Hypoxia-inducible promoters for use in yeast cells and applications thereof

Abstract

The present application provides a low-oxygen-induced promoter for yeast cells and application thereof. The promoter of the present application can achieve appropriate driving of transcription / expression of a target gene under different oxygen environments, and plays a role of driving transcription / expression with medium and high intensity.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and more specifically, this invention relates to a hypoxia-inducible promoter for yeast cells and its application. Background Technology

[0002] Promoters are fundamental gene expression regulatory elements that determine the initiation and level of gene transcription, and are crucial for accurately regulating gene expression levels. Currently, commonly used promoters in yeast expression systems mainly include inducible promoters and constitutive promoters. These promoters each have their own advantages, but also their own drawbacks and limitations. Inducible promoters generally require the addition of exogenous inducers to initiate gene expression, which not only increases costs, but some inducers can also pose safety risks. The transcription regulated by constitutive promoters is difficult to manipulate artificially, cannot respond to changes in culture environment conditions, and overexpression can impose a metabolic burden on cells, even leading to cell death.

[0003] In recent years, environmentally responsive promoters, such as those responsive to pH, temperature, osmotic pressure, and hypoxia, have attracted attention. Compared to traditional promoters that require the addition of exogenous chemical inducers, environmentally responsive promoters have advantages such as low cost, no need to change the culture medium composition, simple operation, and relative safety. However, there are few reports on such promoters that can be used in yeast expression systems.

[0004] PsADH2 is the promoter of Pichia stipites alcoholdehydrogenase II, which is induced to express under hypoxic conditions. It was first used in 2005 to express the VHb heterologous protein in Pichia stipites, showing higher expression levels under hypoxic conditions. In industrial fermentation, high-density culture easily creates a hypoxic environment, providing a natural induction condition for hypoxic-induced promoter expression. However, the reported expression intensity of the PsADH2 promoter is relatively low, making precise regulation of gene expression levels at different intensities impossible.

[0005] Therefore, developing promoters that can effectively / efficiently respond to hypoxic conditions for yeast cells has great application potential for developing high-performance fermentation strains with industrial application value. Summary of the Invention

[0006] The purpose of this invention is to provide a hypoxia-inducible promoter for yeast cells and its application.

[0007] In a first aspect of the invention, a mutant ADH2 promoter is provided, the promoter comprising a promoter selected from the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2; the mutant promoter is responsive to a hypoxic environment, activated by hypoxia, and drives the expression of a target gene.

[0008] In one or more embodiments, the promoter of the nucleotide sequence shown in SEQ ID NO:1 and the promoter of the nucleotide sequence shown in SEQ ID NO:2 have progressively decreasing strengths in promoting the expression of the target gene; preferably, the promoter of the nucleotide sequence shown in SEQ ID NO:1 is a high-intensity enhanced promoter in response to hypoxia, and the promoter of the nucleotide sequence shown in SEQ ID NO:2 is a medium-intensity enhanced (or medium-high intensity enhanced) promoter in response to hypoxia.

[0009] In another aspect of the invention, an expression construct (such as an expression cassette or expression vector) is provided, the expression construct containing the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2 as a promoter element.

[0010] In one or more embodiments, the expression construct further contains a target gene operatively linked to the promoter element.

[0011] In one or more embodiments, the target gene includes (but is not limited to): structural genes and functional genes (genes encoding proteins with specific functions).

[0012] In one or more embodiments, the functional gene includes: a reporter gene, an enzyme.

[0013] In one or more embodiments, the reporter gene includes, but is not limited to, fluorescent protein-encoding genes, luciferase genes, or galactosidase genes.

[0014] In one or more embodiments, the fluorescent protein includes, but is not limited to: green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, blue fluorescent protein, etc., or their enhanced proteins (such as enhanced green fluorescent protein).

[0015] In one or more embodiments, the target gene is located downstream of the promoter element and is spaced less than 2000 bp from the promoter.

[0016] In one or more embodiments, the target gene is located downstream of the promoter element and is spaced less than 1000 bp from the promoter; more preferably less than 500 bp, such as less than 200 bp, less than 100 bp, less than 50 bp, less than 30 bp, less than 20 bp, less than 10 bp, or no space.

[0017] In another aspect of the invention, a genetically engineered host cell is provided, said host cell: containing the expression construct; or having nucleic acid with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2 integrated into its genome; preferably, its genome also integrates a target gene, which is operatively linked to the promoter element.

[0018] In one or more embodiments, the cell is a yeast cell.

[0019] In one or more embodiments, the cells are Pichia Pastoris cells.

[0020] In another aspect of the invention, the use of any of the mutant ADH2 promoters described above is provided for linking to a target gene, responding to a hypoxic environment in the expression setting, being activated by hypoxia and driving the expression of the target gene.

