CN101857845A - Pichia pastoris strain capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein - Google Patents

Abstract

The invention provides a plurality of Pichia pastoris strains capable of utilizing non methyl alcohol carbon source induced AOX1 promoter to express outside source protein. PpHXT1 gene in the Pichia pastoris strain mutates, thus protein can not be coded or the coded protein has no activity, and autophagy gene PpATG30 gene also mutates, thus protein can not be coded or the coded protein has no activity; or PpMIG1 gene in the Pichia pastoris strain mutates, thus protein can not be coded or the coded protein has no activity, and PpMIG2 gene in the Pichia pastoris strain mutates, thus protein can not be coded or the coded protein has no activity. The Pichia pastoris strain of the invention not only has the advantages of the existing Pichia pastoris strain but also can utilize non methyl alcohol carbon source induced AOX1 promoter to express outside source protein, is safe and environmentally friendly, has simple operation and lower cost, also can be applied to production of food or additive and is applicable to large-scale popularization and application.

Description

Can utilize the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source

Technical field

The present invention relates to biotechnology and biological pharmacy technical field, more specifically, relate to the expression strain technical field, be meant especially and can utilize the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source.

Background technology

Till 2006, there be more than 500 kind of foreign protein in pichia spp, to express, these dietary protein origins comprise virus, bacterium, fungi, plant, insect and vertebrates in whole organic sphere.Used promotor is AOX1 (Alcohol oxidase 1 during the Pichia anomala expression foreign protein, the alcohol oxidase gene) promotor, this promoter regulation rule is: methyl alcohol can be induced the expression of AOX1 promotor, other carbon source is (as glucose, fructose, glycerine etc.) suppress the expression of AOX1 promotor.

Why utilizing pichia yeast expression system to express so many foreign protein, is because it has the incomparable advantage of other expression system, simple as the pichia spp genetic manipulation; Exogenous protein expression amount height is expressed in both can born of the same parents, also can secreting, expressing; The foreign protein genes inheritance stability; As eukaryotic expression system, pichia spp has Eukaryotic subcellular structure, has posttranslational modification machining functions such as glycosylation, fatty acidylate, protein phosphorylation; Contain distinctive strong AOX1 promotor, can strictly regulate and control expression of exogenous gene with methyl alcohol.

But present pichia yeast expression system faces many problems in the fermentation amplification process:

Need use methyl alcohol as carbon source when 1, inducing the external source expression of recombinant proteins, methyl alcohol is poisonous and inflammable, and anti-riot design need be carried out in the workshop in suitability for industrialized production, causes cost to increase;

2, the methyl alcohol strong oxygen consumption that ferments, general by the air flow that increases air with improve that rotating speed is difficult to satisfy the demand of oxygen and needs lead to pure oxygen, the more pure oxygens that need of methyl alcohol that consume many more more, this brings very big trouble for actual suitability for industrialized production, the more heats that produce of the methyl alcohol of Xiao Haoing also big more more in addition, and the cooling power of required equipment requires high more;

3, methyl alcohol is unsuitable for being used for the production of food or additive as a kind of petroleum chemicals;

4, methyl alcohol metabolism meeting produces H ₂O ₂, can cause expressed proteic hydrolysis;

5, AOX1 efficiently transcribe too busy to get away methyl alcohol, in other carbon source, suffer carbon source to check effect.

Therefore, if can obtain a strain or some mutant strains by engineered method, make efficiently transcribing of AOX1 promotor not rely on methyl alcohol, and available other carbon source is induced the expression of AOX1 promotor, this has positive effect for the non-methanol induction expression system of exploitation pichia spp.

Summary of the invention

Main purpose of the present invention is exactly the problems and shortcomings at above existence, provide and to utilize the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source, these Pichi strains not only have the advantage of existing Pichi strain, and can adopt non methyl alcohol carbon source induced AOX 1 promoter to express outside source albumen, safety and environmental protection, simple to operate, cost reduces, also can be used for the production of food or additive, be suitable for large-scale promotion application.

To achieve these goals, in a first aspect of the present invention, a kind of proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes is provided, be characterized in, first sudden change takes place in the PpHXT1 gene in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

Preferably, described first sport point mutation, insertion sudden change and/or deletion mutantion.That is to say the method for PpHXT1 transgenation a lot, can be point mutation, can be to insert sudden change, can also be deletion mutantion, as long as the PpHXT1 gene after the sudden change can not proteins encoded or encoded protein do not have active.

Preferably, the sequence of described PpHXT1 gene is the sequence shown in the SEQ ID NO:1.

Preferably, the albumen of described PpHXT1 genes encoding is the sequence shown in the SEQ ID NO:2.

Preferably, the GenBank accession number of described PpHXT1 gene is GU479989, and the hexose carrier protein of encoding is the analogue of yeast saccharomyces cerevisiae HXT gene.

Preferably, second sudden change takes place in autophagy gene PpATG30 in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

More preferably, described second sport point mutation, insertion sudden change and/or deletion mutantion.That is to say the method for PpATG30 transgenation a lot, can be point mutation, can be to insert sudden change, can also be deletion mutantion, as long as the PpATG30 gene after the sudden change can not proteins encoded or encoded protein do not have active.

In a second aspect of the present invention, a kind of proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes also is provided, be characterized in that the 3rd sudden change takes place the PpMIG1 gene in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

Preferably, the described the 3rd sport point mutation, insertion sudden change and/or deletion mutantion.That is to say the method for PpMIG1 transgenation a lot, can be point mutation, can be to insert sudden change, can also be deletion mutantion, as long as the PpMIG1 gene after the sudden change can not proteins encoded or encoded protein do not have active.

Preferably, the sequence of described PpMIG1 gene is the sequence shown in the SEQ ID NO:3.

Preferably, the albumen of described PpMIG1 genes encoding is the sequence shown in the SEQ ID NO:4.

Preferably, the described PpMIG1 gene analogue that is yeast saccharomyces cerevisiae MIG1 gene.

Preferably, the 4th sudden change takes place in the PpMIG2 gene in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

More preferably, the described the 4th sport point mutation, insertion sudden change and/or deletion mutantion.That is to say the method for PpMIG2 transgenation a lot, can be point mutation, can be to insert sudden change, can also be deletion mutantion, as long as the PpMIG2 gene after the sudden change can not proteins encoded or encoded protein do not have active.

More preferably, the sequence of described PpMIG2 gene is the sequence shown in the SEQ ID NO:5.

More preferably, the albumen of described PpMIG2 genes encoding is the sequence shown in the SEQ ID NO:6.

More preferably, the described PpMIG2 gene analogue that also is yeast saccharomyces cerevisiae MIG1 gene.

