WO2014008729A1 - 消除甲醇诱导型启动子单一甲醇碳源依赖性的方法 - Google Patents

消除甲醇诱导型启动子单一甲醇碳源依赖性的方法 Download PDF

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WO2014008729A1
WO2014008729A1 PCT/CN2012/084642 CN2012084642W WO2014008729A1 WO 2014008729 A1 WO2014008729 A1 WO 2014008729A1 CN 2012084642 W CN2012084642 W CN 2012084642W WO 2014008729 A1 WO2014008729 A1 WO 2014008729A1
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methanol
promoter
gene
polypeptide
expression
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PCT/CN2012/084642
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English (en)
French (fr)
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周祥山
张元兴
王锦佳
王小龙
柏鹏
张平
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华东理工大学
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Priority to US14/413,930 priority Critical patent/US9677080B2/en
Priority to EP12881104.9A priority patent/EP2873734B1/en
Publication of WO2014008729A1 publication Critical patent/WO2014008729A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present invention is in the field of bioengineering; more specifically, the present invention relates to a method for eliminating the dependence of a methanol-inducible promoter on a single methanol carbon source to express a foreign polypeptide. Background technique
  • Methanol-nutrient yeast (including corpse c / n' a , Hansenula, Candida, Toru psis, etc.) has been widely used in industrial production, pharmaceuticals and other fields due to its highly efficient expression of exogenous polypeptides. It is characterized in that they have a promoter promoter capable of being efficiently induced by methanol, a DH4S promoter, an FDH promoter, a promoter, a ⁇ promoter, a ZZA1, a PEX5-, a PEX8-, a PEX14-promoter, etc., and these Promoters are strictly dependent on methanol, and other carbon sources (such as glucose, glycerol, etc.) inhibit the expression of these promoters.
  • methylotrophic parent yeast expression system to express so many exogenous peptides is due to its incomparable advantages of other expression systems: simple gene manipulation, high expression of exogenous polypeptides, intracellular expression, It can secrete expression; the foreign polypeptide gene is genetically stable; as a eukaryotic expression system, it has a subcellular structure of eukaryotes, and has post-translational modification processing functions such as glycosylation, fatty acylation, and protein phosphorylation;
  • the object of the present invention is to provide a single methanol carbon source dependence to eliminate methanol-inducible promoters.
  • a method of eliminating the dependence of a methanol-inducible promoter on a single methanol carbon source to drive expression of a foreign polypeptide-encoding gene comprising:
  • methanol-nutrient yeast comprising:
  • Expression cassette 1 which expresses an exogenous Mitl polypeptide
  • Expression cassette 2 comprising a methanol-inducible promoter and a foreign polypeptide encoding gene operably linked thereto;
  • Methanol nutrient yeast (1) is cultured under conditions of no methanol or a single methanol carbon source.
  • the Mitl polypeptide is a transcriptional activation-related factor such that the originally methanol-inducible promoter is no longer dependent on methanol and can also express a foreign polypeptide.
  • the Mitl polypeptide functions as: Activating methanol-inducible promoter expression in activating methanol metabolism.
  • the Mitl is PpMitl.
  • the expression cassette 2 is present in single or multiple copies in methanolic yeast, for example 1-20 copies, such as 15, 10, 8, 6, 5, 3, 2 copies.
  • the methanol inducible promoter includes, but is not limited to: ⁇ ⁇ promoter, DH4S promoter, Z 4S promoter, FDH promoter, FMDH promoter, MOT promoter, AOX2 promoter Sub, ZZA1, PEX5-, PEX8-, cadaverium 4-promoter, corpse kff20 promoter, ⁇ 47 promoter, ⁇ 007 promoter, ⁇ OD2 promoter.
  • the methanolic yeast comprises, but is not limited to, Pichia pastoris (Pc/»' «), Hansenula (H ⁇ m ⁇ /"), Candida (Ow Wa)
  • the Pichia pastoris CPc/»'a) includes, but is not limited to: GS115, Pichia pastoris (A g7A g2), genes and genes are not expressed, NRG1 gene, MIG1 gene And the MIG2 gene does not express Pichia pastoris (AmiglAmig2Anrgl), or the PYS strain which is not expressed by the HYS7 gene.
  • the Mitl polypeptide is selected from the group consisting of:
  • the NCBI Reference Sequence of the gene encoding the Mitl polypeptide is XM-002493021.1.
  • the gene encoding the Mitl polypeptide has the nucleotide sequence shown in SEQ ID NO: 1.
  • the gene encoding the Mitl polypeptide is induced to be expressed in the presence of methanol, and is not expressed in the presence of a carbon source such as glycerol or glucose.
  • the expression cassette 1 comprises a promoter and a coding gene of a Mitl polypeptide operably linked thereto; preferably, the promoter includes, but is not limited to: a constitutive promoter, Inducible promoter, tissue or organ specific promoter, spatiotemporal specific expression promoter.
  • the promoter includes, but is not limited to, a promoter, a promoter, an M/7 promoter, and the like, any promoter capable of overexpressing M/7.
  • the expression cassette 1 is present in single or multiple copies in methanolic yeast, for example 1-20 copies, such as 15, 10, 8, 6, 5, 3, 2 copies.
  • step (2) a yeast culture medium in which glycerin and/or glucose are present is used.
  • a recombinant methanolic yeast comprising:
  • Expression cassette 1 which is capable of expressing an exogenous Mitl polypeptide
  • Expression cassette 2 comprising a methanol-inducible promoter and a foreign polypeptide encoding gene operably linked thereto.
  • the methanolic yeast comprises, but is not limited to, Pichia pastoris (P c/»' «), Hansenula (H ⁇ m «?
  • Pichia pastoris CP c/»'a includes, but is not limited to: GS 1 15 , Pichia pastoris that are not expressed by genes and genes ( A g7A g2), NRG1 gene, MIG1 gene and MIG2 gene are not expressed in Pichia pastoris (AmiglAmig2Anrgl), or the PX yeast strain in which the HXS1 gene is not expressed.
  • nucleotide sequence of the MIG1 gene is as shown in SEQ ID NO: 9.
  • the nucleotide sequence of the gene is set forth in SEQ ID NO: 10; or The nucleotide sequence of the HXS1 gene is shown in SEQ ID NO: 3.
  • the nucleotide sequence of the NRG1 gene is shown in SEQ ID NO: 8 (NCBI Reference Sequence: XM-002493138.1).
  • Figure 1 Schematic diagram of the plasmid map of pGAPZaA.
  • Figure 2 Schematic diagram of the plasmid map of the vector pAG32.
  • FIG. 1 Wild type and GS115-MIT1 were cultured in a medium other than methanol or methanol (glycerol + methanol), and the results of Aox color reaction were carried out.
  • Figure 4 Wild type and GS115-MIT1 were cultured in non-methanol or methanol-containing medium, respectively, and the results of Aox enzyme activity assay were performed.
  • FIG. 5 The strain GS115-MIT1-GFP was pre-incubated overnight in YND liquid medium, and then transferred to non-methanol or methanol-containing medium for analysis. After sampling, the geometric mean fluorescence intensity of GFP in the sample was detected by flow cytometry. .
  • Figure 7 Wild-type and A g7A g2-MIT1 and xy7-MIT1 strains were cultured in non-methanol or methanol-containing medium respectively. After growing to logarithmic growth phase, total protein was sampled and quantified by Bradford method. Live measurement.
  • Figure 8 Wild type and AmigJAmig2-Mm and A/? y7-MIT1 strains were cultured in non-methanol or methanol-containing medium, respectively. After sampling, the geometric mean fluorescence intensity of GFP in the sample was detected by flow cytometry.
  • Figure 9 Schematic diagram of the plasmid map of the vector pPIC3.5K.
  • Figure 10 Schematic diagram of plasmid map of vector pUC18
  • FIG. 11 Wild-type and A g7A g2 ⁇ rg7-MIT1 strains were cultured in non-methanol or methanol-containing medium respectively. After growing to logarithmic growth phase, the total protein was sampled and quantified by Bradford method. Determination.
  • Figure 12 Wild type and ⁇ migJ ⁇ mig2 ⁇ nrgJ-Mm strains were cultured in non-methanol or methanol-containing medium, respectively. After sampling, the geometric mean fluorescence intensity of GFP in the sample was detected by flow cytometry. detailed description
  • promoter refers to a nucleic acid sequence that is typically present upstream (5' end) of the coding sequence of the gene of interest and is capable of directing transcription of the nucleic acid sequence into mRNA.
  • the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the promoter or promoter region includes an active variant of a promoter which may be a naturally occurring allelic variant or a non-naturally occurring variant. Such variants include substitution variants, deletion variants and insertion variants.
  • the "methanol inducible promoter” is a promoter of an enzyme involved in methanol metabolism. In the prior art, these promoters can control the expression of the exogenous polypeptide from the promoter by adding methanol to the growth medium.
  • the "methanol inducible promoter” can be isolated from yeast by a person skilled in the art using conventional techniques.
  • single methanol (carbon source) induction means that the promoter needs to be induced with methanol as the sole carbon source to drive the expression of the gene operably linked thereto, and the non-methanol is the sole carbon source (eg The expression of the gene operably linked to it cannot be driven under the conditions of methanol + glucose.
  • the phrase "elimination of single methanol induction” means that the promoter can drive expression of a gene operably linked thereto without using methanol as the sole carbon source (e.g., methanol + glucose, or glucose, or glycerol).
  • Non-single methanol (carbon source) induction means that in addition to the methanol carbon source, there is at least one non-methanol carbon source.
  • a "constitutive promoter” refers to a class of promoters that have no significant difference in gene expression between different tissues and developmental stages under their regulation.
  • the "inducible promoter” can rapidly induce “on” and “off” or “high” and “low” of gene transcription at a specific cell growth stage or a specific growth environment as needed. Inducible promoters can be divided into naturally occurring promoters and artificially constructed promoters, depending on the source. As used herein, “tissue or organ-specific promoter” refers to a promoter in which gene transcription generally occurs only in certain specific organs or tissues.
  • exogenous or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a gene sequence of interest is not normally present, the promoter is foreign to the gene of interest. A particular sequence is “exogenous” to the cell or organism into which it is inserted.
  • expression cassette refers to a gene expression system comprising all of the necessary elements required for expression of a polypeptide of interest (in the present invention, a foreign polypeptide or a Mitl polypeptide), typically comprising the following elements: a promoter, A gene sequence encoding a polypeptide, a terminator; and optionally a signal peptide coding sequence or the like. These components are operatively connected.
