CN106701787A - Pichia pastoris for expressing foreign proteins, construction method of pichia pastoris and induced expression method of pichia pastoris - Google Patents

Abstract

The present invention provides a Pichia pastoris strain expressing foreign protein and an expression system. The GTP1 gene of the Pichia pastoris strain is mutated, and the mutated Pichia pastoris gene cannot encode a glycerol transporter or the encoded glycerol transporter has no activity, and can The transcription of PAOX1 is activated under the condition of glycerol, thereby increasing the expression level of AOX1 in Pichia pastoris under the condition of glycerol, thereby increasing the expression level of exogenous protein. At the same time, the present invention also provides the construction method of the Pichia pastoris expressing the foreign protein, the induction expression method of the Pichia pastoris expressing the foreign protein, the induction expression method can effectively improve the expression amount of the foreign protein and finally cell concentration.

Description

Pichia pastoris expressing foreign protein, its construction method and its induced expression method

technical field

The invention relates to the technical fields of fermentation engineering and bioengineering, in particular to Pichia pastoris expressing foreign protein, its construction method and its induced expression method.

Background technique

The Pichia pastoris expression system is a new type of exogenous protein expression system developed in the early 1980s. Compared with the E. coli expression system, the Pichia pastoris expression system has obvious advantages, such as: shorter doubling time , the fermentation cycle is short; the genome is simple and easy to operate; the culture conditions are relatively simple, and it is easy to carry out high-density culture, and then can obtain a higher yield of the target protein; foreign proteins can be folded, glycosylated, and disulfide bonded, etc. ; At the same time, it also avoids defects such as poor secretion efficiency of Saccharomyces cerevisiae, unstable expression strains, and easy loss of expression plasmids; The length is 8-14 mannose residues, which is much shorter than the average 50-150 mannose residues per side chain of Saccharomyces cerevisiae, and will not cause excessive glycosylation; There are very few extracellular proteins, which is very beneficial for later protein purification.

The high-density fermentation method using Pichia pastoris as a single-cell protein producer established since the 1980s has matured, and the stem cell yield can reach as high as 100g/L. Various advantages have made Pichia pastoris a very popular host for eukaryotic gene expression systems in recent years. This system has successfully expressed hundreds of proteins from viruses, bacteria, fungi, animals, plants and humans, such as human interleukin, human serum albumin, tumor necrosis factor, hepatitis B surface antigen, hirudin derivatives, etc.

Pichia pastoris is currently one of the most widely used eukaryotic expression systems for exogenous proteins. The system is based on an efficient alcohol oxidase 1 (AOX1) promoter (AOX1 promoter, PAOX1). The transcription of PAOX1 only responds to methanol When methanol was used as the sole carbon source, PAOX1 promoted the expression of exogenous proteins; while when glycerol was present, it inhibited the expression of AOX1.

Contents of the invention

In view of the above problems, the present invention provides Pichia pastoris expressing foreign proteins, which can solve the technical problem of low AOX1 expression in Pichia pastoris under glycerol-induced conditions in the prior art; in addition, the present invention also provides the Pichia pastoris Yeast construction method and inducible expression method.

The present invention provides a mutated Pichia GTP1 gene, characterized in that the mutation causes Pichia to fail to encode a glycerol transporter or the encoded glycerol transporter has no activity, and can initiate the transcription of PAOX1 in the presence of glycerol. The GTP1 gene sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO:2.

Further, the mutation is all or part of sequence deletion mutation, insertion mutation, frameshift mutation or point mutation.

Further, the deletion mutation is the deletion of the entire open reading frame or partial sequence mutation of the GTP1 gene. The sequence of the GTP1 deletion fragment in vitro is shown in SEQ ID NO:3.

The invention also provides a recombinant vector containing the mutant gene. Host cells containing recombinant vectors.

The present invention also provides a Pichia strain expressing a foreign protein, characterized in that the Pichia strain contains the mutated Pichia GTP1 gene according to any one of claims 1-5.

Further, the Pichia strain is obtained by mutating the GTP1 gene of the yeast strain X-33.