Adopt method of the present invention, utilize molecular biological means, carried out genetic engineering modified to the pichia spp wild strain, obtain carbon source and suppress associated gene mutation (comprising point mutation, deletion mutantion and insertion sudden change) thereby the relevant proteic bacterial strain of the carbon source that can't encode correct inhibition, these bacterial strains can utilize non-methyl alcohol carbon source to induce the expression of AOX1 promotor.Problems such as poisonous, inflammable and explosive, the high oxygen consumption that produces in the time of can avoiding adopting methanol induction like this, high heat production have important value at aspects such as Pichia anomala expression recombinant protein, gene pharmacy.

Description of drawings

Fig. 1 is the segmental structure synoptic diagram of a PpHXT1 genetically deficient of first specific embodiment of the present invention.

Fig. 2 is that the Δ hxt1 bacterial strain that first specific embodiment of the present invention makes up adopts different carbon source inductive AOX1 colour developing results respectively with wild type strain WT.Wherein 1 adopt 0.5% methyl alcohol; 2 adopt 1% glucose; 3 adopt 1% glucose+0.5% methyl alcohol; 4 adopt 1% fructose; 5 employing 1% fructose+0.5% methyl alcohol are carbon source; A is a Δ hxt1 strain culturing sample, and B is a wild type strain WT culture sample.

Fig. 3 is that the Δ hxt1 bacterial strain that first specific embodiment of the present invention makes up adopts different carbon source inductive AOX1 enzymes measurement result alive respectively with wild type strain WT.Wherein 1 adopt 0.5% methyl alcohol; 2 adopt 1% glucose; 3 adopt 1% glucose+0.5% methyl alcohol; 4 adopt 1% fructose; 5 employing 1% fructose+0.5% methyl alcohol are carbon source.

Fig. 4 is that the Δ hxt1 bacterial strain that first specific embodiment of the present invention makes up adopts the proteic Western blotting of different carbon source inductive AOX1 detected result respectively with wild type strain WT.Wherein M is albumen marker, and 1-5 is a Δ hxt1 strain culturing sample, and 6-10 is a wild type strain WT culture sample; 1 and 6 adopt 0.5% methyl alcohol respectively; 2 and 7 adopt 1% glucose respectively; 3 and 8 adopt 1% glucose+0.5% methyl alcohol respectively; 4 and 9 adopt 1% fructose respectively; 5 and 10 to adopt 1% fructose+0.5% methyl alcohol respectively be carbon source.

Fig. 5 is the segmental structure synoptic diagram of another PpHXT1 genetically deficient of first specific embodiment of the present invention.

Fig. 6 is the collection of illustrative plates of the pPIC3.5k carrier of first specific embodiment employing of the present invention.

Fig. 7 is that Δ hxt1-GFP bacterial strain, Δ hxt1-GFP-SKL bacterial strain and the Δ atg30 Δ hxt1-GFP-SKL bacterial strain that first specific embodiment of the present invention makes up adopts the proteic Western blotting of different carbon source inductive AOX1 detected result respectively.Wherein A adopts 0.5% methyl alcohol; B adopts 1% fructose; C adopts 1% fructose+0.5% methyl alcohol; D adopts 1% glucose; It is carbon source that E adopts 1% glucose+0.5% methyl alcohol.

Fig. 8 is the segmental structure synoptic diagram of the PpMIG1 genetically deficient of second specific embodiment of the present invention.

Fig. 9 is the segmental structure synoptic diagram of the PpMIG2 genetically deficient of second specific embodiment of the present invention.

Figure 10 is that Δ mig1, the Δ mig2 that second specific embodiment of the present invention makes up adopts different carbon source inductive AOX1 colour developing results respectively with Δ mig1 Δ mig2 and wild type strain WT.Wherein A adopts 1% glycerine; B adopts 1% glycerine+0.5% methyl alcohol; It is carbon source that C adopts 0.5% methyl alcohol; 1 is wild type strain WT; 2 is Δ mig1; 3 is Δ mig2; 4 is Δ mig1 Δ mig2.

Figure 11 is that Δ mig1, the Δ mig2 that second specific embodiment of the present invention makes up adopts the proteic Western blotting of different carbon source inductive AOX1 detected result respectively with Δ mig1 Δ mig2 and wild type strain WT.Wherein A adopts 1% glycerine; B adopts 1% glycerine+0.5% methyl alcohol; It is carbon source that C adopts 0.5% methyl alcohol; 1 is wild type strain WT; 2 is Δ mig1; 3 is Δ mig2; 4 is Δ mig1 Δ mig2.

Embodiment

Materials and methods in the following example is:

The molecule clone technology that is adopted is referring to " the molecular cloning experiment guide " of volumes such as the female Brooker of J. Sa.

Pichia spp total protein extractive technique is extracted handbook with reference to the Yeast protein that cold spring port company provides.

SDS-PAGE, the western blotting handbook that Western blotting technology provides with reference to Millipore company.

All available from TaKaRa biotech firm (Dalian, China), the concrete reaction conditions and the method for use are all with reference to catalogue for employed toolenzyme.

Following commercialization plasmid and bacterial strain are used for gene clone and protein expression:

Plasmid pUC18, plasmid pPIC3.5k, intestinal bacteria Top10, Pichi strain GS115 are available from Invitrogen company, and Pichi strain PPY12 Δ atg30 is provided by the professor SureshSubramani close friend of Univ California-San Diego USA.

Embodiment 1 glucose or fructose can be induced the Pichi strain of AOX1 promoter expression

1.1PpHXT1 the structure of external deletion fragment

PpHXT1 gene length 1614bp (SEQ ID NO:1), 537 amino acid (SEQ ID NO:2) of encoding.Adopt PCR, enzyme is cut the method for connection, with pUC18 be carrier in external structure one section PpHXT1 genetically deficient fragment (as shown in Figure 1), form by PpHXT1 upstream region of gene 200bp, G418 resistant gene 1517bp and PpHXT1 gene downstream 200bp respectively.Gained PpHXT1 genetically deficient fragment deletion the whole open reading frame of this gene.

1.2 electricity changes the screening of pichia spp and the strain of Δ hxt1 disappearance

PpHXT1 deletion fragment electricity is changeed Pichi strain GS115, be applied to 4 YPD flat boards that add G418, be placed on 30 ℃ of incubators and cultivated 48-72 hour.The mono-clonal that grows on the flat board is chosen to 10ml YPD+G418 liquid nutrient medium, after 30 ℃ of shaking tables are cultivated, extracted genome and verify with PCR.The Pichi strain called after Δ hxt1 of correct (promptly containing the PpHXT1 deletion fragment) after PCR check and the sequence verification.

