CN106701787A

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

Info

Publication number: CN106701787A
Application number: CN201610613088.3A
Authority: CN
Inventors: 杨艳坤; 战春君; 张震阳; 白仲虎; 刘秀霞; 戴晓峰; 詹锦玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2016-07-29
Filing date: 2016-07-29
Publication date: 2017-05-24

Abstract

The invention provides a pichia pastoris strain and expression system for expressing foreign proteins. A gene GTP1 of the pichia pastoris strain is mutated, the mutated pichia pastoris gene cannot encode glycerol transport proteins, or encoded glycerol transport proteins are free of activity, and the transcription of PAOX1 can be started in the presence of glycerol, so that the AOX1 expression level of pichia pastoris in the presence of glycerol is increased, and thus, the expression level of the foreign proteins is increased. Meanwhile, the invention further provides a construction method of the pichia pastoris for expressing the foreign proteins and an induced expression method of the pichia pastoris for expressing the foreign proteins. The induced expression method can be used for effectively improving the expression level of the foreign proteins and final cell concentration.

Description

Express Pichia pastoris, its construction method and its derivational expression method of foreign protein

Technical field

The present invention relates to Fermentation Engineering and technical field of bioengineering, and in particular to the Pichia pastoris of expression foreign protein, Its construction method and its derivational expression method.

Background technology

Pichia yeast expression system is a kind of new exogenous protein expression system for growing up the phase at the beginning of the eighties in last century System, for escherichia expression system, pichia yeast expression system has obvious superiority, such as：During multiplication compared with Short, fermentation period is short；Genome is simple, is easy to operation；Condition of culture is relatively easy, and is easy to carry out High Density Cultivation, and then Destination protein yield higher can be obtained；Foreign protein can be folded, it is glycosylation modified, form disulfide bond etc.；Together When, it also avoid, and saccharomyces cerevisiae secernment efficiency is poor, express the defects such as bacterial strain is not sufficiently stable, expression plasmid is easy to lose；Also, just The average length of every side chain of foreign protein is added to for degree of glycosylation, in Pichia pastoris for 8-14 mannose residue, compared with The average 50-150 mannose residues of every side chain of saccharomyces cerevisiae want much shorter, excessive glycosylation phenomenon will not be produced；Additionally, finishing Red yeast its own secretion extracellular protein seldom, this is highly beneficial for the protein purification in later stage.

The fermentation process in high density for producing bacterium as single cell protein using Pichia pastoris of foundation from the eighties in 20th century Ripe, yield of dried cell may be up to 100g/L.A variety of advantages make Pichia pastoris turn into the eucaryon base that pole is favored in recent years Because of expression system host.Hundreds of albumen from virus, bacterium, fungi, animals and plants and people with the system successful expression, Such as human interleukin, human serum albumins, TNF, hepatitis B surface antigen, hirudin derivative.

Pichia pastoris is one of current most widely used foreign protein eukaryotic expression system, and the system is based on one efficiently Alcohol oxidase 1 (alcohol oxidase 1, AOX1) promoter (AOX1promoter, PAOX1), the transcription of PAOX1 only rings The induction of methyl alcohol is answered, when with methyl alcohol as sole carbon source, PAOX1 starts the expression of foreign protein；And in the presence of working as glycerine, suppress The expression of AOX1.

The content of the invention

Regarding to the issue above, the invention provides the Pichia pastoris of expression foreign protein, it can solve to finish in the prior art Red yeast low technical problem of AOX1 expression quantity under glycerine inductive condition；Additionally, present invention also offers the Pichia pastoris Construction method and derivational expression method.

The present invention provides a kind of Pichia pastoris GTP1 genes of mutation, it is characterised in that the mutation causes Pichia pastoris The glycerine transport protein of encoding glycerol transport protein or coding is unable to without activity, and can be started under glycerine existence condition The transcription of PAOX1.The GTP1 gene orders as shown in SEQ ID NO.1, encoding amino acid sequence such as SEQ ID NO:2 institutes Show.

