Method for enhancing transcriptional activation of yeast and application

Abstract

The invention provides a method for enhancing transcriptional activation of yeast and application. The strong activation polypeptide is a yeast source activation polypeptide mutant, a human source activation polypeptide or a fusion thereof, can realize high-intensity transcriptional activation of a promoter in a yeast cell, and can realize high-efficiency expression of an exogenous recombinant protein when the strong activation polypeptide is adaptively used in a related expression tool box. According to the strong activation polypeptide disclosed by the invention, the recombinant expression intensity of pichia pastoris is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering; more specifically, the present invention relates to a strong activating polypeptide for yeast transcription regulation, and a method for establishing and applying the same. Background Art

[0002] In recent years, the recombinant protein industry related to green biomanufacturing has developed rapidly, such as food substitute proteins, bulk industrial enzymes, bulk recombinant protein drugs, biomaterials, etc. Its market size has expanded year by year, and the demand for various proteins in various industries has increased significantly.

[0003] However, the existing recombinant protein expression system is still limited in expression capacity and cannot support the high-yield demand of recombinant proteins in industrial production. In the process of heterologous expression of recombinant proteins in model organisms, the transcription initiation process mediated by transcription activators (peptides) and promoters is a key step in protein expression.

[0004] Therefore, powerful and regulatable transcriptional activators or promoters have always been essential tools for the high-intensity expression and production of exogenous recombinant proteins.

[0005] Yeast expression systems are known as chassis hosts for the expression of foreign proteins. However, compared with other hosts, yeast expression systems have fewer available high-efficiency expression elements, and the expression intensity is difficult to meet the production requirements of proteins and other products. At present, its commercial expression system mainly uses the methanol-inducible alcohol oxidase promoter (P AOX1 ), which is one of the most powerful promoters known to date, but it still has a core bottleneck problem of insufficient expression capacity when used for bulk protein production, limiting the larger-scale application of such expression systems.

[0006] Research institutions and companies around the world have conducted extensive research on the Pichia pastoris expression system, focusing on discovering more natural expression elements or further developing artificial expression elements and tools. The reported expression intensity can reach that of wild-type P. AOX1 The applicant also achieved an expression intensity higher than that of wild-type P AOX1 But overall, its expressive power is still limited. Summary of the invention

[0007] The purpose of the present invention is to provide a strong activating polypeptide for yeast transcription regulation, and a method for establishing and using the same.

[0008] In a first aspect of the present invention, a yeast transcriptional strong activator polypeptide is provided, comprising: (i) a yeast-derived activator polypeptide M1, a truncated form or a conservative variant thereof, (ii) a human-derived activator polypeptide or a conservative variant thereof, or (iii) a fusion of (i) and (ii); the yeast-derived activator polypeptide M1 has an amino acid sequence as shown in SEQ ID NO: 1, and the human-derived activator polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 20, 22, 18, 19, and 21 (Z8, Z9, P1, K1, or Z3).

[0009] In one or more preferred embodiments, the truncated form of the yeast-derived activating polypeptide M1 has an amino acid sequence (M3, M2 or M4-M17) shown in SEQ ID NO: 3, SEQ ID NO: 2 or any one of SEQ ID NOs: 4 to 17;

[0010] In one or more preferred embodiments, the conservative variant is selected from: (a) a derived protein formed by replacing, deleting or adding one or more (such as 1-30 or 1-20; preferably 1-10; preferably 1-5, 1-3 or 1-2) amino acid residues of the yeast-derived activating polypeptide M1 or its truncation, or the human-derived activating polypeptide sequence, and having the function of enhancing gene transcription activation (activating gene expression); (b) a derived protein having more than 80% (preferably more than 85%, more than 90% or more than 95%; such as more than 98% or more than 99%) identity with the sequence of the yeast-derived activating polypeptide M1 or its truncation, or the human-derived activating polypeptide and having the function of enhancing gene transcription activation (activating gene expression).

[0011] In one or more preferred embodiments, the fusion includes: SEQ ID NO: 1 (M1) and SEQ ID NO: 20 fusion (M1Z8), SEQ ID NO: 20 and SEQ ID NO: 1 fusion (Z8M1), SEQ ID NO: 1 and SEQ ID NO: 22 fusion (M1Z9), SEQ ID NO: 22 and SEQ ID NO: 1 fusion (Z9M1), SEQ ID NO: 1 and SEQ ID NO: 21 fusion (M1Z3), or SEQ ID NO: 21 and SEQ ID NO: 1 fusion (Z3M1) SEQ ID NO: 1 and SEQ ID NO: 19 fusion (M1K1), SEQ ID NO: 19 and SEQ ID NO: 1 fusion (K1M1), SEQ ID NO: 1 and SEQ ID NO: 18 fusion (M1P1), SEQ ID NO: 18 and SEQ ID NO: 1 fusion (P1M1).

[0012] In another aspect of the present invention, a polynucleotide is provided, which encodes any of the aforementioned yeast transcriptional strong activator polypeptides.

[0013] In another aspect of the present invention, a vector or a genetically engineered host cell containing the vector is provided, wherein the vector contains a nucleic acid encoding any of the yeast transcriptional strong activator polypeptides described above; or the host cell contains the vector or has any of the yeast transcriptional strong activator polypeptides described above integrated into its genome.

[0014] In one or more preferred embodiments, the host cell includes: a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell includes: a yeast cell, a fungal cell, an insect cell, a mammalian cell, etc.; the prokaryotic host cell includes Escherichia coli, Bacillus subtilis, etc.

[0015] In one or more preferred embodiments, the host cell is a yeast cell.

[0016] In one or more preferred embodiments, in the vector, the 5' end of the polynucleotide further includes a signal peptide, a tag polypeptide, a fluorescent protein and / or a promoter; and / or the 3' end of the polynucleotide further includes a tag polypeptide, a fluorescent protein and / or a terminator.

[0017] In another aspect of the present invention, a method for preparing any of the yeast transcriptional activating polypeptides described above is provided, comprising the steps of: (i) culturing the host cell described above; (ii) collecting the culture containing the yeast transcriptional activating polypeptide; and (iii) isolating the yeast transcriptional activating polypeptide from the culture.

[0018] In another aspect of the present invention, there is provided the use of any of the above-mentioned yeast transcriptional strong activator polypeptides for enhancing the expression of a target gene in yeast cells; or for preparing a construct (such as an expression vector) or a cell for enhancing the expression of a target gene in yeast cells.

[0019] In another aspect of the present invention, a high-intensity expression toolbox (or toolbox, construct or expression cassette combination) of any of the above-mentioned yeast transcriptional strong activator polypeptides is provided, comprising: (1) expression cassette 1, which expresses an exogenous DNA binding protein, any of the above-mentioned yeast transcriptional strong activator polypeptides; and (2) expression cassette 2, which includes, in order from 5' to 3', the following operably linked protein binding sequences and promoters.