[0021] In another aspect of the present invention, a method for driving the expression of a target gene under hypoxic conditions is provided, the method comprising:

[0022] (a) Transforming a host cell with an expression construct (such as an expression cassette or expression vector), the expression construct containing any of the promoters described above and a target gene operatively linked to the promoter;

[0023] (b) The host cell of (a) is placed in a fermentation expression system that can create a hypoxic environment; the promoter responds to the hypoxic environment, is activated by hypoxia and drives the expression of the target gene.

[0024] In one or more embodiments, in the method for expressing the target gene under hypoxic conditions, the fermentation expression system is set to a hypoxic environment in the initial expression stage, or a hypoxic environment is formed during the expression process (early, middle and late stages of expression).

[0025] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0026] Figure 1 Construction process of plasmid pAHg.

[0027] Figure 2 , three The process of constructing recombinant plasmids for promoter regulation of the laZ and vgb genes.

[0028] Figure 3 Construct recombinant plasmid libraries and recombinant bacterial libraries containing mutant promoters.

[0029] Figure 4 High-throughput screening of mutant promoters and promoter library construction process.

[0030] Figure 5 Growth curves of recombinant bacteria with mutant promoters AM23 and AM30 regulating the expression of different reporter genes under different oxygen supply conditions;

[0031] A. Regulates egfp reporter genes;

[0032] B. Regulates the lacZ reporter gene;

[0033] C. Regulates the vgb reporter gene;

[0034] High oxygen conditions are represented by hollow symbols, and low oxygen conditions are represented by solid symbols; each sample is tested in triplicate.

[0035] Figure 6 Hypoxia-inducible properties of AM23 and AM30 promoter mutant sequences;

[0036] A.yEGFP expression level;

[0037] B.egfp gene transcription level;

[0038] Hollow symbols indicate hyperoxic culture conditions, and solid symbols indicate hypooxic culture conditions; transcription levels are referenced to the transcription levels of the ADH2 wild-type promoter at 6h; each sample is tested in triplicate.

[0039] Figure 7 Mutant promoters AM23 and AM30 express the lacZ and vgb genes under different oxygen supply conditions;

[0040] A. β-Gal unit bacterial cell enzyme activity;

[0041] B. VHb cell yield per unit volume;

[0042] C.lacZ gene transcription level;

[0043] D. vgb gene transcription level;

[0044] In this study, hyperoxia conditions are represented by hollow symbols, and hypooxia conditions are represented by solid symbols. The enzyme activity and VHb yield obtained from each sample were normalized to the cell dry weight (g / L) to obtain the β-Gal unit cell enzyme activity and VHb unit cell yield. The transcription level was calculated using a wild-type ADH2 promoter sample under hyperoxia conditions for 6 hours as a reference. Each sample was tested in triplicate.

[0045] Figure 8 The regulatory characteristics of different genes expressed by recombinant bacteria with AM30 promoter mutant sequences during fed-batch shake-flask fermentation;

[0046] A. Protein yield;

[0047] B. Transcription level;

[0048] Protein yield was measured using the 6-hour cell protein expression level of each reporter gene as a reference; relative transcription level was measured using the 6-hour transcription level of each reporter gene as a reference. Detailed Implementation

[0049] Through extensive and in-depth screening studies, the inventors constructed a mutant ADH2 promoter library based on the hypoxia-induced wild-type promoter ADH2 sequence, and screened mutant promoters that possess both hypoxia response (activation) characteristics and general regulatory functions. The promoters of this invention can appropriately drive the transcription / expression of target genes under different oxygen conditions, exerting a medium- to high-intensity driving effect on transcription / expression.

[0050] the term

[0051] As used in this article, "isolated" means that a substance has been separated from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their natural state.

[0052] As used herein, a “promoter” refers to a nucleic acid sequence that is typically located upstream (5' end) of the coding sequence of a target gene and guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.

[0053] As used herein, an "element" refers to a set of functional nucleic acid sequences useful for protein expression, in which the "elements" are systematically constructed to form a nucleic acid construct. The sequences of the "elements" may be those provided in this invention, as well as their variants, as long as these variants substantially retain the function of the "elements".

[0054] As used herein, an "expression cassette" refers to a gene expression system containing all the necessary elements required to express a target gene, typically including the following elements: a promoter, a target gene sequence, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc. These elements are operatively linked to form the expression cassette.

[0055] As used herein, "responding to a hypoxic environment, being activated by hypoxia and driving the transcription / expression of the target gene" means that the promoter of the present invention can sense a hypoxic environment, adapt to the hypoxic environment and be activated by it, thereby playing a more significant role in driving the transcription / expression of the target gene.

[0056] As used in this invention, the term "low-oxygen environment" or "low-oxygen conditions" generally refers to a fermentation broth (culture medium) with an oxygen content lower than that of a conventional culture oxygen environment, for example, less than 80%.