Further, it is described to sport all or part of sequence deletion mutation, insertion mutation, frameshift mutation or point mutation.

Further, described deletion mutation is that the whole ORFs or partial sequence for lacking GTP1 genes are dashed forward Become.The external deletion fragment sequences of described GTP1 such as SEQ ID NO:Shown in 3.

Present invention also offers the recombinant vector comprising the mutator.Host cell comprising recombinant vector.

Present invention also offers a kind of Pichi strain for expressing foreign protein, it is characterised in that the Pichia pastoris Bacterial strain contains the Pichia pastoris GTP1 genes of the mutation described in claim 1-5 any one.

Further, the Pichi strain is by yeast strain X-33 mutation GTP1 gene gained.

Further, the Pichi strain was preserved in Chinese microorganism strain preservation management on 06 28th, 2016 Committee's common micro-organisms center, preserving number is CGMCC No.12718.

Meanwhile, present invention also offers a kind of Pichi strain expression system for expressing foreign protein, it is characterised in that The system includes the Pichi strain CGMCC No.12718 described in (1) claim 1, and (2) are containing promoter The exogenous protein expression carrier of PAOX1；(3) carbon source.

Further, the GTP1 genes of the Pichi strain are undergone mutation, GTP1 gene orders such as SEQ ID NO.1 It is shown so that the Pichia pastoris gene after mutation is unable to encoding glycerol transport protein or the glycerine transport protein of coding is not lived Property.

Further, the carbon source is the mixture of glycerine, methyl alcohol or glycerine and methyl alcohol.Induce the Pichia anomala expression The carbon source of foreign protein is glycerine, and the scope of the volumetric concentration B2 after the glycerine addition is 0<B2≤0.5%.Induction is described to finish The carbon source of red Yeast expression foreign protein is methyl alcohol, and the scope of the volumetric concentration A1 after the methyl alcohol addition is 0<A1≤2%.

It is methyl alcohol and the mixture of glycerine to induce the carbon source of the Pichia anomala expression foreign protein, after the methyl alcohol addition Volumetric concentration A2 scope be 0<A2≤2%, the scope of the volumetric concentration B1 after the glycerine addition is 0<B1≤0.5%. More preferably, the volumetric concentration A2 after the methyl alcohol addition is 1%, and the volumetric concentration B1 after the glycerine addition is 0.25%.

Additionally, present invention also offers it is a kind of express foreign protein Pichia pastoris derivational expression method, it include with Lower step,

Step 1, will convert into Pichia pastoris containing the exogenous protein expression carrier that promoter is PAOX1, described complete red Yeast contains the Pichia pastoris GTP1 genes of mutation；

Step 2, the Pichia pastoris that step 1 is obtained is inoculated in culture medium and is cultivated；

Step 3, the Pichia yeast obtained in centrifugation step 2, and culture in culture to the culture medium containing carbon source again.

Wherein, the GTP1 gene orders are as shown in SEQ ID NO.1, encoding amino acid sequence such as SEQ ID NO:2 institutes Show.It is described to sport all or part of sequence deletion mutation, insertion mutation, frameshift mutation or point mutation.Described deletion mutation To lack whole ORFs or the partial sequence mutation of GTP1 genes.The external deletion fragment sequences of described GTP1 are such as SEQ ID NO:Shown in 3.