1.3AOX1 color reaction

With each bacterial strain (Δ hxt1 bacterial strain and wild type strain WT) respectively at 0.5% methyl alcohol, 1% glucose, 1% glucose+0.5% methyl alcohol, 1% fructose and 1% fructose+0.5% methyl alcohol is to cultivate in the YNB liquid nutrient medium of carbon source, grow into logarithmic growth after date sampling 1ml, add the AOX1 enzyme in the thalline after centrifugal and live the colour developing of colour developing liquid (referring to " StasykO.V.; T.Y.Nazarko, and A.A.Sibirny.2008.Methods of Plate Pexophagy Monitoringand Positive Selection for ATG Gene Cloning in Yeasts.Methods in enzymology.451:229-239 ".Chromogenic substrate is o-dianisidine.)。

After growing in the substratum that contains glucose or fructose as seen from Figure 2, having only has the AOX1 enzyme to live among the bacterial strain Δ hxt1, do not have enzyme to live in the wild strain.

1.4AOX1 the mensuration that enzyme is lived

With each bacterial strain (Δ hxt1 bacterial strain and wild type strain WT) pre-cultivation of in the MGY liquid nutrient medium, spending the night, transfer to respectively then and contain 0.5% methyl alcohol, 1% glucose, 1% glucose+0.5% methyl alcohol, 1% fructose and 1% fructose+0.5% methyl alcohol is in the YNB liquid nutrient medium of carbon source, cultivate sampling extraction total protein after 10 hours, (method is referring to " Verduyn; C.; J.P.van Dijken, and W.A.Scheffers.1984.Colorometric alcohol assays with alcohol oxidase.J.Microbiol.Methods 2:15-25. " to carry out AOX1 enzyme mensuration alive behind Bradford method protein quantification.Chromogenic substrate is 2,2 '-azino-di-(3-ethylbenzthiazoline sulfonate), ABTS.)。

As shown in Figure 3, bacterial strain Δ hxt1 all can induce the expression of AOX1 in the substratum that contains glucose or fructose, and wild strain can not be induced the expression of AOX1.

1.5 the proteic Western blotting of AOX1 detects

With each bacterial strain (Δ hxt1 bacterial strain and wild type strain WT) pre-cultivation of in the MGY liquid nutrient medium, spending the night, transfer to respectively then that to contain 0.5% methyl alcohol, 1% glucose, 1% glucose+0.5% methyl alcohol, 1% fructose and 1% fructose+0.5% methyl alcohol be in the YNB liquid nutrient medium of carbon source, cultivate sampling extraction total protein after 10 hours, behind Bradford method protein quantification, carry out SDS-PAGE, point sample 15 μ g in No. 1 and No. 6 electrophoresis holes, point sample 20 μ g in all the other each electrophoresis holes carry out the proteic Western blotting of AOX1 and detect behind the electrophoresis.

As shown in Figure 4, glucose and fructose all can inducible strain Δ hxt1 be expressed AOX1 albumen, but among the wild strain WT the proteic expression of AOX1 can only to rely on methyl alcohol be sole carbon source.

1.6 the structure of bacterial strain Δ atg30 Δ hxt1

Adopt PCR, enzyme is cut the method for connection, with pUC18 be carrier in external structure other one section PpHXT1 genetically deficient fragment (as shown in Figure 5), form by PpHXT1 upstream region of gene 200bp, Zeocin resistant gene 1321bp and PpHXT1 gene downstream 200bp respectively.Gained PpHXT1 genetically deficient fragment has lacked the whole open reading frame of this gene equally.

This PpHXT1 deletion fragment electricity is changeed Pichi strain PPY12 Δ atg30, be applied to 4 YPD flat boards that add Zeocin, be placed on 30 ℃ of incubators and cultivated 48-72 hour.The mono-clonal that grows on the flat board is chosen to 10ml YPD+Zeocin liquid nutrient medium, after 30 ℃ of shaking tables are cultivated, carried genome and verify with PCR.The Pichi strain called after Δ atg30 Δ hxt1 of correct (promptly containing the PpHXT1 deletion fragment) after PCR check and the sequence verification.

1.7 AOX1 is promotor abduction delivering green fluorescent protein GFP in non-methyl alcohol carbon source

1.7.1 the structure of GFP expression vector

Insert the encoding sequence (SKL is a signal peptide, expressed proteins can be positioned in the peroxysome) of GFP and GFP-SKL in the AOX1 promotor downstream of carrier pPIC3.5k (as shown in Figure 6) respectively, obtain GFP expression vector pP-GFP and pGS respectively.

1.7.2 the screening of the single copy of pichia spp GFP expression strain

Respectively expression vector pP-GFP and pGS electricity are changeed Δ hxt1 bacterial strain, be applied to YND (0.67%YNB+1% glucose+2% agar powder) flat board that does not contain Histidine, be placed on 30 ℃ of incubators and cultivated 48-72 hour.The mono-clonal that grows on the flat board is chosen to liquid nutrient medium, after cultivating, 30 ℃ of shaking tables carry genome, (method is referring to Xuan with real-time PCR checking GFP copy number, Y.J., X.S.Zhou, W.W.Zhang, X.Zhang, Z.W.Song, andY.X.Zhang.2009.An upstream activation sequence controls the expression of AOX1gene in Pichia pastoris.FEMS Yeast Res.9:1271-1282).Real-time PCR is verified as the pichia spp GFP expression strain difference called after Δ hxt1-GFP and the Δ hxt1-GFP-SKL of single copy.In like manner made up expression strain Δ atg30 Δ hxt1-GFP-SKL.

1.7.3 flow cytometer detects the GFP expression amount

With each GFP expression strain (Δ hxt1-GFP, Δ hxt1-GFP-SKL and Δ atg30 Δ hxt1-GFP-SKL) pre-cultivation of in the MGY liquid nutrient medium, spending the night, transfer to respectively then that to contain 0.5% methyl alcohol, 1% fructose, 1% fructose+0.5% methyl alcohol, 1% glucose and 1% glucose+0.5% methyl alcohol be in the YNB liquid nutrient medium of carbon source, the time segment sampling is with the geometric mean fluorescence intensity of GFP in the flow cytometer test sample.

As shown in Figure 7, in bacterial strain Δ hxt1, glucose and fructose all can be induced the expression of GFP, but the GFP (GFP-SKL) that is positioned peroxysome is obviously different with the expression amount that is positioned cytoplasmic GFP, the expression amount of the GFP-SKL trend that significantly decreases at later stages, suspection be since the peroxysome autophagy due to (referring to Farr é, J.C., R.Manjithaya, R.D.Mathewson, and S.Subramani.2008.PpAtg30 TagsPeroxisomes for Turnover by Selective Autophagy.Developmental Cell 14:365-376).