[0057] As used in this invention, the term "high-oxygen environment" or "high-oxygen conditions" generally refers to a fermentation broth (culture medium) with an oxygen content higher than that of a conventional culture oxygen environment, for example, higher than 120%.

[0058] As used herein, “ADH2 promoter assembly” refers to a collection of nucleic acids containing the mutant ADH2 promoters screened by this invention.

[0059] As used herein, the “ADH2 promoter” may also be abbreviated as pADH2 or P. ADH2 .

[0060] As used herein, “operationally linked” or “operationally coupled” refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter, thereby the promoter is “operationally linked” to the nucleic acid sequence.

[0061] As used herein, "target gene" refers to a gene that can be transcribed / expressed under the guidance of the promoter of this invention. This invention does not impose any particular limitation on suitable target genes, including, but not limited to: structural genes, genes encoding proteins with specific functions, enzymes, and reporter genes (such as green fluorescent protein, luciferase genes, or galactosidase genes like LacZ). It is known in the art that yeast cells can be used for large-scale transcription / expression of target genes, and such genes suitable for transcription / expression by yeast can be used as the "target genes" described in this invention.

[0062] As used herein, the term "enhanced ADH2 promoter" refers to a promoter that significantly enhances the transcriptional / expression intensity of the target gene compared to the wild-type ADH2 promoter (whose sequence is shown in SEQ ID NO:8), for example, by more than 10%, preferably more than 20%, even more than 30%, such as more than 50%, more than 80%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 800%, more than 1000%, and so on. The intensity of the target gene transcription / expression can be determined by detecting the amount of the target gene transcribed / expressed, a technique well known to those skilled in the art.

[0063] As used herein, the terms "high-intensity enhanced promoter" or "medium-intensity enhanced promoter" are relative. Generally speaking, the former corresponds to hypoxia and drives the transcription / expression of the target gene significantly more strongly than the latter; and compared with the wild-type ADH2 promoter, they show a significant increase in the intensity of driving the transcription of the target gene or the transcription / expression of the target protein. In some embodiments, the "high-intensity enhanced promoter" is defined as one that, compared with the wild-type ADH2 promoter, shows a very significant increase in the intensity of driving the transcription of the target gene or the expression of the target protein, for example, 1.8 times higher, preferably 2 times higher, more preferably 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times higher, or even higher. In some embodiments, the medium-strength enhanced promoter is defined as one that, compared to the wild-type ADH2 promoter, significantly increases the intensity of transcription of the target gene or transcription / expression of the target protein, for example, by more than 1.1 times, preferably more than 1.2 times, and more preferably more than 1.5 times, 1.8 times, 2 times, 2.5 times, or 3 times. It should be understood that, depending on the target gene / protein, the increase in transcription / expression driven by the high-strength and medium-strength enhanced promoters compared to the wild-type promoter is variable, but the increase in high-strength promoters will be greater than that in medium-strength promoters.

[0064] As used in this article, "exogenous" or "heterogeneous" 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 target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0065] As used in this article, “driving the transcription / expression of the target gene”, “initiating the transcription / expression of the target gene”, and “guiding the transcription / expression of the target gene” can be used interchangeably.

[0066] promoters in response to hypoxic environments

[0067] To obtain promoters responsive to hypoxic conditions, the inventors conducted extensive research. According to a specific embodiment of the present invention, starting with the wild-type ADH2 hypoxia-inducible promoter, the promoter was mutated to obtain a large number of promoter mutant sequences. Using egfp as a reporter gene and Pichia pastoris as the host, a highly efficient high-throughput hypoxia-inducible screening method was established to screen for promoter mutant sequences with different expression intensities, and a yeast ADH2 hypoxia-inducible promoter library was constructed. Furthermore, a medium-intensity enhanced promoter AM23 and a high-intensity enhanced promoter AM30 were selected and ligated to the target gene to verify their promoter characteristics. The results showed that AM23 and AM30 are non-strictly anaerobic promoters that can activate egfp gene expression in response to hypoxia conditions. Compared with the wild-type ADH2 promoter, they are more sensitive to hypoxia and have stronger transcriptional activity. In addition to the egfp gene, AM23 and AM30 can also regulate the expression of lacZ and vgb genes under hypoxia conditions, and can be widely applied to the regulation of different genes (exogenous genes). Furthermore, during conventional culture, as cell density increases, the dissolved oxygen concentration in the fermentation broth decreases. The mutant promoter can then respond to the low-oxygen environment and activate gene transcription. Compared with promoters induced by conventional addition of compounds or changes in culture conditions, this reduces cost and operational complexity.

[0068] Therefore, the inventors screened a group of mutant ADH2 promoters, including promoters with the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2, which can be used independently or in combination. These promoters drive target gene expression at different intensities, exhibiting high-intensity enhanced expression-driving effects and medium-intensity enhanced expression-driving effects, respectively, to guide target gene expression at different intensities under hypoxic conditions. The combined application of two promoters with different intensities ensures gradient regulation of the regulated target gene expression, enabling controllable gene perturbation and systematic analysis.