Further, the Pichia pastoris strain was deposited in the General Microorganism Center of China Committee for Microorganism Culture Collection on June 28, 2016, with the preservation number CGMCC No.12718.

Simultaneously, the present invention also provides a kind of Pichia strain expression system expressing exogenous protein, it is characterized in that, described system comprises (1) Pichia strain CGMCC No.12718 described in claim 1, (2 ) containing a foreign protein expression vector whose promoter is PAOX1; (3) a carbon source.

Further, the GTP1 gene of the Pichia strain is mutated, and the sequence of the GTP1 gene is shown in SEQ ID NO.1, so that the mutated Pichia gene cannot encode a glycerol transporter or the encoded glycerol transporter has no activity.

Further, the carbon source is glycerol, methanol or a mixture of glycerol and methanol. The carbon source for inducing the Pichia pastoris to express the exogenous protein is glycerol, and the range of the volume concentration B2 after the addition of the glycerol is 0<B2≤0.5%. The carbon source for inducing the Pichia pastoris to express the exogenous protein is methanol, and the range of the volume concentration A1 after the addition of the methanol is 0<A1≤2%.

The carbon source for inducing the Pichia pastoris to express the exogenous protein is a mixture of methanol and glycerol, the range of the volume concentration A2 after the addition of the methanol is 0<A2≤2%, and the range of the volume concentration B1 after the addition of the glycerol is 0<B1≤0.5%. More preferably, the volume concentration A2 after the addition of methanol is 1%, and the volume concentration B1 after the addition of glycerin is 0.25%.

In addition, the present invention also provides a method for inducing the expression of Pichia expressing foreign proteins, which includes the following steps,

Step 1, transforming the exogenous protein expression vector containing the promoter PAOX1 into Pichia pastoris, which contains the mutated Pichia pastoris GTP1 gene;

Step 2, inoculating the Pichia pastoris obtained in step 1 in the culture medium;

Step 3, centrifuge the Pichia yeast obtained in step 2, and re-cultivate it in a medium containing carbon source.

Wherein, the GTP1 gene sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO:2. Said mutation is all or part of sequence deletion mutation, insertion mutation, frame shift mutation or point mutation. The deletion mutation is the deletion of the entire open reading frame or partial sequence mutation of the GTP1 gene. The sequence of the GTP1 deletion fragment in vitro is shown in SEQ ID NO:3.

Wherein, the medium described in step 2 is YPD liquid medium, cultured at 30° C. and 230 r/min until the OD ₆₀₀ is 2-6.

Wherein, the carbon source is glycerol, methanol or a mixture of glycerol and methanol. The carbon source for inducing the Pichia pastoris to express the exogenous protein is glycerol, and the range of the volume concentration B2 after the addition of the glycerol is 0<B2≤0.5%. The carbon source for inducing the Pichia pastoris to express the exogenous protein is methanol, and the range of the volume concentration A1 after the addition of the methanol is 0<A1≤2%. The carbon source for inducing the Pichia pastoris to express the exogenous protein is a mixture of methanol and glycerol, the range of the volume concentration A2 after the addition of the methanol is 0<A2≤2%, and the range of the volume concentration B1 after the addition of the glycerol is 0<B1≤0.5%. Preferably, the volume concentration A2 after the addition of methanol is 1%, and the volume concentration B1 after the addition of glycerol is 0.25%.

Wherein, the method of adding the carbon source is, in terms of volume concentration, 0-24h, add 1% glycerol to the liquid medium; 24h-48h, add 1% methanol to the liquid medium; 48h-72h, add 1% methanol to the liquid medium 1% methanol and 0.25% glycerol are added to the medium; 72h to 96h, 1% methanol and 0.25% glycerol are added to the liquid medium.

In addition, the present invention also provides a method for constructing Pichia pastoris expressing foreign proteins, which includes the following steps:

Step 1, constructing a mutated GTP1 gene fragment in vitro, the mutated GTP1 gene fragment cannot encode a glycerol transporter or the encoded glycerol transporter has no activity;

Step 2, transforming the mutated GTP1 gene fragment obtained in step 1 into yeast;

Step 3, screening the yeast obtained in step 2 to obtain a yeast strain with a mutation in the GTP1 gene;

Step 4, screening the yeast obtained in step 3 to obtain a Pichia strain with AOX1 enzyme activity induced under the condition of glycerol or methanol and glycerol mixture.