  • methanol nutrient yeast refers to a yeast that can utilize methanol as the sole carbon source. Including from Haem a, Pichia, CP c/»'a), Saccharomyces
  • operably linked refers to the spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is "operably linked" to the nucleic acid sequence.
  • stringent conditions means: (1) hybridization and elution at lower ionic strengths and higher temperatures, such as 0.2 X SSC, 0.1% SDS, 60 °C; or (2) hybridization. Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 50% identity between the two sequences, Preferably, it is 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more,
  • Hybridization occurs when 85% or more or 90% or more, more preferably 95% or more.
  • containing includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; The subordinate concept consisting of “consisting of”, “consisting of” and “consisting of” with “contains”, “haves” or “includes”.
  • the present invention discloses the use of a Mitl polypeptide to eliminate the single methanol carbon source dependence of a methanol-inducible promoter to drive expression of a foreign polypeptide-encoding gene.
  • a promoter methanol-inducible promoter
  • the inventors unexpectedly discovered that the Mitl polypeptide can deactivate methanol.
  • the dependence of the promoter on methanol allows for the expression of exogenous polypeptides using a non-single or non-methanol carbon source.
  • the invention also encompasses fragments, derivatives and analogs of said Mitl polypeptides.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the MIT1 polypeptide of the present invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example a polypeptide formed by fusion of a polyethylene glycol) or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader or secretion sequence or a sequence or polypeptide sequence used to purify the polypeptide, or fusion) Peptide).
  • conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide
  • the term "Mitl polypeptide” refers to a polypeptide having the sequence of SEQ ID NO: 2 which induces the function of a foreign-inducing polypeptide by a methanol-inducible promoter using a non-methanol carbon source.
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 2 having the function of inducing a methanol-inducible promoter to express a foreign polypeptide using a non-methanol carbon source.
  • variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, still more preferably 1 -8 or 1-5) deletions, insertions and/or substitutions of amino acids, and addition or deletion of one or several at the C-terminus and/or N-terminus (usually within 20, preferably within 10, More preferably, it is 5 or less amino acids.
  • the function of the protein is usually not altered.
  • adding or subtracting one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA encoded by DNA that hybridizes to DNA encoding the Mitl polypeptide under high or low stringency conditions.
  • the invention also provides other polypeptides, such as fusion polypeptides comprising a Mitl polypeptide or a fragment thereof.
  • the invention also provides analogs of Mitl polypeptides or polypeptides.
  • the difference between these analogs and the native Mitl polypeptide may be a difference in amino acid sequence, or may be a difference in the modified form that does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the natural L-amino acid (such as D-amino acids), and classes having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids).
  • the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • the invention also provides polynucleotide sequences encoding a Mitl polypeptide of the invention or a conservative variant polypeptide thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the sequence of SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant in the present invention means that the code has SEQ
  • the present invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the present invention also relates to polynucleotides homologous to the above polynucleotides which have at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80% phase with the above polynucleotides, More preferably at least 90%; more preferably at least 95%; more preferably at least 98% or 99% identical.
  • the protein encoded by these polynucleotides also has the same function as the polypeptide encoded by the aforementioned polynucleotide to induce a methanol-inducible promoter to express a foreign polypeptide using a non-methanol carbon source.
  • the invention also relates to hybridization to the sequences described above and having at least 50% between the two sequences, preferably at least
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • the full-length nucleotide sequence of the Mitl polypeptide of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications can be performed, and then the amplified fragments are spliced together in the correct order.
  • a polynucleotide sequence encoding a Mitl polypeptide can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In short, as long as it can replicate and stabilize in the host, any plasmid and The carrier can be used.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a Mitl polypeptide and a suitable transcriptional/translational control signal. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP).
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP).
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the yeast cell can be overexpressed by the transfer of the expression construct carrying the Mitl polypeptide-encoding gene; or the expression of the Mitl polypeptide can be enhanced by driving with a strong promoter; or the expression of the Mitl polypeptide can be enhanced by an enhancer.
  • Methanol inducible promoter Methanol inducible promoter
  • the methanol-inducible promoter relies on the addition of methanol to the medium to control the expression of the foreign polypeptide by the promoter.
  • the present inventors used the Mitl polypeptide to relieve the dependence of the methanol-induced promoter on methanol.
  • the methanol-inducible promoter includes, but is not limited to: (3 ⁇ 4 promoter, DH4S promoter (or Z ⁇ S promoter), FDH promoter (or FMDH promoter;), ⁇ promoter, A 0X2 promoter Sub, ZZA1, PEX5-, PEX8-, PEX14-J mover, PMP20 promoter, PMP47 promoter, 01)7 promoter, 01)2 promoter.
  • the invention further comprises at least 70%, more preferably at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identity to the polynucleotide sequence of the above promoter.
  • Promoter These promoters are strictly conserved at the sites necessary for initiation of transcription and the location of transcription initiation.
  • the present invention particularly relates to a polynucleotide which hybridizes under stringent conditions to the nucleotide sequence of the methanol-dependent promoter of the present invention, and which also functions as a wild type methanol-dependent promoter. Method for eliminating single methanol carbon source dependence of methanol-inducible promoter and driving expression of exogenous polypeptide encoding gene
  • the present invention also provides a method for eliminating the single methanol carbon source dependence of a methanol-inducible promoter to drive expression of a foreign polypeptide-encoding gene, comprising:
  • methanol-nutrient yeast comprising:
  • Expression cassette 1 which is capable of expressing an exogenous Mitl polypeptide; and expression cassette 2, comprising a methanol-inducible promoter and a foreign polypeptide encoding gene operably linked thereto;
  • the expression cassette 1 and the expression cassette 2 may be present in the same expression vector or may be present in different expression vectors.
  • the expression cassette 1 is present in single or multiple copies in methanolic yeast, for example 1-20 copies, such as 15, 10, 8, 6, 5, 3, 2 copies.
  • the expression cassette 2 may also be present in single or multiple copies in methanolic yeast, for example 1-20 copies, such as 15, 10, 8, 6, 5, 3, 2 copies.
  • a promoter and a coding gene for a Mitl polypeptide operably linked thereto are included. It is to be understood that any promoter which allows recombinant expression of the Mitl polypeptide in methanolic yeast can be used in the present invention. It is not limited to a constitutive promoter, an inducible promoter or a specific promoter. One skilled in the art can make appropriate selections depending on the desired purpose, for example, when it is desired to control the expression or non-expression of the Mitl polypeptide by certain conditional interventions, an inducible promoter can be selected.
  • the promoter comprises: a GAP promoter, a PGK1 promoter, a ⁇ promoter, and the like, any promoter capable of causing sputum expression.
  • methanol-free conditions are easily established by those skilled in the art, that is, in a conventionally used methanol-nutrient yeast culture medium, methanol is not added as a carbon source, and other types of carbon sources are substituted.
  • Commonly used carbon sources are well known to those skilled in the art, such as, but not limited to, glycerin, glucose, starch (containing starch hydrolyzate, tapioca starch, corn starch, cellulosic hydrolyzate, etc.), sucrose, maltose, and the like.
  • the yeast medium is used as a carbon source for glycerol and/or glucose.
  • non-single methanol carbon source conditions are also readily established by those skilled in the art. Recombinant methanolic yeast
  • a recombinant methanol-nutrient yeast comprising: an expression cassette 1 capable of expressing an exogenous MIT1 polypeptide; and an expression cassette 2 comprising methanol An inducible promoter and a foreign polypeptide encoding gene operably linked thereto.
  • the expression cassette 1 and the expression cassette 2 may be present in the same expression vector or may be present in different expression vectors.
  • the methanol-nutrient yeast includes, but is not limited to, Pichia pastoris (Pc/»' «), Hansenula ⁇ Hansenula), Candida (Om Wa), Sagittaria (73 ⁇ 4rw/c ⁇ )
  • the Pichia pastoris CPc/»'a) comprises: GS115, M/G7 gene and Pichia pastoris (A g7A g2) not expressed by MIG2 gene, NRG1 gene, MIG1 gene and MIG2 gene are not expressed Pichia pastoris (A g7A g2A «rg7), or Pichia strains that are not expressed by the gene HXS1.
  • strains capable of expressing a foreign polypeptide by using a non-methanol carbon source-inducing promoter are provided, GS115-MIT1, Amigl Amig2- ⁇ , ⁇ 7- ⁇ 1 and AmiglAmig2Anrgl-MlTl a
  • the wild-type strain GS115, the Pichia pastoris double-deletion strain AmiglAmig2, the Pichia pastoris deletion strain hxsl and the Pichia three-deletion strain migl mig2 nr g l overexpressed the transcriptional activation-related gene ⁇ , and the gene overexpression strain GS115 was constructed.
  • the total protein extraction technology of Pichia pastoris is based on the yeast protein extraction manual provided by Cold Spring Harbor.
  • Plasmids pGAPZaA, plasmid pPIC3.5k, E. coli ⁇ 10, Pichia pastoris strain GS115 were purchased from Invitrogen.
  • the plasmid pAG32 was obtained by excising the P ⁇ BFP-SKL expression system from the plasmid pRDM054 at the cleavage site Bgl II; the plasmid pRDM05 was obtained from the University of California, San Diego.
  • YPD medium 2% peptone, 1% yeast powder, 2% glucose, 2% agar powder; YNB medium: 0.67% YNB; MGY medium: 1% glycerol, 0.67% YNB; YND liquid medium: 1% glucose , 0.67% YNB.
  • pGAPZaA (Fig. 1) was used as a vector to insert the ⁇ gene (SEQ ID NO: 1, full-length 2667 bp) into the two cleavage sites of Asu WlSal I downstream of the GAP promoter.
  • the recombinant plasmid is referred to as the pGM plasmid.
  • the PpM/7 overexpression plasmid (pGMhph) was electrotransformed into Pichia pastoris strain GS115, and applied to 4 pieces of hygromycin-containing YPD solid medium, and cultured in a 30 ° C incubator for 48-72 hours.
  • the monoclonal cloned on the plate was picked into 10 ml of YPD+ hygromycin liquid medium, and cultured at 30 ° C on a shaker, and the genome was verified by PCR.
  • the correct Pichia strain was named GS115-MIT1 after PCR test and sequencing verification. 3.
  • Each strain was cultured in YNB liquid medium supplemented with 0.5% (v/v) methanol, 1% glycerol, 1% glycerol + 0.5% (v/v) methanol as carbon source, and sampled after growth to logarithmic growth phase.