In order to verify that phenomenon that the GFP-SKL expressing quantity descends at later stages rapidly is due to the peroxysome autophagy, we further lack the PpHXT1 gene in strain peroxysome autophagy blocking-up bacterial strain PPY12 Δ atg30, and expressed the GFP-SKL of single copy, found that downtrending obviously relaxes (as shown in Figure 7).

Embodiment 2 glycerine can be induced the Pichi strain of AOX1 promoter expression

2.1 the structure of PpMIG1, the external deletion fragment of PpMIG2

The PpMIG genes encoding of pichia spp be a kind of transcription repression factor, PpMIG1 gene length 1335bp (SEQID NO:3), 444 amino acid (SEQ ID NO:4) of encoding; The long 1365bp of PpMIG2 (SEQ ID NO:5), 454 amino acid (SEQ ID NO:6) of encoding.

Adopt PCR, enzyme is cut the method for connection, with pUC18 be carrier in external structure one section PpMIG1 genetically deficient fragment (as shown in Figure 8), form by PpMIG1 upstream region of gene 728bp, Zeocin resistant gene 1321bp and PpMIG1 gene 3 ' end and downstream 1011bp thereof respectively.Gained PpMIG1 genetically deficient fragment has lacked preceding 315 amino acid of this gene altogether.

Adopting uses the same method has made up PpMIG2 genetically deficient fragment (as shown in Figure 9), and different is that the Zeocin resistant gene has changed G418 resistant gene KAN into, this fragment deletion the whole open reading frame of PpMIG2.

2.2 electricity changes the screening of pichia spp and the strain of Δ mig1, Δ mig2 disappearance

PpMIG1 deletion fragment electricity is changeed Pichi strain GS115, be applied to 4 YPD flat boards that add Zeocin, be placed on 30 ℃ of incubators and cultivated 48-72 hour.The mono-clonal that grows on the flat board is chosen to 10ml YPD+Zeocin liquid nutrient medium, carried genome after 30 ℃ of shaking tables are cultivated, verify with PCR.The Pichi strain called after Δ mig1 of correct (promptly containing the PpMIG1 deletion fragment) after PCR check and the sequence verification.

PpMIG2 deletion fragment electricity is changeed Pichi strain GS115, be applied to 4 YPD flat boards that add G418, be placed on 30 ℃ of incubators and cultivated 48-72 hour.The mono-clonal that grows on the flat board is chosen to 10ml YPD+G418 liquid nutrient medium, carried genome after 30 ℃ of shaking tables are cultivated, verify with PCR.The Pichi strain called after Δ mig2 of correct (promptly containing the PpMIG2 deletion fragment) after PCR check and the sequence verification.

2.3 utilize the method for hybridizing in the classical genetics to make up the two deletion mycopremnas of Δ mig1 Δ mig2

" the Pichia Protocols " that writes referring to James M.Cregg etc.

2.4.AOX1 color reaction

With each bacterial strain (wild type strain WT, Δ mig1 bacterial strain; Δ mig2 bacterial strain and Δ mig1 Δ mig2 bacterial strain) in being the YNB liquid nutrient medium of carbon source, 1% glycerine, 1% glycerine+0.5% methyl alcohol and 0.5% methyl alcohol cultivates respectively, grow into logarithmic growth after date sampling 1ml, add the colour developing of AOX1 enzyme colour developing alive liquid in the thalline after centrifugal.

In the substratum of glycerine and glycerine+methyl alcohol, having only has enzyme to live among bacterial strain Δ mig1 and the Δ mig1 Δ mig2 as seen from Figure 10.

2.5.AOX1 the mensuration that enzyme is lived

With each bacterial strain (wild type strain WT, Δ mig1 bacterial strain; Δ mig2 bacterial strain and Δ mig1 Δ mig2 bacterial strain) in being the YNB liquid nutrient medium of carbon source, 1% glycerine, 1% glycerine+0.5% methyl alcohol and 0.5% methyl alcohol cultivates respectively, grow into the sampling of logarithmic growth after date and extract total protein, behind Bradford method protein quantification, carry out the mensuration that the AOX1 enzyme is lived.

As shown in Table 1, bacterial strain Δ mig1 and the Δ mig1 Δ mig2 expression that in the substratum that contains glycerine or glycerine+methyl alcohol, all can induce AOX1.

Table 1

2.6.AOX1 proteic Western blotting detects

With each bacterial strain respectively at 1% glycerine, 1% glycerine+0.5% methyl alcohol and 0.5% methyl alcohol are to cultivate in the YNB liquid nutrient medium of carbon source, grow into the sampling of logarithmic growth after date and extract total protein, behind Bradford method protein quantification, carry out SDS-PAGE, each electrophoresis hole mid point total protein 20 μ g carries out the proteic Western blotting of AOX1 and detects behind the electrophoresis.

As shown in Figure 11, the proteic expression of AOX1 among inducible strain Δ mig1 that glycerine can be in various degree and the Δ mig1 Δ mig2.

Above-mentioned test-results shows, by knocked out the gene PpHXT1 of several sugared vehicles of coding in pichia spp wild strain GS115, has made up genetically deficient bacterial strain Δ hxt1; Atg30 has lacked PpHXT1 at pichia spp peroxysome autophagy blocking-up bacterial strain PPY12 Δ, has made up two deletion mycopremna Δ atg30 Δ hxt1; Detection alive through the AOX1 enzyme and Western blotting is found, glucose or fructose all can be induced the expression of Δ hxt1 and Δ atg30 Δ hxt1 strains A OX1 promotor, and the abduction delivering amount in non-methyl alcohol carbon source when having detected with flow cytometer that green fluorescent protein GFP is promotor with AOX1; In pichia spp wild strain GS115, lack carbon source inhibitory phase correlation gene PpMIG1 and PpMIG2 respectively, made up genetically deficient bacterial strain Δ mig1 and Δ mig2, and two deletion mycopremna Δ mig1 Δ mig2; Detection alive through the AOX1 enzyme and Western blotting finds that glycerine can be induced the expression of Δ mig1 and Δ mig1 Δ mig2 strains A OX1 promotor.

In sum, the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes of the present invention not only has the advantage of existing Pichi strain, and can adopt non methyl alcohol carbon source induced AOX 1 promoter to express outside source albumen, safety and environmental protection, simple to operate, cost reduces, and also can be used for the production of food or additive, is suitable for large-scale promotion application.

In this specification sheets, the present invention is described with reference to its certain embodiments.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.

