CN1238806A - Preparation of pichia methanolica auxotrophic mutants - Google Patents

Abstract

Methods for preparing Pichia methanolica cells having auxotrophic mutations are disclosed. The methods comprise the steps of: (a) exposing P. methanolica cells to mutagenizing conditions; (b) culturing the cells from step (a) in a rich medium to allow mutations to become established and replicated in at least a portion of the cells; (c) culturing the cells from step (b) in a culture medium deficient in assimilable nitrogen to deplete cellular nitrogen stores; (d) culturing the cells from step (c) in a defined culture medium comprising an inorganic nitrogen source and an amount of nystatin sufficient to kill growing P. methanolica cells to select for cells having a deficiency in a nutritional gene; and (e) culturing the selected cells from step (d) in a rich culture medium.

Description

The preparation of pichia methanolica auxotrophic mutants

Background of invention

Methylotrophic yeast is that those can utilize the yeast of methyl alcohol as sole carbon source and energy source.Have the yeast species that methyl alcohol utilizes necessary biochemical route and can be divided into four genus: Hansenula, Pichia, mycocandida and torulopsis.These yeast belong more or less are based on morphocytology and growth characteristic and synthetic, and do not reflect close genetic affinity (Billon-Grand, Mycotaxon 35:201-204,1989; Kurtzman, Mycologia 84:72-76,1992).In addition.Be not that all species during these belong to can both utilize methyl alcohol as carbon source and energy source.This sorting result is: aspect physiology between single species that belong to and the metabolism great difference is arranged.

Methylotrophic yeast is the attractive candidate that utilizes in recombinant protein generation system.Some methylotrophic yeasts have demonstrated the feature that can grow to high-biomass on basic defined medium fast.Some gene of methylotrophic yeast induce or the depression condition under obtain strict regulate and highly expressing, this promotor that shows these genes is useful for the polypeptide of generation commercial value.Referring to, for example, Faber etc., yeast, 11:1331,1995; Romanos etc., yeast, 8:423,1992; And Cregg etc., biology/technology, 11:905,1993.

The development that is used as host's methylotrophic yeast in reorganization generation system is slowly, and some reasons are owing to lack suitable material (for example promotor, selected marker and mutant host cell) and method (as transformation technology).The methylotrophy type host system of most of high developments utilizes pichia pastoris phaff and multiple-shaped nuohan inferior yeast (Faber etc., Curr.Genet.25:305-310,1994; Cregg etc., the same; Romanos etc., the same; U.S. Patent number 4,855,242; U.S. Patent number 4,857,467; U.S. Patent number 4,879,231 and U.S. Patent number 4,929,555).

This area still needs to transform the method for other species of methylotrophic yeast, and need utilize institute's cell transformed to produce the polypeptide with Economic Importance, comprises industrial enzyme and pharmaceutical protein.The invention provides composition useful in these processes and method and other associated advantages.

Brief summary of the invention

The invention provides the method that preparation has the pichia methanolica cell of auxotrophic mutation.This method comprises step: (a) expose the pichia methanolica cell under mutagenic condition; (b) in rich medium, cultivate the cell that derives from step (a), thereby make that sudden change obtains setting up and duplicating at least a portion cell; (c) cultivation derives from the cell of step (b) in the substratum that lacks assimilable nitrogen, thereby exhausts the nitrogen deposit of cell; (d) cultivate the cell derive from step (c) in defined medium, selecting the damaged cell of vegetative gene, substratum wherein comprises inorganic nitrogen-sourced and is enough to kill the nystatin of the amount of growth pichia methanolica cell; (e) in rich medium, cultivate the cell of the selection derive from step (d).In one embodiment of the invention, the cell that derives from the selection of step (d) is replicated bed board to defined medium, and cultivates it to confirm existing of auxotrophic mutation.In another embodiment, selected cell belongs to the auxotroph of VITAMIN B4.In related embodiment, selected cell lacks Phosphoribosyl-5-aminooimidazole carboxylase.In additional embodiment, mutagenic condition comprises and is exposed in the UV-light or is exposed in the chemical mutagen.In further embodiment, the inorganic nitrogen-sourced ammonium ion that comprises.

In case with reference to following the detailed description and the accompanying drawings, these and other aspect of the present invention will become obvious.

Accompanying drawing is briefly described

Fig. 1 illustrates the influence to the electroporation efficiency of pichia methanolica of strength of electric field and pulse duration.

Fig. 2 is the synoptic diagram of the regrouping process between plasmid pCZR140 and pichia methanolica genomic dna.

Fig. 3 is the synoptic diagram of the regrouping process between plasmid pCZR137 and the pichia methanolica genomic dna.

Detailed description of the Invention

Before illustrating the present invention in more detail, it is useful being defined in earlier some term used herein:

" DNA construct " is a strand or double chain DNA molecule, and this dna molecular obtains modifying by Human disturbance, thereby is included in combination and dna fragmentation arranged side by side in the not naturally occurring arrangement.

" early stage logarithmic phase growth " is the cell vegetative period in cultivation, and cell concn at that time is 2 * 10 ⁶Individual cell/ml to 8 * 10 ⁶Individual cell/ml.

" allogeneic dna sequence DNA " refers to a dna molecular or a group dna molecular, and these dna moleculars are not natural to be present in the given host cell.Can comprise the DNA that derives from these host cell species with the allogenic dna molecular of particular host cell, as long as host DNA combines with nonhost DNA.For example, it is generally acknowledged that the dna molecular (the nonhost DNA sections that comprises coded polypeptide) that is operably connected on the host DNA sections (comprising transcripting promoter) is the allogeneic dna sequence DNA molecule.

" senior eucaryon " organism is the many cells most eukaryotes.This term comprises plant and animal.

" integrating transformant " is that allogeneic dna sequence DNA has been introduced cell wherein, and allogeneic dna sequence DNA wherein has been incorporated in the genomic dna of this cell.

" linear DNA " means has free 5 ' and 3 ' terminal dna molecular, and this dna molecular is non-ring-shaped DNA molecule.Linear DNA can be broken by enzymatic digestion or physics by closed hoop dna molecular (as plasmid) and make.

Term " is operably connected " and means the DNA sections and obtain arranging, so that their consistent its its intended purposes that realizes, for example transcribes with promotor and begins and proceed to terminator by the sections of encoding.

Term " promotor " is used for this paper with its field identification implication, this implication refer to comprise dna sequence dna (provide the combination of RNA polymerase and transcribe initial) a part of gene.Generally but always in 5 ' non-coding region of gene, do not find promoter sequence.Sequential element within the promotor (transcribe initial in work) often classify feature as with total nucleotides sequence.These promoter elements comprise the RNA polymerase binding site; The TATA sequence; The CAAT sequence; Differentiation specificity element (DSEs; McGehee etc., molecular endocrinology, 7:551-560,1993); Ring-type AMP response element (CREs); Serum response element (SREs; Treisman, the symposial of cancer biology, 1:47-58,1990); Glucocorticosteroid response element (GREs); And the binding site of other transcription factor, as CRE/ATF (O ' Reilly etc., journal of biological chemistry, 267:19938-19943,1992), AP2 (Ye etc., journal of biological chemistry, 269:25728-25734,1994), SP1.cAMP response element binding protein (CREB; Loeken, genetic expression, 3:253-264,1993) and the eight aggressiveness factors.Put it briefly, referring to editors such as Watson, the molecular biology of gene, the 4th edition, Benjamin/Cummings publishes limited-liability company, Menlo park, CA, 1987; And Lemaigre and Rousseau, journal of biological chemistry, 303:1-14,1994.

" preventing carbon source " is metabolizable carbon compound, and when unrestricted, this compound is prevented the expression of the needed gene of other carbon source katabolism in organism." limited " refers to that carbon source can not be utilized or can be so that its a kind of like this speed utilization that is consumed immediately, and therefore the general concentration of the carbon source in the organism environment says to be zero from validity.The carbon source of preventing that can be used for the present invention comprises hexose and ethanol.Glucose is particularly preferred.

" enriching " substratum is those substratum based on nutraceutical complex source, typically is cell or tissue extract or protein hydrolyzate.Rich medium changes between criticizing aspect the composition and criticizing to some extent, and this is because due to the variation aspect the composition of nutrition source.

As mentioned above, the invention provides the method that preparation has the pichia methanolica cell of auxotrophic mutation.Can transform the auxotrophic mutation body of pichia methanolica with homologous dna (deriving from the DNA of host species) and allogeneic dna sequence DNA, and the transformant that is produced can be used for a large amount of different biologic applications.Mutant cell of the present invention especially can be well suited for the conversion with allogeneic dna sequence DNA, and wherein cell transformed can be used for producing polypeptide and protein, comprises polypeptide and the protein of senior organism (comprising the mankind).Can transform auxotroph pichia methanolica cell with other dna molecular (comprising DNA library and synthetic dna molecule).Therefore the invention provides a kind of like this host cell, this host cell can be used for the diversified library of expressing gene, thereby produce the product that goes out for new bioactivity screening, this host cell can be used as the target of library of compounds screening in genetically engineered, can also obtain genetic modification, thereby improve their utilizations in other method.

Generally have a sudden change with the allogeneic dna sequence DNA cell transformed, this sudden change can by the gene on the allogeneic dna sequence DNA molecule (" selected marker ") complementation.This selected marker is grown institute's cell transformed under certain condition, and this condition i.e. wherein no transformed cells can not be multiplied (" optionally condition ").The rule of selecting is known in the art.Usually the selected marker of utilizing is the gene of coding synthesizing amino acid or the desired enzyme of Nucleotide.In these genes, there is the cell (nutrition falls into the type mutant) of sudden change in the substratum that lacks specific amino acid or Nucleotide, can not grow, unless it is complementary to sport selected marker institute.The stable maintenance of the allogeneic dna sequence DNA within host cell is guaranteed in the utilization of " selectivity " substratum like this.The preferred selected marker that is used for this type of pichia methanolica is a pichia methanolica ADE2 gene, this genes encoding Phosphoribosyl-5-aminooimidazole carboxylase (AIRC; EC4.1.1.21).When the ADE2 gene transformation was in the ade2 host cell, this gene made cell be grown lacking under the situation of VITAMIN B4.The coding strand of representative pichia methanolica ADE2 gene order is presented among the SEQ ID NO:1.Illustrated sequence comprises 5 '-1006 Nucleotide and 3 of non-coding sequence '-442 Nucleotide of non-coding sequence, they all have initial ATG codon at Nucleotide 1007-1009.In a preferred embodiment of the invention, the DNA sections that comprises Nucleotide 407-2851 is used as selected marker, though also can utilize longer or shorter sections (as long as encoding part is operably connected on promotor and the terminator sequence).It will be appreciated by those skilled in the art that this sequence provided herein and other sequence represent the single allele of gene separately, and the expectation allelic variation exists.Any functional ADE2 allelotrope may be used among the present invention.Other trophicity mark that can be used for the present invention comprises pichia methanolica ADE1, HIS3 and LEU2 gene, and these genes allow to select lacking respectively under VITAMIN B4, Histidine and the leucic situation.Heterologous gene (as derive from other fungi gene) also can be used as selected marker.For large-scale commercial run (wherein needing to reduce to greatest extent utilization), preferably utilize wherein two host cells that methyl alcohol utilizes gene (AUG1 and AUG2) to lack to methyl alcohol.For the proteinic generation of excretory, the host cell that lacks vacuole protein enzyme gene (PEP4 and PRB1) is preferred.Can use the mutant of currently known methods (as site-directed mutagenesis) preparation gene defect.Can be based on cloning the pichia methanolica gene with their homology of counterpart yeast saccharomyces cerevisiae.Provide the definition of ADE2 gene disclosed herein based on such homology.