[0020] In one or more preferred embodiments, the expression cassette 1 further comprises a promoter, and the promoter comprises: a constitutive promoter or an inducible promoter; preferably, the constitutive promoter comprises: a GAP promoter, a TEF1 promoter, a GCW14 promoter; preferably, the inducible promoter comprises: an AOX1 promoter, a DAS1 promoter, a FLD1 promoter.

[0021] In one or more preferred embodiments, in expression cassette 1, the DNA binding protein includes: LacI, AraC, LexA, and FapR.

[0022] In one or more preferred embodiments, in expression cassette 2, the protein binding sequence includes: lacO, araI, lexO, fapO; preferably, the protein binding sequence exists in 1 to 15 copies (such as 13, 11, 9, 7, 5, 3, 1 copies), more preferably 1 to 9 copies.

[0023] In one or more preferred embodiments, in expression cassette 2, the promoter includes a core promoter; preferably, the core promoter includes: ADH2 core promoter, AOX1 core promoter, GAP core promoter, DAS2 core promoter, GCW14 core promoter; preferably includes ADH2 core promoter.

[0024] In one or more embodiments, the encoding nucleotide sequence of LacI is as shown in SEQ ID NO: 23 or a degenerate sequence thereof.

[0025] In one or more embodiments, the encoding nucleotide sequence of AraC is as shown in SEQ ID NO: 24 or a degenerate sequence thereof.

[0026] In one or more embodiments, the encoding nucleotide sequence of LexA is as shown in SEQ ID NO: 25 or a degenerate sequence thereof.

[0027] In one or more embodiments, the encoding nucleotide sequence of FapR is as shown in SEQ ID NO: 26 or a degenerate sequence thereof.

[0028] In one or more embodiments, the nucleotide sequence of lacO is as shown in SEQ ID NO: 27 or a degenerate sequence thereof.

[0029] In one or more embodiments, the nucleotide sequence of araI is as shown in SEQ ID NO: 28 or a degenerate sequence thereof.

[0030] In one or more embodiments, the nucleotide sequence of lexO is as shown in SEQ ID NO: 29 or a degenerate sequence thereof.

[0031] In one or more embodiments, the nucleotide sequence of fapO is as shown in SEQ ID NO: 30 or a degenerate sequence thereof.

[0032] In one or more embodiments, the nucleotide sequence of the ADH2 core promoter is as shown in SEQ ID NO: 31 or a degenerate sequence thereof.

[0033] In one or more embodiments, the nucleotide sequence of the AOX1 core promoter is as shown in SEQ ID NO: 32 or a degenerate sequence thereof.

[0034] In one or more embodiments, the nucleotide sequence of the GAP core promoter is as shown in SEQ ID NO: 33 or a degenerate sequence thereof.

[0035] In one or more embodiments, the nucleotide sequence of the DAS2 core promoter is as shown in SEQ ID NO: 34 or a degenerate sequence thereof.

[0036] In one or more embodiments, the nucleotide sequence of the GCW14 core promoter is as shown in SEQ ID NO: 35 or a degenerate sequence thereof.

[0037] In one or more preferred embodiments, in the high-intensity expression toolbox, the promoter of the expression cassette 2 is connected downstream to the target gene, and the two are operably connected.

[0038] In another aspect of the present invention, there is provided a use of any of the high-intensity expression toolkits described above for recombinantly expressing a target gene (such as a foreign protein encoding gene) in yeast.

[0039] In another aspect of the present invention, an isolated polynucleotide (ADH2 core promoter) is provided, which is a polynucleotide with a nucleotide sequence shown in SEQ ID NO: 31, and has the function of driving efficient gene expression.

[0040] In another aspect of the present invention, there is provided the use of the isolated polynucleotide described above as a promoter (core promoter) to drive efficient gene expression.

[0041] In another aspect of the present invention, a method for recombinantly expressing a target gene in a yeast cell is provided, comprising: (S1) providing any of the high-intensity expression toolboxes described above; (S2) connecting the target gene downstream of the promoter of the expression cassette 2 of the high-intensity expression toolbox (S1), and the two are operably connected; (3) introducing the high-intensity expression toolbox carrying the target gene of (S2) into the yeast cell to recombinantly express the target gene.

[0042] In one or more preferred embodiments, the target gene includes (but is not limited to): a gene encoding a biologically or pharmaceutically active protein, a genetic defect gene, a gene encoding a diagnostically active protein, a gene encoding an immunogenic protein, a gene encoding a structural protein, an enzyme encoding gene, and a reporter gene; preferably, the target gene includes a lactoglobulin encoding gene; preferably, the 5' end of the target gene is operably linked to: a natural signal peptide encoding sequence, an exogenous signal peptide encoding sequence, or a signal peptide-free encoding sequence.

[0043] In another aspect of the present invention, a yeast engineering bacterium is provided, comprising any of the high-intensity expression toolboxes described above.

[0044] In one or more preferred embodiments, the yeast includes (but is not limited to): methylotrophic yeast, Kluyveromyces, Saccharomyces; preferably, the methanol-trophic yeast includes: Pichia, Komagataella, Hansenula, Candida, Torulopsis.

[0045] In one or more preferred embodiments, the methanol-trophic yeast comprises: Pichia pastoris (Pichiapastoris or Komagataella phaffii).

[0046] In one or more embodiments, the Pichia pastoris includes NRRL-Y 11430 or a derivative thereof; preferably, the Pichia pastoris is a GS115 strain.

[0047] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 , GS115-pPAG, P GAP LM-lacO1cAG,P GAP LM-lacO1cADH2G,P GAP LM1-lacO1cADH2G and 16 M1 mutants P GAP LMn-lacO1cADH2G has a total of 20 strains with unit cell fluorescence intensity.

[0049] Figure 2 , GS115-pPAG, P GAP LM-lacO1cAG,P GAPLM-lacO1cADH2G,P GAP LP1-lacO1cADH2G, P GAP LK1-lacO1cADH2G、P GAP LZ3-lacO1cADH2G、P GAP LZ8-lacO1cADH2G、P GAP LZ9-lacO1cADH2G has a total of 8 strains with unit cell fluorescence intensity.

[0050] Figure 3 , GS115-pPAG, P GAP LM-lacO1cAG,P GAP LM1-lacO1cADH2G,P GAP LM1P1-lacO1cADH2G,P GAP LP1M1-lacO1cADH2G,P GAP LM1K1-lacO1cADH2G,P GAP LK1M1-lacO1cADH2G,P GAP LM1Z3-lacO1cADH2G,P GAP LZ3M1-lacO1cADH2G、P GAP LM1Z8-lacO1cADH2G,P GAP LZ8M1-lacO1cADH2G、P GAP LM1Z9-lacO1cADH2G,P GAP LZ9M1-lacO1cADH2G has a total of 13 strains with unit cell fluorescence intensity.