  • Ml adding Aoxl enzyme activity to the color of the cells after centrifugation (see Stasyk OV, TY Nazarko, and AA Sibirny. 2008. Methods of Plate Pexophagy Monitoring and Positive Selection for ATG Gene Cloning in Yeasts. Methods in enzymology .451: 229-239.
  • the chromogenic substrate is o-dianisidine).
  • Each strain was pre-incubated overnight in MGY liquid medium and then transferred to contain 0.5% (v/v) methanol, 1% glycerol, 1% glycerol + 0.5% (v/v) methanol, 0.5% (v/v)
  • the YNB liquid medium containing methanol as a carbon source was sampled and extracted for 10 hours, and the total protein was sampled and quantified by Bradford method. The method was carried out to determine the activity of Aoxl (see Verduyn, C., JP van Dijken, and WA Scheffers. 1984). J. Microbiol. Methods 2:15_25.
  • the chromogenic substrate is 2,2, -azino-di-(3-ethylbenzthiazoline sulfonate), ABTS).
  • the strain GS115-MIT1 can induce the expression of Aoxl in the medium containing glycerol, and the wild strain could not induce the expression of Aox.
  • the GFP gene (full length 714 bp) was inserted into the S «aB I restriction site downstream of the ⁇ promoter of the vector pPIC3.5k (Fig. 9) to obtain the GFP expression vector pP-GFP.
  • the expression vector pP-GFP was electroporated with GS115-MIT1 strain, and applied to a histidine-free YND plate, and cultured in a 30 °C incubator for 48-72 hours. The monoclonal cloned on the plate was picked into a liquid medium, and the genome was shaken at 30 ° C, and the GFP copy number was verified by real-time PCR (for the method, see Xuan, YJ, XS Zhou, WW Zhang, X. Zhang). , ZW Song, and YX Zhang. 2009. An upstream activation sequence controls the expression of AOXl gene in Pichia pastoris. FEMS Yeast Res. 9: 1271-1282). The real-time PCR test was performed as a single copy of the Pichia pastoris GFP expression strain, which was named GS115-MIT1-GFP.
  • strain A g7A g2-MITl-GFP and A/zx ⁇ -MITl-GFP were constructed in the same manner.
  • the construction method of the expression strain A g7A g2-MIT1-GFP was specifically as follows: The expression vector pP-GFP and the expression vector pGMhph were co-transformed into the A g7A g2 strain (see Chinese Laid-Open Patent No. CN101857845A).
  • the construction method of the expression strain A/?x?7-MITl-GFP was specifically as follows: the expression vector pP-GFP and the expression vector pGMhph were co-transformed into the ⁇ / ⁇ - ⁇ strain.
  • the strain GS115-MIT1-GFP was pre-incubated overnight in YND liquid medium, and then transferred with 0.5% ( ⁇ / ⁇ ) methanol, 1% glycerol, 1% glycerol + 0.5% ( ⁇ / ⁇ ) methanol as carbon source.
  • the medium was cultured in a liquid medium, and the geometric mean fluorescence intensity of GFP in the sample was measured by flow cytometry after sampling.
  • GS115-MIT1 can induce expression of exogenous polypeptide GFP in the presence of glycerol.
  • Example 2 Glycerol or glucose can induce the expression of the P promoter of Pichia pastoris
  • the Zeocin resistance gene Sh ble fragment was excised from the plasmid pGAPZaA by B-111 and Sal I enzymes, and the Zeocin resistance gene was inserted into the pUC18 plasmid (Fig. 10) and the Zeocin resistance gene was inserted at BamH I and Sal I.
  • the /e fragment was named pUC18-b/e plasmid.
  • primers HS1-5F/HS1-5R were used to amplify the 5'-end peripheral promoter region of the PpH gene (starting codon ATG), and the size of the amplified product was 728 bp.
  • the primers HS1-3F/HS1-3R were used to amplify the 3'-end region of the PpH ⁇ S7 gene (downstream of the stop codon), and the size of the amplified product was 1011 bp, and the size fraction was cut by gel.
  • the 5'-end peripheral promoter DNA fragment of the recovered PpH W gene was digested with coRI and BamlU, and the fragment was recovered.
  • the pUC18-b/e plasmid was digested with coRI and S , and the fragment was recovered.
  • the recovered two fragments were ligated overnight, transformed into E. coli, and positive clones were verified by PCR, and named pUC18-(HS7 5'-ble
  • the plasmids Sa/I and S W were double-digested in the previous step, and the fragments were recovered.
  • the 3'-end region DNA fragment of the PpH ⁇ S7 gene was also digested with SpW. The two fragments were ligated overnight and transformed into E. coli, a positive PCR strain, and the positive strain was named pUC18-(HS7 '-ble-HSl 3').
  • the pUC18-(HS75 '-b!e-HSl 3 ') plasmid was digested with coRI and SpW, and the 3,350 bp PpH ⁇ S7 knockout fragment HS75'-b!e-HSl 3 ' was recovered by gel.
  • the recovered 20 ⁇ PpH S7 knockout fragment HS75′-ble-HSl 3′ (concentration greater than 50 ng/ ⁇ ) was mixed with 80 ⁇ Pichia pastoris GS115 competent on ice, and electroporation was carried out according to the method provided in the experimental procedure. .
  • the cells were cultured in YP+Glycerol liquid medium containing Zeocin for 2 to 3 days. PCR verified the positive strain of PpHXSJ knockout, named Ahxs
  • the plasmid pGMhph was electrotransformed into Pichia pastoris strains migl mi g 2 and hxsl, and applied to 4 hygromycin-loaded YPD plates, and cultured in a 30 ° C incubator for 48-72 hours.
  • the monoclonal cloned on the plate was picked into 10 ml of YPD + hygromycin liquid medium, and cultured at 30 ° C on a shaker, and the extracted genome was verified by PCR.
  • the correct Pichia strains were named A g7A g2-MITl and Ahxsl-MIT ⁇ 0 after PCR test and sequencing verification.
  • the medium was cultured in a liquid medium, and 1 ml was sampled after growing to a logarithmic growth phase, and Aoxl enzyme activity coloring solution was added to the cells after centrifugation to develop color.
  • Each strain was used as a carbon source in 1% glycerol, 1% glycerol + 0.5% (v/v) methanol, 1% glucose, 1% glucose + 0.5% (v/v) methanol and 0.5% (v/v) methanol.
  • the YNB liquid medium was cultured, and after growing to the logarithmic growth phase, total protein was sampled and extracted, and the protein was quantified by Bradford method to measure the activity of Aoxl.
  • the strain AmigJ ⁇ mig2-M and AhxsJ-MlTl can induce the expression of Aoxl in the medium containing glycerol or glucose, and the wild strain could not induce the expression of Aox.
  • AmigJ mig2-MVT1 and ⁇ 7- ⁇ 1 can induce expression of exogenous polypeptide GFP in the presence of glycerol or glucose.
  • Example 5 Pichia pastoris strain Amigl Amig2 Anrgl-MIT 1 in which glycerol or glucose can induce AOX1 promoter expression
  • primers 5, NRG1-F/5, NRG1-R were used to amplify the 5'-end region of the PpNIG1 gene to obtain a 320 bp fragment, and the amplification product was named 5, brain 1, and gel recovery 5WRG7. Fragment.
  • the 5WRG7 fragment obtained above was digested with Sac I and S al, and ligated with the same Sac I and Sma I digested pUC18 plasmid to construct pUC18 (S ⁇ 3cI-5, NRG1-S aI).
  • the vector pUC18 (SacI-5'NRG1-S aI) was transformed into E. coli competent state, and colonies were screened for positive clones by PCR.
  • the plasmid pRDM054 was used as a template to amplify the hygromycin B resistance gene, which was 1648 bp in size.
  • the HPH fragment obtained above was digested with Sma I and ⁇ , and ligated with the same Sma I and H double-digested vector pUC18 (SacI-5'NRG1-S aI) to construct vector pUC 18 (SacI-5'NG 1 -Smal -UPU-Xbal).
  • pUC 18 (SacI-5 'NRG 1 -Smal- ⁇ -baI) was transformed into E. coli, and colonies were screened for positive clones by PCR.
  • NRG1-F/NRG1-A1 the plasmid pUC18 (SacI-5, NRG1-S aI-HPH- ⁇ 3 ⁇ 4aI) was used as a template to obtain the fragment Overlap-A.
  • the fragment Overlap-B was amplified by using the primers NRG1-B2/3, NRG1-R and the GS115 genome as a template. Fragments A and B were then linked by the Overlap PCR method to obtain 5, NRG1-HPH-3, NRG1 fragments.
  • This fragment was ligated to the pMD19-T vector to obtain a knockout plasmid pMD 19-T (SacI-5 'NRG 1 -SmaI-HPH-3 'NRG 1 -Sph ⁇ ).
  • MIT1 overexpression plasmid in three deletion strains
  • the pPG plasmid was obtained by inserting the GAP promoter into the Sac 1/BamH I two cleavage sites with pPIC3.5k as a vector.
  • the PpMPP1 gene sequence was inserted between the two cleavage sites of BamHI/Notl, and the resulting recombinant plasmid was designated as pPGPP1 plasmid.
  • the plasmid pPGPP1 was electrotransformed into Pichia pastoris strain mi g l mi g 2 g l and applied to 4 without his
  • the YND plate was placed in a 30 ° C incubator for 48-72 hours.
  • the monoclonal cloned on the plate was picked into 10 ml of YND liquid medium, and cultured at 30 ° C on a shaker, and the extracted genome was verified by PCR.
  • the correct Pichia strains were identified as migl Am ig2 Anrgl -MIT 1 0 after PCR test and sequencing verification.
  • the expression vector pP-GFP was electroporated with GS11+-MIT1 strain, and applied to a histidine-free YND plate, and cultured in a 30 °C incubator for 48-72 hours. The monoclonal cloned on the plate was picked into MGY liquid medium, and green fluorescence was observed under a fluorescence microscope. The fluorescent transformant was cultured at 30 ° C shaker and the genome was extracted, and the GFP copy number was verified by real-time PC. (See Xuan, YJ, XS Zhou, WW Zhang, X. Zhang, ZW Song, and YX Zhang. 2009. An upstream activation sequence controls the expression of AO J gene in Pichia pastoris. FEMS Yeast Res.9: 1271-1282 ). The real-time PC was tested as a single copy of the Pichia pastoris GFP expression strain named Am igl Amig2 Anrgl - MIT 1 -GFP.