The pichia methanolica bacterial strain can derive from American type culture collection, and (Rockville MD) with other storage place, and can be used as starting material in the present invention.In order to prepare the auxotrophic mutation body of pichia methanolica, at first make cellular exposure under mutagenic condition (promptly causing the envrionment conditions of the genetic mutation of cell).The method that is used for mutagenized cell is known in the art, and it comprises chemical treatment, is exposed under the ultraviolet ray, is exposed under the X-ray and retrovirus insertion mutagenesis.Chemical mutagen comprises ethyl methane sulfonate (EMS), N-methyl-N '-nitro-N-nitrosoguanidine, 2-methoxyl group-6-chloro-9-[3-(ethyl-2-chloroethyl) aminopropyl amino] acridine 2HCl, 5-bromouracil, acridine and aflatoxin.Referring to Lawrence, Enzymology method, 194:273-281,1991.The ratio of the mutagenized cell that obtains is the intensity of mutagenic compound (cellular exposure in wherein) or the function of consumption.Low-level mutagenic compound produce the mutant cell of small proportion.High-caliber mutagenic compound produce the mutant cell of vast scale, but also kill a large amount of cells.Therefore be necessary balance mutagenesis and lethal effect, so that obtain the mutant cell of fair amount.According to experience, thereby, realize balance generally by making cellular exposure under different condition, set up a destruction curve.Usually make cellular exposure in mutagenic condition and cultivated one day, check their viability thereafter according to standard determination method.In the present invention, preferably utilize to cause the mutagenesis level of 20-50% mortality ratio, can adjust as required, if when for example handling with a large number of cell though those skilled in the art recognize that this value.

In rich medium, cultivate the cell of mutagenesis then, thereby make the sudden change at least a portion cell colony be set up and duplicate.This step makes cell (genome wherein changes) be duplicated sudden change, and this sudden change is passed to their filial generation, sets up sudden change thus in colony.

Then with cell transfer to the substratum that lacks assimilable nitrogen, so that exhaust cell nitrogen deposit.Wherein " lack assimilable nitrogen " and mean the nitrogen amount that substratum lacks is enough to the sustenticular cell growth.The exhaustion of cell nitrogen deposit generally needs about 12 to 24 hours incubation, under household condition with just being enough to exhaust cell nitrogen deposit in 16 hours.After the nitrogen deposit is exhausted, in defined medium, cultivate this cell, the antifungal antibiotic that this substratum comprises inorganic nitrogen-sourced and a certain amount of (being enough to kill growth pichia methanolica cell).Preferred microbiotic is nystatin (Stamicin).Preferred inorganic nitrogen-sourced is those materials that comprise ammonium ion, as ammonium sulfate.Usually, substratum contains the 10-200mM ammonium, is preferably about 60mM ammonium.The concentration of the nystatin that is comprised is 0.1-100mg/L, is preferably 0.5-20mg/L, more preferably 2mg/L (10 units/L).Utilize the processing of nystatin to carry out ten minutes to six hours, be preferably about 1 hour.It will be appreciated by those skilled in the art that to be easy to rule of thumb to determine to kill former foster cell required actual antibiotic concentration and exposure duration, and need carry out certain adjustment so that the variation aspect the specific activity of compensation between each batch microbiotic.By exhausting cell nitrogen deposit and culturing cell in defined medium (comprising inorganic nitrogen-sourced and microbiotic) then, auxotrophic cell with amino acid or Nucleotide biosynthesizing effect is still survived, and this is because they can not be grown in defined medium.Grown cell is killed by microbiotic.After antibiotic treatment, with cell transfer to rich medium.

Confirm and identify auxotrophic mutation by the nutritional requirement of measuring handled cell.Usually replica plating is used for this mensuration.With the cell plating at rich medium with lack on the substratum of specific nutrition thing.The cell of not growing on specific plate is to lack nutraceutical auxotroph cell.Complementation analysis can be used for further evaluation.

Allogeneic dna sequence DNA can be introduced in pichia methanolica cell by any of several currently known methodss, and these methods comprise that lithium transforms (Hiep etc., yeast, 9:1189-1197,1993; Tarutina and Tolstorukov are about the special symposial summary in the 15th world of zymic, Riea (USSR), 1991,137; Ito etc., bacteriology magazine, 153:163,1983; Bogdanova etc., yeast, 11:343,1995), spheroplast transforms (Beggs, nature, 275:104,1978; Hinnen etc., institute of NAS newspaper, 75:1929,1978; Cregg etc., molecular cytobiology, 5:3376,1985), the freeze thawing polyoxyethylene glycol transforms (Pichia expression test kit Guide Book, Invitrogen Corp., San Diego, CA, catalog number (Cat.No.) K1710-01) or electroporation, a kind of method in back is preferred.Electroporation is a kind of like this method, and the pulsed electrical field that it utilizes instantaneousization cytolemma makes macromole (as DNA) be entered cell.Existing bibliographical information the electroporation that utilizes together with host cell such as (for example Meilhoc, biology/technology, 8:223-227,1990) of mammiferous (Neumann etc. for example, EMBO J.1:841-845,1982) and fungi.Yet, shift real mechanism and the insufficient understanding that enters cell for DNA.For the conversion of pichia methanolica, people find: when cellular exposure was in the pulsed electrical field of exponential attenuation (having 2.5 to 4.5kV/cm strength of electric field and 1 to 40 millisecond time constant (τ)), electroporation was very effective.Timeconstant is defined as initial crest voltage V ₀Reduce to V ₀Required time during/e value.Can come constant computing time according to the total electrical resistance of pulsing circuit and the product of electric capacity, i.e. τ=R * C.Typically, can preset or select resistance and electric capacity by the user, this depends on selected electroporation equipment.Under any circumstance, the above-mentioned strength of electric field that provides according to manufacturer and the specification sheets of attenuation parameter come configuration device.(BioRad laboratory for example, Hercules CA) provides electroporation equipment by supplier.

The dna molecular that generally will be used to transform pichia methanolica is prepared into double-stranded cyclic plasmid, and preferably, this plasmid is linearized before transforming.Produce for polypeptide or protein, except that selected marker described above, dna molecular also comprises expression cassette, and it comprises transcripting promoter, the interested polypeptide of coding or protein DNA sections (as cDNA) and transcription terminator.These elements are operatively coupled on together, thereby transcribing of interested DNA sections is provided.Preferably, promotor and terminator are the promotor and the terminators of pichia methanolica gene.Useful promotor comprises that those derive from the promotor of composing type and methanol inducible promoters.Promoter sequence usually is included within the 1.5kb of encoding sequence upstream of gene, within be everlasting 1kb or scope still less.In general, owing to there is regulatory element, the adjusting promotor is bigger than constitutive promoter.The methanol inducible promoters that comprises the positive and negative regulatory element can stretch the 1kb that surpasses initial ATG upstream.By function identification promotor, and can be according to the currently known methods cloning promoter.

Particularly preferred methyl inducible promoter is the promotor that pichia methanolica alcohol utilizes gene.The representative coding strand sequence of such gene A UG1 is presented among the SEQ ID NO:2.In SEQ ID NO:2, initial ATG codon is positioned at Nucleotide 1355-1357.The Nucleotide 1-23 right and wrong AUG1 polylinker sequence of SEQ ID NO:2.Particularly preferably be utilize the Nucleotide 24-1354 comprise SEQ IDNO:2 sections as promotor, though additional upstream sequence is also includable.Pichia methanolica contains second alcohol and utilizes gene A UG2, and the promotor of this gene can be used for the present invention.An AUG2 clone's partial dna sequence is presented among the SEQ ID NO:9.The AUG2 promotor sections that is used for the present invention generally comprises the Nucleotide 91-169 of SEQ ID NO:9, does not have promoter function though do not wish the fragment of blocking for a short time on 3 ' end.Other useful promotor comprises the promotor of those dihydroxyacetone synthases (DHAS), hydrogenlyase (FMD) and catalase (CAT) gene.Derive from the existing bibliographical information of gene of the encoding such enzymes of other species, their sequence is obtainable (for example, Janowicz etc., nucleic acids research, 13:2043,1985; Hollenberg and Janowicz, EPO publication 0 299 108; Didion and Roggenkamp, FEBS Lett.303:113,1992).By utilizing known array as probe, perhaps by the contrast known array, based on sequence to recently designing primer and by polymerase chain reaction (PCR) amplification pichia methanolica DNA, can these proteinic genes of clones coding.

Constitutive promoter is that those are not activated by envrionment conditions or the promotor of deactivation; They always have active aspect transcribing.The preferred constitutive promoter that is used for the present invention comprises that those derive from the promotor of the phosphoglyceric kinase gene of glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase and pichia methanolica.By the complementary action in the host cell (as brewing yeast cell) of the sudden change aspect having corresponding gene, can clone these genes.The mutant of this type is known in the art.Referring to, for example, Kawasaki and Fraenkel, the communication of biological chemistry biophysical studies, 108:1107-1112,1982; McKnight etc., cell, 46:143-147,1986; Aguilera and Zimmermann, molecular gene genetics, 202:83-89,1986.

Dna molecular also comprises makes evaluation, selection and transformant keep the selected marker that is achieved.Dna molecular can also contain additional element, and such replication orgin and selected marker make the amplification of the DNA in alternate host (for example intestinal bacteria) and keep and be achieved.Be incorporated in the host chromosome in order to help DNA, preferably dna molecular has the whole expression sections that side is connected in the two ends of host DNA sequence, comprises promotor-gene of interest-terminator and selected marker.By comprising 3 ' untranslated dna sequence dna on the downstream end of sections and depend on promoter sequence on 5 ' end expressing, can realize this purpose expediently.When utilizing linear DNA, express sections side is connected cleavage site, thereby make molecule be able to linearizing and make the expression sections be able to separate with other sequence (for example bacterium replication orgin and selected marker).Preferred such cleavage site is that those are the cleavage site of discerning without the restriction enzyme that cuts in the dna sequence dna of being everlasting, as the cleavage site (as the Not I) of those identification 8-base target sequence.

The protein that can produce in pichia methanolica comprises protein of interest matter on industry and the medicine.Such protein comprises the senior eukaryotic protein that derives from plant and animal (especially as mammiferous vertebrates), though some protein that derives from microorganism also has very high value.Can utilize the protein of the inventive method preparation to comprise enzyme (as lipase, cellulase and proteolytic enzyme); Enzyme inhibitors (comprising proteinase inhibitor); Somatomedin (as platelet-derived somatomedin, fibroblast growth factor and Urogastron); Cytokine (as erythropoietin and thrombopoietin) and hormone (as Regular Insulin, leptin and hyperglycemic-glycogenolytic factor).

In order to produce polypeptide, under about 25-35 ℃ of temperature, in the substratum that comprises abundant carbon source, nitrogenous source and trace nutritive substance, cultivate the pichia methanolica cell.Provide liquid culture by conventional means (as shake bottle or spray fermentor tank) with fully ventilating.Preferred substratum is YEPD (table 1).Can make passage by dilution in fresh culture, perhaps cell can be under freezing on flat board short-term storage.For prolonged storage, cell preferably remains in 50% glycerine solution under-70 ℃.