Sequence table

＜110〉East China University of Science

 

＜120〉can utilize the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source

 

<160>6

 

<210>1

<211>1614

<212>DNA

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<220>

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<222>(1)…(1614)

＜223〉the PpHXT1 gene in the Pichi strain

 

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atgagttcaa?cagatatcca?aggtgatcaa?ggtgacaatg?aaaagatata?cgccattgag 60

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aaagtgagag?ttggtttaat?tgtttctatt?ttcaacattg?gttgtgctat?cggaggtgtc 360

actctaggta?aacttggtga?catttggggt?agaaagaagg?ctttgatgtt?cgtcatggtc 420

atctatatgg?tcggtatttt?gattcaaatt?gcttccattg?acaaatggta?ccagtatttc 480

attggaagaa?ttattgcagg?tctggccgtc?ggtgcagttt?ccgttttatc?ccccatgttc 540

atcagtgaga?cttctcctaa?acacatcaga?ggttccttag?tctcctgcta?ccaattaatg 600

attacagccg?gtattttctt?gggttactgt?accacttacg?gaaccaagac?ttacaccgac 660

tccacccaat?ggagagttcc?tttgggattg?tgtttcgctt?gggccattct?gatgattgtt 720

ggtatgacct?tcatgccaga?gtccccacgt?ttcttggttg?aggttaacag?agtcgacgag 780

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gagatgagac?ttatttctga?cggtattgag?aaggagaagg?aggctggtag?cgtttcttgg 900

ggtgaactgt?tcactggtaa?gccaaagatt?ttctaccgtc?tattgattgg?tattttcatg 960

caatctttgc?aacaattgac?cggtaacaac?tatttcttct?actacggaac?taccattttc 1020

aaggctgtcg?gattggacga?ttctttccaa?acttctatca?ttcttggtgt?tgtcaatttt 1080

gcttccacat?tcctaggtat?ctacaccatg?gataaatttg?gtagaagaag?aacactttta 1140

ggaggttctg?gagccatggt?tgtttgtttg?gtcattttca?gttccgttgg?tgtcaagtct 1200

ctttatgaga?acggtaagga?tgatccatcc?aaaccagcag?gtaacgccat?gattgtcttc 1260

acctgtctgt?tcattttctt?ctttgcatgt?acctgggctc?caggtgtttt?cgtcgttgtg 1320

tctgaaacct?acccacttag?aattagatcc?aagggtatgg?ccatcgctca?aggttccaat 1380

tggctttggg?gtttcctcat?tgccttcttc?actccattta?tctcaggtgc?cattgatttc 1440

gcctacggtt?acgtctttat?gggatgtact?ctgttcgcct?tcttctttgt?gtacttcttc 1500

gttcctgaaa?ccaagggtct?gtcgctggaa?gacgttgatg?aagtctatga?gaaccttacc 1560

ttcggaagag?catatgcata?cagccacacg?attaaagaca?agggcgccct?ataa 1614

 

<210>2

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Met?Ser?Ser?Thr?Asp?Ile?Gln?Gly?Asp?Gln?Gly?Asp?Asn?Glu?Lys?Ile