  • the methanol was cultured in a YNB liquid medium of carbon source, and after growing to a logarithmic growth phase, total protein was sampled and extracted, and the protein was quantified by Bradford method to measure the activity of Aoxl.
  • strain AmigJAmig2AnrgJ- ⁇ can induce the expression of Aoxl in the medium containing glycerol or glucose, and the wild strain can not induce the expression of Aox.
  • a g7A g2A «rg7-MITl-GFP was pre-incubated overnight in liquid medium and then transferred to methanol containing 0.5% (v/v), 1% glycerol, 1% glycerol + 0.5% (v/v) methanol, respectively. 1% glucose and 1% glucose + 0.5% (v/v) methanol was used as the carbon source in YNB liquid medium. After sampling, the geometric mean fluorescence intensity of GFP in the sample was detected by flow cytometry.
  • a g7A g2A «rg7-MITl can induce expression of the exogenous polypeptide GFP in the presence of glycerol or glucose.
  • Table 3 Primers used for N/G7 knockout
  • NRG1-A1 21 5

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Abstract

提供了一种消除甲醇诱导型启动子单一甲醇碳源依赖性而表达外源多肽的方法。该方法通过在甲醇营养型酵母细胞内增加Mit1多肽的表达量，激活需要甲醇诱导的启动子的表达，使得原本依赖甲醇诱导的启动子不再依赖单一性甲醇也可以表达外源多肽。

Description

消除甲醇诱导型启动子单一甲醇碳源依赖性的方法
技术领域
本发明属于生物工程领域； 更具体地， 本发明涉及一种消除甲醇诱导型启动 子单一甲醇碳源依赖性而表达外源多肽的方法。 背景技术
甲醇营养型酵母（包括尸c/n'a， Hansenula, Candida, Toru psis等）彔达系统 由于其高效的外源多肽表达能力已经在工业生产、制药等领域被非常广泛的运用。 其特点在于， 它们拥有能够被甲醇高效诱导的启动子 启动子、 DH4S启动 子、 FDH启动子、 启动子、 ^ Ώ启动子、 ZZA1、 PEX5-、 PEX8-、 PEX14- 启动子等)， 并且这些启动子严格依赖甲醇， 其它碳源 (如葡萄糖， 甘油等)会抑制 这些启动子的表达。
随着石油危机的爆发， 利用甲醇生产单细胞蛋白， 成本变高。 之所以利用甲 基营养型母酵母表达系统表达如此之多的外源多肽， 是因为其具有其他表达系统 不可比拟的优势： 基因操作简单， 外源多肽表达量高， 既可以胞内表达， 也可以 分泌表达； 外源多肽基因遗传稳定； 作为真核表达系统， 具有真核生物的亚细胞 结构， 具有糖基化、 脂肪酰化、 蛋白磷酸化等翻译后修饰加工功能；
由于被利用的范围越来越广，在实际的发酵放大过程中遇到很多问题： (1 ) 表 达所用的启动子需要甲醇的诱导， 甲醇有毒易燃， 在大规模工业发酵中需要进行 特别的防暴设计； （2) 甲醇发酵强耗氧， 一般靠增大空气的通气量和提高转速很 难满足氧气的需求而需要通纯氧， 甲醇代谢需要氧气的量是葡萄糖为碳源所需氧 气量的三至四倍。 消耗的甲醇越多需要的纯氧越多， 这给实际的工业化生产带来 很大的麻烦。 另外消耗的甲醇越多所产的热量也越大， 所需设备的冷却能力要求 越高；（3) 甲醇作为一种石油化学产品，不适合于一些食品领域添加剂的生产； （4) 甲醇代谢会产生 ¾02， 会导致所表达多肽的水解。
因此， 如果得到一种能够利用非甲醇诱导甲醇诱导型启动子表达的方法， 使 原本依赖甲醇诱导的启动子的高效转录不依赖于甲醇， 而可用其他碳源来诱导这 些启动子的表达，这对于实现工业上利用非甲醇高效表达外源多肽具有积极意义。 发明内容
本发明的目的在于提供一种消除甲醇诱导型启动子单一甲醇碳源依赖性而驱 动甲醇诱导型启动子表达外源多肽的方法。
本发明的另一目的还在于提供重组的甲醇营养型酵母， 其可以利用消除甲醇 诱导型启动子单一甲醇碳源依赖性来表达外源多肽。
在本发明的第一方面， 提供一种消除甲醇诱导型启动子单一甲醇碳源依赖性 而驱动外源多肽编码基因表达的方法， 包括：
(1) 提供甲醇营养型酵母， 所述甲醇营养型酵母含有：
表达盒 1， 其表达外源的 Mitl多肽； 以及
表达盒 2， 包括甲醇诱导型启动子及与之操作性连接的外源多肽编码基因；
(2) 在无甲醇条件或非单一甲醇碳源条件下培养 (1)的甲醇营养型酵母。
在一个优选例中， 所述的 Mitl多肽是转录激活相关因子， 使得原本依赖甲醇 诱导的启动子不再依赖甲醇也可表达外源多肽。
在另一优选例中， 所述的 Mitl多肽的功能为： 激活甲醇代谢中需要甲醇诱导 的启动子表达。
在另一优选例中， 所述的 Mitl是 PpMitl。
在另一优选例中，所述表达盒 2在甲醇营养型酵母中以单拷贝或多拷贝存在， 例如 1-20拷贝， 如 15， 10， 8， 6， 5， 3， 2拷贝。
在另一优选例中， 所述的甲醇诱导型启动子包括（但不限于）： ^ Ώ启动子、 DH4S启动子、 Z 4S启动子、 FDH启动子、 FMDH启动子、 MOT启动子、 AOX2 启动子、 ZZA1、 PEX5-, PEX8-、 尸 ^ 4-启动子、 尸kff20启动子、 ΡΑίΡ47启动子、 ^007启动子、 ^OD2启动子。
在另一优选例中，所述的甲醇营养型酵母包括 （但不限于)：毕赤酵母 (Pc/»'«)， 汉逊酵母 (H<m ^ /")， 假丝酵母 (Ow Wa), 球拟酵母 较佳地， 所述 的毕赤酵母 CPc/»'a)包括 (但不限于)： GS115, 基因和 基因不表达的毕 赤酵母 (A g7A g2)， NRG1 基因、 MIG1 基因和 MIG2 基因不表达的毕赤酵母 (AmiglAmig2Anrgl), 或 HYS7基因不表达的毕赤酵母菌株。
在另一优选例中， 述的 Mitl多肽选自下组：
(a) SEQ ID NO: 2所示氨基酸序列的多肽； 或
(b) 将 SEQ ID NO: 2所示氨基酸序列经过一个或多个 (如 1-30个，更佳地 1-20 个， 更佳地 1-15个， 更佳地 1-10个， 更佳地 1-5个或 1-3个)氨基酸残基的取代、 缺失或添加而形成的， 且具有利用非甲醇碳源诱导甲醇诱导型启动子表达外源多 肽功能的由 衍生的多肽； (c) 与 (a)限定的序列在具有 70%以上 (较佳地 80%以上， 更佳地 90%以上， 更 佳地 95%以上， 更佳地 98%以上， 如 99%或更高)的序列相同性的， 且具有利用非 甲醇碳源诱导甲醇诱导型启动子表达外源多肽功能的由 (a)衍生的多肽。
在另一优选例中，所述的 Mitl多肽的编码基因的 NCBI Reference Sequence为 XM— 002493021.1。
在另一优选例中，所述的 Mitl多肽的编码基因具有 SEQ ID NO: 1所示的核苷 酸序列。
在另一优选例中， 所述的 Mitl 多肽的编码基因在甲醇存在的情况下诱导表 达， 在甘油、 葡萄糖等碳源存在的情况下不表达。
在另一优选例中，所述的表达盒 1中，包括启动子以及与之操作性连接的 Mitl 多肽的编码基因； 较佳地， 该启动子包括 (但不限于)： 组成型启动子， 诱导型启 动子， 组织或器官特异性启动子， 时空特异性表达启动子。
在另一优选例中，表达盒 1中，所述启动子包括 (但不限于）： 启动子、 启动子、 M/7 启动子等任意能使 M/7 过表达的启动子。
在另一优选例中， 所述的表达盒 1在甲醇营养型酵母中以单拷贝或多拷贝存 在， 例如 1-20拷贝， 如 15， 10， 8， 6， 5， 3， 2拷贝。
在另一优选例中， 步骤 (2)中， 釆用存在甘油和 /或葡萄糖的酵母培养基。
在本发明的另一方面， 提供 Mitl多肽或其编码基因的用途， 用于消除甲醇诱 导型启动子单一甲醇碳源依赖性而驱动外源多肽编码基因表达。
在本发明的另一方面， 提供一种重组的甲醇营养型酵母， 所述甲醇营养型酵 母含有：
表达盒 1， 其能够表达外源的 Mitl多肽； 以及
表达盒 2， 包括甲醇诱导型启动子及与之操作性连接的外源多肽编码基因。 在一个优选例中，所述的甲醇营养型酵母包括 （但不限于)：毕赤酵母 (P c/»'«)， 汉逊酵母 (H<m«? /")， 假丝酵母 (Ow Wa), 球拟酵母 (rorw/o W; 较佳地， 所述 的毕赤酵母 CP c/»'a)包括 (但不限于)： GS 1 15 , 基因和 基因不表达的毕 赤酵母 (A g7A g2)， NRG1 基因、 MIG1 基因和 MIG2 基因不表达的毕赤酵母 (AmiglAmig2Anrgl), 或 HXS1基因不表达的毕赤酵母菌株。
在一个优选例中， 所述的所述的 MIG1基因的核苷酸序列如 SEQ ID NO: 9所 示；
所述的 基因的核苷酸序列如 SEQ ID NO: 10所示； 或 所述的 HXS1基因的核苷酸序列如 SEQ ID NO: 3所示。
所述的 NRGl 基因的核苷酸序列如 SEQ ID NO: 8 所示 (NCBI Reference Sequence: XM— 002493138.1)。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易 见的。 附图说明
图 1、 pGAPZaA的质粒图谱示意图。