[0069] The promoter of this invention can be operatively linked to a target gene, which can be exogenous (heterologous) relative to the promoter. The target gene can typically be any nucleic acid sequence (such as a structural nucleic acid sequence), and preferably encodes a protein with a specific function, such as a protein with important properties or functions.

[0070] For example, when used to study promoter expression intensity under different environments (such as different oxygen environments), the target gene includes, but is not limited to, green fluorescent protein (GFP), enhanced GFP, luciferase gene, or galactosidase gene LacZ. "Green fluorescent protein" possesses an endogenous fluorescent group and can efficiently emit clearly visible green light when excited by ultraviolet or blue light, and it is not easily quenched by light. "Enhanced GFP" is a modified version of GFP. "Luciferase," as a representative tool for indicating gene expression status, can effectively indicate promoter-guided gene expression.

[0071] As a preferred embodiment of the present invention, promoters of appropriate strength can be selected from the promoter combination of the present invention, and they can be operatively linked to the target gene to be studied, or the target gene and the promoters can be operatively linked into a suitable vector and introduced into host cells in an appropriate manner, thereby obtaining a series of cells with different target gene expression levels. The function or use of the target gene can be determined by analyzing the metabolic status, phenotypic changes, protein expression or interaction status, and changes in various signaling molecules of these cells under different environments (such as different oxygen environments).

[0072] As a preferred embodiment of the present invention, the target gene may be a gene that is missing or underexpressed in a certain cell. The target gene may be operatively linked to the enhanced ADH2 promoter of the present invention, or the target gene may be operatively linked to the promoter of the present invention into a suitable vector and introduced into the cell in an appropriate manner, thereby expressing the target gene at a high level.

[0073] In a preferred embodiment of the present invention, the host cell expressing the target gene can be a type of cell that grows densely (high-density growth) as the expression process proceeds. This dense growth can lead to hypoxia in the entire or part of the fermentation broth (culture medium) system. In this case, the promoter of the present invention will respond to hypoxia, enhancing gene transcription / expression with high intensity or with moderate intensity.

[0074] The promoter of this invention can also be operatively linked to a modified target gene sequence, which is exogenous (heterologous) relative to the promoter. The target gene can be modified to produce various desired properties. For example, the target gene can be modified to increase the content of essential amino acids, improve the translation of amino acid sequences, alter post-translational modifications (such as phosphorylation sites), transport translation products extracellularly, improve protein stability, insert or delete cellular signals, etc.

[0075] Furthermore, promoters and target genes can be designed to downregulate specific genes. This is typically achieved by linking a promoter to the target gene sequence, which is then directed in an antisense manner. This antisense technique is familiar to those skilled in the art. Any nucleic acid sequence can be modulated in this way.

[0076] Expression constructs containing promoters

[0077] Any promoter and / or target gene sequence derived from the promoter combinations of the present invention may be included in the expression construct. The expression construct includes an expression vector or expression cassette, etc., containing the necessary expression elements.

[0078] In one approach, an expression construct (expression vector) is provided, which includes the promoter of the present invention, and downstream of the promoter comprises a multiple cloning site or at least one restriction enzyme site. When it is necessary to express a target gene, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter.

[0079] In one manner, the expression construct (expression vector) comprises (from 5' to 3' direction): a promoter to guide the transcription of the target gene, and the target gene. If desired, the recombinant vector may further include a 3' transcription terminator, a 3' polynucleotide signal, other untranslated nucleic acid sequences, transport and target nucleic acid sequences, resistance selection markers, enhancers, or operators.

[0080] The methods used to prepare recombinant vectors are well known to those skilled in the art. The term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within a host cell. Preferably, the expression vector is an expression vector suitable for yeast cells.

[0081] Methods well known to those skilled in the art can be used to construct expression vectors containing the promoter and / or target gene sequence described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0082] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting the transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein (GFP).

[0083] In addition to the promoter of this invention, the recombinant vector may also contain one or more other promoters. These other promoters may be, for example, tissue-specific, constitutive, or inducible promoters.

[0084] Vectors containing the appropriate promoters and target genes described above can be used to transform suitable host cells so that they can express proteins.

[0085] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include yeast, Escherichia coli, animal tissue cells, and plant cells. Those skilled in the art will understand how to select an appropriate carrier and host cell. As a preferred embodiment of the present invention, the host cell is a yeast cell, and more preferably, the host cell is a Pichia pastoris cell.

[0086] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0087] The main advantages of this invention are:

[0088] (1) This invention obtained promoters whose expression ability is enhanced by low temperature through extensive screening, and their ability to promote transcription / expression is significant.