Further, the GTP1 gene sequence of the Pichia pastoris strain is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO:2. The mutation in the GTP1 gene of the Pichia pastoris strain is a deletion mutation, an insertion mutation, a frameshift mutation or a point mutation. Preferably, the mutation in the GTP1 gene of the Pichia strain is a deletion mutation.

Further, the deletion mutation is deletion of the entire open reading frame or partial sequence of the GTP1 gene, and the sequence of the GTP1 deletion fragment in vitro is shown in SEQ ID NO:3.

After adopting the present invention, by mutating the GTP1 gene of Pichia pastoris, the mutated Pichia pastoris gene cannot encode glycerol transporter or the encoded glycerol transporter has no activity, and it is proved by experiments that the Pichia pastoris after the mutation of GTP1 gene Yeast can induce the transcription of PAOX1 in glycerol or a mixed medium of glycerol and methanol, thereby enabling the expression of foreign proteins. At the same time, by adopting the culture method of continuously adding carbon source in the present invention, when ΔGTP1-EGFP is finally harvested, the cell concentration is high and the amount of EGFP induced to express is high.

It should be noted that the Pichia pastoris involved in the present invention is Pichia pastoris (Pichia pastoris), and it was preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on June 28, 2016, and the address is Beijing No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, and the preservation number is CGMCC No.12718.

Description of drawings

Figure 1-1 is a gene structure diagram of the vector pPICZB used in the examples of the present invention.

Figures 1-2 are gene structure diagrams of the vector pGAPZB used in the examples of the present invention.

Fig. 2 is a graph showing the measurement results of AOX1 enzyme activity induced by different carbon sources for the ΔGTP1 strain constructed in the embodiment of the present invention and the wild-type strain X-33 respectively. Among the figure 1 adopts 0.5% methanol; 2 adopts 0.25% glycerin; 3 adopts 0.5% glycerin; 4 adopts 0.25% glycerin and 0.5% methanol; Rear volume concentration meter.

Fig. 3 is a graph showing the measurement results of the fluorescence values at 24h, 48h, 72h and 96h for the expression strains ΔGTP1-EGFP, x-EGFP and x-GGEFP constructed in the embodiment of the present invention respectively cultured with different carbon sources. Each letter in the figure represents as shown in Table 1, and methanol and glycerin in Table 1 are all calculated by volume concentration after addition.

Table 1

Fig. 4 is a graph showing the strain growth curves of the ΔGTP1-EGFP strain and the wild-type strain x-GGEFP induced by different carbon sources in the specific example of the present invention, where each letter represents as shown in Table 1.

Fig. 5 is a graph showing the growth curve of the ΔGTP1 strain constructed in a specific example of the present invention under optimized culture conditions and induced by 0.25% glycerol and 1% methanol as a carbon source, wherein the addition of methanol and glycerol is based on volume concentration; The optimized culture conditions are shown in Table 2.

Table 2

0～24h 24h～48h 48h～72h 72h～96h 1% glycerin 1% Methanol 1% methanol and 0.25% glycerol 1% methanol and 0.25% glycerol

Fig. 6 is a graph showing the measurement results of the fluorescence value of EGFP expression induced by the ΔGTP1 bacterial strain constructed in the specific embodiment of the invention under optimized culture conditions and 0.25% glycerol and 1% methanol as carbon source, wherein, sampling at 96h, adding methanol and glycerol All in volume concentration.

Figure 7 is a comparison of the biomass (OD600) of the mutant strain ΔGTP1-EGFP under the new culture method and the wild-type x-GEFP under the traditional culture method during the whole fermentation cycle (0-96h).

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1 Construction method of ΔGTP1 strain

The test materials used and their sources include:

DNA Marker, protein Marker, restriction enzymes (EcoRI, BamHI, HindIII, XbaI, KpnI, AvrII), T4 DNA ligase, and LA Taq DNA polymerase were all purchased from TaKaRa Company.