[0051] Figure 4 , GS115-pPAG, P GAP LM-lacO1cAG,P GAP LM1Z8-lacOncADH2G,P GAP CM1Z8-araIncADH2G、P GAP Fluorescence intensity per unit cell of AM1Z8-LexOncADH2G strain.

[0052] Figure 5 , GS115-pPA-BLG and P GAP Production of β-lactoglobulin by shake flask fermentation of LM1Z8-lacO5cADH2-BLG strain. DETAILED DESCRIPTION

[0053] After in-depth research, the inventors have revealed for the first time a class of strong activating polypeptides for yeast transcriptional regulation. The strong activating polypeptide is a yeast-derived activating polypeptide mutant, a human-derived activating polypeptide or a fusion thereof, which can achieve high-intensity transcriptional activation of the promoter in yeast cells. It is adapted for use in relevant expression toolboxes to achieve efficient expression of exogenous recombinant proteins. The strong activating polypeptide of the present invention can make the recombinant expression intensity of Pichia pastoris reach up to about 14 times that of the natural AOX1 promoter under methanol induction conditions.

[0054] Transcription activating polypeptides and the transcription pre-initiation complexes they recruit and assemble are essential and critical steps in initiating protein expression. In this process, transcription activating polypeptides can recruit RNA polymerase. The activation ability of the polypeptide has a decisive influence on the initiation and intensity of transcription, and further affects the subsequent protein translation flux and even the final total expression. However, the discovery of high-intensity natural transcription activating polypeptides faces huge challenges. It is urgent to develop polypeptide elements with high-intensity transcription activation ability through artificial design and modification to enhance the recombinant expression ability of exogenous proteins, which has positive significance for the biomanufacturing production of bulk industrial proteins.

[0055] In order to overcome the strength limitation of expression elements in Pichia pastoris and further enhance the recombinant protein expression capacity of the Pichia pastoris system, the inventors conducted in-depth and systematic research and constructed an artificial transcription activator polypeptide adapted to yeast, so that the Pichia pastoris system can express heterologous products more efficiently.

[0056] As used herein, the "activator polypeptide" refers to a polypeptide that can independently recruit RNA polymerase and transcription initiation complex, which usually binds to a specific promoter nucleic acid sequence to activate the transcription process. In this article, the activator polypeptide includes an active variant of the activator polypeptide, which may be a naturally occurring allelic variant or a non-naturally occurring variant. The variant includes a substitution variant, a deletion variant, an insertion variant or a fusion.

[0057] As used herein, the "promoter" refers to a nucleic acid sequence that is usually present in the upstream (5' end) of the target gene coding sequence and can guide the transcription of the nucleic acid sequence into mRNA. Generally, the promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for the correct initiation of transcription. In this article, the promoter or promoter region includes an active variant of the promoter, which can be a naturally occurring allelic variant or a non-natural variant. The variant includes a substitution variant, a deletion variant, an insertion variant, and a fusion variant.

[0058] As used herein, the "target gene" may also be referred to as a "target gene", which refers to a gene of interest that can be expressed using the high-intensity expression toolbox of the present invention. In the present invention, the "target gene" may be various and is not limited to those listed in the examples.

[0059] As used herein, the "constitutive promoter" refers to a type of promoter under which there is no obvious difference in gene expression in different tissues, organs and developmental stages.

[0060] As used herein, the "inducible promoter" can rapidly induce the "on" and "off" or "high" and "low" of gene transcription in a specific cell growth stage or a specific growth environment as needed. According to the source, inducible promoters can be divided into naturally occurring promoters and artificially constructed promoters.

[0061] As used herein, the "core promoter" refers to the minimum DNA sequence necessary to ensure the normal initiation of transcription by RNA polymerase, including the transcription start site and the TATA region upstream of the transcription start site.

[0062] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene of interest sequence does not normally occur in nature. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0063] As used herein, the "methanol-trophic yeast" refers to yeast that can use methanol as the sole carbon source, including (but not limited to) yeast from Pichia, Komagataella, Hansenula, Candida, and Torulopsis.

[0064] As used herein, the Kluyveromyces yeast includes, but is not limited to, Kluyveromyces marxianus or Kluyveromyces lactis, which are generally closer relatives.

[0065] As used herein, the brewer's yeast includes (but is not limited to) conventional yeast of the genus Saccharomyces used in brewing, baking, etc., typically Saccharomyces cerevisiae.

[0066] As used herein, the term "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence, so that transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operably linked" to the nucleic acid sequence.

[0067] As used herein, the terms “containing”, “having” or “including” include “comprising”, “mainly consisting of…”, “substantially consisting of…”, and “consisting of…”; “mainly consisting of…”, “substantially consisting of…” and “consisting of…” are subordinate concepts of “containing”, “having” or “including”.

[0068] The present invention relates to a strong activating polypeptide for yeast transcription regulation, and a method for establishing and using the same. The inventors obtained yeast-derived polypeptide variants and human-derived polypeptides with high-intensity transcriptional activation ability in Pichia pastoris, and constructed an artificial strong activating polypeptide fusion, which can successfully achieve a substantial increase in protein expression level after being applied to an expression toolbox.

[0069] In the present invention, the methanol-nourishing yeast refers to yeast that can use methanol as the sole carbon source, including (but not limited to) yeast from the genera Pichia, Komagataella, Hansenula, Torulopsis, Candida, etc.; the Kluyveromyces mainly includes Kluyveromyces marxianus or Kluyveromyces lactis; the Saccharomyces cerevisiae mainly refers to the conventional model yeast (Saccharomyces cerevisiae) used in brewing, baking, etc.

[0070] In the present invention, the yeast-derived activating polypeptide includes (but is not limited to): Pichia pastoris-derived activating polypeptide M1 and its mutants M2 to M17; preferably, M1. The M1 has the nucleotide sequence shown in SEQ ID NO: 1, and the M1 mutants M2 to M17 have the nucleotide sequence shown in SEQ ID NO: 2 to 17. The present invention also relates to the degenerate sequences of these polynucleotides. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of the polypeptides having the same amino acid sequence as those encoded by the above-mentioned nucleotides. These nucleotide variants include substitution variants, deletion variants, insertion variants and fusion variants. The present invention also relates to polynucleotides homologous to the above-mentioned polynucleotides, preferably with a homology of more than 70%, more than 80%, more than 90%, more than 93%, more than 95% or more than 97%, and the polypeptides encoded by these polynucleotides also have the same functions as M1 and its mutants.