[0089] (2) The mutant promoter of the present invention is non-strictly anaerobic in response to oxygen and can adapt to the conventional environment. As the oxygen consumption of the cells increases and the dissolved oxygen decreases with cell growth, it can spontaneously activate the expression of a variety of target genes without additional induction conditions. This characteristic has great application potential in industrial fermentation.

[0090] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0091] Materials and methods

[0092] 1. Strains, plasmids, and primers

[0093] The strains used in this invention are shown in Table 1.

[0094] Table 1. Strains and Characteristics

[0095]

[0096] The plasmids used in this invention are shown in Table 2.

[0097] Table 2. Plasmids and their characteristics

[0098]

[0099]

[0100] The primers used in this invention are shown in Table 3.

[0101] Table 3. Primers

[0102]

[0103]

[0104] The promoter sequences obtained by screening in this invention are shown in Table 4.

[0105] Table 4. Sequences of mutant promoters AM23 and AM30

[0106]

[0107] 2. Main reagents and instruments

[0108] The molecular biology tools, including restriction endonucleases, ligases, DNA markers, reverse transcription kits, and RT-PCR enzymes, were purchased from Dalian TaKaRa Biotechnology Co., Ltd.

[0109] The molecular cloning kits, including those for plasmid extraction, PCR purification, and gel extraction, were purchased from Axygen.

[0110] Yeast genomic DNA extraction kit and yeast RNA extraction kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0111] The Varioskan LUX Microplate Reader was purchased from Thermo Fisher Scientific.

[0112] 3. Culture medium

[0113] For YPD and MD media, please refer to the Invitrogen Pichia pastoris manual.

[0114] The glucose content in the carbon-limited YPD medium used for high-throughput screening was 0.2% (w / v).

[0115] BMGT medium (%, w / v): 1.34% YNB, 1% glycerol, 2% tryptone, 4 × 10⁻⁶ -5 % Biotin, 100mM phosphate buffer (pH 6.0).

[0116] 4. Plasmid extraction, DNA fragment purification, and gel recovery

[0117] Plasmid extraction, PCR product purification, enzyme digestion product purification, and gel recovery were all performed according to the instructions of the relevant kits from Axygen.

[0118] For other basic molecular cloning experiments, such as routine PCR, enzyme digestion reactions, ligation reactions, and Pichia pastoris-related molecular manipulations, please refer to *Molecular Cloning: A Laboratory Manual* and the Invitrogen Pichia pastoris manual. For Pichia pastoris genome extraction, total RNA extraction, reverse transcription of cDNA, and RT-PCR, please refer to the instructions for the relevant kits and enzyme tools.

[0119] 5. Error-prone PCR reaction (EP-PCR)

[0120] 50μL reaction system:

[0121]

[0122] Commonly misused PCR reaction parameters:

[0123]

[0124]

[0125] 6. Construction of recombinant plasmids and corresponding recombinant bacteria

[0126] (1) Construction of pAHg recombinant plasmid and G / AHg recombinant bacteria

[0127] First, the recombinant plasmid pAHg was constructed using the hypoxia-inducible promoter ADH2. Using egfp as the reporter gene and pGHg as the vector backbone, the PGAP promoter in the vector was replaced with the hypoxia-inducible promoter ADH2. The basic expression vector construction process is as follows: Figure 1 As shown, the vector pGHg was digested at the SpeⅠ / NotⅠ site, and the large vector backbone was recovered by gel extraction. Then, the ADH2 promoter fragment was amplified from the Pichia pastoris genome using primers ADH2-F / ADH2-R. The vector backbone and the ADH2 promoter fragment were then ligated to obtain the basic expression vector pAHg.

[0128] Plasmid pAHg was linearized at the SalⅠ site and transformed into Pichia pastoris GS115 competent cells by electroporation. Transformants were obtained by plate culture. Positive transformants were randomly picked from the plates, and genomic DNA was extracted from them. RT-PCR was performed using arg4 as an internal reference gene, with primers qarg4-F / qarg4-R for the arg4 gene and qegfp-F / qegfp-R for the egfp gene to determine copy number. Single-copy strains were selected and named G / AHg.

[0129] (2) Construction of recombinant plasmids and recombinant bacteria containing the mutant promoter sequences AM23 and AM30

[0130] After large-scale screening and comparison, the inventors selected and obtained the mutant promoter sequences AM23 and AM30.

[0131] The wild-type ADH2 promoter was replaced at the SpeⅠ / NotⅠ site in the pAHg recombinant plasmid to obtain recombinant plasmids pAM23g and pAM30g containing the mutant promoter sequences AM23 and AM30, which regulate the egfp reporter gene.

[0132] The recombinant plasmid was transformed into Pichia pastoris, and single-copy positive transformants were selected and named recombinant strains G / AM23g and G / AM30g, respectively.