Plasmid extraction kits, gel recovery kits, PCR product column purification kits, protein quantification kits, kanamycin, bleomycin, and G418 antibiotics were purchased from Shanghai Sangong Bioengineering Company.

Primers were synthesized by Shanghai Sangong Bioengineering Company; yeast extract, peptone (OXOID, UK), and other reagents were domestic analytical grade.

DNA electrophoresis instrument, gel imager, protein electrophoresis instrument (Beijing Liuyi Instrument), PCR instrument (Hangzhou Longji Scientific Instrument Co., Ltd.), spectrophotometer (Shanghai Minorda Instrument Co., Ltd.), ultrasonic breaker (Ningbo New Zhi Biotechnology Co., Ltd.), microplate reader (Sunnyvale CA US Patent), etc.

Strains, plasmids and media:

Cloning host strain E.coli DH5a was purchased from Shanghai Sangon Company; Pichia pastoris X-33 (P.pastoris), Pichia pastoris expression vector pGAPZB, pPICZB were purchased from Invitrogen Company, wherein Pichia pastoris X- 33 is wild type, hereinafter referred to as wild type X-33, E. coli growth medium LB and screening medium low-salt LLB, Pichia growth medium YPD, induction medium BMMY, BMGY, BMMGY are all operated by the author with reference to Invitrogen Manual preparation.

1.1 Construction of GTP1 deletion fragment in vitro

The GTP1 gene is 1593bp long (SEQ ID NO:1) and encodes 530 amino acids (SEQ ID NO:2). The in vitro deletion fragment of GTP1 was constructed in vitro with pMD ^TM 19-T as a vector by PCR and restriction enzyme digestion ( SEQ ID NO:3), consisting of GTP1 gene upstream fragment 410bp (SEQ ID NO:4), G418 resistance gene 1464bp (SEQ ID NO:5) and GTP1 gene downstream fragment 363bp (SEQ ID NO:6), the obtained The GTP1 in vitro deletion fragment deletes the entire open reading frame of the gene, avoiding the production of the protein encoded by the GTP1 gene.

The specific operation is as follows:

a. Using the genome of the wild-type X-33 strain as a template, amplify the upstream segment of the GTP1 gene; two segments have EcoRI and BamHI restriction sites, and then double-digest the upstream segment and the plasmid pMD ^TM 19-T (TAKARA), T4 The ligase was ligated overnight at 16°C to generate the PGTup plasmid.

Primers are

GTP1 upstream F: 5'-GCGAATTCCCGACAGAAGCAACCTCAGATCAACC-3'

GTP1 upstream R: 5'-AGGGATCCATGGAGCGTTAATCCGGAGTGTAAGAG-3'

b. Using the genome of the wild-type X-33 strain as a template, amplify the downstream fragment of the GTP1 gene, the two fragments have XbaI and HindIII restriction sites, then double-enzyme digest the downstream fragment and the PGTup plasmid, and connect with ligase to generate the PGT up-down plasmid .

Primers are

GTP1 downstream F: 5'-GCTCTAGAAACATCTCGTTTCGTGTGCTTGTGG-3'

GTP1 downstream R: 5'-GCTAAGCTTCTTGCATTCGCTCAGGGCTCATTAC-3'

c. Use the pFA6a-KanMX6 plasmid as a template to amplify the G418 resistance gene fragment, two segments with BamHI and XbaI restriction sites, and use these two enzymes to double-digest the G418 resistance gene fragment and PGT up-down plasmid , ligated to generate the knockout vector pMD ^™ 19-T-GTP1-del.

Primers are

kanF (G418): 5'-GCGGATCCCCGGTTAATTAA-3'

kanR(G418): 5'-GCTCTAGAGAGCTCGTTTAAAC-3'

1.2 Electroporation of wild-type X-33 strain and screening of GTP1 deletion fragment in vitro

Electroporation: Under 2.0Kv pulse, the GTP1 deletion fragment was electroporated into the wild-type X-33 strain.