[0071] In the present invention, the human activating polypeptide includes (but is not limited to): P1, K1, Z8, Z3, Z9; preferably P1. The P1 has the nucleotide sequence shown in SEQ ID NO:18. The K1 has the nucleotide sequence shown in SEQ ID NO:19. The Z8 has the nucleotide sequence shown in SEQ ID NO:19. The Z3 has the nucleotide sequence shown in SEQ ID NO:20. The Z9 has the nucleotide sequence shown in SEQ ID NO:21. The present invention also relates to the degenerate sequences of these polynucleotides. The present invention also relates to variants of the above polynucleotides, which encode polypeptides or fragments, analogs and derivatives of the polypeptides having the same amino acid sequence as those encoded by the above nucleotides. These nucleotide variants include substitution variants, deletion variants, insertion variants and fusion variants. The present invention also relates to polynucleotides homologous to the above-mentioned polynucleotides, preferably with a homology of more than 70%, more than 80%, more than 90%, more than 93%, more than 95% or more than 97%, and the polypeptides encoded by these polynucleotides also have the same functions as P1, K1, Z8, Z3 or Z9.

[0072] In the present invention, the DNA binding protein includes but is not limited to: LacI, AraC, LexA, FapR; the protein binding sequence includes but is not limited to: lacO, araI, lexO, fapO. Moreover, when the DNA binding protein uses LacI, the corresponding protein binding sequence uses lacO; when the DNA binding protein uses AraC, the corresponding protein binding sequence uses araI; when the DNA binding protein uses lexA, the corresponding protein binding sequence uses lexO; when the DNA binding protein uses fapR, the corresponding protein binding sequence uses fapO.

[0073] The coding nucleotide sequence of LacI can be shown in SEQ ID NO:23; the coding nucleotide sequence of AraC can be shown in SEQ ID NO:24; the coding nucleotide sequence of LexA can be shown in SEQ ID NO:25; the coding nucleotide sequence of FapR can be shown in SEQ ID NO:26. The nucleotide sequence of lacO is shown in SEQ ID NO:27; the nucleotide sequence of araI is shown in SEQ ID NO:28; the nucleotide sequence of lexO is shown in SEQ ID NO:29; the nucleotide sequence of fapO is shown in SEQ ID NO:30. The present invention also relates to the degenerate sequence of the above polynucleotide. The present invention also relates to variants of the above polynucleotide, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the above nucleotides. These nucleotide variants include substitution variants, deletion variants, insertion variants and fusion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it. The present invention also relates to polynucleotides homologous to the above-mentioned polynucleotides, preferably with a homology of more than 70%, more than 80%, more than 90%, more than 93%, more than 95% or more than 97%, and the polypeptides encoded by these polynucleotides also have the same functions as the polypeptides encoded by the aforementioned polynucleotides.

[0074] The expression cassette 1 also includes a promoter element that allows the fusion expression of the DNA binding protein and the activation polypeptide. Any promoter that can express a large amount of the fusion polypeptide can be applied to the expression cassette 1. The promoter can be: a constitutive promoter, an inducible promoter, a tissue or organ-specific promoter, a time-space specific expression promoter, etc. Preferably, the promoter includes (but is not limited to): a constitutive and an inducible promoter; preferably, the constitutive promoter includes (but is not limited to): a GAP promoter, a TEF1 promoter, a GCW14 promoter; preferably, the inducible promoter includes (but is not limited to): an AOX1 promoter, a DAS1 promoter, a FLD1 promoter. Similarly, the expression cassette 1 also includes a suitable terminator, which is an element well known to those skilled in the art for constructing a gene expression cassette.

[0075] When driving the expression of the target gene, a variety of promoters known in the art or under development can be used, preferably a core promoter. The promoter can also be: a constitutive promoter, a tissue or organ specific promoter, a time-space specific expression promoter, etc.

[0076] As a preferred embodiment of the present invention, the promoters used in the expression cassette for expressing the exogenous DNA binding protein include: GAP promoter, AOX1 promoter and the like.

[0077] As a preferred embodiment of the present invention, the promoters used in the expression cassette for expressing the target gene include: ADH2 core promoter, AOX1 core promoter, GAP core promoter, DAS2 core promoter, GCW14 core promoter; preferably includes ADH2 core promoter. The ADH2 core promoter is isolated for the first time, and it shows a better effect of driving the expression of the target gene than other core promoters.

[0078] The present invention also includes variant promoters (core promoters) or promoter fragments that drive expression functions identical to the promoters of the nucleotide sequences specifically listed in the embodiments. The hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. The present invention also relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. The present invention also includes nucleic acids that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, and most preferably 95% or more) identity with any promoter sequence of the present invention, and the nucleic acids also have the function of directing the specific expression of the target gene. "Identity" refers to the level of similarity (i.e., sequence homology, similarity or identity) between two or more nucleic acids according to the percentage of identical positions.

[0079] As a preferred embodiment of the present invention, in expression cassette 2, the protein binding sequence exists in single copy or multiple copies upstream of the core promoter, preferably 1 to 15 copies, more preferably 1-9 copies, such as 9, 7, 5, 3, 1 copies.

[0080] In the present invention, the yeast cell comprising the two expression cassettes can be obtained by introducing the combination of the expression cassettes of the present application, or a nucleic acid construct or a recombinant vector comprising the combination of the expression cassettes of the present application into a eukaryotic cell.

[0081] In the present invention, the combination of expression cassettes can be integrated into the yeast cell genome or exist episomally in the yeast cell.

[0082] Based on the high-intensity expression toolbox (or toolbox, construct or expression box combination) of the novel yeast transcription strong activator polypeptide constructed by the present invention, the present invention also provides a kit containing the expression toolbox. The kit may also contain Pichia pastoris cells. The kit may also contain other reagents such as culture medium and inducer required for yeast expression.

[0083] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

[0084] Material

[0085] The seamless cloning kit of Novazome Biotech was used for the plasmid construction.

[0086] The tool enzymes used were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), and the specific reaction conditions refer to the corresponding instructions.

[0087] The following commercial plasmids and strains were used for gene cloning and protein expression: plasmid pGAPZαA, plasmid pPIC3.5K, Escherichia coli Top10, and Pichia pastoris strain GS115, all purchased from Invitrogen.

[0088] Plasmid pPAG was obtained by inserting the GFP gene (full length 714 bp, sequence see position 80 to 793 in GenBank accession number AY656807.1) into the SnaBI restriction site downstream of the AOX1 promoter of plasmid pPIC 3.5K.

[0089] Plasmid pGGLacIMit1AD (i.e., pGGLacIM, containing GAP promoter-LacI-Mit1AD expression cassette): see Chinese patent application 201710352843.1 (expression driven by constitutive promoter GAP).

[0090] Plasmid pPlacO1cAG (containing lacO-AOX1 core promoter (cA)-GFP (G) expression cassette): see Chinese patent application 201710352843.1. The number 1 in lacO1 indicates a single copy; the subsequent number 2 indicates a double copy, and so on.

[0091] Establishment of plasmid pPcADH2G (containing AOX1 core promoter-GFP expression cassette): The AOX1 promoter of the pPAG plasmid was replaced with the ADH2 core promoter (SEQ ID NO: 31).