Wherein, culture medium described in step 2 is YPD fluid nutrient mediums, is cultivated to OD in 30 DEG C, 230r/min₆₀₀For 2~ 6。

Wherein, the carbon source is the mixture of glycerine, methyl alcohol or glycerine and methyl alcohol.Induce the Pichia anomala expression external source The carbon source of albumen is glycerine, and the scope of the volumetric concentration B2 after the glycerine addition is 0<B2≤0.5%.Induction is described to finish red ferment Matrix is methyl alcohol up to the carbon source of foreign protein, and the scope of the volumetric concentration A1 after the methyl alcohol addition is 0<A1≤2%.Induction institute The carbon source for stating Pichia anomala expression foreign protein is the mixture of methyl alcohol and glycerine, the volumetric concentration A2's after the methyl alcohol addition Scope is 0<A2≤2%, the scope of the volumetric concentration B1 after the glycerine addition is 0<B1≤0.5%.Preferably, the methyl alcohol Volumetric concentration A2 after addition is 1%, and the volumetric concentration B1 after the glycerine addition is 0.25%.

Wherein, the addition manner of the carbon source is that in terms of volumetric concentration, 0~24h is sweet to addition 1% in fluid nutrient medium Oil；24h~48h, to adding 1% methyl alcohol in fluid nutrient medium；48h~72h, to added in fluid nutrient medium 1% methyl alcohol and 0.25% glycerine；72h~96h, to adding 1% methyl alcohol and 0.25% glycerine in fluid nutrient medium.

Additionally, the construction method of the Pichia pastoris present invention also offers expression foreign protein, it is comprised the following steps：

Step 1, the GTP1 genetic fragments that external structure is undergone mutation, the GTP1 genetic fragments undergone mutation can not encode sweet The glycerine transport protein of oily transport protein or coding is without activity；

Step 2, the GTP1 genetic fragments undergone mutation that will be obtained in step 1 are converted into saccharomycete；

Step 3, screens to the saccharomycete that step 2 is obtained, and obtains the yeast strains that GTP1 genes are undergone mutation；

Step 4, screens to the saccharomycete that step 3 is obtained, and obtains and is induced under glycerine or methyl alcohol, glycerine mixing condition Pichia pastoris strain with AOX1 enzymatic activitys.

Further, the GTP1 gene orders of the Pichi strain are as shown in SEQ ID NO.1, coded amino acid sequence Row such as SEQ ID NO:Shown in 2.Deletion mutation that what the GTP1 genes of the Pichi strain occurred sport, insertion mutation, Frameshift mutation or point mutation.Preferably, what the GTP1 genes of the Pichi strain occurred sports deletion mutation.

Further, described deletion mutation is the whole ORFs or partial sequence for lacking GTP1 genes, GTP1 External deletion fragment sequence such as SEQ ID NO:Shown in 3.

After using the present invention, undergone mutation by the GTP1 genes to Pichia pastoris so that the Pichia pastoris base after mutation Because being unable to the glycerine transport protein of encoding glycerol transport protein or coding without activity, and the experiment proved that mutated GTP1 genes Pichia pastoris afterwards can induce the transcription of PAOX1 in glycerine or glycerine, methyl alcohol mixed culture medium, and then cause external source egg Expressed in vain.Meanwhile, using the training method of continuous addition carbon source of the invention, make Δ GTP1-EGFP in final results, carefully Born of the same parents' concentration is high and EGFP of induced expression amount is high.

It should be noted that Pichia pastoris involved in the present invention is pichia pastoris phaff (Pichia pastoris), And China Committee for Culture Collection of Microorganisms's common micro-organisms center was preserved on 06 28th, 2016, address is Beijing The institute 3 of city Chaoyang District North Star West Road 1, preserving number is CGMCC No.12718.

Brief description of the drawings

Fig. 1-1 is the gene structure figure of carrier pPICZB used in embodiments of the invention.

Fig. 1-2 is the gene structure figure of carrier pGAPZB used in embodiments of the invention.

Fig. 2 is that the Δ GTP1 bacterial strains that embodiments of the invention build and wild-type strain X-33 are respectively adopted different carbon source and lure Lead the measurement result figure of AOX1 enzyme activity.1 uses 0.5% methyl alcohol in figure；2 use 0.25% glycerine；3 use 0.5% glycerine；4 adopt With 0.25% glycerine and 0.5% methyl alcohol；5 to use 0.5% glycerine and 0.5% methyl alcohol be carbon source, and above-mentioned methyl alcohol, glycerine are adding Volumetric concentration meter afterwards.