Screening: Spread the electrotransferred wild-type X-33 strain on a YPD plate supplemented with 100ug/ml G418, and culture it in a 30°C incubator for 48 hours. The single clones grown on the plate were picked into YPD liquid medium supplemented with 100ug/ml G418, cultured on a shaker at 30°C, and the genome was extracted and verified by PCR. The correct strain after PCR test and sequencing verification was named ΔGTP1.

Example 2 Determination of AOX1 enzyme activity

The ΔGTP1 strain and wild-type X-33 stored on the YPD plate were inoculated in the YPD liquid medium for activation, and all the bacteria were collected by centrifugation in the logarithmic phase, and resuspended in a solution containing 0.5% methanol, 0.5% glycerol, 0.25% glycerol, 0.5% glycerol and 0.5% methanol, 0.25% glycerol and 0.5% methanol as carbon source YNB liquid medium, after 48 hours of culture, the protein was extracted, and the AOX1 enzyme activity was determined after protein quantification by Bradford method.

The determination of AOX1 enzyme activity is as follows: add 100uL of 100μL alcohol oxidase detection solution to the microplate well, then add 5μL of extracted protein, add 3μL of methanol to start the reaction, react at room temperature for 15-30 minutes, until the color develops, add 20μL 2M sulfuric acid (containing 0.1M sodium sulfite) terminates the reaction, and measures the absorbance at 492nm. Fig. 2 is a measurement result figure, and its specific data are as shown in Table 3. The ΔGTP1 bacterial strain can induce the expression of AOX1 in glycerol and glycerol-methanol medium. And it is obviously improved when the glycerin content is 0.25%.

table 3

0.5% Methanol 0.25% glycerin 0.5% glycerin 0.25% glycerol and 0.5% methanol 0.5% glycerol and 0.5% methanol X-33 0.171 0.011 0.0001 0.059 0.0002 ΔGTP1 0.166 0.064 0.036 0.161 0.038

Example 3 Induced expression of foreign protein

3.1 Construction of EGFP expression vector

Using EGFP as the foreign protein, as shown in Figure 1-1 and Figure 1-2, the EGFP coding sequence was inserted downstream of the AOX1 promoter and GAP promoter of the vectors pPICZB and pPGAPZB to obtain EGFP expression vectors, respectively PAOX1- EGFP and Pgap-EGFP.

The specific operation is as follows: the plasmids pMD ^TM 19-T-EGFP, pPICZB, and Pgapzb were double-digested with EcoRI and XhoI respectively; the pMD ^TM 19-T-EGFP digestion product EGFP was recovered and purified with a gel recovery kit, and the yeast expression vector pPICZB and The Pgapzb digested product was recovered with a PCR purification kit; the fragment and the plasmid were ligated with T4 ligase to obtain EGFP expression vectors Paox1-EGFP and Pgap-EGFP, respectively, and transformed into competent E.coli DH5α, and then bleomycin-resistant LLB plate screening.

3.2 Screening of expression strains

Under 2.0Kv pulse, the expression vector PAOX1-EGFP was electrotransformed into the ΔGTP1 strain and the wild-type X-33 strain respectively, spread on the YPD plate containing bleomycin resistance, and placed in a 30°C incubator for 48 hours; grow the plate out The strains were picked into YPD liquid medium, and the genome was extracted after cultivation, and the correct strains were named ΔGTP1-EGFP and x-EGFP by PCR; the expression vector Pgap-EGFP was electrotransformed into wild-type X-33 in the same way, and finally the The correct strain was named x-GGEFP.

3.3 Determination of copy number

The three expression strains constructed above were coated with different concentrations of bleomycin (100 μg/mL, 200 μg/mL, 300 μg/mL, 500 μg/mL, 800 μg/mL, 1000 μg/mL), and finally picked Three strains growing on plates at 800 μg/mL and very slowly at 1000 μg/m.