图 2、 载体 pAG32的质粒图谱示意图。
图 3、 野生型及 GS115-MIT1分别在非甲醇或含甲醇 (甘油 +甲醇)的培养基中 培养， 并进行 Aox显色反应的结果。
图 4、 野生型及 GS115-MIT1 分别在非甲醇或含甲醇的培养基中培养， 并进 行 Aox酶活测定的结果。
图 5、菌株 GS115-MIT1-GFP在 YND液体培养基中过夜预培养， 然后分别转 移至非甲醇或含甲醇的培养基中培养， 取样后用流式细胞仪检测样品中 GFP的几 何平均荧光强度。
图 6、 野生型及 AmigJAmig2-Mm和 A/? y7-MITl菌株分别在非甲醇或含甲 醇的培养基中培养， 生长到对数生长期后取样 lml， 在离心后的菌体中加入 Aox 酶活显色液显色。
图 7、 野生型及 A g7A g2-MITl和 xy7-MIT1菌株分别在非甲醇或含甲 醇的培养基中培养， 生长到对数生长期后取样提取总蛋白， 经 Bradford法蛋白定 量后进行 Aox酶活的测定。
图 8、 野生型及 AmigJAmig2-Mm和 A/? y7-MITl菌株分别在非甲醇或含甲 醇的培养基中培养， 取样后用流式细胞仪检测样品中 GFP的几何平均荧光强度。
图 9、 载体 pPIC3.5K的质粒图谱示意图。
图 10、 载体 pUC18的质粒图谱示意图
图 11、野生型及 A g7A g2^rg7-MITl菌株分别在非甲醇或含甲醇的培养 基中培养， 生长到对数生长期后取样提取总蛋白， 经 Bradford法蛋白定量后进行 Aox酶活的测定。
图 12、野生型及 ^migJ^mig2^nrgJ-Mm菌株分别在非甲醇或含甲醇的培养 基中培养， 取样后用流式细胞仪检测样品中 GFP的几何平均荧光强度。 具体实施方式
为了克服目前甲醇诱导型启动子在发酵时对于甲醇诱导的依赖性， 本发明人 经过深入的研究，揭示了一种消除甲醇诱导型启动子单一甲醇碳源依赖性的方法， 通过在甲醇营养型酵母细胞内增加 Mitl (甲醇诱导的转录因子 1； Methanol Induced Transcription Factor 1)多肽的表达量，激活甲醇代谢中需要单一甲醇诱导的启动子 表达， 以此使得原本依赖甲醇诱导的启动子不再依赖甲醇也可表达外源多肽。 术语
如本文所用， 所述的 "启动子" 是指一种核酸序列， 其通常存在于目的基因 编码序列的上游 (5'端）， 能够引导核酸序列转录为 mRNA。 一般地， 启动子或启 动子区提供 RNA 聚合酶和正确起始转录所必需的其它因子的识别位点。 在本文 中， 所述的启动子或启动子区包括启动子的活性变异体， 该变异体可以是天然发 生的等位变异体或非天然发生的变异体。 所述变异体包括取代变异体、 缺失变异 体和插入变异体。
如本文所用， 所述的 "甲醇诱导型启动子"是与甲醇代谢有关的酶的启动子。 在现有技术中， 这些启动子通过向生长培养基中添加甲醇， 可以控制外源多肽由 所述启动子的表达。 所述的 "甲醇诱导型启动子" 可以由本领域技术人员使用常 规技术从酵母中分离获得。
如本文所用， 所述的 "单一甲醇 (碳源)诱导" 是指启动子需要以甲醇为唯一 碳源进行诱导来驱动与之操作性连接的基因的表达，在非甲醇为唯一碳源 (如甲醇 +葡萄糖)的条件下不能驱动与之操作性连接的基因的表达。 所述的 "消除单一甲 醇诱导" 是指使得启动子在不以甲醇为唯一碳源 (如甲醇 +葡萄糖， 或葡萄糖， 或 甘油)的条件下就可驱动与之操作性连接的基因的表达。 "非单一甲醇 (碳源)诱导" 是指除了甲醇碳源外， 还存在至少一种非甲醇的碳源。
如本文所用， 所述的 "组成型启动子" 是指在其调控下不同组织器官和发育 阶段的基因表达没有明显差异的一类启动子。
如本文所用， 所述的 "诱导型启动子" 可根据需要在特定细胞生长阶段或特 定生长环境下， 快速诱导基因转录的 "开" 与 "关" 或者 "高" 与 "低" 。 根据 来源， 可将诱导型启动子分为天然存在的启动子和人工构建的启动子。 如本文所用， 所述的 "组织或器官特异性启动子" 是指基因转录一般只发生 在某些特定器官或组织中的启动子。
如本文所用， "外源的" 或 "异源的" 是指来自不同来源的两条或多条核酸 或蛋白质序列之间的关系。 例如， 如果启动子与目的基因序列的组合通常不是天 然存在的， 则启动子对于该目的基因来说是外源的。 特定序列对于其所插入的细 胞或生物体来说是 "外源的" 。
如本文所用， 所述的 "表达盒"是指包含有表达目的多肽 (本发明中为外源多 肽或 Mitl 多肽)所需的所有必要元件的基因表达系统， 通常其包括一下元件： 启 动子、 编码多肽的基因序列， 终止子； 此外还可选择性包括信号肽编码序列等。 这些元件是操作性相连的。
如本文所用， 所述的 "甲醇营养型酵母" 是指可利用甲醇作为唯一碳源的酵 母。 包括来自汉逊酵母属（Ha em a) , 毕赤酵母属 CP c/»'a)， 球拟酵母属
(Torulopsis) , 假丝酵母属（Ow Wa)等的酵母。
如本文所用， 所述的 "可操作性连接" 是指两个或多个核酸区域或核酸序列 的功能性的空间排列。 例如： 启动子区被置于相对于目的基因核酸序列的特定位 置， 使得核酸序列的转录受到该启动子区域的引导， 从而， 启动子区域被 "可操 作地连接" 到该核酸序列上。
如本文所用， 术语 "严格条件"是指： （1)在较低离子强度和较高温度下的杂 交和洗脱， 如 0.2 X SSC , 0.1%SDS， 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(v/v) 甲酰胺， 0.1 %小牛血清 /0.1 %Ficoll， 42°C等； 或 (3)仅在两条序列之间的相同性至 少在 50%， 优选 55%以上、 60%以上、 65%以上、 70%以上、 75%以上、 80%以上、
85%以上或 90%以上， 更优选是 95%以上时才发生杂交。
如本文所用， 所述的 "含有" ， "具有" 或 "包括"包括了 "包含" 、 "主 要由 构成" 、 "基本上由 ......构成" 、 和 "由 构成" ； "主要由 ......构 成" 、 "基本上由 ......构成"和 "由 ......构成" 属于 "含有" 、 "具有 " 或 "包 括" 的下位概念。
Mitl多肽
本发明揭示了 Mitl 多肽在消除甲醇诱导型启动子单一甲醇碳源依赖性而驱 动外源多肽编码基因表达中的应用。在对依赖甲醇控制外源多肽表达的启动子（甲 醇诱导型启动子)的研究工作中， 本发明人意外发现 Mitl 多肽可以解除甲醇诱导 型启动子对于甲醇的依赖性， 使其可利用非单一甲醇碳源或非甲醇碳源进行外源 多肽的表达。
本发明还包括所述的 Mitl多肽的片段、 衍生物和类似物。 如本文所用， 术语 "片段" 、 "衍生物" 和 "类似物" 是指基本上保持本发明的 MIT1多肽相同的 生物学功能或活性的多肽。 本发明的多肽片段、 衍生物或类似物可以是 (i)有一个 或多个保守或非保守性氨基酸残基 (优选保守性氨基酸残基)被取代的多肽， 而这 样的取代的氨基酸残基可以是也可以不是由遗传密码编码的， 或 (ii)在一个或多个 氨基酸残基中具有取代基团的多肽， 或 (iii)成熟多肽与另一个化合物 (比如延长多 肽半衰期的化合物， 例如聚乙二醇)融合所形成的多肽， 或 (iv)附加的氨基酸序列 融合到此多肽序列而形成的多肽 (如前导序列或分泌序列或用来纯化此多肽的序 列或多肽原序列， 或融合多肽)。 根据本文的定义这些片段、 衍生物和类似物属于 本领域熟练技术人员公知的范围。
在本发明中， 术语 "Mitl多肽"指具有利用非甲醇碳源诱导甲醇诱导型启动 子表达外源多肽功能的 SEQ ID NO: 2序列的多肽。该术语还包括具有利用非甲醇 碳源诱导甲醇诱导型启动子表达外源多肽功能的、 SEQ ID NO: 2序列的变异形式。 这些变异形式包括 (但并不限于)： 若干个 (通常为 1-50个， 较佳地 1-30个， 更佳 地 1-20个， 最佳地 1-10个， 还更佳如 1-8个或 1-5个)氨基酸的缺失、 插入和 /或 取代， 以及在 C末端和 /或 N末端添加或缺失一个或数个 (通常为 20个以内， 较佳 地为 10个以内， 更佳地为 5个以内)氨基酸。 例如， 在本领域中， 用性能相近或 相似的氨基酸进行取代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 / 或 N末端添加或减少一个或数个氨基酸通常也不会改变蛋白质的功能。
多肽的变异形式包括： 同源序列、 保守性变异体、 等位变异体、 天然突变体、 诱导突变体、 在高或低的严紧度条件下能与编码 Mitl多肽的 DNA杂交的 DNA 所编码的多肽、 以及利用抗 Mitl多肽的抗血清获得的多肽或蛋白。本发明还提供 了其他多肽， 如包含 Mitl多肽或其片段的融合多肽。
本发明还提供 Mitl多肽或多肽的类似物。 这些类似物与天然 Mitl多肽的差 别可以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者 兼而有之。 这些多肽包括天然或诱导的遗传变异体。 诱导变异体可以通过各种技 术得到， 如通过辐射或暴露于诱变剂而产生随机诱变， 还可通过定点诱变法或其 他已知分子生物学的技术。 类似物还包括具有不同于天然 L-氨基酸的残基 (如 D- 氨基酸)的类似物， 以及具有非天然存在的或合成的氨基酸 (如 β、 Υ -氨基酸)的类 似物。 应理解， 本发明的多肽并不限于上述例举的代表性的多肽。
本发明还提供了编码本发明 Mitl多肽或其保守性变异多肽的多核苷酸序列。 本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、基 因组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编 码链或非编码链。编码成熟多肽的编码区序列可以与 SEQ ID NO: 1序列相同或者 是简并的变异体。 如本文所用， "简并的变异体"在本发明中是指编码具有 SEQ
ID NO: 2序列或其变异形式的蛋白，但与 SEQ ID NO: 1中的编码区序列有差别的 核酸序列。
本发明还涉及上述多核苷酸的变异体， 其编码与本发明有相同的氨基酸序列 的多肽或多肽的片段、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的 等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变 异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质上改变其编码的 多肽的功能。