1 5 10 15

Tyr?Ala?Ile?Glu?Ser?Ser?Pro?Ser?Asn?Glu?Gln?Ile?Lys?Asp?Ile?His

20 25 30

Glu?Ala?Pro?Ala?Asp?Asn?Lys?Ser?Glu?Leu?Asp?Ile?Pro?Val?Lys?Pro

35 40 45

Lys?Gly?Ser?Tyr?Ile?Leu?Val?Ser?Val?Leu?Cys?Leu?Leu?Val?Ala?Phe

50 55 60

Gly?Gly?Phe?Val?Phe?Gly?Trp?Asp?Thr?Gly?Thr?Ile?Ser?Gly?Phe?Val

65 70 75 80

Asn?Met?Ser?Asp?Phe?Thr?Arg?Arg?Phe?Gly?Gln?Phe?Asn?Gly?Glu?Thr

85 90 95

Tyr?Tyr?Leu?Ser?Lys?Val?Arg?Val?Gly?Leu?Ile?Val?Ser?Ile?Phe?Asn

100 105 110

Ile?Gly?Cys?Ala?Ile?Gly?Gly?Val?Thr?Leu?Gly?Lys?Leu?Gly?Asp?Ile

115 120 125

Trp?Gly?Arg?Lys?Lys?Ala?Leu?Met?Phe?Val?Met?Val?Ile?Tyr?Met?Val

130 135 140

Gly?Ile?Leu?Ile?Gln?Ile?Ala?Ser?Ile?Asp?Lys?Trp?Tyr?Gln?Tyr?Phe

145 150 155 160

Ile?Gly?Arg?Ile?Ile?Ala?Gly?Leu?Ala?Val?Gly?Ala?Val?Ser?Val?Leu

165 170 175

Ser?Pro?Met?Phe?Ile?Ser?Glu?Thr?Ser?Pro?Lys?His?Ile?Arg?Gly?Ser

180 185 190

Leu?Val?Ser?Cys?Tyr?Gln?Leu?Met?Ile?Thr?Ala?Gly?Ile?Phe?Leu?Gly

195 200 205

Tyr?Cys?Thr?Thr?Tyr?Gly?Thr?Lys?Thr?Tyr?Thr?Asp?Ser?Thr?Gln?Trp

210 215 220

Arg?Val?Pro?Leu?Gly?Leu?Cys?Phe?Ala?Trp?Ala?Ile?Leu?Met?Ile?Val

225 230 235240

Gly?Met?Thr?Phe?Met?Pro?Glu?Ser?Pro?Arg?Phe?Leu?Val?Glu?Val?Asn

245 250 255

Arg?Val?Asp?Glu?Ala?Met?Lys?Ser?Ile?Ala?Arg?Val?Asn?Lys?Val?Ser

260 265 270

Ile?Asp?Asp?Pro?Ser?Val?Tyr?Asn?Glu?Met?Arg?Leu?Ile?Ser?Asp?Gly

275 280 285

Ile?Glu?Lys?Glu?Lys?Glu?Ala?Gly?Ser?Val?Ser?Trp?Gly?Glu?Leu?Phe

290 295 300

Thr?Gly?Lys?Pro?Lys?Ile?Phe?Tyr?Arg?Leu?Leu?Ile?Gly?Ile?Phe?Met

305 310 315 320

Gln?Ser?Leu?Gln?Gln?Leu?Thr?Gly?Asn?Asn?Tyr?Phe?Phe?Tyr?Tyr?Gly

325 330 335

Thr?Thr?Ile?Phe?Lys?Ala?Val?Gly?Leu?Asp?Asp?Ser?Phe?Gln?Thr?Ser

340 345 350

Ile?Ile?Leu?Gly?Val?Val?Asn?Phe?Ala?Ser?Thr?Phe?Leu?Gly?Ile?Tyr

355 360 365

Thr?Met?Asp?Lys?Phe?Gly?Arg?Arg?Arg?Thr?Leu?Leu?Gly?Gly?Ser?Gly

370 375 380

Ala?Met?Val?Val?Cys?Leu?Val?Ile?Phe?Ser?Ser?Val?Gly?Val?Lys?Ser

385 390 395 400

Leu?Tyr?Glu?Asn?Gly?Lys?Asp?Asp?Pro?Ser?Lys?Pro?Ala?Gly?Asn?Ala

405 410 415

Met?Ile?Val?Phe?Thr?Cys?Leu?PheIl?e?Phe?Phe?Phe?Ala?Cys?Thr?Trp

420 425 430

Ala?Pro?Gly?Val?Phe?Val?Val?Val?Ser?Glu?Thr?Tyr?Pro?Leu?Arg?Ile

435 440 445

Arg?Ser?Lys?Gly?Met?Ala?Ile?Ala?Gln?Gly?Ser?Asn?Trp?Leu?Trp?Gly

450 455 460

Phe?Leu?Ile?Ala?Phe?Phe?Thr?Pro?Phe?Ile?Ser?Gly?Ala?Ile?Asp?Phe

465 470 475 480

Ala?Tyr?Gly?Tyr?Val?Phe?Met?Gly?Cys?Thr?Leu?Phe?Ala?Phe?Phe?Phe

485 490 495

Val?Tyr?Phe?Phe?Val?Pro?Glu?Thr?Lys?Gly?Leu?Ser?Leu?Glu?Asp?Val

500 505 510

Asp?Glu?Val?Tyr?Glu?Asn?Leu?Thr?Phe?Gly?Arg?Ala?Tyr?Ala?Tyr?Ser

515 520 525

His?Thr?Ile?Lys?Asp?Lys?Gly?Ala?Leu

530 535

 

<210>3

<211>1335

<212>DNA

<213>Pichia?pastoris

 

<220>

<221>gene

<222>(1)…(1335)

＜223〉the PpMIG1 gene in the Pichi strain

 

<400>3

atgaccactg?cctatcccaa?cgcacgtgtg?gcaagtgtca?gatcgtcggg?ggactctgtg 60

ggtgatatca?aagtgggctc?caggaagtcc?gcggagtctt?ctaacgcaaa?ctccagctcc 120

agctctaaaa?aatcagagtt?atcacgcccc?taccaatgtc?ccatgtgtga?aaaggctttc 180

cacagattag?agcatcaaac?aagacacata?cggactcaca?cgggtgagaa?gccccaccac 240

tgtaattatc?ccggctgttt?caagaagttt?tcaaggtcgg?atgagttgac?cagacattcc 300

agaatacaca?acaacccaaa?cccaaggaaa?agggggccag?gtgttgtccc?caagaagact 360

cctagaccaa?agatggcagg?caagtcagcc?tcatatagtg?acgagggcaa?ctactcttta 420

ggcgaaggcc?atcagcagtt?ttacggcagt?gatgacgctt?cctctttccc?gttggtgtcg 480

attccaactg?cagcagcaac?taccacggtg?ggaaaaccaa?caaccccaac?tccgagctca 540

aagtcgtctc?agagccagac?agctgaggcc?aagttcaacc?ctctaagaag?tgcgtctact 600

ctgagcatca?acttgctggc?tactgctgcc?tctcaggaac?tacaagagtt?aagggctgca 660

gaggaaaact?cttcaagatt?gcaacaagta?aaatctctac?cttcgttaac?tcaatacttc 720

attgcttcgg?aagactcctc?tcatggtgga?aaccctccgt?tatctcatcc?gaagccattc 780

agcagtctaa?gcggacttaa?aagaatgacc?ccaataaacc?cttcttcttc?ttcctcttcg 840

tccggaggtg?tttcaatcaa?caaatcaata?tctgttactt?cgttgactcg?aaccttttcc 900

aacacagaga?ttgctgacga?attcactact?cctccgacaa?tgttgaaaaa?atcccgacca 960

aattcgcctg?tgctcacagg?acgatctcca?acgacttttc?aacaatacca?acaatcacag 1020

catacatcta?acttggcaca?gcagcaatct?aagggtaact?ttcctccatc?ccatattgat 1080

tccaaggttt?catcagcggc?tctgcatctt?ttaggacttg?ggctgaacca?tgccactcct 1140

gaagtcaccc?ctttgcaaac?ccctgcagtg?tctcccaaac?tctttccaaa?atccgtttcc 1200

aataatagct?tggagacact?tcacaaagct?ttggatgaga?ccactgaagc?agagaacctc 1260

caaagcacag?cactaccttc?tctgagctct?ctaaatttac?cttcaacctc?tctggaaaat 1320

caagaaaaga?aatga 1335

<210>4

<211>444

<212>PRT

<213>Pichia?pastoris

 

<220>

<221>peptide

<222>(1)…(444)

＜223〉albumen of the PpMIG1 genes encoding in the Pichi strain

 