[0133] (3) Constructing recombinant plasmids for lacZ and vgb reporter genes and recombinant bacteria

[0134] Plasmids pAHg, pAM23g, and pAM30g were digested with NotI / HindIII to obtain three vector backbones containing different promoters. The lacZ gene was amplified from plasmid pPIC9KL using primers lacZ-F / lacZ-R, and the vgb gene was amplified from plasmid pPIC9K-vgb using primers vgb-F / vgb-R. These amplifications were then inserted into the NotI / HindIII sites of the three different promoter vector backbones to construct recombinant plasmids pAHl, pAM23l, and pAM30l for the lacZ gene, and recombinant plasmids pAHb, pAM23b, and pAM30b for the vgb gene. Figure 2 ).

[0135] The recombinant plasmid was transformed into Pichia pastoris, and single-copy positive transformants were selected to obtain recombinant strains G / AHl, G / AM23l and G / AM30l containing different promoters to regulate lacZ gene expression, as well as recombinant strains G / AHb, G / AM23b and G / AM30b containing different promoters to regulate vgb gene expression.

[0136] 7. Strains Culture Methods

[0137] (1) Shake flask culture under different oxygen supply levels

[0138] Differences in the oxygen environment under shake flask culture conditions are achieved by controlling the shake flask volume, liquid volume, rotation speed, and sealing method. The specific culture method is as follows:

[0139] (1) Seed culture. Single colonies of recombinant Pichia pastoris were picked from the plate and inoculated into 3 mL of YPD medium. The culture was carried out overnight at 30°C and 220 rpm.

[0140] (2) Enrichment culture. Take 1 mL of seed culture and inoculate it into a 500 mL shake flask containing 100 mL of YPD medium. Incubate at 30°C and 220 rpm until OD reaches 100%. 600 =0.5.

[0141] (3) The enriched bacterial culture was divided into two equal volumes, 50 mL each, and further cultured under two different oxygen supply conditions. High oxygen condition: 500 mL shake flask, final volume 10%, sealed with eight layers of medical gauze, rotation speed 220 rpm (oxygen content of the culture medium is above 120% of the conventional culture oxygen environment); Low oxygen condition: 100 mL shake flask, final volume 50%, sealed with microporous rubber stopper, rotation speed 150 rpm (oxygen content of the culture medium is below 80% of the conventional culture oxygen environment). During the culture process, samples were taken periodically to detect the expression levels and transcription levels of yEGFP, β-Gal, and VHb per unit bacterial protein.

[0142] (2) Culture under conventional non-oxygen-limiting conditions

[0143] Recombinant Pichia pastoris strains containing mutant promoters of different reporter genes were inoculated into 3 mL of YPD medium and cultured overnight. Subsequently, a 1% inoculum was transferred to 250 mL shake flasks containing 50 mL of YPD medium and cultured at 220 rpm. At 48 h and 72 h, 2 mL of 2×YPD liquid medium was added. During the culture process, samples were taken periodically to detect the expression levels and transcriptional levels of yEGFP, β-Gal, and VHb per unit cell.

[0144] 8. Detection of reporter genes

[0145] (1) Detection of yEGFP fluorescent protein

[0146] Sample preparation: During high-throughput screening, after plate culture, the culture medium was diluted appropriately with PBS buffer. During shake-flask culture, 1 mL of bacterial culture was centrifuged, the supernatant was discarded, and the culture was diluted appropriately with PBS buffer. Fluorescence intensity was detected using a multi-mode microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The OD600 value was also measured, and the fluorescence intensity per bacterial cell was calculated.

[0147] (2) Detection of β-galactosidasey (β-Gal) enzyme activity

[0148] For detailed experimental procedures and related solution preparation, please refer to the Pichia pastoris manual. Use a multi-functional microplate reader to read the OD value of each well. 420 Numerical value. Calculate the enzyme activity per unit cell using the following formula.

[0149]

[0150] (3) Determination of VHb activity by CO difference spectroscopy

[0151] Take a certain volume of crude intracellular extract, add excess Na2S2O4 (final concentration 10 mg / mL), mix thoroughly, and reduce for 10 min. Divide the reduced crude intracellular extract into two equal portions, each placed in a 10 mL test tube. One portion serves as a control sample, while the other portion is bubbled with CO at 2-3 bubbles per second for 3 min to allow CO to complex with the reduced VHb. After aeration, allow to stand in the dark for 5 min, then immediately perform differential spectroscopy. Use a multi-mode microplate reader to scan in the wavelength range of 380-500 nm, recording the absorbance values ​​at 419 nm and 436 nm. The extinction coefficient E419-436 = 274 nm is used. - 1 cm -1 The expression level was calculated. Three parallel tests were performed at each time point.

[0152] Example 1: Establishment of a high-throughput screening method and screening of mutant promoters

[0153] 1. High-throughput screening

[0154] Creating a hypoxic condition suitable for high-throughput culture to simultaneously activate the transcriptional regulation of large numbers of recombinant bacterial mutant promoters, thereby enabling the expression and detection of reporter genes, is one of the most critical factors in promoter library construction.