Table 1YEPD

2%D-glucose

2%Bact ^TMPeptone (the Difco laboratory, the Detroit, MI)

1%Bacto ^TMYeast extract (Difco laboratory)

0.004% VITAMIN B4

0.006%L-leucine ADE D

The 0.056%-Ade-Trp-Thr powder

0.67% no amino acid whose yeast nitrogen matrix

2%D-glucose

0.5%200X tryptophane, Threonine solution A DE DS

The 0.056%-Ade-Trp-Thr powder

0.67% no amino acid whose yeast nitrogen matrix

2%D-glucose

0.5%200X tryptophane, Threonine solution

18.22%D-Sorbitol Powder LEU D

The 0.052%-Leu-Trp-Thr powder

0.67% no amino acid whose yeast nitrogen matrix

2%D-glucose

0.5%200X tryptophane, Threonine Solution H IS D

The 0.052%-His-Trp-Thr powder

0.67% no amino acid whose yeast nitrogen matrix

2%D-glucose

0.5%200X tryptophane, Threonine solution URA D

The 0.056%-Ura-Trp-Thr powder

0.67% no amino acid whose yeast nitrogen matrix

2%D-glucose

0.5%200X tryptophane, Threonine solution URA DS

The 0.056%-Ura-Trp-Thr powder

0.67% no amino acid whose yeast nitrogen matrix

2%D-glucose

0.5%200X tryptophane, Threonine solution

18.22%D-Sorbitol Powder-Leu-Trp-Thr powder

By in conjunction with the prepared meal of following material: 4.0g VITAMIN B4,3.0g arginine, 5.0g aspartic acid, 2.0g Histidine, 6.0g Isoleucine, 4.0g Methionin, 2.0g methionine(Met), 6.0g phenylalanine, 5.0g Serine, 5.0g tyrosine, 4.0g uridylic and 6.0g Xie Ansuan (all L-amino acid)-His-Trp-Thr powder

By in conjunction with the prepared meal of following material: 4.0g VITAMIN B4,3.0g arginine, 5.0g aspartic acid, 6.0g Isoleucine, 8.0g leucine, 4.0g Methionin, 2.0g methionine(Met), 6.0g phenylalanine, 5.0g Serine, 5.0g tyrosine, 4.0g uridylic and 6.0g Xie Ansuan (all L-amino acid)-Ura-Trp-Thr powder

By in conjunction with the prepared meal of following material: 4.0g VITAMIN B4,3.0g arginine, 5.0g aspartic acid, 2.0g Histidine, 6.0g Isoleucine, 8.0g leucine, 4.0g Methionin, 2.0g methionine(Met), 6.0g phenylalanine, 5.0g Serine, 5.0g tyrosine and 6.0g Xie Ansuan (all L-amino acid)-Ade-Trp-Thr powder

By in conjunction with the prepared meal of following material: 3.0g arginine, 5.0g aspartic acid, 2.0g Histidine, 6.0g Isoleucine, 8.0g leucine, 4.0g Methionin, 2.0g methionine(Met), 6.0g phenylalanine, 5.0g Serine, 5.0g tyrosine, 4.0g uridylic and 6.0g Xie Ansuan (all L-amino acid) 200X tryptophane, Threonine solution

3.0%L-Threonine, 0.8% is at H ₂L-tryptophane among the O adds 1.8%Bacto for flat board ^TMAgar (Difco laboratory)

Carry out the electroporation of pichia methanolica on the cell in the preferred logarithmic phase growth in early days.Go up at solid medium (being preferably solid YEPD) and to be single bacterium colony with cell is streak culture.After 30 ℃ were grown about 2 days down, utilize the single bacterium colony that derives from fresh flat board to inoculate the rich medium (as YEPD) of aequum and make cell density reach about 5-10 * 10 ⁵Cell/ml.Under about 25-35 ℃ (being preferably 30 ℃),, be in early stage logarithmic phase up to them by acutely shaking culturing cell.Then by as 2-3 minute of 3000 * g is centrifugal comes harvested cell and this cell of resuspending.By the disulfide linkage of reduction in the cell walls, in solion (being fit to the electroporation condition) balance they and cool off them, make cell be in electroreception attitude (electrocompetent).Typically,, make cell be in the electroreception attitude, help picked-up thereafter with the reduction cell wall protein DNA by in pH6-8 damping fluid (comprising reductive agent), cultivating them as dithiothreitol (DTT) (DTT) or beta-mercaptoethanol (BME).With regard to this point, preferably to cultivate damping fluid be pH7.5, comprise the fresh solution of the 50mM potassium phosphate buffer agent of 25mM DTT.At under about 30 ℃ in this damping fluid (utilize typically former cultivation amount 1/5th) the about 5-30 of culturing cell minute, be preferably about 15 minutes.Then harvested cell and in suitable electroporation damping fluid (when it utilizes be through ice-cold) this cell of washing.Based on this consideration, suitable damping fluid comprises and comprises weak buffer reagent, divalent cation (as Mg ⁺⁺, Ca ⁺⁺) and the pH6-8 solution of permeating stablizer (for example sugar).After washing, in damping fluid in a small amount, its time is the time that reaches the electroreception attitude with the cell resuspending, and this cell can directly be utilized or five equilibrium freezes under (being preferably-70 ℃) with being stored in.Preferred electroporation damping fluid is STM (270mM sucrose, 10mM Tris, pH7.5,1mM MgCl ₂).In preferred scheme, make cell accept twice washing, at first use the ice-cold damping fluid of former cultivation amount, use half former cultivation amount then.After washing for the second time, results and resuspending cell utilize about 3-5ml damping fluid (for the former cultivation amount of 200ml) typically.

Utilize a small amount of electroreception attitude cell (typically being about 100 μ l) and the linear DNA molecules of 1/10th amounts at the most, implement electroporation.For example, the cell suspending liquid in damping fluid (its ionic strength is no more than 50mM) of 0.1ml is mixed with the DNA of 0.1-10 μ g (volume≤10 μ l).This mixed solution is placed among the ice-cold electroporation Xiao Chi, and make it to be subjected to pulsed electrical field in being exponential attenuation, strength of electric field is 2.5 to 4.5kV/cm, be preferably about 3.75kV/cm, time constant is 1 to 40 millisecond, be preferably the 10-30 millisecond, more preferably the 15-25 millisecond most preferably is about 20 milliseconds.In order to reach the required pulse parameter, the physical device setting that is utilized is by the equipment decision that is utilized.The BioRad (Hercules, CA) the Gene Pulser that have 2mm electroporation Xiao Chi when utilization ^TMDuring electroporation apparatus, resistance is made as 600 ohm or bigger, is preferably " infinity ", and resistance value, electric capacity then are made as 25 μ F, so that obtain required electric field feature.After pulse, the about 10X of cell dilution is become 1ml YEPD meat soup, and under 30 ℃ with its incubation one hour.

Then harvested cell and with its plating on selective medium.In preferred embodiments, (6.7g/L does not have amino acid whose yeast nitrogen matrix, Difco laboratory, Detroit with the 1X yeast nitrogen matrix of a small amount of (equaling the electroporation of cells of amount of dilution), MI) disposable washed cell, and with its plating on basic selective medium.Can will have cell (the transforming) plating of ade2 sudden change on the minimum medium (as ADE D (table 1) or ADE DS (table 1)) that lacks VITAMIN B4 with the ADE2 selected marker.Use typical method, can be with the branch platings such as 250 μ l of cell on 4 independent ADE D or ADE DS flat board, so that select Ade ⁺Cell.

Pichia methanolica is discerned some few sequence that exists, promptly so-called autonomously replicating sequence (ARS) as the dna replication dna starting point, and these sequences can betide in the dna molecular that is used for transforming by accident, and transfering DNA is remained on outside the karyomit(e).Yet.Integrating transformant generally is preferred in the protein generation system.Comprising on the selective medium of Sorbitol Powder as carbon source, the integration transformant has the extremely strong growth vigor above the ARS transformant, and the method that is used for selecting from cell transformed colony of institute the integration transformant is provided thus.Have been found that the ARS sequence is present in the ADE2 gene, and also might be present in the AUG1 gene of pichia methanolica.Therefore ade2 host cell with the pichia methanolica of ADE2 gene transformation can become Ade by at least two kinds of different modes ⁺ARS in the ADE2 gene can make to keep outside the unstable karyomit(e) of transfering DNA and be had (Hiep etc., yeast, 9:1189-1197,1993).The bacterium colony of such transformant be characterized as growth velocity and pink color (because so-called Ade slowly ^-Due to a large amount of generations of filial generation).Transfering DNA also can be integrated in the host genome, but generation is growth fast, white and can not produce detection limit Ade ^-The stable conversion body of filial generation.ADE D flat board makes institute's cell transformed be able to the most promptly grow, and unstable and stable conversion body is also grown with roughly the same speed.Under 30 ℃ on ADE D flat board after incubation 3-5 days, stable conversion body bacterium colony is white, and roughly is the twice of the size of unsettled pink transformant.ADE DS flat board for the stable conversion body especially optionally, this transformant formed big (≈ 5mm) bacterium colony after 5-7 days, unstable (ARS maintenance) bacterium colony then much smaller (≈ 1mm).Therefore have more optionally the ADEDS substratum and preferably identify and select the stable conversion body.For some application, as the genic rare combination of screening from the genetic diversity library, need to screen a large amount of unstable transformant sometimes, the quantity that people have observed unstable transformant surpasses about 100 times of the quantity of stable conversion body.In such a case, those skilled in the art will recognize that plating upward transforms somatic purposes at less selective medium (as ADE D).

Integrating transformant is preferred in the protein production method.Can breed such cell and need not the pressure of Continuous Selection, this is because DNA seldom loses from genome.Can confirm that DNA is incorporated in the host chromosome by the Southern engram analysis.In brief, with restriction endonuclease digestion that transform with unconverted host DNA, electrophoretic separation it, its trace to supporting film, and is surveyed it with suitable host DNA sections.The fragment schema that observes in unconverted and cell transformed different are shown with in advance to integrate and transform.Can the selectional restriction enzyme and probe so that in genomic fragment, determine transfering DNA sections (as promotor, terminator, allogeneic dna sequence DNA and selected marker sequence).

The difference of heterologous protein expression level results from some factors like this, as the copy number of integration site and expression cassette and the difference of the promoter activity aspect between each isolate.Therefore it is favourable selecting some isolates according to difference expression horizontal screen before selecting to produce bacterial strain.There are various suitable screening methods available.For example, make the transformant colony growth on the flat board that the film (for example nitrocellulose) with conjugated protein covers.By secretion or thereafter cracking and with the combining of film, protein is discharged from cell.Can utilize currently known methods (comprising immunoassay) to analyze bonded protein then.By suitably culturing cell and suitably analysis condition substratum or cellular lysate in the liquid medium within, can obtain more accurate expression level analysis.From substratum and lysate, concentrate and partly determine by protein of interest matter with method for purifying proteins.Such method is easy to be selected and practice by the technician.

For small-scale protein production (for example dull and stereotyped or shake bottle production), under the situation of other carbon source (for example glucose) that has methyl alcohol and shortage interference volume, make the pichia methanolica transformant growth of the expression cassette that carries the promotor (as the AUG1 promotor) that comprises the methyl alcohol adjusting.For small-scale experiment (preliminary screening that comprises expression level), transformant is grown down at 30 ℃ on solid medium, this solid medium comprises, and for example 20g/L Bacto agar (Difco), 6.7g/L do not have yeast nitrogen matrix, 10g/L methyl alcohol, 0.4 μ g/L vitamin H and the 0.56g/L-Ade-Thr-Trp powder of amino acid (Difco).Because methyl alcohol is volatile carbon source, so it loses when long-time incubation easily.By at the methanol aqueous solution of being inverted placement 50% on the dull and stereotyped lid, can continuously supply with methyl alcohol, the evaporation transmission is transferred to methyl alcohol on the grown cell thus.Generally speaking, the dull and stereotyped methyl alcohol that utilizes less than 1mL of every 100mm.Utilization is grown in the culture that shakes in the bottle and can tests slightly on a large scale.Use a typical method, on aforesaid basic methyl alcohol flat board 30 ℃ of following culturing cells two days, then, bacterium colony is used for inoculating a spot of basic methyl alcohol substratum (6.7g/L does not have amino acid whose yeast nitrogen matrix, 10g/L methyl alcohol, 0.4 μ g/L vitamin H), its cell density is about 1 * 10 ⁶Individual cell/ml.Make cell 30 ℃ of growths down.Be grown on the methyl alcohol cell high oxygen requirement is arranged, and need acutely shake in the training period.Replenish methyl alcohol (typically being 50% methyl alcohol of 1/100 amount every day) every day.