<400>4

Met?Thr?Thr?Ala?Tyr?Pro?Asn?Ala?Arg?Val?Ala?Ser?Val?Arg?Ser?Ser

1 5 10 15

Gly?Asp?Ser?Val?Gly?Asp?Ile?Lys?Val?Gly?Ser?Arg?Lys?Ser?Ala?Glu

20 25 30

Ser?Ser?Asn?Ala?Asn?Ser?Ser?Ser?Ser?Ser?Lys?Lys?Ser?Glu?Leu?Ser

35 40 45

Arg?Pro?Tyr?Gln?Cys?Pro?Met?Cys?Glu?Lys?Ala?Phe?His?Arg?Leu?Glu

50 55 60

His?Gln?Thr?Arg?His?Ile?Arg?Thr?His?Thr?Gly?Glu?Lys?Pro?His?His

65 70 75 80

Cys?Asn?Tyr?Pro?Gly?Cys?Phe?Lys?Lys?Phe?Ser?Arg?Ser?Asp?Glu?Leu

85 90 95

Thr?Arg?His?Ser?Arg?Ile?His?Asn?Asn?Pro?Asn?Pro?Arg?Lys?Arg?Gly

100 105 110

Pro?Gly?Val?Val?Pro?Lys?Lys?Thr?Pro?Arg?Pro?Lys?Met?Ala?Gly?Lys

115 120 125

Ser?Ala?Ser?Tyr?Ser?Asp?Glu?Gly?Asn?Tyr?Ser?Leu?Gly?Glu?Gly?His

130 135 140

Gln?Gln?Phe?Tyr?Gly?Ser?Asp?Asp?Ala?Ser?Ser?Phe?Pro?Leu?Val?Ser

145 150 155 160

Ile?Pro?Thr?Ala?Ala?Ala?Thr?Thr?Thr?Val?Gly?Lys?Pro?Thr?Thr?Pro

165 170 175

Thr?Pro?Ser?Ser?Lys?Ser?Ser?Gln?Ser?Gln?Thr?Ala?Glu?Ala?Lys?Phe

180 185 190

Asn?Pro?Leu?Arg?Ser?Ala?Ser?Thr?Leu?Ser?Ile?Asn?Leu?Leu?Ala?Thr

195 200 205

Ala?Ala?Ser?Gln?Glu?Leu?Gln?Glu?Leu?Arg?Ala?Ala?Glu?Glu?Asn?Ser

210 215 220

Ser?Arg?Leu?Gln?Gln?Val?Lys?Ser?Leu?Pro?Ser?Leu?Thr?Gln?Tyr?Phe

225 230 235 240

Ile?Ala?Ser?Glu?Asp?Ser?Ser?His?Gly?Gly?Asn?Pro?Pro?Leu?Ser?His

245 250 255

Pro?Lys?Pro?Phe?Ser?Ser?Leu?Ser?Gly?Leu?Lys?Arg?Met?Thr?Pro?Ile

260 265 270

Asn?Pro?Ser?Ser?Ser?Ser?Ser?Ser?Ser?Gly?Gly?Val?Ser?Ile?Asn?Lys

275 280 285

Ser?Ile?Ser?Val?Thr?Ser?Leu?Thr?Arg?Thr?Phe?Ser?Asn?Thr?Glu?Ile

290 295 300

Ala?Asp?Glu?Phe?Thr?Thr?Pro?Pro?Thr?Met?Leu?Lys?Lys?Ser?Arg?Pro

305 310 315 320

Asn?Ser?Pro?Val?Leu?Thr?Gly?Arg?Ser?Pro?Thr?Thr?Phe?Gln?Gln?Tyr

325 330 335

Gln?Gln?Ser?Gln?His?Thr?Ser?Asn?Leu?Ala?Gln?Gln?Gln?Ser?Lys?Gly

340 345 350

Asn?Phe?Pro?Pro?Ser?His?Ile?Asp?Ser?Lys?Val?Ser?Ser?Ala?Ala?Leu

355 360 365

His?Leu?Leu?Gly?Leu?Gly?Leu?Asn?His?Ala?Thr?Pro?Glu?Val?Thr?Pro

370 375 380

Leu?Gln?Thr?Pro?Ala?Val?Ser?Pro?Lys?Leu?Phe?Pro?Lys?Ser?Val?Ser

385 390 395 400

Asn?Asn?Ser?Leu?Glu?Thr?Leu?His?Lys?Ala?Leu?Asp?Glu?Thr?Thr?Glu

405 410 415

Ala?Glu?Asn?Leu?Gln?Ser?Thr?Ala?Leu?Pro?Ser?Leu?Ser?Ser?Leu?Asn

420 425 430

Leu?Pro?Ser?Thr?Ser?Leu?Glu?Asn?Gln?Glu?Lys?Lys

435 440

 

<210>5

<211>1365

<212>DNA

<213>Pichia?pastoris

 

<220>

<221>gene

<222>(1)…(1365)

＜223〉the PpMIG2 gene in the Pichi strain

 

<400>5

atgactactg?ctcccccaac?gaagcccaat?gataggccct?accagtgccc?catgtgcgac 60

aaggccttcc?accggttgga?acatcaaaca?agacacatta?gaacacacac?aggggaaaaa 120

cctcaccctt?gcacgttccc?tggatgccca?aagaagtttt?ccaggtctga?tgaattgacc 180

agacacttga?ggatacacac?aaacccaact?gtgagaaagg?gcagaaagaa?gaagcggaag 240

gacgaagaac?aagctgtgga?gttgccacct?cagaataacg?aggtacatct?cgttcccatg 300

gggaacgatc?aaatgggaca?accaatatac?acgcaggcgg?ttcctgttta?ttgggttcca 360

tctggtgctg?caaacggcga?acaaggccag?tatttgatgc?ctccgctttt?ttccttacaa 420

ccaagacagg?tgatggcagg?gacttctcaa?accagtttga?atggtgtaga?tgctcaacaa 480

cagcagcagc?agcagcagca?gcagcaacaa?caacaacaac?aacaacaaca?acaaccacag 540

caacaaccac?agcaacaacc?gccactgcaa?ccacaaccac?ttcaaccaca?accacaggcc 600

cagcaacaat?ttggatttgc?tcaagatcaa?agaaacctgg?cacccgctaa?tcagcaacag 660

cacagattct?ccccaccatt?ttctgcatct?tcaaggaccc?attcagccaa?ttcattgttt 720

tctctcaact?caaatggatc?cacgccttca?gggtcatatc?aacagttgaa?ctctttatct 780

cttttacaca?gaatcactcc?aatcaggact?ccaagcagta?atagcctgtt?gacaaaatct 840

aataaccagt?cgatgacatc?aatagtcacg?ttgagcgacc?aacaacagga?ttttgtttcc 900

agaaaaaagt?caagacccaa?ctcaccaaca?gttccaaact?ctccaacaat?ttcaaacttg 960

gtctcgcccg?ctgatacgcc?tttaactact?ccgttgcaat?cgcctacact?gaagcccgca 1020

atgcccagca?acgtacaact?tccaccaata?agatcactgt?taaatttgga?agaacttccc 1080

tcggaaccat?tgcagcaacc?agccaatgtc?tctactgaca?ataaagtgaa?gacaatgttg 1140

aacaaatctt?cgtccaacgt?cactttgagt?aaatcgtttt?cttcacaaga?cattcgtctg 1200

gggaccaaga?gaaagtctga?taccaacctc?tctgcattag?attctaccaa?tgctattcgc 1260

aagcctgccc?tatcaccgtt?ggctcctctg?tcagtctcat?cagatcgatt?taccaagaga 1320

aagaacaact?tcacgatagg?caatatcatg?aactcggact?cttga 1365

 

<210>6

<211>454

<212>PRT

<213>Pichia?pastoris

 

<220>

<221>peptide

<222>(1)…(454)

＜223〉albumen of the PpMIG2 genes encoding in the Pichi strain

 