本发明还涉及与上述多核苷酸同源的多核苷酸， 它们是与上述多核苷酸具有 至少 50%，较佳地至少 60%，更佳地至少 70%，更佳地至少 80%相，更佳地至少 90%; 更佳地至少 95%; 更佳地至少 98%或 99%相同性的多核苷酸。这些多核苷酸所编码 的蛋白也具有与前述多核苷酸所编码的多肽相同的利用非甲醇碳源诱导甲醇诱导 型启动子表达外源多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少
70%， 更佳地至少 80%相同性的多核苷酸。 本发明特别涉及在严格条件下与本发 明所述多核苷酸可杂交的多核苷酸。
本发明的 Mitl多肽核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组 法或人工合成的方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸 序列， 尤其是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术 人员已知的常规方法所制备的 cDNA库作为模板， 扩增而得有关序列。 当序列较 长时， 可进行两次或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序拼接 在一起。
本发明中，编码 Mitl多肽的多核苷酸序列可插入到重组表达载体中。术语"重 组表达载体" 指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺 乳动物细胞病毒或其他载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和 载体都可以用。 表达载体的一个重要特征是通常含有复制起点、 启动子、 标记基 因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含 Mitl多肽编码 DNA序列和合适 的转录 /翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成 技术、 体内重组技术等。 所述的 DNA序列可有效连接到表达载体中的适当启动 子上， 以指导 mRNA合成。 表达载体还包括翻译起始用的核糖体结合位点和转录 终止子。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转 化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以 及绿色荧光蛋白 (GFP)。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于 转化适当的宿主细胞， 以使其能够表达蛋白质。
增加 Mitl多肽表达的方法是本领域周知的。 例如， 可通过转入携带 Mitl多 肽编码基因的表达构建物使酵母细胞过表达 Mitl多肽；或可通过用强启动子驱动 从而增强 Mitl多肽的表达； 或者通过增强子来增强该 Mitl多肽的表达。 甲醇诱导型启动子
在现有常规技术中， 所述的甲醇诱导型启动子依赖于在培养基中添加甲醇， 从而控制外源多肽由所述启动子的表达。而本发明人利用 Mitl多肽解除了甲醇诱 导型启动子对于甲醇的依赖。
本领域技术人员熟悉甲醇依赖型启动子，可使用常规技术从酵母中分离获得。 由于甲醇依赖型启动子具有基本相同的工作机制和工作原理， 本发明对于甲醇依 赖型启动子的种类没有特别的限制。 例如， 所述的甲醇诱导型启动子包括但不限 于： (¾ 启动子、 DH4S启动子 (或者 Z^S启动子）、 FDH启动子 (或者 FMDH启 动子；)、 ΜλΤ启动子、 A 0X2启动子、 ZZA1、 PEX5-, PEX8-、 PEX14-J 动子、 PMP20 启动子、 PMP47启动子、 01)7启动子、 01)2启动子。
本发明还包括与上述启动子的多核苷酸序列具有至少 70%， 更佳地至少 80% (例如 85%、 90%、 95%、 96%、 97%、 98%、 或 99%) 相同性的启动子。 这些启动 子在引发转录的必要位点及转录起始点的位置上是严格保守的。 本发明特别涉及 在严格条件下与本发明所述甲醇依赖型启动子的核苷酸序列可杂交的多核苷酸， 且该多核苷酸也具有野生型甲醇依赖型启动子的功能。 消除甲醇诱导型启动子单一甲醇碳源依赖性而驱动外源多肽编码基因表达 的方法
本发明还提供了一种消除甲醇诱导型启动子单一甲醇碳源依赖性而驱动外源 多肽编码基因表达的方法， 包括：
(1) 提供甲醇营养型酵母， 所述甲醇营养型酵母含有：
表达盒 1， 其能够表达外源的 Mitl多肽； 以及表达盒 2， 包括甲醇诱导型启 动子及与之操作性连接的外源多肽编码基因；
(2) 在无甲醇条件或非单一甲醇碳源条件培养 (1)的甲醇营养型酵母。
所述的表达盒 1和表达盒 2可以存在于同一表达载体中， 也可以存在于不同 的表达载体中。
所述表达盒 1在甲醇营养型酵母中以单拷贝或多拷贝存在， 例如 1-20拷贝， 如 15， 10， 8， 6， 5， 3， 2拷贝。 所述的表达盒 2在甲醇营养型酵母中也可以单 拷贝或多拷贝存在， 例如 1-20拷贝， 如 15， 10， 8， 6， 5， 3， 2拷贝。
所述的表达盒 2中，包括启动子以及与之操作性连接的 Mitl多肽的编码基因。 应理解，任何可使得 Mitl多肽在甲醇营养型酵母中重组表达的启动子都可用于本 发明。 不限于组成型启动子、 诱导型启动子或特异性启动子。 本领域技术人员可 以根据所需达到的目的进行合适的选择， 例如当希望通过一定的条件干预来控制 Mitl多肽的表达或不表达， 则可以选择诱导型启动子。 较佳地， 表达盒 2中， 所 述启动子包括： GAP启动子、 PGK1启动子、 ΜΓΠ启动子等任意能够使得 ΜΓΠ过 表达的启动子。
步骤 (2)中， "无甲醇的条件"是本领域技术人员易于建立的， 也即， 在常规 应用的甲醇营养型酵母培养基中， 不加入甲醇作为碳源， 取代以其它类型的碳源， 常用的碳源是本领域技术人员熟知的， 例如但不限于： 甘油、 葡萄糖、 淀粉 (含淀 粉水解液， 木薯淀粉， 玉米淀粉， 纤维素类水解液等)、 蔗糖、 麦芽糖等。 较佳地， 釆用以甘油和 /或葡萄糖为碳源的酵母培养基。 类似地， "非单一甲醇碳源条件" 也是本领域技术人员易于建立的。 重组甲醇营养型酵母
基于本发明的新发现， 还提供了一种重组的甲醇营养型酵母， 所述甲醇营养 型酵母含有： 表达盒 1， 其能够表达外源的 MIT1多肽； 以及表达盒 2， 包括甲醇 诱导型启动子及与之操作性连接的外源多肽编码基因。 附加条件是， 所述的表达 盒 1和表达盒 2可以存在于同一表达载体中， 也可以存在于不同的表达载体中。
任何甲醇营养型酵母均可被应用于本发明中， 以构建上述重组的甲醇营养型 酵母。 例如， 所述的甲醇营养型酵母包括但不限于： 毕赤酵母 (Pc/»'«)， 汉逊酵母 {Hansenula), 假丝酵母 (Om Wa), 球拟酵母 (7¾rw/c^^); 较佳地， 所述的毕赤酵 母 CPc/»'a)包括： GS115，M/G7基因和 MIG2基因不表达的毕赤酵母 (A g7A g2)， NRGl基因、 MIGl基因和 MIG2基因不表达的毕赤酵母 (A g7A g2A«rg7)， 或 基因 HXS1不表达的毕赤酵母菌株。
在本发明的具体实施例中， 提供了四株能够利用非甲醇碳源诱导 启动 子表达外源多肽的菌株， GS115-MIT1、 Amigl Amig2 -ΜΙΎΙ、 Δ^7-ΜΙΤ1 和 AmiglAmig2Anrgl-MlTl a 在毕赤酵母野生菌株 GS115、 毕赤酵母双缺失菌株 AmiglAmig2,毕赤酵母缺失菌株 hxsl和毕赤酵母三缺失菌株 migl mig2 nrgl 中分别过表达了转录激活相关基因 ΜΓΠ, 构建了基因过表达菌株 GS115-MIT1、 Amigl Amig2-MIT\, Am igl Amig2Anrgl -MIT 1禾口 A/z ^-MITl。 经 Aoxl酶活检测 发现， 甘油可以诱导 GS115-MIT1 的 (¾ 启动子的表达， 甘油或者葡萄糖均能 诱导 A g7A g2-MITl、
Figure imgf000012_0001
Ι禾卩 Amig i Amig2 Anrg 1-ΜΓ 1的 ^ΟΏ启动子 表达。 并且用流式细胞仪检测了绿色荧光蛋白 GFP以 ^ Ώ为启动子时在非甲醇 碳源中的诱导表达量。
所述的 ^ixsl菌株中， 与酿酒酵母己糖感应体 Snf3/Rgt2有很高相似性的一 个基因 HYS7(GenBank登录号： 8197942)的编码区缺失。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本 发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通 常按照常规条件如 J.萨姆布鲁克等编著， 分子克隆实验指南， 科学出版社， 2002 中所述的条件， 或按照制造厂商所建议的条件。 除非另外说明， 否则百分比和份 数按重量计算。 材料
毕赤酵母总蛋白提取技术参照冷泉港公司提供的酵母蛋白提取手册。
所使用的工具酶均购自 TaKaRa生物公司（大连， 中国），具体的反应条件和使 用的方法均参考商品说明书。 下面的商品化质粒和菌株用于基因克隆和蛋白表达： 质粒 pGAPZaA、 质粒 pPIC3.5k、 大肠杆菌 Τορ10、 毕赤酵母菌株 GS115均购自 Invitrogen公司。
质粒 pAG32由质粒 pRDM054在酶切位点 Bgl II处切除 P^ BFP-SKL表达 体系得到； 质粒 pRDM05获自加州大学圣地亚哥分校。
YPD培养基： 2% 蛋白胨， 1% 酵母粉， 2% 葡萄糖， 2% 琼脂粉； YNB培 养基： 0.67% YNB; MGY培养基： 1% 甘油， 0.67% YNB; YND液体培养基： 1% 葡萄糖， 0.67% YNB。
配制以上培养基时， 葡萄糖 115°C高压灭菌 20min， 甲醇在使用时添加。 其 它成分 121°C高压灭菌 20 min。 固体培养基加 2% 琼脂粉。 实施例 1、 甘油可诱导 AOX1启动子表达的毕赤酵母菌株 GS115-MIT1 1. PpMITl过表达质粒的构建