Fig. 3 is that expression bacterial strain Δ GTP1-EGFP, x-EGFP and x-GGEFP that embodiments of the invention build are respectively adopted Different carbon source culture, respectively to the measurement result figure of 24h, 48h, 72h and 96h fluorescent value.Each letter is represented such as the institute of table 1 in figure Show, methyl alcohol, glycerine are in terms of the volumetric concentration after addition in table 1.

Table 1

Fig. 4 is the Δ GTP1-EGFP bacterial strains that specific embodiment of the invention builds and wild-type strain x-GGEFP in difference Strain growth curve map under carbon source induction, wherein each letter representative is as shown in table 1.

Fig. 5 is the Δ GTP1 bacterial strains of specific embodiment structure of the invention under optimum culture condition and in 0.25% glycerine It is the strain growth curve map under carbon source induction with 1% methyl alcohol, wherein, methyl alcohol, glycerine addition are in terms of volumetric concentration；Optimization training The condition of supporting is as shown in table 2.

Table 2

0~24h	24h~48h	48h~72h	72h~96h
				1% glycerine	1% methyl alcohol	1% methyl alcohol and 0.25% glycerine	1% methyl alcohol and 0.25% glycerine

Fig. 6 be the Δ GTP1 bacterial strains that build of specific embodiment of invention under optimum culture condition and in 0.25% glycerine and 1% methyl alcohol is the measurement result figure of the fluorescent value of induction EGFP expression under carbon source, wherein, 96h samplings, methyl alcohol, glycerine addition are In terms of volumetric concentration.

Fig. 7 be at whole fermentation period (0-96h), mutant strain Δ GTP1-EGFP under new training method with it is wild The comparing of type x-GEFP biomass (OD600) under traditional training method.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.These embodiments be merely to illustrate the present invention and without In restriction the scope of the present invention.

The construction method of the Δ GTP1 bacterial strains of embodiment 1

Test material and its source used includes:

DNA Marker, albumen Marker, restriction enzyme (EcoRI, BamHI, HindIII, XbaI, KpnI, AvrII), T4 DNA ligases, LA Taq archaeal dna polymerases, are purchased from TaKaRa companies.

Plasmid extraction kit, glue reclaim kit, PCR primer Column kit, protein quantification kit blocks that mould Element, bleomycin, G418 antibiotic is purchased from Shanghai Sheng Gong bio-engineering corporations.

Primer is synthesized by Shanghai Sheng Gong bio-engineering corporations；Yeast extract, peptone (OXOID, UK), remaining reagent is state Produce analysis pure.

DNA electrophoresis apparatuses, gel imaging instrument, protein electrophoresis instrument (instrument of Beijing 6 1), PCR instrument (the bright base scientific instrument in Hangzhou Co., Ltd), spectrophotometer (Shanghai Mei Nuoda Instrument Ltd.), Ultrasonic Cell Disruptor (the new sesame biotechnology share in Ningbo Co., Ltd), ELIASA (Sunnyvale CA US Patent) etc..

Bacterial strain, plasmid and culture medium:

Cloning host bacterium E.coli DH5a, purchased from Shanghai Sangon companies；Pichia pastoris Pichia pastoris X-33 (P.pastoris), yeast expression vector pGAPZB, pPICZB, is purchased from Invitrogen companies, wherein Pichia pastoris Pichia pastoris X-33 are wild type, hereinafter referred to as wild type X-33, Escherichia coli Growth culture medium LB and screening Culture medium less salt LLB, Pichia pastoris growth medium YPD, inducing culture BMMY, BMGY, BMMGY are referred to by author Invitrogen operation manuals are prepared.