[0155] First, the recombinant bacteria G / AHg was used as the research object and inoculated into a 96-well culture plate containing 500 μL of YPD liquid medium in each well. The culture was carried out at 30°C and 220 rpm for 24 h. This stage was seed culture.

[0156] Subsequently, the pre-culture stage began. 200 μL of seed culture was aspirated from each well of the seed culture plate and inoculated into 800 μL of YPD medium containing 0.2% glucose (w / v). The main purpose of the pre-culture stage was to promote further cell growth, provide sufficient cell concentration for the final hypoxia induction stage, and ensure that the cells in each well were growing synchronously.

[0157] After the pre-culture is completed, the induction culture stage begins. The culture plate is centrifuged at 5000 rpm for 5 min, the supernatant is discarded, and the bacterial cells are resuspended in 1 mL of BMGT medium in each well. After thorough mixing, the cells are transferred to a 96-well deep culture plate. Low-oxygen induction culture is achieved by using a high liquid volume and bacterial concentration, as well as low rotation speed and low aeration.

[0158] Subsequently, the hypoxia-induced culture plates were incubated at 30°C and 150 rpm for 72 h. After the incubation period, the fluorescence intensity of the reporter gene egfp expressed protein was detected.

[0159] 2. Obtaining mutant promoters

[0160] The wild-type sequence of the ADH2 promoter was randomly mutated using error-prone PCR (EP-PCR). To obtain a higher mutation rate, the ion concentration in the EP-PCR reaction system and the number of EP-PCR rounds were increased. The purified error-prone PCR product from each round was used as the template for the next round of reaction. The promoter mutant sequence was obtained through three consecutive rounds of EP-PCR.

[0161] After three rounds of EP-PCR reactions, the mutation rate increased from 0.47% in the first round to 1.52% in the third round. The mixed plasmid carrying the mutant promoter sequence from the third round of EP-PCR was used as a recombinant plasmid library with the mutant promoter. This mixed recombinant plasmid library was linearized at the SalⅠ site and electroporated into Pichia pastoris GS115 to obtain a recombinant bacterial library containing the ADH2 promoter mutant sequence, which will be further screened using high-throughput PCR. Figure 3 ).

[0162] 3. High-throughput screening of promoters with different mutant sequences

[0163] Single colonies of the recombinant bacterial library were picked from plates and cultured in high-throughput plates using a high-throughput screening method. The fluorescence intensity of the recombinant bacterial yEGFP was detected using a microplate reader. The relative fluorescence intensity of the mutant promoters was calculated using the G / AHg ratio as a reference to represent the relative activity of the mutant promoters. Finally, mutant promoters with different relative activities were selected to construct hypoxia-inducible promoter libraries. Figure 4 ).

[0164] 4. Construction of hypoxia-induced promoter library

[0165] A total of 12,442 strains were screened and tested. Recombinant bacteria with different fluorescence intensities were selected, genomic DNA was extracted and copy number was detected. After confirming single-copy integration, the mutant promoter sequences were determined, 30 promoters with different mutant sequences were identified, and a hypoxia-inducible promoter library was constructed.

[0166] Example 2: Shake-flask culture verification of the hypoxia-inducible properties of mutant promoters AM23 and AM30

[0167] After comprehensive analysis, the inventors selected two mutant promoters, AM23 and AM30, and their corresponding recombinant bacteria, G / AM23g and G / AM30g, from 30 promoters. Two oxygen supply methods, hyperoxia and hypooxia, were used in shake-flask culture to verify the results of the high-throughput screening. To eliminate differences caused by varying bacterial growth, the inventors used the ratio of total fluorescence intensity to bacterial growth, i.e., the relative fluorescence intensity per unit bacterial cell, to represent the protein expression level regulated by the promoter.

[0168] The growth status of the three recombinant bacteria was analyzed under two oxygen supply conditions, and the results are as follows: Figure 5 As shown in Figure A, the three recombinant bacteria exhibited significant differences in cell growth under two different oxygen supply conditions. The overall growth curves revealed that the three recombinant bacteria showed higher cell concentrations under hyperoxia conditions compared to hypooxia. Furthermore, the three recombinant bacteria maintained the same growth trend under each oxygen supply condition, indicating that the expression of the egfp reporter gene by the mutant promoter does not impose an additional growth burden on the strains.

[0169] like Figure 6 As shown in Figure A, the expression intensity order of the three promoters under the two oxygen supply conditions is consistent with the results of high-throughput screening, i.e., AM30 is the highest, and the wild-type ADH2 promoter is the lowest. Under hypoxic conditions, the expression levels of all mutant promoters in recombinant bacteria are higher than their respective levels under hyperxic conditions, indicating that the mutant promoters are further activated and induced to increase protein expression levels under hypoxic conditions. Compared with their respective hyperxic conditions, the relative fluorescence intensities regulated by the three promoters under hypoxic conditions increased by 1.5-fold, 1.6-fold, and 1.6-fold, respectively. The highest relative fluorescence intensities obtained by the mutant promoters AM23 and AM30 under hypoxic conditions were 4.7-fold and 8.1-fold higher than those of the wild-type ADH2 promoter under hypoxic conditions, respectively, and 3.5-fold and 5.5-fold higher than those under the plate culture conditions during high-throughput screening.