釆用 PCR， 酶切连接的方法， 以 pGAPZaA (图 1)为载体在 GAP启动子下游的 Asu WlSal I两个酶切位点插入 ΡρΜΙΊΊ基因（SEQ ID NO: 1， 基因全长 2667bp)， 得 到的重组质粒称为 pGM质粒。
以 M1-GAP5和 M1-AOX1TT为引物 (表 1)， 通过 PCR的方法从 pGM质粒上 扩增得到 pGAP-PpMITl-AOXITT的表达体系（全长 3615 bp), 以 pAG32(图 2)为 载体在 Sac 1/Spe I处插入该表达体系。 得到重组质粒为 pGMhph。
表 1.引物列表
Figure imgf000013_0001
2. 电转毕赤酵母和 GS115-MIT1菌株的筛选
将 PpM/7 过表达质粒 (pGMhph)电转毕赤酵母菌株 GS115,涂于 4块加潮霉素 的 YPD固体培养基上， 放在 30°C培养箱培养 48-72小时。将平板上长出的单克隆 挑至 10ml YPD+ 潮霉素液体培养基中，30°C摇床培养后，提基因组用 PCR验证。 把 PCR检验及测序验证后正确的毕赤酵母菌株命名为 GS115-MIT1。 3. Aox显色反应
将各菌株分别在 0.5% (v/v) 甲醇， 1% 甘油， 1% 甘油 +0.5% (v/v) 甲醇为碳 源的 YNB液体培养基中培养， 生长到对数生长期后取样 1 ml, 在离心后的菌体 中加入 Aoxl酶活显色液显色 （参见 Stasyk O. V., T. Y. Nazarko, and A. A. Sibirny. 2008. Methods of Plate Pexophagy Monitoring and Positive Selection for ATG Gene Cloning in Yeasts. Methods in enzymology.451:229-239。显色底物为 o-dianisidine)。
结果如图 3， 可以看到在含有甘油的培养基中生长后， 只有菌株 GS115-MIT1 中有 Aox酶活， 野生菌株中均没有酶活。
4. Aox酶活的测定
将各菌株在 MGY液体培养基中过夜预培养，然后分别转移到含有 0.5% (v/v) 甲醇， 1% 甘油， 1% 甘油 +0.5% (v/v) 甲醇， 0.5% (v/v) 甲醇为碳源的 YNB液 体培养基中，培养 10小时后取样提取总蛋白，经 Bradford法蛋白定量后进行 Aoxl 酶活的测定（方法参见 Verduyn, C.， J. P. van Dijken, and W. A. Scheffers. 1984. Colorometric alcohol assays with alcohol oxidase. J. Microbiol. Methods 2:15_25。 显 色底物为 2,2， -azino-di-(3-ethylbenzthiazoline sulfonate), ABTS)。
由图 4可知， 菌株 GS115-MIT1在含有甘油的培养基中均可以诱导 Aoxl 的 表达， 野生菌株均不能诱导 Aox的表达。
5、 毕赤酵母 GFP单拷贝表达菌株的筛选
在载体 pPIC3.5k (图 9) 的 ^ Ώ启动子下游的 S«aB I酶切位点处插入 GFP 基因（全长 714 bp), 得到 GFP表达载体 pP-GFP。
6、 毕赤酵母 GFP单拷贝表达菌株的筛选
分别将表达载体 pP-GFP电转 GS115-MIT1菌株， 涂于不含组氨酸的 YND平 板，放在 30°C培养箱培养 48-72小时。将平板上长出的单克隆挑至液体培养基中， 30°C摇床培养后提基因组，用 real-time PCR验证 GFP拷贝数 (方法参见 Xuan， Y. J., X. S. Zhou, W. W. Zhang, X. Zhang, Z. W. Song, and Y. X. Zhang. 2009. An upstream activation sequence controls the expression of AOXl gene in Pichia pastoris. FEMS Yeast Res. 9: 1271-1282)。 把 real-time PCR检验为单拷贝的毕赤酵母 GFP 表达菌株分别命名为 GS115-MIT1-GFP。
同理构建了表达菌株 A g7A g2-MITl-GFP禾卩 A/zx^-MITl-GFP。
表达菌株 A g7A g2-MITl-GFP构建方法具体如下： 将表达载体 pP-GFP和 表达载体 pGMhph共转 A g7A g2菌株 （参见中国公开专利 CN101857845A)。 表达菌株 A/?x?7-MITl-GFP构建方法具体如下：将表达载体 pP-GFP和表达载 体 pGMhph共转 Δ/ζ ^-ΜΙΤΙ菌株。
7、 流式细胞仪检测 GFP表达量
将菌株 GS115-MIT1-GFP在 YND液体培养基中过夜预培养， 然后分别转移 含有 0.5% (ν/ν) 甲醇， 1%甘油， 1%甘油 +0.5% (ν/ν) 甲醇为碳源的 ΥΝΒ液体培 养基中培养， 取样后用流式细胞仪检测样品中 GFP的几何平均荧光强度。
如图 5所示， GS115-MIT1可以在甘油存在的情况下诱导外源多肽 GFP表达。 实施例 2、 甘油或者葡萄糖可诱导 O 启动子表达的毕赤酵母菌株
l.^X^W基因敲除质粒的构建
用 B謹 111和 Sal I酶将 Zeocin抗性基因 Sh ble片段从质粒 pGAPZaA上切下， 以 pUC18质粒（图 10)为载体， 在 BamH I和 Sal I处酶切连接插入了 Zeocin抗性 基因 b/e片段， 命名为 pUC18-b/e质粒。 以 GS115基因组为模板， 首先使用引 物 HS1-5F/HS1-5R扩增 PpH 基因的 5'端外围启动子区域 (起始密码子 ATG上 游）， 扩增产物的大小为 728 bp。 引物 HS1-3F/HS1-3R用于扩增 PpH¥S7基因的 3' 端的区域 (终止密码子下游)， 扩增产物的大小为 1011 bp, 分别切胶回收目的大小 片段。
将回收的 PpH W基因的 5'端外围启动子 DNA片段， 用 coRI和 BamlU双 酶切后， 回收片段。 pUC18-b/e质粒、 用 coRI和 S ΗΙ双酶切， 回收片段。 将 回收的两片段连接过夜， 转化大肠杆菌， PCR验证阳性克隆， 命名为 pUC18-(HS7 5'-ble
将上一步构建好质粒 Sa/I和 S W双酶切， 回收片段。 PpH¥S7基因的 3'端区 域 DNA片段也用 和 SpW双酶切。 两片段连接过夜， 转化大肠杆菌， PCR阳 性菌株， 阳性菌株命名为 pUC18-(HS7 '-ble-HSl 3')。
表 2、 PpH S7敲除使用的引物
引物 SEQID 、
5 ' -CCGGAATTCTAGTCACGGATTGCTTC-3 '
5 ' -CGCGGATCCAGTCTGAAATAGGCTCAGACAT-3 ' ' -ACGCGTCGAC ATGCCTATTGAACAAGACTATT-3 ' 5'-ACATGCATGCGTTCCCAATAGATTTCACAA-3 ' 2. \hxsl基因缺失菌株的构建
将 pUC18-(HS75 '-b!e-HSl 3 ')质粒用 coRI和 SpW双酶切，胶回收大小为 3350 bp的 PpH¥S7敲除片段 HS75'-b!e-HSl 3 '。
将回收的 20 μΐ PpH S7敲除片段 HS75'-ble-HSl 3 '(浓度大于 50 ng/μΐ)与 80 μΐ 毕赤酵母 GS115感受态在冰上混匀， 按照实验步骤提供的方法进行电转化。将 50 μΐ复苏的电转化菌液涂于添加 Zeocin抗生素的 YP+Glycerol固体培养基平板， 将 筛选平板倒置于 30°C培养箱培养 2〜3天， 当有肉眼可见菌落长出时， 将菌落挑 至含 Zeocin的 YP+Glycerol液体培养基中培养 2〜3天。 PCR验证 PpHXSJ敲除 的阳性菌株， 命名为 Ahxs
3. \migl\mig2-MlTl和 Α/ιχ -ΜΙΤ1的构建
将质粒 pGMhph电转毕赤酵母菌株 migl mig2和 hxsl, 涂于 4块加潮霉素 的 YPD平板，放在 30°C培养箱培养 48-72小时。将平板上长出的单克隆挑至 10 ml YPD+潮霉素液体培养基中， 30°C摇床培养后， 抽提基因组用 PCR验证。 把 PCR 检验及测序验证后正确的毕赤酵母菌株分别命名为 A g7A g2-MITl 和 Ahxsl-MIT\0
4. Aox显色反应
将 AmigJ Z^mig2-Mm禾卩
Figure imgf000016_0001
Ι分别在 1% 甘油， 1% 甘油 +0.5% (v/v) 甲醇， 1% 葡萄糖， 1% 葡萄糖 +0.5% (v/v) 甲醇和 0.5% (v/v) 甲醇为碳源的 YNB 液体培养基中培养， 生长到对数生长期后取样 lml， 在离心后的菌体中加入 Aoxl 酶活显色液显色。
结果如图 6， 可以看到在含有甘油或者葡萄糖的培养基中， 只有菌株 AmiglAmig2-MlTl禾口 ΔΛ ^-ΜΙΤΙ中有酶活。
5. Aox酶活的测定
将各菌株分别在 1% 甘油， 1%甘油 +0.5% (v/v) 甲醇， 1% 葡萄糖， 1% 葡萄 糖 +0.5% (v/v) 甲醇和 0.5% (v/v) 甲醇为碳源的 YNB液体培养基中培养， 生长到 对数生长期后取样提取总蛋白， 经 Bradford法蛋白定量后进行 Aoxl酶活的测定。
由图 7可知， 菌株 AmigJ ^mig2-M 禾 Π AhxsJ-MlTl在含有甘油或者葡萄糖 的培养基中均可以诱导 Aoxl的表达， 野生菌株均不能诱导 Aox的表达。
6. 流式细胞仪检测 A ^7A g2-MITl和 Δ/ιχ^-ΜΙΤΙ的 GFP表达量 将 A g7A g2-MITl-GFP和 A/zx^-MITl-GFP在液体培养基中过夜预培养， 然后分别转移到含有 0.5% (v/v) 甲醇， 1% 甘油， 1% 甘油 +0.5% (v/v) 甲醇， 1% 葡萄糖和 1% 葡萄糖 +0.5% (v/v) 甲醇为碳源的 YNB液体培养基中， 取样后用流 式细胞仪检测样品中 GFP的几何平均荧光强度。