Cultivate for industrial scale, with the fresh culture thing that shakes bottle preparation high yield clone.Then, the culture that is produced is used for inoculation medium in fermentor tank.Typically, by thermal agitation, the 500ml culture in YEPD (growing 1-2 days down at 30 ℃) is used for inoculating 5 liters of fermentor tanks.Cell is grown in the suitable medium (comprise salt, glucose, vitamin H and trace elements and＞30% dissolved oxygen).After the glucose that has consumed (reducing as indication) filling just, glucose/methanol feeding is sent in the container so that cause the generation of protein of interest matter with oxygen-consumption.Because under restriction carbon condition, realize large-scale fermentation, so methanol inducible promoters is not prevented in the existence of glucose in charging.The utilization of the glucose that combines with methyl alcohol under the glucose limitation condition produces quick growth, carbon to the effective conversion of biomass and the rapid change of physiology growth conditions, and fully inducing of methanol evoked gene promoter still is provided.In exemplary fermentation process, obtainable cell density is the every liter about 80 moist cell mashed prod to about 400 grams." moist cell mashed prod " refers to the cell mass that obtains by from fermentor tank harvested cell (typically passing through the centrifugal of culture).

The present invention is further specified by following non-limiting examples.EXAMPLE Example 1

By UV expose mutagenesis pichia methanolica cell (derive from the CBS6515 bacterial strain of American type culture collection, Rockville, MD).By with about 200-250 cell/flat board with cell inoculation on several flat boards, at first produce destruction curve.Then, utilize the G8T5 germicidal lamp (Sylvania) that is suspended on from planar surface 25cm place, under the UV radiation, the time length is as shown in table 2 with plate exposure.Protect flat board then in order to avoid visible light source irradiation and 30 ℃ of following incubations two days.

Table 2

Dull and stereotyped 1 dull and stereotyped 2 mean values of the cell time that can survive 0 second 225 229 2271 seconds 200 247 2232 seconds 176 185 1814 seconds 149 86 1188 seconds 20 7 1416 seconds 021

Utilize 2 seconds UV to expose then and carry out large scale mutagenesis, so that about 20% kill to be provided.With about 10 ⁴Individual cell/flat board with the cell plating on eight YEPD flat boards, these flat boards replenish with uridylic, VITAMIN B4 and leucine (every kind of material respectively is 100mg/L), add these materials so that give the auxotroph growth feed supplement of the possible relevant nutritive substance of shortage.After UV exposes, wrap up these flat boards with paper tinsel, and 30 ℃ of one nights of following incubation.Second day, the bacterium colony on the flat board (is total up to about 10 ⁵) resuspending in water, and wash with water once.Be enough to provide 0.1-0.2OD ₆₀₀A certain amount of cell suspending liquid be used for inoculating the basic meat soup of 500ml (making) with no amino acid or the yeast nitrogen matrix of not having ammonia, wherein use 1% glucose and 400 μ g/L vitamin H feed supplements.Culture is placed in the 2.8L baffle plate type Bell bottle (baffled Bell flask), and 30 ℃ of one nights of following thermal agitation.Second day, cell reached about 1.0-2.0OD ₆₀₀Make the cell glomeration, and with its resuspending in the basic meat soup of 500ml with the feed supplement of 5g/L ammonium sulfate.Cell suspending liquid is placed in the 2.8L baffle plate type Bell bottle, and 30 ℃ of following thermal agitations 6 hours.The 50ml culture is placed in the 250ml bottle and is shelved on one side in contrast, and in the culture of rest part, add 1mg nystatin (Sigma chemical company, St.Louis, MO), so that select nutritional mutant (Snow, nature, 211:206-207,1966).Came the incubation culture in additional one hour by vibrating.The cell of handling by centrifugal results control cells and nystatin then, and wash with water three times.Thereby the cell that resuspending washed obtains 1.0OD in 50% glycerine ₆₀₀And freeze it.The cell that the nystatin of representing with colony-forming unit was handled discloses with respect to the titration of control cells: but the nystatin enrichment makes the quantity of survivaling cell reduce 10 ⁴Doubly.

The 10-2 diluent plating of the cell that nystatin was handled is on 15 YEPD flat boards.With the bacterium colony replica plating on dull and stereotyped substantially (2% agar, 1X YNB, 2% glucose, 400 μ g/L vitamin Hs).Auxotrophic frequency is about 2-4%.Select about 180 auxotroph bacterium colonies to YEPD+Ade, Leu, in the Ura flat board, and with its replica plating on various drops (dropout) flat board.All auxotrophs all are Ade ^-Wherein 30 present significant pink (LEU D, HIS D etc.: referring to table 1) on the drop flat board.In 30 pink mutant, select 21 and further study, surplus person for the growth on ADE D flat board be seepage or polluted by wild-type cell.

Then to Ade ^-Mutant carries out complementation analysis and phenotype check.Quantity for the locus determining to be limited by mutant is paired into single peach Ade with all 21 mutant ^-Check bacterial strain (bacterial strain 2#).By cell mixing suspension (OD ₆₀₀=1) on the YEPD flat board, realizes pairing and 10 μ l aliquots containig platings of mixture.Then with cellular replication to the SPOR substratum (0.5% sodium acetate, 1%KCl, 1% glucose, 1% agar), and 30 ℃ of one nights of following incubation.Then cellular replication is plated on the ADE D flat board to estimate phenotype.Just as shown in table 3, some mutant combinations do not provide Ade ⁺Bacterium colony (may limit with bacterial strain #2 in identical genetic loci), the combination of other mutant then produces many Ade ⁺Bacterium colony (may limit independent genetic loci).Because when matching with #2, mutant #3 provides Ade ⁺Bacterium colony is so repeat complementary check with mutant #3.If should limit two genetic locis by the group mutant, when matching with #3, (when matching with bacterial strain #2, it does not provide Ade to all mutant so ⁺Bacterium colony) all should provide Ade ⁺Bacterium colony.The result of hybridization is presented in the table 3.

Table 3

Mutant	X mutant #2	X mutant #3
			#1	+	-
#3	+	-
			#10	+	-
#15	+	-
			#18	+	-
#24	+	-
			#28	+	-
#30	+	-
			#2	-	+
#6	-	+
			#8	-	+
#9	-	+
			#11	-	+
#17	-	+
			#19	-	+
#20	-	+
			#22	-	+
#27	-	+
			#4	+	+
#12	+	+
			#16	+	+

Just as shown in table 3, most of mutant belong to one of two groups, this and the idea unanimity that two kinds of VITAMIN B4 biosynthesis genes (when the disappearance, it produces pink bacterium colony on limited VITAMIN B4 substratum) arranged.Three bacterium colonies (#4, #12 and #16) can limit the 3rd locus or demonstrate intragenic complementation.Selection derives from two kinds of mutant (#3 and #10 with strong chromogenesis of each group of two complementary groups; #6 and #11) further identify.Additional analysis revealed Ade ^-It is the unique auxotroph that is present in these bacterial strains.

Make up the pichia methanolica clone bank with carrier pRS426 (a kind of shuttle vectors that comprises 2 μ and yeast saccharomyces cerevisiae URA3 sequence, URA3 sequence wherein makes carrier pRS426 be able to breed) in yeast saccharomyces cerevisiae.Prepare genomic dna according to standard method by bacterial strain CBS6515.In brief, at one night of culturing cell in rich medium,, and use the SDS cracking with the nodularization of enzymolysis enzyme protoplasma.With ethanol deposit D NA from lysate, and extract it, use ammonium acetate and ethanol sedimentation then with the phenol/chloroform mixture.The gel electrophoresis of DNA prepared product demonstrates the existence of the RNA of complete high-molecular-weight DNA and considerable amount.Cultivate this DNA when existing, with Sau 3A part dna digestion by a series of diluents when this enzyme.By the sample of electrophoretic analysis Digestive system, so that determine segmental size distribution.From gel cutting down 4 and 12kb between the DNA that moves, and extract it from gel slice.Then the DNA of size fractionation is connected on the pRS426 that digested with Bam H I and crossed with alkaline phosphatase treatment.According to the method that manufacturer is recommended, utilize BioRad Gene Pulser ^TMDevice, the aliquots containig of electroporation reaction mixture in intestinal bacteria MC1061 cell.

Genomic library cause electroporation (Becker and Guarente, Enzymology method, 194:182-187,1991) transformed saccharomyces cerevisiae bacterial strain HBY21A (ade2 ura3).Make the cell resuspending in the 1.2M Sorbitol Powder, and with the aliquots containig plating of six 300 μ l on ADE D, ADE DS, URA D and URA DS flat board (table 1).Dull and stereotyped 4-5 days of 30 ℃ of following incubations.On ADE D or ADE DS flat board, be not recovered to any Ade ⁺Bacterium colony.The bacterium colony replica plating that derives from URA D and URA DS flat board on ADE D flat board, can be obtained two tight alternate white colonies.These bacterium colonies of ruling again, and determine that it is Ura ⁺And Ade ⁺The bacterial strain line of these two kinds of called after Ade1 and Ade6 is being comprised 5FOA (5 fluoro vitamin B13s; Sikorski and Boeke, Enzymology method is on substratum 194:302-318).Be found to be Ade when obtaining replica plating ^-Ura ^-Bacterium colony.These results show: Ade ⁺Complementary activity is connected on the URA3 mark that carries plasmid by gene.As if the plasmid that derives from yeast strain Ade1 and Ade6 consistent with restriction enzyme digestion mapping as described below.With these genomic clones difference called after pADE1-1 and pADE1-6.

Total DNA is separated with Ade6 with HBY21A transformant Ade1, and it is used for transformed into escherichia coli bacterial strain MC1061 is Amp ^R2 Amp by Ade1 ^R3 Amp of bacterium colony and Ade6 ^RBacterium colony prepares DNA.With Pst I, Sca I and Pst I+Sca I dna digestion, and by gel electrophoresis analysis it.All five isolates produce identical estriction map.

(be also referred to as and be ADE1 by pichia methanolica P ADE2 gene; Hiep etc., yeast, 9:1251-1258,1993) announcement sequences Design PCR primer.Primer 9080 (SEQ ID NO:3) is designed to guide on the 406-429 base of ADE2 DNA (SEQ ID NO:I), and primer 9079 (SEQ ID NO:4) is designed to guide on the 2852-2829 base.Two kinds of primers that comprised can not be introduced Avr II and Spe I site at the extension increasing sequence two ends.The predictor of the PCR clip size that is produced is 2450bp.

Utilize plasmid DNA to carry out PCR with five ADE2 clones that infer as template.100 μ l reaction mixtures comprise 1 * Taq PCR damping fluid (Boehringer Mannheim, Indianapolis, IN), every kind of primer and the 1 μ l Taq polysaccharase (Boehringer Mannheim) of 10-100ng plasmid DNA, 0.25mM dNTPs, 100pmol.PCR is included in the circulation of carrying out 30 times 30 seconds under 94 ℃, is carrying out 30 times 60 seconds circulation under 50 ℃, carries out 30 times 120 seconds circulation under 72 ℃.Each of five ADE2 genomic clones of inferring all produces the PCR product of required size (2.4kb).When with Bgl II or the digestion of Sal I, the restriction map that derives from the dna fragmentation of a reaction provides the fragment of required size.

