solution is 2300-2560 unit of activity, and wherein the enzyme activity in the supernatant liquor is 91% of a total activity, and intracellular amylase activity only is 9% of a total activity.

Embodiment 4, the secreting, expressing of phytase in debaryomyces hansenii bacterium (Hansenula) YH-11CGMCC No.2976

1) plasmid YEp352 (Hill JE, Meyers AM, Koerner TJ, Tzagoloff A.Yeast/E.colishuttle vectors with multiple unique restriction sites.Yeast, 1993,9:163-167.) be the carrier that sets out, with EcoRI and SalI double digestion plasmid YEp352, reclaim the 5.2kb linear DNA fragment, reclaim dna fragmentation (MOXp-MOXt) and the debaryomyces hansenii autonomously replicating sequence HARS that contains methanol oxidase gene promoter and terminator sequence and multiple clone site respectively by embodiment 3 described methods, at first linear YEp352 and HARS are carried out ligation, 16 ℃ of reactions are after 8 hours, add the MOXp-MOXt dna fragmentation in the reaction system again, 16 ℃ are continued reaction 8 hours, connect product transformed into escherichia coli competent cell, by the white bacterium colony screening positive clone of indigo plant, white transformant being inoculated in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivated after 12-24 hour, use the alkaline lysis method of extracting plasmid, obtain recombinant vectors pYEAM1.

According to the report intestinal bacteria phytase gene appA nucleotide sequence design and synthesize primer AP1 (5 '-CAA CTGCAGATGAAAGCGATCTTAATCCCAT-3 ', the line part is the PstI recognition site) and AP2 (5 '-CCA GAATTCATTACAAACTGCACGCCGGT-3 ', the line part is the EcoRI recognition site), be template with intestinal bacteria (China Committee for Culture Collection of Microorganisms common micro-organisms center) genomic dna, carry out the phytase gene appA of PCR reaction amplification 1.3kb.With EcoRI digested plasmid pYEAM1, reclaim the 7.5kb linear DNA fragment, cut the PCR product with PstI and EcoRI enzyme, the 1.3kbappA gene is reclaimed in the low melting-point agarose gel electrophoresis, above-mentioned two kinds of dna fragmentations connect with α-factor signal peptide sequence MF α 1s of the 0.3kb that embodiment 3 reclaims, connect product transformed into escherichia coli competent cell, transformant being inoculated in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivated after 6 hours, the rapid extraction plasmid, electrophoresis detection, with the correct transformant of plasmid size be inoculated in the LB liquid nutrient medium that contains penbritin (50 μ g/ml) 37 ℃ cultivate 16 hours after, use the alkaline lysis method of extracting plasmid, obtain recombinant plasmid pMOX α-appA, the physical map of this plasmid as shown in Figure 5, this plasmid has the selection markers of URA3 gene as the transformed yeast bacterium;

Step 2)-6) identical with embodiment 2;

7) after cultivation finishes, 5000 rev/mins of centrifugal 5 minutes collecting cells and supernatant liquor, measure the inside and outside phytase activity of cell according to a conventional method, show under these conditions, the wet cell weight of every liter of nutrient solution is the 83-87 gram, phytase in every liter of nutrient solution is a 1700-1800 unit of activity, and wherein 92% phytase is secreted in the nutrient solution.

After the preferred embodiment that describes in detail, being familiar with this technology personage can be well understood to, can carry out various variations and modification not breaking away under above-mentioned claim and the spirit, all foundations technical spirit of the present invention all belongs to the scope of technical solution of the present invention to any simple modification, equivalent variations and modification that above embodiment did.And the present invention also is not subjected to the restriction of the embodiment that gives an actual example in the specification sheets.

Sequence table

＜110〉unit's sky

＜120〉uracil auxotroph Hansenula yeast and construction process thereof and application

<160>2

<210>1

<211>29bp

<212>DNA

＜213〉artificial sequence

<220>

<221>29bp

<222>(1)..(29)

<400>1

ccaggatcct?caacatttcc?ctgaataat 29

<210>2

<211>29bp

<212>DNA

＜213〉artificial sequence

<220>

<221>29bp

<222>(1)..(29)

<400>2

cgagaattct?cactagtatt?cccgcgact 29

Claims (4)

1, uracil auxotroph Hansenula Yeast gene engineering host bacterium debaryomyces hansenii bacterium (Hansenula) YH-11CGMCC NO.2976.

2, a kind of method that makes up the described uracil auxotroph Hansenula yeast of claim 1, carry out according to the following steps:

1) structure contains the recombinant plasmid p18HURA of vitamin B13 glycosides-5-phosphate decarboxylase gene (HURA3) of debaryomyces hansenii bacterium;

2) make up the ruined recombinant plasmid pHURK of HURA3 gene;

With step 2) plasmid pHURK transform the debaryomyces hansenii bacterium with yeast intact cell conversion method, obtain the uracil auxotrophy bacterial strain through screening.

3, the application of the described uracil auxotroph Hansenula Yeast gene engineering of claim 1 host bacterium debaryomyces hansenii bacterium (Hansenula) YH-11CGMCC NO.2976 in producing foreign protein.

4, application as claimed in claim 3, wherein said foreign protein are amylase or phytase.

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