The structure of the external deletion fragments of 1.1 GTP1

GTP1 gene lengths 1593bp (SEQ ID NO:1) 530 amino acid (SEQ ID NO, are encoded:2), using PCR, enzyme The method for cutting connection, with pMD^TM19-T constructs the external deletion fragments of GTP1 (SEQ ID NO in vitro for carrier:3), respectively by GTP1 upstream region of gene fragments 410bp (SEQ ID NO:4), G418 resistant genes 1464bp (SEQ ID NO:5) with GTP1 genes Segments downstream 363bp (SEQ ID NO:6) constitute, the external deletion fragments of gained GTP1 have lacked the whole open reading of the gene Frame, it is to avoid the generation of GTP1 gene coded proteins.

Concrete operations are as follows：

A, with wild type X-33 strain gene groups as template, expand GTP1 upstream region of gene fragments；Two sections with EcoRI and BamHI restriction enzyme sites, then double digestion fragment upstream and plasmid pMD^TM19-T (TAKARA), 16 DEG C of T4 ligases are connected overnight, Produce PGTup plasmids.

Primer is

GTP1 upstreams F：5‘-GCGAATTCCCGACAGAAGCAACCTCAGATCAACC-3’

GTP1 upstreams R：5‘-AGGGATCCATGGAGCGTTAATCCGGAGTGTAAGAG-3'

B, with wild type X-33 strain gene groups as template, expand GTP1 downstream of gene fragments, two sections with XbaI and HindIII restriction enzyme sites and then double digestion segments downstream and PGTup plasmids, ligase connection produce PGT up-down plasmids.

Primer is

GTP1 downstreams F：5‘-GCTCTAGAAACATCTCGTTTCGTGTGCTTGTGG-3‘

GTP1 downstreams R：5‘-GCTAAGCTTCTTGCATTCGCTCAGGGCTCATTAC-3’

C, with pFA6a-KanMX6 plasmids as template, amplify G418 resistance gene fragments, two sections carry BamHI and XbaI Restriction enzyme site, with both enzyme double digestion G418 resistance gene fragments and PGT up-down plasmids, connection produces knockout carrier pMD^TM19-T-GTP1-del。

Primer is

kanF(G418)：5‘-GCGGATCCCCGGTTAATTAA-3’

kanR(G418)：5‘-GCTCTAGAGAGCTCGTTTAAAC-3’

The external deletion fragment electricity of 1.2 GTP1 turns wild type X-33 bacterial strains, screening

Electricity turns：Under 2.0Kv pulses, GTP1 deletion fragment electricity is turned into wild type X-33 bacterial strains.

Screening：Wild type X-33 bacterial strains after electricity is turned are applied to the YPD flat boards of addition 100ug/ml G418, are placed on 30 DEG C Incubator culture 48 hours.The monoclonal that will be grown on flat board is chosen into the YPD fluid nutrient mediums of addition 100ug/ml G418, After 30 DEG C of shaking table cultures, extract genome and verified with PCR.It is Δ correct Strain Designation after PCR inspections and sequence verification GTP1。

The measure of the AOX1 enzyme activity of embodiment 2

The Δ GTP1 bacterial strains and wild type X-33 that YPD flat boards will be stored in are inoculated in YPD fluid nutrient mediums and are activated, and select Select logarithmic phase and whole thalline are collected by centrifugation, in terms of volumetric concentration, be resuspended in containing 0.5% methyl alcohol, 0.5% glycerine, 0.25% sweet Oil, 0.5% glycerine and 0.5% methyl alcohol, 0.25% glycerine and 0.5% methyl alcohol are in the YNB fluid nutrient mediums of carbon source, culture 48 is small When after leach protein, must be determined by carrying out AOX1 enzyme activity after Bradford method protein quantifications.