Concentrate positive PCR reactant and digest it with the Spe I.With Spe I digested vector pRS426, and handle it with the calf intestinal Phosphoric acid esterase.Utilize conventional condition of contact that 4 μ l PCR fragments are combined with 1 μ l carrier DNA.By the DNA that gel electrophoresis analysis connected.Analyze Spe I Digestive system so that determine to be carried at the plasmid of the subclone of the ADE2 gene within the pRS426.With correct plasmid called after pCZR118.

Because by pcr amplification the ADE2 gene among the pCZR118, so possiblely be to produce the sudden change that the functional performance that makes gene can not exist.In order to check such sudden change, will have the segmental subclone of required insertion and be transformed into individually among the Wine brewing yeast strain HBY21A.Make cell be in the electroreception attitude, and transform it according to standard method.With the transformant plating on URA D and ADE D flat board.Three phenotype groups obtain identifying.Clone 1,2,11 and 12 produces the transformant of many healthy and strong growths on ADED.Transformation frequency and Ura ⁺The frequency of transformant is suitable.Clone 6,8,10 and 14 also provides to Ura ⁺And Ade ⁺High efficiency conversion, but Ade ⁺Bacterium colony is slightly littler than those bacterium colonies in first group.Clone 3 produces many Ura ⁺Bacterium colony, but do not produce Ade ⁺Bacterium colony, this shows that clone 3 carries non-functional ade2 sudden change.Concentrate clone 1,2,11 and 12.

In order to determine the complementary group of pichia methanolica ade2, derive from two representative mutant (#3 and #10 of each complementary group with clone's ADE gene transformation; #6 and #11), this mutant, when it was grown on the restriction VITAMIN B4, formation was selected based on deep erythronium.By the 200ml culture of the early stage logarithmic phase cell of centrifugal (3000 * g, 3 minutes) results, and with the fresh KD damping fluid (50mM potassium phosphate buffer, pH7.5 comprise 25mM DTT) of its resuspending in 20ml.Under 30 ℃ in this damping fluid incubation cell 15 minutes.Harvested cell then, and with its resuspending in the ice-cold STM of 200ml (270mM sucrose, 10mM Tris, pH7.5,1mMMgCl ₂) in.Harvested cell, and with its resuspending in the ice-cold STM of 100ml.Harvested cell again, and with its resuspending in the ice-cold STM of 3-5ml.The aliquots containig of 100 μ l of the electroreception attitude cell that derives from every kind of culture is mixed with the pADE1-1 DNA of Not I digestion.Cell/DNA mixture is placed among the 2mm electroporation Xiao Chi, and utilizes the BioRad Gene Pulser that is set to 1000 Ω resistance and 25 μ F electric capacity ^TMThis mixture is subjected in the pulsed electrical field of 5kV/cm.After pulse, come diluting cells by adding 1ml YEPD, and 30 ℃ of following incubations one hour.Then by gentle centrifugal cell harvesting, and resuspending is in the basic selective medium of 400 μ l (ADE D) that lacks VITAMIN B4.The resuspending sample is divided into the aliquots containig of 200 μ l, and with its plating on ADE D and ADE DS flat board.Dull and stereotyped 4-5 days of 30 ℃ of following incubations.Mutant #6 and #11 provide Ade ⁺Transformant.When DNA is omitted, do not observe any Ade ⁺Transformant, as if therefore, two isolates limit the complementary group of ade2.The ADE2 sequence is presented among the SEQ ID NO:1.Embodiment 2

The pichia methanolica clone bank that is disclosed among the embodiment 1 is used as the source that clone's alcohol utilizes gene (AUG1).Clone bank is as independent library storage, and wherein about 200-250 individual genomic clone represented in each storehouse.0.1 μ l " in a small amount preparation " DNA that derives from each storehouse utilize the PCR primer (8784, SEQ ID NO:5; 8787, SEQ ID NO:6) be used as template in the polymerase chain reaction, this PCR primer is designed by the arrangement of the conserved sequence in the alcohol oxidase gene of multiple-shaped nuohan inferior yeast, Candida boidinii and pichia pastoris phaff.Amplified reaction carries out as follows: 94 ℃ of following 30 circulations of 30 seconds, 50 ℃ of following 30 circulations of 30 seconds, 72 ℃ of following 30 circulations of 60 seconds; Subsequently 72 ℃ of following incubations 7 minutes.A storehouse (#5) provides about 600bp band.The dna sequencing of this PCR product discloses: its a kind of like this aminoacid sequence of encoding, this aminoacid sequence and for about 70% sequence identity property being arranged between the pichia pastoris alcohol oxidase of AOX1 coded by said gene, and about 85% sequence identity property is arranged between the multiple-shaped nuohan inferior yeast alcohol oxidase of MOX1 coded by said gene.The sequence of clone's AUG1 gene is presented among the SEQ IDNO:2.

Utilize with initially increase in used identical primer by the Ya Ku in pcr analysis #5 storehouse.Further decompose an inferior storehouse of the positive so that determine positive bacterium colony.This positive bacterium colony of line on flat board, and prepare DNA by individual bacterium colony.After with the digestion of Cla I, three bacterium colonies provide same pattern.

The restriction map of genomic clone and PCR product discloses: the AUG1 gene is positioned at the 7.5kb genome and inserts on the fragment, and the site within the PCR fragment can be inserted at genome and obtained within the fragment uniquely determining.Because the orientation of the gene within the PCR fragment is known, therefore a back information is provided at apparent position and the transcriptional orientation that genome inserts the AUG1 gene within the fragment.Disclose gene at the dna sequencing within this zone and on the amino acid levels and between other known alcohol oxidase gene very high sequence similarity is being arranged.Embodiment 3

With comprising AUG1 promotor and terminator, people GAD65 DNA (Karlsen etc., institute of NAS newspaper, 88:8337-8341,1991) and the expression vector of ADE2 selected marker, basically process is as described above transformed the pichia methanolica cell of ade2 sudden change by electroporation.Bacterium colony is gone up at the basic methyl alcohol flat board of agar (10 to 100 bacterium colonies of every 100mm flat board) formed diaphragm, this flat board comprises 20g/L Bacto ^TMAgar (Difco), 6.7g/L do not have amino acid whose yeast nitrogen matrix (Difco), 10g/L methyl alcohol and 0.4 μ g/L vitamin H.Cover agar with nitrocellulose, and flat board is upside down on the lid that comprises 1ml 50% methyl alcohol (in water), and 30 ℃ of following incubations 3 to 5 days.Then film is transferred on the filter that is immersed in 0.2M NaOH, 0.1%SDS, the 35mM dithiothreitol (DTT) so that the cracking adhesive cell.After 30 minutes, with distilled water from filter flushing cell debris down, and in coming in 30 minutes by flushing in 0.1M acetate and filter.

Analyze the adhesion protein matter in the filter then.By rinsing 30 minutes in the TTBS-NFM (20mMTris pH7.4,0.1%Tween 20,160mM NaCl, 5% nonfat milk powder) at room temperature, the binding site of blockading and not occupying.Then filter is transferred in the solution that comprises GAD6 monoclonal antibody (Chang and Gottlieb, J.Neurosci, 8:2123-2130,1988), the dilution ratio of this monoclonal antibody in TTBS-NFM is 1: 1000.In this antibody-solutions, filter is carried out incubation with at least one hour gentle stirring, use TTBS (20mM TrispH7.4,0.1%Tween 20,160mM NaCl) washed twice then, each five minutes.Use the goat anti-mouse antibody incubation filter puted together with horseradish peroxidase (1 μ g/ml TTBS-NFM) then at least one hour, and used the TTBS washing nozzle then three times, each 5 minutes.Make filter be exposed to commercially available chemical illuminating reagent (ECL then ^TMAmersham Inc., Arlington Heigths, IL) under.On the X-ray film, detect the light that produces by positive diaphragm.

In order to detect the GAD65 expression levels more accurately, in the shake-flask culture thing, cultivate candidate clone.Method was being grown two days bacterium colony on basic methyl alcohol flat board under 30 ℃ as described above.Bacterium colony is used for inoculating the basic methyl alcohol substratum of 20ml (the no amino acid whose yeast nitrogen matrix of 6.7g/L, 10g/L methyl alcohol, 0.4 μ g/L vitamin H), and its cell density is 1 * 10 ⁶Individual cells/ml.Culture was grown 1-2 days down at 30 ℃.In every kind of culture, add 50% methyl alcohol of 0.2ml every day.By centrifugal cell harvesting, and it is suspended in ice-cold lysis buffer (20mM Tris pH8.0,40mM NaCl, 2mM PMSF, 1mM EDTA, 1 μ g/ml leupeptin, 1 μ g/ml pepstatin, 1 μ g/ml aprotinin) in, its final volume is every g cell mashed prod 10ml lysis buffer.The suspension that 2.5ml produced is added to the 400-600 micron, ice-cold, the granulated glass sphere (it places the 15ml container) that acid elution is crossed of 2.5ml, and vigorous stirring mixture one minute, then incubation on ice 1 minute.Repeat this process and be stirred five minutes altogether up to cell.Remove big fragment and not broken cell by centrifugal (1000 * g, 5 minutes).Then clarifying lysate is poured in the clean container.Dilute clarifying lysate with sample buffer (5%SDS, 8M urea, 100mM Tris pH6.8,10% glycerine, 2mM EDTA, 0.01% tetrabromophenol sulfonphthalein), and (Novex, San Diego CA) go up electrophoresis at 4-20% acrylamide gradient gel.With western blotting to nitrocellulose, and with aforesaid GAD6 antibody test it.

Under the high cell density fermentation condition, the clone of the high expression level of the exogenous protein that demonstrates methanol induction in shake-flask culture is carried out more deep analysis.At first under vigorous stirring under 30 ℃ in 0.5 liter of YEPD meat soup culturing cell 1-2 days, use it to inoculate 5 liters of fermentation units (BioFlow III for example then; New Brunswick Scientific Co., Inc., Edison, NJ).At first by adding 57.8g (NH ₄) ₂SO ₄, 68g KH ₂PO ₄, 30.8g MgSO ₄7H ₂O, 8.6g CaSO ₄2H ₂O, 2.0g NaCl and 10ml defoamer (PPG) are loaded fermenting container with mineral salt.Add H ₂O to be forming the 2.5L volume, and with solution autoclaving 40 minutes.After cooling, add 50% glucose of 350ml, the 10X trace elements (table 4) of 250ml, 200 μ g/ml vitamin Hs and the 250ml cell inoculation thing of 25ml.

Table 410 X trace elements:

FeSO ₄·7H ₂O??100mM????27.8g/L

CuSO ₄·5H ₂O??2mM??????0.5g/L

ZnCl ₂?????????8mM??????1.09g/L

MnSO ₄·H ₂O???8mM??????1.35g/L

CoCl ₂·6H ₂O??2mM??????0.48g/L

Na ₂MoO ₄·2H ₂O1mM?????0.24g/L

H ₃BO ₃????????8mM??????0.5g/L

KI?????????????0.5mm?????0.08g/L

Vitamin H 5mg/L

VitB1 0.5g/L adds 1-2 milliliter H for every liter ₂SO ₄So that make compound formation solution

With fermenting container be arranged on 28 ℃, pH5.0 and＞operation down of 30% dissolved oxygen amount.Cell will consume initial glucose and load thing (this oxygen requirement during by consumption of glucose sharply increase show), and thereafter, oxygen-consumption reduces after glucose is depleted.After initial glucose filling thing is used up, will add NH ₄ ⁺Be transported in the container with the glucose-methanol feeding of trace elements, wherein 0.2% (w/v) glucose, 0.2% (w/v) methanol feeding are 5 hours, are thereafter 0.1% (w/v) glucose, 0.4% (w/v) methanol feeding 25 hours.In pure methyl alcohol, utilize the initial delivery speed of 12.5ml/hr and the final speed of 25ml/hr to supply with 550 gram methyl alcohol altogether by a port of container.Utilization comprises 175 gram glucose, 250ml 10X trace elements and 99g (NH ₄) ₂SO ₄700ml solution, supply with glucose by second port.Under these conditions, glucose and methyl alcohol obtain utilizing simultaneously, in case and follow glucose-methanol feeding to begin, just induce GAD ₆₅Express.The method that is used for shake-flask culture as described above, analysis derives from the GAD of the cell of fermenting container ₆₅Express.