3.4 Detection of EGFP expression by multifunctional microplate reader

Pick single colonies of ΔGTP1-EGFP, x-EGFP and x-GGEFP obtained in 1.4.2, inoculate them in a 250mL shake flask filled with YPD liquid medium, and culture them at 30°C and 230r/min until the _OD600 is 2-6 Centrifuge at 4000r/min for 5min to collect the bacteria, resuspend in BMY containing 0.5% methanol, 0.5% glycerol, 0.5% glycerol and 0.5% methanol, 0.25% glycerol and 0.5% methanol and 0.25% glycerol and 1% methanol as carbon source In the liquid medium, add carbon source again every 24 hours. The amount of carbon source added each time is equal to the amount of carbon source contained for the first time. Samples are taken, and the fluorescence intensity is measured after OD quantification. The excitation wavelength is 488nm and the emission wavelength is 505nm , and the results are shown in Table 4 and Figure 3.

Table 4

It can be seen from the above table and Figure 3 that the wild-type strain can only induce high expression of EGFP in AOX1 when methanol is the carbon source; the strain ΔGTP1-EGFP can induce AOX1 to express EGFP in glycerol medium, while the wild-type strain has low expression ; In the case of 0.25% glycerol and 0.5% methanol as the carbon source, ΔGTP1-EGFP induced AOX1 expression of EGFP was significantly higher than that of the wild-type strain, especially at 48h, it was nearly 6 times higher; ΔGTP1-EGFP at 96 hours, Its expression of AOX1 induced by 0.25% glycerol and 1% methanol was 1.6 times higher than that of 0.25% glycerol and 0.5% methanol as a carbon source; at the same time, it can be seen that the inducible promoter AOX1 is significantly higher than the constitutive promoter GAPDH Much higher when expressing EGFP.

3.5 Growth curves of expression strains under different carbon sources

Pick single colonies of ΔGTP1-EGFP, x-EGFP and x-GGEFP obtained in 1.4.2, inoculate them in a 250mL shake flask filled with YPD medium, and cultivate them at 30°C and 230r/min until the _OD600 is 2-6, 4000r /min centrifuged for 5min to collect the thalline, then transferred to the liquid culture medium of YNB and YP with different carbon sources, as shown in Table 5, the initial quantitative OD600 was 0.1, and then samples were taken every 8 hours to measure the OD600 value, the result As shown in Figure 4.

table 5

It can be seen from Figure 4 that the final OD of the wild-type strain x-GGEFP can reach 32.88 when using 0.5% glycerol as the carbon source, but it can be seen from Figure 3 that the amount of expressed EGFP is small; The EGFP that induces the expression of AOX1 is higher, but its final OD is 19.65, which is 13 worse than that of 0.5% glycerol as carbon source; ΔGTP1-EGFP is induced when 0.25% glycerol and 1% methanol are used as carbon source The expression of EGFP was higher, and the final OD was 26.84.

Example 4 Optimizing the culture method of ΔGTP1-EGFP

Based on the experimental results above, a new culture method was designed so that when ΔGTP1-EGFP was finally harvested, the cell concentration was high and the amount of induced expression of EGFP was high. Therefore, a new culture method was designed, as shown in Table 2, in terms of volume concentration , 0～24h, add 1% glycerol to the liquid medium; 24h～48h, add 1% methanol to the liquid medium; 48h～72h, add 1% methanol and 0.25% glycerol to the liquid medium; 72h～96h , add 1% methanol and 0.25% glycerol to the liquid medium. At the same time, the wild-type strain x-EGFP was cultivated by the traditional culture method, that is, adding 1% glycerol in the first 24 hours, and then adding 1% methanol every 24 hours to induce the expression of EGFP.

It can be seen from Figure 6 that at 96 hours, the unit OD600 fluorescence value was 10% higher than that of the optimal culture when the carbon source was 0.25% glycerol and 1% methanol, but as shown in Figure 5, the final cell concentration was higher than that of the optimal culture when the final cell concentration was 1 When % methanol and 0.25% glycerol were used as carbon sources, it was 17% higher, that is, the total expression level of EGFP after the final optimized culture was 6.36% higher than when 1% methanol and 0.25% glycerol were used as carbon sources.

It can be seen from Figure 7 that during the entire fermentation period (0-96h), the biomass (OD600) of the mutant strain ΔGTP1-EGFP in the new culture method was significantly different from that of the wild-type x-GEFP in the traditional culture method, and the final optimal culture The total expression of EGFP was 23% higher than that of wild bacteria in traditional culture conditions.