AOX1 enzyme activity must determine specific as follows：100uL100 μ L alcohol oxidases detection solution is added in ELISA Plate hole, so Add the μ L of albumen 5 of extraction afterwards, add 3 μ L methyl alcohol to start reaction, in room temperature reaction 15 minutes~30 minutes, until colour developing, plus Enter 20 μ L 2M sulfuric acid (sodium sulfite containing 0.1M) terminating reactions, and absorbance is measured in 492nm, Fig. 2 is measurement result figure, its As shown in table 3, Δ GTP1 bacterial strains can induce the expression of AOX1 to specific data in containing glycerine and glycerine methanol medium, and And significantly improved when glycerol content is 0.25%.

Table 3

	0.5% methyl alcohol	0.25% glycerine	0.5% glycerine	0.25% glycerine and 0.5% methyl alcohol	0.5% glycerine and 0.5% methyl alcohol
						X-33	0.171	0.011	0.0001	0.059	0.0002
ΔGTP1	0.166	0.064	0.036	0.161	0.038

The induced expression foreign protein of embodiment 3

The structure of 3.1 EGFP expression vectors

With EGFP as foreign protein, as shown in Fig. 1-1, Fig. 1-2, in the AOX1 promoters of carrier pPICZB and pPGAPZB The coded sequence of EGFP is inserted in downstream with GAP promoters, obtains EGFP expression vectors, respectively PAOX1-EGFP and Pgap- EGFP。

Concrete operations are as follows：With EcoRI and XhoI respectively by plasmid pMD^TMThe double enzymes of 19-T-EGFP, pPICZB, Pgapzb Cut；pMD^TM19-T-EGFP digestion products EGFP glue reclaim kit recovery purifyings, Yeast expression carrier pPICZB and Pgapzb Digestion products are reclaimed with PCR purification kits；With T4 ligases junction fragment and plasmid, EGFP expression vectors are respectively obtained Paox1-EGFP and Pgap-EGFP, transformed competence colibacillus E.coli DH5 α, then with the LLB flat screens containing blasticidin resistance Choosing.

The screening of 3.2 expression bacterial strains

Under 2.0Kv pulses, expression vector PAOX1-EGFP electricity is turned into Δ GTP1 bacterial strains and wild type X-33 bacterial strains respectively, applied In on the YPD flat boards containing blasticidin resistance, being placed on 30 DEG C of incubators 48 hours；The bacterial strain that flat board grows is chosen to YPD In fluid nutrient medium, genome is carried after culture, verify that correct Strain Designation is Δ GTP1-EGFP and x-EGFP with PCR；With Expression vector Pgap-EGFP electricity is turned wild type X-33 by same method, and most correct Strain Designation is x-GGEFP at last.

The determination of 3.3 copy numbers

By above-mentioned build 3 expression bacterial strains be respectively coated on containing various concentrations bleomycin (100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 500 μ g/mL, 800 μ g/mL, 1000 μ g/mL), final picking is grown in 800 μ g/mL and 1000 μ g/m grow three plants of bacterial strains on very slow flat board.

3.4 multi-function microplate readers detect EGFP expression quantity

Δ GTP1-EGFP, x-EGFP and x-GGEFP single bacterium colony that picking 1.4.2 is obtained, is inoculated in equipped with YPD liquid The shaking flask of the 250mL of culture medium, 30 DEG C, 230r/min cultivated to OD₆₀₀It is 2~6,4000r/min centrifugation 5min collects thallines, Be resuspended in containing 0.5% methyl alcohol, 0.5% glycerine, 0.5% glycerine and 0.5% methyl alcohol, 0.25% glycerine and 0.5% methyl alcohol and During 0.25% glycerine and 1% methyl alcohol are for the BMY fluid nutrient mediums of carbon source, carbon source is added again every 24h, every time the carbon source amount of addition It is equal, the first carbon source amount for containing is, and sample is taken, and fluorescence intensity is surveyed after OD is quantitative, excitation wavelength is 488nm, transmitted wave A length of 505nm, its result is as shown in table 4 and Fig. 3.