Remove cell from fermenting container at interval with certain hour, and subsequently it is analyzed.Observe low GAD at the growing period that utilizes glucose ₆₅Express.Exhausting of glucose causes GAD ₆₅Proteinic low expression level; During the charging of fermenting culture, can improve expression level by adding MeOH.The adding of methyl alcohol is to the people GAD by the reactive AUG1 promoters driven of methyl alcohol ₆₅Expression tangible hormesis is arranged.Embodiment 4

The research conversion condition is so that definite current field condition, DNA topological framework and DNA concentration that is suitable for effective conversion of pichia methanolica most.All experiments all utilize pichia methanolica ade2 bacterial strain #11.Prepare competent cell according to method as previously mentioned.Utilize BioRad GenePulser ^TMImplement electroporation.

Three electric pulse field parameters influence the transformation efficiency of electroporation: electric capacity, strength of electric field and pulse duration.Strength of electric field is by the voltage decision of electricimpulse, and the pulse duration is then determined by the resistance that instrument is provided with.In this group experiment, checked one group of strength of electric field setting under various resistance.In all experiments, all utilized the maximum capacity setting (25 μ F) of instrument.100 μ l aliquots containigs of electroreception attitude cell are mixed with 10 μ l DNA, and this DNA comprises the ADE2 plasmid pCZR133 of about 1 μ g, and this plasmid has been used the linearizing of restriction enzyme Not I.Cell and DNA are transferred to electroporation Xiao Chi (the BTX Corp. of 2mm, San Diego, CA) in, and make it to accept the electricimpulse that strength of electric field is 0.5kV (2.5kV/cm), 0.75kV (3.75kV/cm), 1.0kV (5.0kV/cm), 1.25kV (6.25kV/cm) and 1.5kV (7.5kV/cm).Under the various pulse durations, check these field conditions.By instrument resistance being changed into 200 ohm, 600 ohm or " " ohmic value is regulated and control the pulse duration to infinity.With the cell suspension of pulse in YEPD, and 30 ℃ of following incubations one hour, and results, resuspending and plating it.Carrying out three groups independently tests.In each group, all find:, provide high transformation efficiency (referring to Fig. 1) in the electroporation conditions of " infinity " 0.75kV under the resistance of ohmic value (3.75kV/cm) than other condition of being checked.

After setting up the suitableeest impulsive condition, the researching DNA topological framework is to the influence of transformation efficiency.Electroreception attitude cell is mixed with the uncut ring-type pCZR133 of 1 μ g, and perhaps the pCZR133 with the digestion of 1 μ g Not I mixes.In three independent experiments, reclaim average about 25 transformant with cyclic DNA, and linear DNA produces average near 1 * 10 ⁴Individual transformant.These data show: than cyclic DNA, linear DNA transforms pichia methanolica with much bigger efficient.

At last, the relation between researching DNA concentration and the transformation efficiency.Make the aliquots containig (1ng, 10ng, 100ng and 1 μ g are in 10 μ l water) and 100 μ l electroreception attitude cytomixis of line style pCZR133 DNA, and implement electroporation under the ohmic value at 3.75kV/cm and " infinity ".The number change of transformant in about 10 (1ng DNA) to 10 ⁴Between (1 μ g DNA), and find that this quantity is directly proportional with DNA concentration.Embodiment 5

By relatively deriving from the DNA of wild-type cell and stable white transformant bacterium colony, detect the genome that transfering DNA is integrated into pichia methanolica.Identify two classes and integrate transformant.In the first kind, find that transfering DNA is integrated into homologous site.In second class, find that transfering DNA replaces endogenous AUG1 open reading frame.Though do not wish to be bound by theory, it is generally acknowledged that the appearance of the second class transformant is " displaced type recombination event " (Rothstein, Enzymology method, 194:281-301,1991), wherein transfering DNA replaces endogenous dna via dual group of incident.

The pCZR140 that digested with the Asp I transforms into Ade with pichia methanolica ade2 bacterial strain #11 ⁺PCZR140 is a kind of based on Bluescript  (Stratagene cloning system, La Jolla, carrier CA), this carrier comprises the mutant of pichia methanolica ADE2 gene and AUG1, and wherein the whole open reading frame between promotor and terminator zone is all by disappearance (Fig. 2).By wild-type cell with at the transformant cell preparation genomic dna of on the YEPD flat board, growing two days under 30 ℃.With about 100-200 μ l cell suspension in 1ml H ₂Among the O, in Eppendorf centrifuge centrifugal 30 seconds then.Reclaim cell precipitation, and with its resuspending in 400 μ l SCE+DTT+ enzymolysis enzymes (1.2M Sorbitol Powder, 10mM Trisodium Citrate, 10mM EDTA, 10mM DTT, 1-2mg/ml enzymolysis enzyme 100T), and at 37 ℃ of following incubation 10-15 minutes.The 1%SDS that adds 400 μ l, and mixing solutions is up to clarification.The 5M potassium acetate (pH8.9) that adds 300 μ l, mixing solutions and in Eppendorf centrifuge centrifugal at full speed five minutes.750 μ l supernatant liquors are transferred in the new test tube, and extracted it with the equal-volume phenol/chloroform.Reclaim the supernatant liquor that 600 μ l are produced, and by adding 2 times of volume of ethanol and in the cold house, coming deposit D NA in centrifugal 15 minutes.Under 65 ℃, in 50mlTE (10mM Tris pH8,1mM EDTA)+100 μ g/ml RNA enzyme, DNA was precipitated resuspending about 1 hour.Under 37 ℃, be used in one night of Eco R I (5 μ l) digestion 10 μ l DNA samples in the 100 μ l reacting weights.Use ethanol sedimentation DNA, by centrifugal recovery it, and with its resuspending in 7.5 μ l TE+2.5 μ l 5X application of sample dyestuffs.Whole 10ml amounts are applied on a swimming lane of 0.7% agarose in 0.5 * TBE (10 * TBE is 108g/L Tris alkali 7-9,55g/L boric acid, 8.3g/L EDTA disodium) gel.In comprising 0.5 * TBE of ethidium bromide, under 100V, move gel.Take pictures to gel, and under 400mA, 20mV, the DNA electrophoretic transfer is arrived positive deutero-nylon membrane (Nytran  N+, Schleicher ﹠amp with 30 minutes; Schuell, Keene, NH) on.Use 2 * SSC to wash this film then, this film trace five minutes on denaturing soln with 2 * SSC neutralization, is provided with crosslinked damping according to it then automatically in UV linking agent (Stratalinker , Stratagene cloning system).Utilize commercially available test kit (ECL ^TMTest kit, Amersham Corp., Arlington Heigthts IL), makes blot hybridization to the AUG1 promoter probe of PCR generation.The result shows: transfering DNA changes the structure of AUG1 promoter DNA, and this is consistent with homology integration incident (Fig. 2).

In second experiment, the pCZR137 that digested with the Not I transforms into Ade with pichia methanolica ade2 bacterial strain #11 ⁺, pCZR137 promptly comprises the carrier (Fig. 3) of people GAD65 cDNA between AUG1 promotor and terminator.Method as described above is by wild-type cell and stable white Ade ⁺Transformant prepares genomic dna, and digests it with the EcoR I.Go out the DNA that is digested by electrophoretic separation, and with its trace to film.Use by PCR probe that produce, corresponding with AUG1 open reading frame or AUG1 promotor and survey trace.The result shows: disappearance AUG1 open reading frame DNA in the transformant bacterial strain, and the AUG1 promoter region has lived through significant rearrangement.These results are (Fig. 3) consistent with dual group of incident (displaced type) between transfering DNA and the host genome.Embodiment 6

The AUG1 strain growth that makes pichia methanolica is under the high density fermentation condition.By adding 57.8g (NH ₄) ₂SO ₄, 46.6gKCl, 30.8gMgSO ₄7H ₂O, 8.6gCaSO ₄2H ₂O, 2.0gNaCl and 10ml defoamer (PPG) are loaded fermenting container with mineral salt.Add H ₂O to be producing the volume of 2.5L, and with solution autoclaving 40 minutes.After cooling, 30% sodium phosphate, 25ml 200 μ g/ml vitamin Hs and the 250ml cell inoculation thing of 50% glucose of adding 350ml, 250ml 10X trace elements (table 4), 210ml.At three phases to cell batch feed glucose or glucose/methyl alcohol.In the stage 1, cell receives 0.4%/L/ hour glucose (the whole amount of fermentation of w/v) 25 hours, and wherein per 1.5 liters are utilized 750g glucose, 110g (NH ₄) ₂SO ₄And 278ml 10X trace elements.Give the transition charging of cell then, last 5 hours with 0.2% glucose, 0.2% methyl alcohol/L/ hour.Last glucose complementarity methanol feeding comprises 0.1% glucose, 0.4% methyl alcohol/L/ hour, lasts 25 hours.Last biomass is about 300g/L cell mashed prod.Embodiment 7

For the fermentation of pichia methanolica aug1 Δ bacterial strain, fermenting container is filled with mineral salt, glucose, phosphoric acid salt, trace elements and vitamin H as described in example 6 above at first.Add 250ml cell inoculation thing.Per 1.2 liters are utilized 600g glucose, 108g (NH ₄) ₂SO ₄And the 273ml10X trace elements, the charging of preparation glucose.At three phases to the cell batch feed.In the fs, cell received glucose 12-25 hour with 0.4%/L/ hour.Adding weight percent by bolus then is that 1% methyl alcohol comes inducing cell, and utilizes the stage by carrying out the transition to methyl alcohol in 10 hours with blended 0.2% glucose/0.1% methanol feeding.In the phase III, carry the parallel feeding 15 hours of 0.2% glucose, 0.2% methyl alcohol.Embodiment 8

Pichia methanolica cell (AUG1 gene wherein breaks for the insertion of GAD65 expression construct) still remains on the energy for growth on the methyl alcohol, and this shows one second alcohol oxidase gene of existence.Identify second gene (called after AUG2) by PCR.The sequential analysis of 5 ' coding region of gene shows: coded proteinic N-end is similar to those known alcohol oxidase genes.

Bacterial strain MC GAD8 (a kind of very poor transformant of growing on basic methyl alcohol meat soup) is used as the source of clone AUG2 gene.Prepare genomic dna by MC GAD8, and be specific to the AUG1 open reading frame (8784, SEQ ID NO:5; 8787, SEQ ID NO:6) justice and this genomic dna of antisense PCR primer amplification arranged.Obtain consistent with the AUG1 product in size but show extremely low intensive product on the gel analyzing.

Digest the AUG2 PCR product of inferring with one group of restriction enzyme.EcoR I and Pvu I show its part digestion and the existence in several Bgl II site: DNA is polluted by a spot of AUG1.In order to remove the AUG1 DNA depollute, with EcoR I cutting PCR mixture and gel-purified it.Because as if MC GAD8 product does not have EcoR I site, so it is unaffected.The DNA of the gel-purified that produced of amplification again, and by restrictive diges-tion it is analyzed again.DNA provides and the different restriction map of AUG1 PCR product.