[0092] Establishment of plasmid pPlacO1cADH2G (containing lacO-ADH2 core promoter (cADH2)-GFP expression cassette): The recombinant plasmid pPlacO1cAG was double-digested at the restriction sites ApaⅠ / BamHI to linearize it, and then seamlessly assembled with the ADH2 core promoter sequence (SEQID NO:31) to obtain the recombinant plasmid pPlacO1cADH2G.

[0093] Establishment of plasmid pPlexO1cADH2G (containing lexO-ADH2 core promoter-GFP expression cassette): The recombinant plasmid pPcADH2G was double-digested at the restriction sites SacI / XhoI to linearize it, and then ligated with the lexO fragment (SEQ ID NO: 29) to obtain the recombinant plasmid pPlexO1cADH2G.

[0094] Establishment of plasmid pParaI1cADH2G (containing araI-ADH2 core promoter-GFP expression cassette): The recombinant plasmid pPcADH2G was double-digested at the restriction sites SacI / XhoI to linearize it, and then ligated with the araI fragment (SEQ ID NO: 28) to obtain the recombinant plasmid pParaI1cADH2G.

[0095] YPD medium: 2% peptone, 1% yeast powder, 2% glucose;

[0096] YNB medium: 0.67% YNB; YND liquid medium: 1% glucose, 0.67% YNB;

[0097] BMDY medium: 2% peptone, 1% yeast powder, 1.34% YNB, 100mmol / L pH6.0 phosphate buffer, 2% glucose;

[0098] BMMY medium: 2% peptone, 1% yeast powder, 1.34% YNB, 100 mmol / L pH 6.0 phosphate buffer, 0.5% methanol;

[0099] When preparing the above culture medium, glucose was sterilized at 115°C for 30 minutes and methanol was added when used. Other ingredients were sterilized at 121°C for 20 minutes. 2% agar powder was added to the solid culture medium.

[0100] Example 1. Pichia pastoris expression system based on yeast / human activating polypeptide (including mutants)

[0101] 1. Construction of pPlacO1cADH2G plasmid

[0102] The recombinant plasmid pPlacO1cAG was double-digested at the restriction sites ApaⅠ / BamHI to linearize it, and then seamlessly assembled with the ADH2 core promoter sequence (SEQ ID NO: 31) to obtain the recombinant plasmid pPlacO1cADH2G (containing the lacO-ADH2 core promoter (cADH2)-GFP (G) expression cassette).

[0103] 2. Construction of yeast-derived activation polypeptide expression plasmid

[0104] Using pGAP-F (SEQ ID NO: 36) and LacI-GAP-R (SEQ ID NO: 37) as primers, GAP promoter and resistance gene expression cassette and other fragments were amplified from pGAPZαA by PCR; using primers LacI-M1-F (SEQ ID NO: 38) and GAP-M1-R (SEQ ID NO: 39) to amplify the M1 fragment of Mit1AD (encoding SEQ ID NO: 1) from the genome of Pichia pastoris GS115, and seamlessly assembled with the plasmid fragment and LacI fragment, respectively, to obtain the recombinant plasmid pGGLacIM1.

[0105] Using the recombinant plasmid pGGLacIM1 as a template, two fragments (encoding SEQ ID NO: 2; Δ2-75aa) were amplified from pGGLacIM1 by PCR using primer pairs LacI-M1-F (SEQ ID NO: 40), GAP-M1-R (SEQID NO: 39) and pGAP-F (SEQ ID NO: 36), LacI-R (SEQ ID NO: 41), and the recombinant plasmid pGGLacIM2 was obtained by seamless assembly.

[0106] Similarly, recombinant plasmids pGGLacIM3 to pGGLacIM17 (where M3 to M17 encode proteins of SEQ ID NOs: 3 to 17, respectively) can be constructed.

[0107] 3. Construction of human activating polypeptide expression plasmid

[0108] The DNA fragments of human activating polypeptides P1 (SEQ ID NO: 18), K1 (SEQ ID NO: 19), Z3 (SEQ ID NO: 21), Z8 (SEQ ID NO: 20), and Z9 (SEQ ID NO: 22) were artificially synthesized by GenScript Biotech Co., Ltd.

[0109] Using the recombinant plasmid pGGLacIM1 as a template, primers pGAP-F (SEQ ID NO: 36) and LacI-R (SEQ ID NO: 41) were used to amplify a fragment not containing the activation polypeptide from pGGLacIM1 by PCR, and the fragment was seamlessly assembled with the P1 fragment to obtain the recombinant plasmid pGGLacIP1.

[0110] Similarly, the corresponding primers can be used to construct recombinant plasmids pGGLacIK1, pGGLacIZ3, pGGLacIZ8, and pGGLacIZ9.

[0111] 4. Construction of GFP expression strains based on yeast and human activating polypeptides

[0112] The recombinant plasmids pPAG, pPlacO1cAG or pPlacO1cADH2G were introduced into the Pichia pastoris strain GS115 by electroporation, spread on YND plates and cultured in a 30°C incubator for 2-3 days. Single colonies on the plates were picked into YPD medium and cultured in a 30°C shaker for about 2 days, the genome was extracted, and the copy number was verified by real-time PCR. The Pichia pastoris expression strains with a single copy of GFP (G) were named GS115-pPAG, GS115-lacO1cAG and GS115-lacO1cADH2G.

[0113] The plasmid pGGLacIM was introduced into the GS115-lacO1cAG and GS115-lacO1cADH2G strains by electroporation, and the other yeast or human activating polypeptide expression plasmids were introduced into the GS115-lacO1cADH2G strain by electroporation, and the plasmids were spread on YPD plates with bleomycin and placed in a 30°C incubator for 2-3 days. The single clones on the plate were picked into YPD medium and cultured in a 30°C shaker for about 2 days, the genome was extracted, and the copy number was verified by real-time PCR. The single-copy Pichia expression strains were named P GAP LM-lacO1cAG,P GAP LM-lacO1cADH2G (M represents Mit1AD, L represents LacI), P GAP LMn-lacO1cADH2G (n is a positive integer selected from 1 to 17, representing M1 or different mutants), P GAP LP1-lacO1cADH2G, P GAP LK1-lacO1cADH2G、P GAP LZ3-lacO1cADH2G、P GAP LZ8-lacO1cADH2G、P GAPLZ9-lacO1cADH2G.

[0114] 5. Detect GFP fluorescence intensity using an ELISA reader

[0115] The strain GS115-pPAG, P GAP LM-lacO1cAG,P GAP LM-lacO1cADH2G,P GAP LMn-lacO1cADH2G,P GAP LP1-lacO1cADH2G, P GAP LK1-lacO1cADH2G、P GAP LZ3-lacO1cADH2G、P GAP LZ8-lacO1cADH2G、P GAP LZ9-lacO1cADH2G was pre-cultured in YPD medium, the cells were collected by centrifugation, washed twice with sterile water, and then transferred to YNM medium or YPD medium supplemented with methanol for culture. After sampling, the fluorescence intensity of different samples was detected by a microplate reader.