Table 4

As can be seen that wild-type strain can only could induce AOX1 expression high when methyl alcohol is carbon source from upper table and Fig. 3 EGFP；Bacterial strain Δ GTP1-EGFP can be in glycerin medium induction AOX1 expression EGFP, and wild-type strain low expression；With In the case that 0.25% glycerine and 0.5% methyl alcohol are carbon source, the EGFP of Δ GTP1-EGFP induction AOX1 expression substantially compares wild type Bacterial strain is high, especially in 48h, is higher by nearly 6 times；At 96, it is being Δ GTP1-EGFP with 0.25% glycerine and 1% methyl alcohol Carbon source is than 0.25% glycerine and 0.5% methyl alcohol for carbon source induced expression AOX1 is higher by 1.6 times；Opened it can be seen that carrying out induction type simultaneously Mover AOX1 substantially just expresses high during EGFP than constitutive promoter GAPDH.

Growth curve of the 3.5 expression bacterial strains in different carbon source

Δ GTP1-EGFP, x-EGFP and x-GGEFP single bacterium colony that picking 1.4.2 is obtained, is inoculated in equipped with YPD culture mediums 250mL shaking flask, 30 DEG C, 230r/min cultivated to OD₆₀₀It is 2~6,4000r/min centrifugation 5min collects thallines, Ran Houzhuan It is connected in the fluid nutrient medium of the YNB of different carbon source and YP, as shown in table 5, it is 0.1 initially to quantify OD600, then small every 8 When sample, survey OD600 values, its result is as shown in Figure 4.

Table 5

As shown in Figure 4, wild-type strain x-GGEFP is being that final OD can reach 32.88 by carbon source of 0.5% glycerine, but It is that the EGFP amounts expressed as shown in Figure 3 are less；Although Δ GTP1-EGFP is being induction AOX1 expression by carbon source of 1% methyl alcohol EGFP is higher, but its final OD be 19.65 compare 0.5% glycerine be carbon source in the case of OD differed from 13；ΔGTP1- EGFP induced expression EGFP amounts when with 0.25% glycerine and 1% methyl alcohol as carbon source are higher, and final OD is 26.84.

The training method of the optimization Δ of embodiment 4 GTP1-EGFP

The experimental result done more than designs new training method, makes Δ GTP1-EGFP in final results, cell concentration The EGFP amounts of high and induced expression are high, therefore devise new training method, as shown in table 2, in terms of volumetric concentration, 0~ 24h, to adding 1% glycerine in fluid nutrient medium；24h~48h, to adding 1% methyl alcohol in fluid nutrient medium；48h~72h, to 1% methyl alcohol and 0.25% glycerine are added in fluid nutrient medium；72h~96h, to added in fluid nutrient medium 1% methyl alcohol and 0.25% glycerine.Meanwhile, it is with traditional training method culture wild-type strain x-EGFP:1% glycerine is added in preceding 24h, with Afterwards a 1% methanol induction EGFP expression is added every 24h.

It will be appreciated from fig. 6 that in 96h, unit OD600 fluorescent values are when with 0.25% glycerine and 1% methyl alcohol as carbon source than excellent Change culture and be higher by 10%, but as shown in figure 5, final cell concentration optimization culture when ratio with 1% methyl alcohol and 0.25% glycerine be carbon To be higher by during source the total expression quantity ratios of the EGFP after 17%, i.e. final optimization pass culture with 1% methyl alcohol and 0.25% glycerine be carbon source When be higher by 6.36%.

As shown in Figure 7, at whole fermentation period (0-96h), mutant strain Δ GTP1-EGFP compares under new training method Wild type x-GEFP under traditional training method biomass (OD600) there were significant differences, the EGFP after final optimization pass culture is total Expression quantity be higher by 23% in traditional culture conditions than wild mushroom.

More than experiment prove the expression foreign protein that the application construction method is obtained Pichia pastoris can in glycerine or The induced expression side after PAOX1 expression foreign protein, and the above-mentioned optimization of the application is induced in glycerine methyl alcohol mixed culture medium Method can effectively improve final cell concentration.

Claims

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.