Utilize AUG1 or AUG2 open reading frame PCR fragment as probe, the genomic dna that derives from MCGAD8 and wild-type cell is carried out the Southern engram analysis.The AUG2 probe under the condition of severity difference with AUG1 locus hybridization, under severity difference and strong condition with second locus hybridization.The AUG1 probe all combines with two locus under the condition of severity difference, but mainly combines with the AUG1 locus under the strong condition of severity.These data show: the new PCR product type that derives from MC GAD8 is similar to but is different from AUG1.Sequential analysis demonstrates 83% identity property between AUG1 and AUG2 gene product.

In order to clone the genomic locus of AUG2, by former AUG2 PCR fragment design PCR primer.Primer 9885 (SEQ ID NO:7) and 9883 (SEQ ID NO:8) are used for screening the pichia methanolica genomic library.Survey the positive colony storehouse with former MC GAD8 PCR product then.With about 5000 bacterium colony/flat boards with the cell plating on 10 flat boards and grow a night, use then filter disc (Hybond-N, Amersham Corp., Arlington Heigthts, IL) cover dull and stereotyped.Make the bacterium colony sex change, neutralize it, and it is crosslinked that it is carried out UV.Wash bacterial debris down with 5X SSC from filter, and crosslinked again filter.In the 25ml hybridization buffer 42 ℃ of following paired cross traces 1 hour.The probe that in every pair of filter, adds about 250ng then.Implement hybridization 4 hours down at 42 ℃.Use 0.1X SSC, 6M urea and the 0.4%SDS of 500ml to wash trace 10 minutes down at 42 ℃ then, amount to four times.At room temperature use among the 2X SSC of 500ml then and trace 5 minutes, wherein wash twice.Then trace is immersed in the 100ml developping agent (ECL, Amersham Corp.).

Select positive bacterium colony, and utilize PCR primer 9885 (SEQ ID NO:7) and 9883 (SEQID NO:8) to increase this positive bacterium colony to determine their identity property.The positive storehouse of line on flat board, and rescreen menu one bacterium colony by PCR.Select a bacterium colony to be used for further analysis (doing restriction map and order-checking).The partial sequence of AUG2 gene is presented among the SEQ ID NO:9.Shown in SEQID NO:9, the AUG2 sequence starts from the Nucleotide 91 on the Hind III site.Nucleotide in this position upstream is the carrier sequence.Encoding sequence starts from Nucleotide 170.

The growth of pair cell on methyl alcohol of breaking of AUG2 gene almost do not have influence.The cell that lacks functional AUG1 and AUG2 gene product can not be grown on methyl alcohol.Thereafter analysis shows: the AUG1 gene product is that unique in the cell in growing in fermentor tank can detected alcohol oxidase.

According to the above, be apparent that: though, can carry out various modifications and not depart from spirit of the present invention and scope it in order to illustrate that this paper of the present invention has described specific embodiments of the present invention.Therefore, except that the qualification of appending claims, the present invention does not have other restriction.

Sequence table (1) general information: (ⅰ) applicant: ZymoGenetics, Inc.

1201?Eastlake?Avenue?East

Seattle,Washington?98102

The United States of America (ⅱ) denomination of invention: the preparation of pichia methanolica auxotrophic mutants (ⅲ) sequence number: 9 (ⅳ) address:

(A) addressee: ZymoGenetics, Inc.

(B) street: 1201 Eastlake Avenue East

(C) city: Seattle

(D) state: WA

(E) country: the U.S.

(F) ZIP:98102 (ⅴ) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, the current request for data of version #1.25 (ⅵ):

(A) application number:

(B) applying date:

(C) classification: (ⅷ) lawyer/proxy's information:

(A) name: Parker, Gary E

(B) registration number: 31-648

(C) reference/certificate number: 96-17 (ⅸ) contact details:

(A) phone: 206-442-6673

(B) fax: 206-442-6678 (2) is about information (ⅰ) sequence signature of SEQ ID NO:1:

(A) length: 3077 base pairs

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅴ ) ： ( ⅵ ) ： ( ⅹⅰ ) ：SEQ ID NO:1:CAGCTGCTCT GCTCCTTGAT TCGTAATTAA TGTTATCCTT TTACTTTGAA CTCTTGTCGG 60TCCCCAACAG GGATTCCAAT CGGTGCTCAG CGGGATTTCC CATGAGGTTT TTGACAACTT 120TATTGATGCT GCAAAAACTT TTTTAGCCGG GTTTAAGTAA CTGGGCAATA TTTCCAAAGG 180CTGTGGGCGT TCCACACTCC TTGCTTTTCA TAATCTCTGT GTATTGTTTT ATTCGCATTT 240TGATTCTCTT ATTACCAGTT ATGTAGAAAG ATCGGCAAAC AAAATATCAA CTTTTATCTT 300GAACGCTGAC CCACGGTTTC AAATAACTAT CAGAACTCTA TAGCTATAGG GGAAGTTTAC 360TGCTTGCTTA AAGCGGCTAA AAAGTGTTTG GCAAATTAAA AAAGCTGTGA CAAGTAGGAA 420CTCCTGTAAA GGGCCGATTC GACTTCGAAA GAGCCTAAAA ACAGTGACTA TTGGTGACGG 480AAAATTGCTA AAGGAGTACT AGGGCTGTAG TAATAAATAA TGGAACAGTG GTACAACAAT 540AAAAGAATGA CGCTGTATGT CGTAGCCTGC ACGAGTAGCT CAGTGGTAGA GCAGCAGATT 600GCAAATCTGT TGGTCACCGG TTCGATCCGG TCTCGGGCTT CCTTTTTTGC TTTTTCGATA 660TTTGCGGGTA GGAAGCAAGG TCTAGTTTTC GTCGTTTCGG ATGGTTTACG AAAGTATCAG 720CCATGAGTGT TTCCCTCTGG CTACCTAATA TATTTATTGA TCGGTCTCTC ATGTGAATGT 780TTCTTTCCAA GTTCGGCTTT CAGCTCGTAA ATGTGCAAGA AATATTTGAC TCCAGCGACC 840TTTCAGAGTC AAATTAATTT TCGCTAACAA TTTGTGTTTT TCTGGAGAAA CCTAAAGATT 900TAACTGATAA GTCGAATCAA CATCTTTAAA TCCTTTAGTT AAGATCTCTG CAGCGGCCAG 960TATTAACCAA TAGCATATTC ACAGGCATCA CATCGGAACA TTCAGAATGG ACTCGCAAAC 1020TGTCGGGATT TTAGGTGGTG GCCAACTTGG TCGTATGATC GTTGAAGCTG CACACAGATT 1080GAATATCAAA ACTGTGATTC TCGAAAATGG AGACCAGGCT CCAGCAAAGC AAATCAACGC 1140TTTAGATGAC CATATTGACG GCTCATTCAA TGATCCAAAA GCAATTGCCG AATTGGCTGC 1200CAAGTGTGAT GTTTTAACCG TTGAGATTGA ACATGTTGAC ACTGATGCGT TGGTTGAAGT 1260TCAAAAGGCA ACTGGCATCA AAATCTTCCC ATCACCAGAA ACTATTTCAT TGATCAAAGA 1320TAAATACTTG CAAAAAGAGC ATTTGATTAA GAATGGCATT GCTGTTGCCG AATCTTGTAG 1380TGTTGAAAGT AGCGCAGCAT CTTTAGAAGA AGTTGGTGCC AAATACGGCT TCCCATACAT 1440GCTAAAATCT AGAACAATGG CCTATGACGG AAGAGGTAAT TTTGTTGTCA AAGACAAGTC 1500ATATATACCT GAAGCTTTGA AAGTTTTAGA TGACAGGCCG TTATACGCCG AGAAATGGGC 1560TCCATTTTCA AAGGAGTTAG CTGTTATGGT TGTGAGATCA ATCGATGGCC AAGTTTATTC 1620CTACCCAACT GTTGAAACCA TCCACCAAAA CAACATCTGT CACACTGTCT TTGCTCCAGC 1680TAGAGTTAAC GATACTGTCC AAAAGAAGGC CCAAATTTTG GCTGACAACG CTGTCAAATC 1740TTTCCCAGGT GCTGGTATCT TTGGTGTTGA AATGTTTTTA TTACAAAATG GTGACTTATT 1800AGTCAACGAA ATTGCCCCAA GACCTCACAA TTCTGGTCAC TATACCATCG ACGCTTGTGT 1860CACCTCGCAA TTTGAAGCTC ATGTTAGGGC CATTACTGGT CTACCCATGC CGAAGAACTT 1920CACTTGTTTG TCGACTCCAT CTACCCAAGC TATTATGTTG AACGTTTTAG GTGGCGATGA 1980GCAAAACGGT GAGTTCAAGA TGTGTAAAAG AGCACTAGAA ACTCCTCATG CTTCTGTTTA 2040CTTATACGGT AAGACTACAA GACCAGGCAG AAAAATGGGT CACATTAATA TAGTTTCTCA 2100ATCAATGACT GACTGTGAGC GTAGATTACA TTACATAGAA GGTACGACTA ACAGCATCCC 2160TCTCGAAGAA CAGTACACTA CAGATTCCAT TCCGGGCACT TCAAGCAAGC CATTAGTCGG 2220TGTCATCATG GGTTCCGATT CGGACCTACC AGTCATGTCT CTAGGTTGTA ATATATTGAA 2280GCAATTTAAC GTTCCATTTG AAGTCACTAT CGTTTCCGCT CATAGAACCC CACAAAGAAT 2340GGCCAAGTAT GCCATTGATG CTCCAAAGAG AGGGTTGAAG TGCATCATTG CTGGTGCTGG 2400TGGTGCCGCT CATTTACCGG GAATGGTTGC GGCGATGACG CCGCTGCCTG TTATTGGTGT 2460CCCTGTTAAA GGCTCTACTT TGGATGGTGT TGATTCACTA CACTCCATCG TTCAAATGCC 2520AAGAGGTATT CCTGTTGCTA CTGTGGCTAT TAACAATGCT ACTAACGCTG CCTTGCTAGC 2580TATCACAATC TTAGGTGCCG GCGATCCAAA TACTTGTCTG CAATGGAAGT TTATATGAAC 2640AATATGGAAA ATGAAGTTTT GGGCAAGGCT GAAAAATTGG AAAATGGTGG ATATGAAGAA 2700TACTTGAGTA CATACAAGAA GTAGAACCTT TTATATTTGA TATAGTACTT ACTCAAAGTC 2760TTAATTGTTC TAACTGTTAA TTTCTGCTTT GCATTTCTGA AAAGTTTAAG ACAAGAAATC 2820TTGAAATTTC TAGTTGCTCG TAAGAGGAAA CTTGCATTCA AATAACATTA ACAATAAATG 2880ACAATAATAT ATTATTTCAA CACTGCTATA TGGTAGTTTT ATAGGTTTGG TTAGGATTTG 2940AGATATTGCT AGCGCTTATC ATTATCCTTA ATTGTTCATC GACGCAAATC GACGCATTTC 3000CACAAAAATT TTCCGAACCT GTTTTTCACT TCTCCAGATC TTGGTTTAGT ATAGCTTTTG 3060ACACCTAATA CCTGCAG 3077 ( 2 ) SEQ ID NO:2： ( ⅰ ) ：