如图 8所示， AmigJ mig2-MVTl禾 Π Δ^7-ΜΙΤ1可以在甘油或者葡萄糖存在 的情况下诱导外源多肽 GFP表达。 实施例 5、 甘油或者葡萄糖可诱导 AOX1 启动子表达的毕赤酵母菌株 Amigl Amig2 Anrgl -MIT 1
1. NRG J基因敲除质粒的构建
以 GS115基因组为模板， 使用引物 5，NRG1-F/5，NRG1-R扩增 PpNIGl基因 的 5'端外围区域得到大小为 320 bp 片段， 将扩增产物命名为 5,腦1， 胶回收 5WRG7片段。 以上所得到的 5WRG7片段用 Sac I和 S al双酶切， 与同样 Sac I 和 Sma I 双酶切的 pUC18 质粒连接， 构成 pUC18(S<3cI-5，NRGl-S aI)。 载体 pUC18(SacI-5'NRGl-S aI)转化大肠杆菌感受态， 菌落 PCR筛选阳性克隆。
然后， 使用弓 I物 HYG-F和 HYG-R, 以质粒 pRDM054为模板， 扩增潮霉素 B 抗性基因 ΗΡΗ, 大小为 1648 bp。 以上所得 HPH片段用 Sma I和 αί双酶切， 与 同样 Sma I 和 H 双酶切的载体 pUC18(SacI-5'NRGl-S aI)相连， 构成载体 pUC 18(SacI-5'N G 1 -Smal-UPU-Xbal)。 pUC 18(SacI-5 'NRG 1 -Smal- ΗΡΗ- baI)转化 入大肠杆菌， 菌落 PCR筛选阳性克隆。
用引物 5，NRG1-F/NRG1-A1 以质粒 pUC18(SacI-5，NRGl-S aI-HPH-^¾aI)为 模板扩增得到片段 Overlap-A。 用引物 NRG1-B2/3，NRG1-R， 以 GS115基因组为 模板， 扩增得到片段 Overlap-B。 然后通过 Overlap PCR的方法链接片段 A、 B， 得到 5，NRG1-HPH-3，NRG1片段。将该片段连接到 pMD19-T载体上， 得到敲除质 粒 pMD 19-T(SacI-5 'NRG 1 -SmaI-HPH-3 'NRG 1 -Sph\)。
2. Amigl \mig2\nrgl菌株的构建
将得到的 S<3cI-5，NRGl-S aI-HPH-3，NRGl-S^I片段与 migl mig2双缺失菌 株感受态共转， 电转后迅速加入 700ul预冷的 1 mol/L山梨醇， 然后转移至 EP管 中， 并加入 700 ul YPD液体培养基， 30°C和 200 r/min摇床复苏培养 1〜2 h。 最 后将菌液涂于添加 Zeocin、 G418和 Hygromycin抗生素的 YPD固体平板。 30°C培 养箱倒置培养 2〜3天。 将阳性转化子提基因组 PCR验证， 得到的阳性菌株命名 为 migl mig2 t rglo
3. 三缺失菌株中 MIT1过表达质粒的构建 以 pPIC3.5k为载体在 Sac 1/BamH I两个酶切位点插入 GAP启动子得到 pPG 质粒。 再于 BamHI/Notl两个酶切位点间插入 PpMPPl基因序列， 得到的重组质 粒称为 pPGPPl质粒。
4. \migl\mig2 \nrgl -ΜΙΎί的构建
将质粒 pPGPPl 电转毕赤酵母菌株 migl mig2 gl， 涂于 4块不加 his的
YND平板， 放在 30°C培养箱培养 48-72小时。 将平板上长出的单克隆挑至 10 ml YND液体培养基中， 30°C摇床培养后， 抽提基因组用 PCR验证。 把 PCR检验及 测序验证后正确的毕赤酵母菌株分别命名为 migl Am ig2 Anrgl -MIT 10
5. 毕赤酵母 GFP单拷贝表达菌株的筛选
分别将表达载体 pP-GFP电转 GS11+-MIT1菌株， 涂于不含组氨酸的 YND平 板， 放在 30°C培养箱培养 48-72小时。 将平板上长出的单克隆挑至 MGY液体培 养基中， 在荧光显微镜下观察是否有绿色荧光， 有荧光的转化子 30°C摇床培养后 提基因组，用 real-time PC 验证 GFP拷贝数 (方法参见 Xuan, Y. J.， X. S. Zhou, W. W. Zhang, X. Zhang, Z. W. Song, and Y. X. Zhang. 2009. An upstream activation sequence controls the expression of AO J gene in Pichia pastoris. FEMS Yeast Res.9: 1271-1282)。 把 real-time PC 检验为单拷贝的毕赤酵母 GFP 表达菌株命名为 Am igl Amig2Anrgl -MIT 1 -GFP。
6. Aox酶活的测定
将 A g7A g2AMrg7-MITl菌株分别在 1% 甘油， 1%甘油 +0.5% (v/v) 甲醇， 1% 葡萄糖， 1% 葡萄糖 +0.5% (v/v) 甲醇和 0.5% (v/v) 甲醇为碳源的 YNB液体 培养基中培养， 生长到对数生长期后取样提取总蛋白， 经 Bradford法蛋白定量后 进行 Aoxl酶活的测定。
由图 11可知，菌株 AmigJAmig2AnrgJ -ΜΓΠ在含有甘油或者葡萄糖的培养基 中均可以诱导 Aoxl的表达， 野生菌株均不能诱导 Aox的表达。
7. 流式细胞仪检测 A ^7A g2Am^7-MITl的 GFP表达量
将 A g7A g2A«rg7-MITl-GFP在液体培养基中过夜预培养， 然后分别转移 到含有 0.5% (v/v) 甲醇， 1% 甘油， 1% 甘油 +0.5% (v/v) 甲醇， 1% 葡萄糖和 1% 葡萄糖 +0.5% (v/v) 甲醇为碳源的 YNB液体培养基中， 取样后用流式细胞仪检测 样品中 GFP的几何平均荧光强度。
如图 12所示， A g7A g2A«rg7-MITl可以在甘油或者葡萄糖存在的情况下 诱导外源多肽 GFP表达。 表 3、 N/G7敲除使用的引物
引物名称 SEQ ID NO: 序列
5'NRG1-F 15 5，- CGAGCTCCTGTGCCTATTACCCCCCTT -3'
5'NRG1-R 16 5，- TCCCCCGGGAACAGATAACCAAAACGGACG -3 '
3'NRG1-F 17 5- GCTCTAGAGTATTTATTTACGGATTGGA -3'
3'NRG1-R 18 5，- ACATGCATGCCACCACTTTTTGAATCTCGG -3 '
HYG-F 19 5，- TCCCCCGGGAGCTTGCCTTGTCCCCGCCG -3 '
HYG-R 20 5，- GCTCTAGATCGACACTGGATGGCGGCGT -3'
NRG1-A1 21 5， -TCCAATCCGTAAATAAATACTCGACACTGGATGGCGGCGT-3'
NRG1-B2 22 5' -ACGCCGCCATCCAGTGTCGAGTATTTATTTACGGATTGGA-3' 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

m ^
1. 一种消除甲醇诱导型启动子单一甲醇碳源依赖性而驱动外源多肽编码基 因表达的方法， 包括：
(1) 提供甲醇营养型酵母， 所述甲醇营养型酵母含有：
表达盒 1， 其表达外源的 Mitl多肽； 以及
表达盒 2， 包括甲醇诱导型启动子及与之操作性连接的外源多肽编码基因； (2) 在无甲醇条件或非单一甲醇碳源条件下培养 (1)的甲醇营养型酵母。
2. 如权利要求 1所述的方法， 其特征在于， 所述的甲醇诱导型启动子包括：
^ Ώ启动子、 DH4S启动子、 Z 4S启动子、 FDff启动子、 FMDff启动子、 MOX 启动子、 ^ Ώ启动子、 ΖΖΑ1、 ΡΕΧ5-, ΡΕΧ8-、 启动子、 启动子、 启动子、 ( Z)7启动子、 ( Z)2启动子。
3. 如权利要求 1所述的方法， 其特征在于， 所述的甲醇营养型酵母包括： 毕 赤酵母 CP c/? a)，汉逊酵母 (Hrn^e /")，假丝酵母（Om Wa),球拟酵母 (7¾rw/¾w ); 较佳地， 所述的毕赤酵母包括： GS 1 15 , 基因和 基因不表达的毕赤酵 母， NRG7基因、 基因和 基因不表达的毕赤酵母， 或 H 基因不表 达的毕赤酵母菌株。
4. 如权利要求 1所述的方法， 其特征在于， 所述的 Mitl多肽选自下组：
(a) SEQ ID NO: 2所示氨基酸序列的多肽； 或
(b) 将 SEQ ID NO: 2所示氨基酸序列经过一个或多个氨基酸残基的取代、 缺 失或添加而形成的， 且具有利用非甲醇碳源诱导甲醇诱导型启动子表达外源多肽 功能的由 (a)衍生的多肽；
(c) 与 (a)限定的序列在具有 70%以上的序列相同性的， 且具有利用非甲醇碳 源诱导甲醇诱导型启动子表达外源多肽功能的由 (a)衍生的多肽。
5. 如权利要求 1所述的方法， 其特征在于， 所述的表达盒 1中， 包括启动子 以及与之操作性连接的 Mitl多肽的编码基因； 较佳地， 该启动子包括： 组成型启 动子， 诱导型启动子， 组织或器官特异性启动子， 时空特异性表达启动子。
6. 如权利要求 1 所述的方法， 其特征在于， 步骤 (2)中， 釆用存在甘油和 /或 葡萄糖的酵母培养基。
7. Mitl多肽或其编码基因的用途， 用于消除甲醇诱导型启动子单一甲醇碳源 依赖性而驱动外源多肽编码基因表达。
8. 一种重组的甲醇营养型酵母， 所述甲醇营养型酵母含有：
表达盒 1， 其能够表达外源的 Mitl多肽； 以及
表达盒 2， 包括甲醇诱导型启动子及与之操作性连接的外源多肽编码基因。
9. 如权利要求 8所述的重组的甲醇营养型酵母， 其特征在于， 所述的甲醇营 养型酵母包括： 毕赤酵母 汉逊酵母 (H»we聰 /")， 假丝酵母 (Ow Wa), 球 拟酵母（7¾rw/ W; 较佳地， 所述的毕赤酵母 CP c/»'a)包括： GS 1 15 , MIG1 基因 和 基因不表达的毕赤酵母， NRG7基因、 基因和 基因不表达的 毕赤酵母， 或 H 基因不表达的毕赤酵母菌株。
10. 如权利要求 9所述的方法， 其特征在于， 所述的 基因的核苷酸序 列如 SEQ ID NO: 9所示；
所述的 基因的核苷酸序列如 SEQ ID NO: 10所示； 或
所述的 HXS1基因的核苷酸序列如 SEQ ID NO: 3所示。
所述的 NRG1基因的核苷酸序列如 SEQ ID NO:8所示。
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