<400>6

Met?Thr?Thr?Ala?Pro?Pro?Thr?Lys?Pro?Asn?Asp?Arg?Pro?Tyr?Gln?Cys

1 5 10 15

Pro?Met?Cys?Asp?Lys?Ala?Phe?His?Arg?Leu?Glu?His?Gln?Thr?Arg?His

20 25 30

Ile?Arg?Thr?His?Thr?Gly?Glu?Lys?Pro?His?Pro?Cys?Thr?Phe?Pro?Gly

35 40 45

Cys?Pro?Lys?Lys?Phe?Ser?Arg?Ser?Asp?Glu?Leu?Thr?Arg?His?Leu?Arg

50 55 60

Ile?His?Thr?Asn?Pro?Thr?Val?Arg?Lys?Gly?Arg?Lys?Lys?Lys?Arg?Lys

65 70 75 80

Asp?Glu?Glu?Gln?Ala?Val?Glu?Leu?Pro?Pro?Gln?Asn?Asn?Glu?Val?His

85 90 95

Leu?Val?Pro?Met?Gly?Asn?Asp?Gln?Met?Gly?Gln?Pro?Ile?Tyr?Thr?Gln

100 105 110

Ala?Val?Pro?Val?Tyr?Trp?Val?Pro?Ser?Gly?Ala?Ala?Asn?Gly?Glu?Gln

115 120 125

Gly?Gln?Tyr?Leu?Met?Pro?Pro?Leu?Phe?Ser?Leu?Gln?Pro?Arg?Gln?Val

130 135 140

Met?Ala?Gly?Thr?Ser?Gln?Thr?Ser?Leu?Asn?Gly?Val?Asp?Ala?Gln?Gln

145 150 155 160

Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln?Gln

165 170 175

Gln?Gln?Pro?Gln?Gln?Gln?Pro?Gln?Gln?Gln?Pro?Pro?Leu?Gln?Pro?Gln

180 185 190

Pro?Leu?Gln?Pro?Gln?Pro?Gln?Ala?Gln?Gln?Gln?Phe?Gly?Phe?Ala?Gln

195 200 205

Asp?Gln?Arg?Asn?Leu?Ala?Pro?Ala?Asn?Gln?Gln?Gln?His?Arg?Phe?Ser

210 215 220

Pro?Pro?Phe?Ser?Ala?Ser?Ser?Arg?Thr?His?Ser?Ala?Asn?Ser?Leu?Phe

225 230 235 240

Ser?Leu?Asn?Ser?Asn?Gly?Ser?Thr?Pro?Ser?Gly?Ser?Tyr?Gln?Gln?Leu

245 250 255

Asn?Ser?Leu?Ser?Leu?Leu?His?Arg?Ile?Thr?Pro?Ile?Arg?Thr?Pro?Ser

260 265 270

Ser?Asn?Ser?Leu?Leu?Thr?Lys?Ser?Asn?Asn?Gln?Ser?Met?Thr?Ser?Ile

275 280 285

Val?Thr?Leu?Ser?Asp?Gln?Gln?Gln?Asp?Phe?Val?Ser?Arg?Lys?Lys?Ser

290 295 300

Arg?Pro?Asn?Ser?Pro?Thr?Val?Pro?Asn?Ser?Pro?Thr?Ile?Ser?Asn?Leu

305 310 315 320

Val?Ser?Pro?Ala?Asp?Thr?Pro?Leu?Thr?Thr?Pro?Leu?Gln?Ser?Pro?Thr

325 330 335

Leu?Lys?Pro?Ala?Met?Pro?Ser?Asn?Val?Gln?Leu?Pro?Pro?Ile?Arg?Ser

340 345 350

Leu?Leu?Asn?Leu?Glu?Glu?Leu?Pro?Ser?Glu?Pro?Leu?Gln?Gln?Pro?Ala

355 360 365

Asn?Val?Ser?Thr?Asp?Asn?Lys?Val?Lys?Thr?Met?Leu?Asn?Lys?Ser?Ser

370 375 380

Ser?Asn?Val?Thr?Leu?Ser?Lys?Ser?Phe?Ser?Ser?Gln?Asp?Ile?Arg?Leu

385 390 395 400

Gly?Thr?Lys?Arg?Lys?Ser?Asp?Thr?Asn?Leu?Ser?Ala?Leu?Asp?Ser?Thr

405 410 415

Asn?Ala?Ile?Arg?Lys?Pro?Ala?Leu?Ser?Pro?Leu?Ala?Pro?Leu?Ser?Val

420 425 430

Ser?Ser?Asp?Arg?Phe?Thr?Lys?Arg?Lys?Asn?Asn?Phe?Thr?Ile?Gly?Asn

435 440 445

Ile?Met?Asn?Ser?Asp?Ser

450

Claims (17)

1. one kind can be utilized the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source, it is characterized in that, first sudden change takes place the PpHXT1 gene in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

2. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 1 is characterized in that, described first sports point mutation, insertion sudden change and/or deletion mutantion.

3. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 1 is characterized in that the sequence of described PpHXT1 gene is the sequence shown in the SEQ ID NO:1.

4. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 1 is characterized in that the albumen of described PpHXT1 genes encoding is the sequence shown in the SEQ ID NO:2.

5. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 1, it is characterized in that, the GenBank accession number of described PpHXT1 gene is GU479989, and the hexose carrier protein of encoding is the analogue of yeast saccharomyces cerevisiae HXT gene.

6. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 1, it is characterized in that, second sudden change takes place in autophagy gene PpATG30 in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

7. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 6 is characterized in that, described second sports point mutation, insertion sudden change and/or deletion mutantion.

8. one kind can be utilized the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source, it is characterized in that the 3rd sudden change takes place the PpMIG1 gene in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

9. the proteic Pichi strain of the utilized non methyl alcohol carbon source induced AOX 1 promoter to express outside source of stating according to Claim 8 is characterized in that, the described the 3rd sports point mutation, insertion sudden change and/or deletion mutantion.

10. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 8 is characterized in that the sequence of described PpMIG1 gene is the sequence shown in the SEQ ID NO:3.

11. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 8 is characterized in that the albumen of described PpMIG1 genes encoding is the sequence shown in the SEQ ID NO:4.

12. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 8 is characterized in that described PpMIG1 gene is the analogue of yeast saccharomyces cerevisiae MIG1 gene.

13. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 8, it is characterized in that, the 4th sudden change takes place in the PpMIG2 gene in the described Pichi strain, thus can not proteins encoded or encoded protein do not have activity.

14. the proteic Pichi strain of utilized non methyl alcohol carbon source induced AOX 1 promoter to express outside source according to claim 13 is stated is characterized in that, the described the 4th sports point mutation, insertion sudden change and/or deletion mutantion.

15. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 13 is characterized in that the sequence of described PpMIG2 gene is the sequence shown in the SEQ ID NO:5.

16. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 13 is characterized in that the albumen of described PpMIG2 genes encoding is the sequence shown in the SEQ ID NO:6.

17. the proteic Pichi strain of non methyl alcohol carbon source induced AOX 1 promoter to express outside source that utilizes according to claim 13 is characterized in that described PpMIG2 gene also is the analogue of yeast saccharomyces cerevisiae Mig1 gene.

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