[0116] The results are as follows Figure 1 and Figure 2 Compared with before transformation, M1-M17, Z8, Z9, P1, K1 or Z3 can significantly promote the expression of the target protein (GFP), with M1 being the most effective among yeast-derived peptides and P1 being the most effective among human-derived peptides. The expression enhancement factor of the target protein in strains containing M1-M17, Z8, Z9, P1, K1 or Z3 is shown in Table 1.

[0117] Table 1

[0118] Multiples of GS115-pPAG <![CDATA[Multiple of P GAP LM-lacO1cAG]]> <![CDATA[P GAP LM1-lacO1cADH2G]]> 9.4 2.3 <![CDATA[P GAP LM2-lacO1cADH2G]]> 7.5 1.8 <![CDATA[P GAP LM3-lacO1cADH2G]]> 8.8 2.1 <![CDATA[P GAP LM4-lacO1cADH2G]]> 8.7 2.1 <![CDATA[P GAP LM5-lacO1cADH2G]]> 8.7 2.1 <![CDATA[P GAP LM6-lacO1cADH2G]]> 8.1 2.0 <![CDATA[P GAP LM7-lacO1cADH2G]]> 5.2 1.3 <![CDATA[P GAP LM8-lacO1cADH2G]]> 6.8 1.6 <![CDATA[P GAP LM9-lacO1cADH2G]]> 6.2 1.5 <![CDATA[P GAP LM10-lacO1cADH2G]]> 7.7 1.8 <![CDATA[P GAP LM11-lacO1cADH2G]]> 7.5 1.8 <![CDATA[P GAP LM12-lacO1cADH2G]]> 8.1 1.9 <![CDATA[P GAP LM13-lacO1cADH2G]]> 7.5 1.8 <![CDATA[P GAP LM14-lacO1cADH2G]]> 7.8 1.9 <![CDATA[P GAP LM15-lacO1cADH2G]]> 6.5 1.6 <![CDATA[P GAP LM16-lacO1cADH2G]]> 7.6 1.8 <![CDATA[P GAP LM17-lacO1cADH2G]]> 6.9 1.6 <![CDATA[P GAP LP1-lacO1cADH2G]]> 10.1 2.6 <![CDATA[P GAP LK1-lacO1cADH2G]]> 6.9 1.8 <![CDATA[P GAP LZ3-lacO1cADH2G]]> 6.2 1.6 <![CDATA[P GAP LZ8-lacO1cADH2G]]> 6.8 1.8 <![CDATA[P GAP LZ9-lacO1cADH2G]]> 6.5 1.7

[0119] Example 2: Pichia pastoris expression system based on yeast-human fusion activation polypeptide

[0120] 1. Construction of fusion activation polypeptide expression plasmid

[0121] Using the recombinant plasmid pGGLacIM1 as a template, primers pGAP-F (SEQ ID NO: 36) and M1-R (SEQ ID NO: 42) were used to amplify a fragment from pGGLacIM1 by PCR; using the recombinant plasmid pGGLacIP1 as a template, primers GAP-M1-F (SEQ ID NO: 43) and GAP-M1-R (SEQ ID NO: 44) were used to amplify a fragment from pGGLacIP1 by PCR, and the two fragments were seamlessly assembled to obtain the recombinant plasmid pGGLacIM1P1. Similarly, the recombinant plasmids pGGLacIM1K1, pGGLacIM1Z3, pGGLacIM1Z8, and pGGLacIM1Z9 can be constructed.

[0122] Using the recombinant plasmid pGGLacIM1 as a template, primers LacI-R (SEQ ID NO: 41) and M1-F (SEQ ID NO: 45) were used to amplify a fragment from pGGLacIM1 by PCR; using the recombinant plasmid pGGLacIP1 as a template, primers GAP-LacI-F (SEQ ID NO: 46) and P1M1-R (SEQ ID NO: 47) were used to amplify a fragment from pGGLacIM1 by PCR, and the two fragments were seamlessly assembled to obtain the recombinant plasmid pGGLacIP1M1. Similarly, the recombinant plasmids pGGLacIK1M1, pGGLacIZ3M1, pGGLacIZ8M1, and pGGLacIZ9M1 can be constructed.

[0123] 2. Construction of GFP expression strain based on fusion activation polypeptide

[0124] The fusion polypeptide expression plasmids were introduced into the GS115-lacO1cADH2G strain by electroporation, spread on YPD plates with bleomycin and cultured in a 30°C incubator for 2-3 days. Single clones on the plates were picked into YPD medium and cultured in a 30°C shaker for about 2 days, the genome was extracted, and the copy number was verified by real-time PCR.

[0125] The Pichia pastoris expression strains tested as single copies were named P GAP LM1P1-lacO1cADH2G,P GAP LP1M1-lacO1cADH2G,P GAP LM1K1-lacO1cADH2G,P GAP LK1M1-lacO1cADH2G,P GAP LM1Z3-lacO1cADH2G,P GAP LZ3M1-lacO1cADH2G、PGAP LM1Z8-lacO1cADH2G,P GAP LZ8M1-lacO1cADH2G、P GAP LM1Z9-lacO1cADH2G,P GAP LZ9M1-lacO1cADH2G.

[0126] 3. Detect GFP fluorescence intensity with an enzyme-labeled instrument

[0127] The strain GS115-pPAG, P GAP LM-lacO1cAG,P GAP LM1-lacO1cADH2G,P GAP LM1P1-lacO1cADH2G,P GAP LP1M1-lacO1cADH2G,P GAP LM1K1-lacO1cADH2G,P GAP LK1M1-lacO1cADH2G,P GAP LM1Z3-lacO1cADH2G,P GAP LZ3M1-lacO1cADH2G、P GAP LM1Z8-lacO1cADH2G,P GAP LZ8M1-lacO1cADH2G、P GAP LM1Z9-lacO1cADH2G,P GAP LZ9M1-lacO1cADH2G were pre-cultured in YPD medium, the cells were collected by centrifugation, washed twice with sterile water, and then transferred to YNM medium or YPD medium supplemented with methanol for culture. After sampling, the fluorescence intensity of different samples was detected by a microplate reader.

[0128] The results are as follows Figure 3 As shown. Compared with before transformation, the fusion activation polypeptide can significantly promote the expression of the target protein (GFP), and the fusion M1Z8 is the best. The expression enhancement factor of the target protein in the strains containing the coding sequence of each fusion activation polypeptide is shown in Table 2.