The above experiments prove that Pichia pastoris expressing exogenous protein obtained by the construction method of this application can induce PAOX1 to express exogenous protein in glycerol or glycerol-methanol mixed medium, and the above-mentioned optimized induction expression method of this application can effectively improve the final cell concentration.

Claims (21)

1. Pichia pastoris GTP1 genes of a kind of mutation, it is characterised in that the mutation causes Pichia pastoris to be unable to encoding glycerol The glycerine transport protein of transport protein or coding can start the transcription of PAOX1 without activity under glycerine existence condition.

2. Pichia pastoris GTP1 genes of mutation according to claim 1, it is characterised in that the GTP1 gene orders are such as Shown in SEQ ID NO.1, encoding amino acid sequence such as SEQ ID NO：Shown in 2.

3. Pichia pastoris GTP1 genes of mutation according to claim 2, it is characterised in that described to sport whole or portion Sub-sequence deletion mutation, insertion mutation, frameshift mutation or point mutation.

4. Pichia pastoris GTP1 genes of mutation according to claim 3, it is characterised in that described deletion mutation is scarce Lose whole ORFs or the partial sequence mutation of GTP1 genes.

5. Pichia pastoris GTP1 genes of mutation according to claim 4, it is characterised in that described GTP1 is lacked in vitro Fragment sequence such as SEQ ID NO：Shown in 3.

6. the recombinant vector of gene described in claim 1-5 any one is included.

7. the host cell of recombinant vector described in claim 6 is included.

8. it is a kind of express foreign protein Pichi strain, it is characterised in that the Pichi strain contains claim The Pichia pastoris GTP1 genes of the mutation described in 1-5 any one.

9. Pichi strain according to claim 8, it is characterised in that the Pichi strain is by yeast strain X-33 mutation GTP1 gene gained.

10. Pichi strain according to claim 9, it is characterised in that be preserved in China on 06 28th, 2016 Microbiological Culture Collection administration committee common micro-organisms center, preserving number is CGMCC No.12718.

11. a kind of Pichi strain expression systems for expressing foreign protein, it is characterised in that the system includes (1) right It is required that the Pichi strain in 8-10 described in any one, the exogenous protein expression carrier of (2) containing promoter, (3) carbon source.

12. Pichi strain expression systems according to claim 11, it is characterised in that the promoter is PAOX1.

13. Pichi strain expression systems according to claim 11, it is characterised in that the carbon source is glycerine, first The mixture of alcohol or glycerine and methyl alcohol.

14. Pichi strain expression systems according to claim 13, it is characterised in that the induction Pichia pastoris table Carbon source up to foreign protein is glycerine, and the scope of the volumetric concentration B2 after the glycerine addition is 0 ＜ B2≤0.5%.

15. Pichi strain expression systems according to claim 13, it is characterised in that the induction Pichia pastoris table Carbon source up to foreign protein is methyl alcohol, and the scope of the volumetric concentration A1 after the methyl alcohol addition is 0 ＜ A1≤2%.

16. Pichi strain expression systems according to claim 13, it is characterised in that the induction Pichia pastoris table Carbon source up to foreign protein is methyl alcohol and the mixture of glycerine, and the scope of the volumetric concentration A2 after the methyl alcohol addition is 0 ＜ A2 The scope of≤2%, the volumetric concentration B1 after glycerine addition is 0 ＜ B1≤O.5%.

17. Pichi strain expression systems according to claim 16, it is characterised in that：Body after the methyl alcohol addition Product concentration A2 is 1%, and the volumetric concentration B1 after the glycerine addition is 0.25%.

The Pichia pastoris GTP1 genes of the mutation described in 18. claim 1-5 any one express foreign protein in yeast strain Application in matter.

The application of recombinant vector or host cell described in 19. claim 6-7 in exogenous proteins are expressed.

The application of Pichi strain described in 20. claim 8-10 in exogenous proteins are expressed.

The application of Pichi strain expression system described in 21. claim 11-17 in exogenous proteins are expressed.