(A) length: 3386 base pairs

(B) type: nucleic acid

(C) chain: two strands

( D ) ： ( ⅱ ) ：DNA ( ⅲ ) ： ( ⅳ ) ： ( ⅴ ) ： ( ⅵ ) ： ( ⅹⅰ ) ：SEQ ID NO:2:GAATTCCTGC AGCCCGGGGG ATCGGGTAGT GGAATGCACG GTTATACCCA CTCCAAATAA 60AAGTGTAGTA GCCGGACTGA AAGGTTTTAG GAGTCTGTTT GTTTGTTCAT GTGCATCATT 120CCCTAATCTG TTAACAGTCT CGGAGTATAC AAAAAAGTAA GTCAAATATC AAGGTGGCCG 180GGGGCAGCAT CGAGACTCGA GATGGTACAT ACTTAAAAGC TGCCATATTG AGGAACTTCA 240AAGTTTTATC TGTTTTTAGA ATTAAAAGAC GATTGTTGTA ACAAAACGTT GTGCCTACAT 300AAACTCAAAT TAATGGAAAT AGCCTGTTTT GAAAAATACA CCTTCTTAAG TACTGACAAA 360GTTTTGTTAA ATGACTATCG AACAAGCCAT GAAATAGCAC ATTTCTGCCA GTCACTTTTA 420ACACTTTCCT GCTTGCTGGT TGACTCTCCT CATACAAACA CCCAAAAGGG AAACTTTCAG 480TGTGGGGACA CTTGACATCT CACATGCACC CCAGATTAAT TTCCCCAGAC GATGCGGAGA 540CAAGACAAAA CAACCCTTTG TCCTGCTCTT TTCTTTCTCA CACCGCGTGG GTGTGTGCGC 600AGGCAGGCAG GCAGGCAGCG GGCTGCCTGC CATCTCTAAT CGCTGCTCCT CCCCCCTGGC 660TTCAAATAAC AGCCTGCTGC TATCTGTGAC CAGATTGGGA CACCCCCCTC CCCTCCGAAT 720GATCCATCAC CTTTTGTCGT ACTCCGACAA TGATCCTTCC CTGTCATCTT CTGGCAATCA 780GCTCCTTCAA TAATTAAATC AAATAAGCAT AAATAGTAAA ATCGCATACA AACGTCATGA 840AAAGTTTTAT CTCTATGGCC AACGGATAGT CTATCTGCTT AATTCCATCC ACTTTGGGAA 900CCGCTCTCTC TTTACCCCAG ATTCTCAAAG CTAATATCTG CCCCTTGTCT ATTGTCCTTT 960CTCCGTGTAC AAGCGGAGCT TTTGCCTCCC ATCCTCTTGC TTTGTTTCGG TTATTTTTTT 1020TTCTTTTGAA ACTCTTGGTC AAATCAAATC AAACAAAACC AAACCTTCTA TTCCATCAGA 1080TCAACCTTGT TCAACATTCT ATAAATCGAT ATAAATATAA CCTTATCCCT CCCTTGTTTT 1140TTACCAATTA ATCAATCTTC AAATTTCAAA TATTTTCTAC TTGCTTTATT ACTCAGTATT 1200AACATTTGTT TAAACCAACT ATAACTTTTA ACTGGCTTTA GAAGTTTTAT TTAACATCAG 1260TTTCAATTTA CATCTTTATT TATTAACGAA ATCTTTACGA ATTAACTCAA TCAAAACTTT 1320TACGAAAAAA AAATCTTACT ATTAATTTCT CAAAATGGCT ATTCCAGATG AATTTGATAT 1380TATTGTTGTC GGTGGTGGTT CCACCGGTTG TGCTCTTGCT GGTAGATTAG GTAACTTGGA 1440CGAAAACGTC ACAGTTGCTT TAATCGAAGG TGGTGAAAAC AACATCAACA ACCCATGGGT 1500TTACTTACCA GGTGTTTATC CAAGAAACAT GAGATTAGAC TCAAAGACTG CTACTTTTTA 1560CTCTTCAAGA CCATCACCAC ACTTGAACGG TAGAAGAGCT ATTGTTCCAT GTGCTAACAT 1620CTTGGGTGGT GGTTCTTCCA TCAACTTCTT GATGTACACC AGAGCCTCTG CCTCCGATTA 1680CGATGATTGG GAATCTGAAG GTTGGACTAC CGATGAATTA TTACCACTAA TGAAGAAGAT 1740TGAAACTTAT CAAAGACCAT GTAACAACAG AGAATTGCAC GGTTTCGATG GTCCAATTAA 1800GGTTTCATTT GGTAACTATA CTTATCCAAA CGGTCAAGAT TTCATTAGAG CTGCCGAATC 1860TCAAGGTATT CCATTTGTTG ATGATGCTGA AGATTTGAAA TGTTCCCACG GTGCTGAGCA 1920CTGGTTGAAG TGGATCAACA GAGACTTAGG TAGAAGATCC GATTCTGCTC ATGCTTACAT 1980TCACCCAACC ATGAGAAACA AGCAAAACTT GTTCTTGATT ACTTCCACCA AGTGTGAAAA 2040GATTATCATT GAAAACGGTG TTGCTACTGG TGTTAAGACT GTTCCAATGA AGCCAACTGG 2100TTCTCCAAAG ACCCAAGTTG CTAGAACTTT CAAGGCTAGA AAGCAAATTA TTGTTTCTTG 2160TGGTACTATC TCATCACCAT TAGTTTTGCA AAGATCTGGT ATCGGTTCCG CTCACAAGTT 2220GAGACAAGTT GGTATTAAAC CAATTGTTGA CTTACCAGGT GTTGGTATGA ACTTCCAAGA 2280TCACTACTGT TTCTTCACTC CATACCATGT CAAGCCAGAT ACTCCATCAT TCGATGACTT 2340TGTTAGAGGT GATAAAGCTG TTCAAAAATC TGCTTTCGAC CAATGGTATG CTAACAAGGA 2400TGGTCCATTA ACCACTAATG GTATTGAGGC AGGTGTTAAG ATTAGACCAA CTGAAGAAGA 2460ATTAGCCACT GCTGATGACG AATTCAGAGC TGCTTATGAT GACTACTTTG GTAACAAGCC 2520AGATAAGCCA TTAATGCACT ACTCTCTAAT TTCTGGTTTC TTTGGTGACC ACACCAAGAT 2580TCCAAACGGT AAGTACATGT GCATGTTCCA CTTCTTGGAA TATCCATTCT CCAGAGGTTT 2640CGTTCACGTT GTTTCTCCAA ACCCATACGA TGCTCCTGAC TTTGATCCAG GTTTCATGAA 2700CGATCCAAGA GATATGTGGC CAATGGTTTG GTCTTACAAG AAGTCCAGAG AAACTGCCAG 2760AAGAATGGAC TGTTTTGCCG GTGAAGTTAC TTCTCACCAC CCACACTACC CATACGACTC 2820ACCAGCCAGA GCTGCTGACA TGGACTTGGA AACTACTAAA GCTTATGCTG GTCCAGACCA 2880CTTTACTGCT AACTTGTACC ACGGTTCATG GACTGTTCCA ATTGAAAAGC CAACTCCAAA 2940GAACGCTGCT CACGTTACTT CTAACCAAGT TGAAAAACAT CGTGACATCG AATACACCAA 3000GGAGGATGAT GCTGCTATCG AAGATTACAT CAGAGAACAC ACTGAAACCA CATGGCATTG 3060TCTTGGTACT TGTTCAATGG CTCCAAGAGA AGGTTCTAAG GTTGTCCCAA CTGGTGGTGT 3120TGTTGACTCC AGATTAAACG TTTACGGTGT TGAAAAGTTG AAGGTTGCTG ATTTATCAAT 3180TTGCCCAGAT AATGTTGGTT GTAACACTTA CTCTACTGCT TTGTTAATCG GTGAAAAGGC 3240TTCTACCTTA GTTGCTGAAG ACTTGGGCTA CTCTGGTGAT GCTTTGAAGA TGACTGTTCC 3300AAACTTCAAA TTGGGTACTT ATGAAGAAGC TGGTCTAGCT AGATTCTAGG GCTGCCTGTT 3360TGGATATTTT TATAATTTTT GAGAGT 3386 ( 2 ) SEQ ID NO:3： ( ⅰ ) ：

(A) length: 38 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅹ ⅰ) sequence description: SEQ ID NO:3:TGATCACCTA GGACTAGTGA CAAGTAGGAA CTCCTGTA 38 (2) information: (ⅰ) sequence signature about SEQ ID NO:4:

(A) length: 39 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅹ ⅰ) sequence description: SEQ ID NO:4:CAGCTGCCTA GGACTAGTTT CCTCTTACGA GCAACTAGA 39 (2) information: (ⅰ) sequence signature about SEQ ID NO:5:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ⅹ ⅰ) sequence description: SEQ ID NO:5:TGGTTGAAGT GGATCAA 17 (2) information: (ⅰ) sequence signature about SEQ ID NO:6:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅹ ⅰ) sequence description: SEQ ID NO:6:GTGTGGTCAC CGAAGAA 17 (2) information: (ⅰ) sequence signature about SEQ ID NO:7:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC9885 (ⅹ ⅰ) sequence description: SEQ ID NO:7:GTTGTTCCTT CCAAACCATT GAAC 24 (2) information: (ⅰ) sequence signature about SEQ ID NO:8:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style (ⅶ) direct sources:

(B) clone: ZC9883 (ⅹ ⅰ) sequence description: SEQ ID NO:8:AAAGTAAGAA GCGTAGCCTA GTTG 24 (2) information: (ⅰ) sequence signature about SEQ ID NO:9:

(A) length: 329 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological structure: line style (ⅹ ⅰ) sequence description: SEQ ID NO:9:GACCATGATT ACGCCAAGCG CGCAATTAAC CCTCACTAAA GGGAACAAAA GCTGGGTACC 60GGGCCCCCCC TCGAGGTCGA CGGTATCGAT AAGCTTTATT ATAACATTAA TATACTATTT 120TATAACAGGA TTGAAAATTA TATTTATCTA TCTAAAACTA AAATTCAAAA TGGCTATTCC 180TGAAGAATTC GATATCATTG TTGTCGGTGG TGGTTCTGCC GGCTGTCCTA CTGCTGGTAG 240ATTGGCTAAC TTAGACCCAA ATTTAACTGT TGCTTTAATC GAAGCTGGTG AAAACAACAT 300TAACAACCCA TGGGTCTACT TACCAGGCG 329

Claims (9)

1. method for preparing pichia methanolica cell with auxotrophic mutation, this method comprises:

(a) expose the pichia methanolica cell under mutagenic condition;

(b) in rich medium, cultivate the cell that derives from step (a), thereby make that sudden change obtains setting up and duplicating at least a portion cell;

(c) cultivation derives from the cell of step (b) in the insufficient substratum of assimilable nitrogen, thereby exhausts the nitrogen deposit of cell;

(d) cultivate the cell derive from step (c) in defined medium, selecting the damaged cell of vegetative gene, substratum wherein comprises inorganic nitrogen-sourced and is enough to kill the nystatin of the amount of growth pichia methanolica cell;

(e) in rich medium, cultivate the cell of the selection derive from step (d).

2. according to the process of claim 1 wherein that the cellular replication with deriving from the selection of step (e) is plated in the defined medium, and cultivate this cell to determine existing of auxotrophic mutation.

3. according to the process of claim 1 wherein that selected cell belongs to the auxotroph of VITAMIN B4.

4. according to the method for claim 3, wherein selected cell lacks Phosphoribosyl-5-aminooimidazole carboxylase.

5. comprise 2mg/L nystatin according to the defined medium that the process of claim 1 wherein.

6. comprise according to the mutagenic condition that the process of claim 1 wherein and being exposed under the ultraviolet light.

7. comprise according to the mutagenic condition that the process of claim 1 wherein and being exposed under the chemical mutagen.

8. according to the inorganic nitrogen-sourced ammonium ion that comprises that the process of claim 1 wherein.

9. according to the method for claim 8, wherein inorganic nitrogen-sourced is ammonium sulfate.

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