[0129] Table 2

[0130] Multiples of GS115-pPAG <![CDATA[Multiple of P GAP LM-lacO1cAG]]> <![CDATA[P GAP LM1P1-lacO1cADH2G]]> 5.3 1.3 <![CDATA[P GAP LP1M1-lacO1cADH2G]]> 7.7 1.8 <![CDATA[P GAP LM1K1-lacO1cADH2G]]> 8.8 2.1 <![CDATA[P GAP LK1M1-lacO1cADH2G]]> 10.4 2.5 <![CDATA[P GAP LM1Z3-lacO1cADH2G]]> 12.2 2.8 <![CDATA[P GAP LZ3M1-lacO1cADH2G]]> 10.4 2.4 <![CDATA[P GAP LM1Z8-lacO1cADH2G]]> 12.7 2.9 <![CDATA[P GAP LZ8M1-lacO1cADH2G]]> 10.8 2.6 <![CDATA[P GAP LM1Z9-lacO1cADH2G]]> 11.2 2.6 <![CDATA[P GAP LZ9M1-lacO1cADH2G]]> 12.4 2.8

[0131] Example 3. Pichia pastoris expression system containing different DNA binding protein / binding sequence combinations and binding sequence copy numbers

[0132] 1. Construction of plasmids containing different copy numbers of protein binding sequences

[0133] Plasmids containing protein binding sequences with different copy numbers can be obtained by enzyme digestion. Taking plasmids containing different copies of lacO as an example, the plasmid pPlacO1cADH2G (containing 1 copy of lacO) was linearized by double enzyme digestion with SacⅠ / XhoⅠ, and then ligated with the lacO fragment to obtain the recombinant plasmid pPlacO2cADH2G (containing two copies of lacO). This plasmid contains GFP expressed by a synthetic promoter consisting of the protein binding sequence lacO (2 copies) and the ADH2 core promoter (cADH2).

[0134] Similarly, the recombinant plasmid can be obtained as follows:

[0135] pPlacOncADH2G: A synthetic promoter consisting of lacO (n copies, n=a positive integer from 3 to 9) and cADH2 drives the expression of GFP.

[0136] Similarly, plasmids containing different copy numbers of lexO and araI can be obtained as follows:

[0137] pPlexOncADH2G: A synthetic promoter composed of lexO (n copies, n=a positive integer from 1 to 9) and cADH2 drives the expression of GFP.

[0138] pParaIncADH2G: A synthetic promoter composed of araI (n copies, n=a positive integer from 1 to 9) and cADH2 drives the expression of GFP.

[0139] 2. Construction of constitutive promoter expressing activating polypeptide plasmid

[0140] Using the recombinant plasmid pGGLacIM1Z8 as a template, the primer pairs AraC-GAP-R (SEQ ID NO: 48), AraC-M1Z8-F (SEQ ID NO: 49) and LexA-GAP-R (SEQ ID NO: 50), LexA-M1Z8-F (SEQ ID NO: 51) were used to amplify the fragments from pGGLacIM1Z8 by PCR; the fragments were seamlessly assembled with the AraC and LexA fragments to obtain the recombinant plasmids pGGAraCM1P1Z8 and pGGLexAM1P1Z8.

[0141] 3. Construction of GFP expression strain based on fusion activation polypeptide

[0142] The recombinant plasmids pGGLacIM1Z8, pGGAraCM1P1Z8, and pGGLexAM1P1Z8 were introduced into the Pichia pastoris strain GS115 by electroporation, spread on YPD plates with bleomycin, and cultured in a 30°C incubator for 2-3 days. Single clones on the plates were picked into YPD medium and cultured in a 30°C shaker for about 2 days. The genome was extracted and the copy number was verified by real-time PCR. The Pichia pastoris expression strain with a single copy was named GS115-P GAP LM1Z8, GS115-P GAP CM1Z8 (C refers to AraC), GS115-P GAP AM1Z8.

[0143] The above plasmids containing different copy numbers of protein binding sequences were electroporated into strains containing the corresponding DNA binding proteins, spread on YND plates and placed in a 30°C incubator for 2-3 days. Single colonies on the plates were picked into YPD medium and cultured in a 30°C shaker for about 2 days, the genome was extracted, and the copy number was verified by real-time PCR. The Pichia pastoris expression strain with a single copy of GFP was named P GAP LM1Z8-lacOncADH2G,P GAP CM1Z8-araIncADH2G、P GAP AM1Z8-LexOncADH2G.

[0144] 4. Detect GFP fluorescence intensity using an ELISA reader

[0145] The strain GS115-pPAG, P GAP LM-lacO1cAG,P GAP LM1Z8-lacOncADH2G,P GAP CM1Z8-araIncADH2G、P GAP AM1Z8-LexOncADH2G were pre-cultured in YPD medium, the cells were collected by centrifugation, washed twice with sterile water, and then transferred to YNM medium or YPD medium supplemented with methanol for culture. After sampling, the fluorescence intensity of different samples was detected by an ELISA instrument.

[0146] The results are as follows Figure 4 It can be seen that the Pichia pastoris expression system containing different DNA binding protein / binding sequence combinations can achieve effective enhanced expression, among which lacO or lexO has a better effect.

[0147] According to the expression of the Pichia pastoris expression system with different binding sequence copy numbers, it can be seen that when the copy number is 3-9 copies, the expression is further enhanced to a certain extent compared with the low copy number.

[0148] Example 4: Recombinant expression of milk protein using a Pichia pastoris expression system based on high-intensity activated polypeptides

[0149] 1. Construction of lactoglobulin expression plasmid

[0150] The DNA fragment of lactoglobulin (β-BLG) (SEQ ID NO: 52) was artificially synthesized by GenScript Biotech Corporation.

[0151] Using plasmid pPAG as a template and primers AOX1-BLG-F / R (SEQ ID NO: 53-54), a fragment not containing fluorescent protein was amplified from pPAG by PCR, and seamlessly assembled with the β-BLG fragment to obtain the recombinant plasmid pPA-BLG expressing β-BLG using the conventional AOX1 promoter (the DNA fragment of β-BLG was inserted downstream of the AOX1 promoter of pPIC 3.5K).

[0152] Using the recombinant plasmid pPlacO5cADH2G as a template and primer pair AOX1-BLG-F / cADH2-BLG-R (SEQ ID NO: 55), a fragment not containing fluorescent protein was amplified from pPlacO5cADH2G by PCR, and the fragment was seamlessly assembled with the β-BLG fragment to obtain the recombinant plasmid pPlacO5cADH2-BLG expressing β-BLG using the lacO5cADH promoter developed by the present invention.

[0153] 2. Construction of Pichia pastoris expression strain for lactoglobulin

[0154] The recombinant plasmid pPA-BLG was introduced into the Pichia pastoris strain GS115 by electroporation, spread on a YND plate and placed in a 30°C incubator for 2-3 days. The single clone on the plate was picked into the YPD medium and cultured in a 30°C shaker for about 2 days, the genome was extracted, and the copy number was verified by real-time PCR. The Pichia pastoris expression strain with a single copy was named GS115-pPA-BLG.

[0155] The recombinant plasmid pPlacO5cADH2-BLG was introduced into the Pichia pastoris strain GS115-P by electroporation. GAP LM1Z8, spread on YND plates and placed in a 30°C incubator for 2-3 days. Pick the single colony on the plate into YPD medium and culture it in a 30°C shaker for about 2 days, extract the genome, and verify the copy number using real-time PCR. The Pichia pastoris expression strain that was tested as a single copy was named P GAP LM1Z8-lacO5cADH2-BLG.

[0156] 3. Lactoglobulin shake flask fermentation production

[0157] The strains GS115-pPA-BLG, P GAP LM1Z8-lacO5cADH2-BLG was pre-cultured in YPD medium, the cells were collected by centrifugation, washed twice with sterile water, and then transferred to BMMY or BMDY liquid medium for shake flask culture for 96 hours, during which the corresponding carbon source was added to the medium every 24 hours. After the fermentation, the supernatant was centrifuged and the protein content was determined.

[0158] like Figure 5 As shown, when lactoglobulin is expressed based on high-intensity fusion activation polypeptide, P AOX1 The promoter expression level can be increased by about 6 to 7 times.

[0159] The sequence information of each element in the above embodiments is shown in Table 3.

[0160] Table 3

[0161]

[0162]

[0163]

[0164] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Yeast transcriptional strong activator polypeptide, including: (i) yeast activating polypeptide M1, a truncated form or a conservative variant thereof, (ii) a human activating polypeptide or a conservative variant thereof, or (iii) a fusion of (i) and (ii); the yeast activating polypeptide M1 has the amino acid sequence shown in SEQ ID NO: 1, and the human activating polypeptide has the amino acid sequence shown in any one of SEQ ID NOs: 20, 22, 18, 19, and 21.

2. The polypeptide according to claim 1, characterized in that The truncated form of the yeast-derived activating polypeptide M1 has an amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 4 to 17; Preferably, the conservative variant is selected from: (a) a derived protein having the function of enhancing gene transcription activation formed by replacing, deleting or adding one or more amino acid residues of the yeast activating polypeptide M1 or its truncated form, or the sequence of the human activating polypeptide; (b) A protein derived from the yeast activating polypeptide M1 or its truncated form, or the human activating polypeptide having a sequence identity of more than 80% and having the function of enhancing gene transcription activation.

3. The polypeptide according to claim 1, characterized in that The fusions include: SEQ ID NO: 1 and SEQ ID NO: 20, SEQ ID NO: 20 and SEQ ID NO: 1, SEQ ID NO: 1 and SEQ ID NO: 22, SEQ ID NO: 22 and SEQ ID NO: 1, SEQ ID NO: 1 and SEQ ID NO: 21, SEQ ID NO: 21 and SEQ ID NO: 1, SEQ ID NO: 1 and SEQ ID NO: 19, SEQ ID NO: 19 and SEQ ID NO: 1, SEQ ID NO: 1 and SEQ ID NO: 18, or SEQ ID NO: 18 and SEQ ID NO:

1.

4. A polynucleotide encoding the yeast transcriptional strong activator polypeptide according to any one of claims 1 to 3.

5. A vector or a genetically engineered host cell containing the vector, wherein the vector contains the nucleic acid encoding the yeast transcriptional strong activator polypeptide according to any one of claims 1 to 3; or the host cell contains the vector or the nucleic acid encoding the yeast transcriptional strong activator polypeptide according to any one of claims 1 to 3 is integrated into its genome.

6. A method for preparing the yeast transcriptional activator polypeptide according to any one of claims 1 to 3, comprising the steps of: (i) culturing the host cell according to claim 5; (ii) collecting the culture containing the yeast transcriptional activator polypeptide; and (iii) isolating the yeast transcriptional activator polypeptide from the culture.

7. Use of the yeast transcriptional activator polypeptide according to any one of claims 1 to 3 for enhancing the expression of a target gene in yeast cells; or for preparing a construct or cell for enhancing the expression of a target gene in yeast cells.

8. A high-intensity expression toolbox based on the yeast transcriptional strong activator polypeptide according to any one of claims 1 to 3, comprising: (1) An expression cassette 1, which expresses an exogenous DNA binding protein, a yeast transcriptional strong activator polypeptide according to any one of claims 1 to 3; and (2) Expression cassette 2, including in order from 5' to 3', operably linked: a protein binding sequence, a promoter.

9. The high-intensity expression toolbox according to claim 8, characterized in that: The expression cassette 1 also includes a promoter, and the promoter includes: a constitutive promoter or an inducible promoter; preferably, the constitutive promoter includes: a GAP promoter, a TEF1 promoter, a GCW14 promoter; preferably, the inducible promoter includes: an AOX1 promoter, a DAS1 promoter, a FLD1 promoter.

10. The high-intensity expression toolbox according to claim 8, characterized in that: In expression cassette 1, the DNA binding protein includes: LacI, AraC, LexA, FapR; or In expression cassette 2, the protein binding sequence includes: lacO, araI, lexO, fapO; preferably, the protein binding sequence exists in 1 to 15 copies, more preferably 1 to 9 copies; or In expression cassette 2, the promoter includes a core promoter; preferably, the core promoter includes: ADH2 core promoter, AOX1 core promoter, GAP core promoter, DAS2 core promoter, GCW14 core promoter; preferably includes ADH2 core promoter.

11. The high-intensity expression toolbox according to any one of claims 8 to 10, characterized in that: The promoter of the expression cassette 2 is connected downstream to the target gene, and the two are operably connected.

12. Use of the high-intensity expression toolbox according to any one of claims 8 to 11 for recombinant expression of a target gene in yeast.

13. An isolated polynucleotide, which is a polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 31, and has the function of driving efficient gene expression.

14. Use of the isolated polynucleotide according to claim 13 as a promoter to drive efficient gene expression.

15. A method for recombinantly expressing a target gene in a yeast cell, comprising: (S1) providing the high-intensity expression toolbox according to any one of claims 8 to 10; (S2) connecting the target gene downstream of the promoter of expression cassette 2 of the high-intensity expression toolbox (S1), and the two are operably connected; (3) Introducing the high-intensity expression toolbox carrying the target gene in (S2) into yeast cells to recombinantly express the target gene.

16. The method according to claim 15, characterized in that The target gene includes: a gene encoding a biologically or pharmaceutically active protein, a genetic defect gene, a gene encoding a diagnostically active protein, a gene encoding an immunogenic protein, a gene encoding a structural protein, an enzyme encoding gene, and a reporter gene; preferably, the target gene includes a lactoglobulin encoding gene; preferably, the 5' end of the target gene is operably connected to: a natural signal peptide encoding sequence, an exogenous signal peptide encoding sequence, or a signal peptide-free encoding sequence.

17. An engineered yeast comprising the high-intensity expression toolbox according to any one of claims 8 to 11.

18. The engineered yeast according to claim 17, characterized in that: The yeast includes: methylotrophic yeast, Kluyveromyces, Saccharomyces; preferably, the methanol-trophic yeast includes: Pichia, Komagataella, Hansenula, Candida, Torulopsis.

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