[0170] Subsequently, the transcriptional levels of the two mutant promoters were examined, with the transcriptional level of the wild-type ADH2 promoter under 6 hours of hyperoxia as a control. The results are as follows: Figure 6B. Overall, the transcriptional levels of all promoters under hypoxic conditions were higher than those under hyperxic conditions. The transcriptional levels of the three promoters gradually increased before 24 hours under both oxygen supply conditions, reaching their maximum at 24 hours. Furthermore, the order of transcriptional levels was consistent with the protein expression levels, with the mutant promoter AM30 showing the highest level and the wild-type ADH2 promoter showing the lowest. In the early stage of culture (6 hours), the transcriptional level of the mutant promoter AM30 was higher than the other two promoters, indicating that the mutant promoter AM30 was more sensitive to hypoxia. Under hypoxic culture conditions, the highest transcriptional levels of the mutant promoters AM30, AM23, and wild-type ADH2 promoter were 1.4, 1.5, and 1.9 times higher than those under hyperxic conditions, respectively. Simultaneously, the highest transcriptional levels of the mutant promoters AM30 and AM23 reached 3.6 and 7.6 times that of the wild-type ADH2 promoter, respectively, indicating a significant increase in transcriptional levels.

[0171] Based on the above experimental results, by setting two oxygen supply conditions under shake-flask culture conditions, the high-throughput screening method was verified to obtain effective promoters. Specifically, under the same culture conditions, the mutant promoters AM23 and AM30 exhibited higher induction strength than the wild-type ADH2 promoter. The experimental results also demonstrated that the mutant promoters showed higher protein expression and transcription levels under hypoxic conditions than under hyperxic conditions. Furthermore, under the same culture conditions, as the bacterial cell mass increased and the oxygen content in the culture environment decreased, the promoters of this invention responded to the hypoxic environment, further activating and spontaneously inducing to achieve even higher induction strength.

[0172] Example 3: Applicability of the hypoxia regulation characteristics of mutant promoters

[0173] To investigate the applicability of hypoxia-induced promoters to express multiple heterologous proteins, the gene lacZ encoding β-Gal and the gene vgb encoding VHb were selected as two other reporter genes. Recombinant bacteria were constructed using the wild-type ADH2 promoter and the mutant promoters AM23 and AM30 to regulate the expression of the two heterologous proteins and to investigate the hypoxia-regulated characteristics of the mutant promoters.

[0174] First, the cell growth of recombinant bacteria was investigated when the mutant promoter regulated the lacZ and vgb genes. Figure 5 B and Figure 5(C) For the three recombinant bacteria expressing β-Gal, the cell mass increased almost synchronously, indicating that the two mutant promoters AM23 and AM30 did not adversely affect the growth of the recombinant bacteria during heterologous expression of the lacZ gene. For the three recombinant bacteria expressing VHb, at the end of 48 hours of culture, the cell dry weight of the AM30 recombinant bacteria under hyperoxia conditions was approximately 8.7% higher than the other two recombinant bacteria, and under hypoxia conditions, it was approximately 7.7% higher. This indicates that the vgb gene, under the regulation of the strongest AM30 mutant promoter, expressed more VHb protein. During cell growth, VHb protein promotes the binding of oxygen to cells, thereby promoting cell growth.

[0175] Regarding protein expression, the three promoters regulated the expression intensity of two reporter genes under two different oxygen supply conditions, as follows: Figure 7 A and Figure 7 As shown in Figure B, the protein expression levels of all three promoters were higher under hypoxic conditions than under hyperxic conditions, indicating that all three promoters can respond to hypoxia to further induce protein expression. Regarding β-Gal expression ( Figure 7 A) The expression levels of the three promoters consistently increased under hypoxic conditions, with the mutant promoter AM30 exhibiting strong hypoxia-induced expression characteristics. Compared to the highest expression levels of each promoter under hyperoxia conditions, the β-Gal expression levels of promoters ADH2, AM23, and AM30 increased by 1.4, 1.9, and 1.9 times, respectively, under hypoxia conditions. Simultaneously, under hypoxia conditions, the highest expression levels reached by the mutant promoters AM23 and AM30 in recombinant bacteria at the culture endpoint (48 h) were 2.6 times and 10.1 times that of the wild-type ADH2 promoter, respectively. Regarding VHb protein expression... Figure 7 (B) Under two different oxygen supply conditions, the expression level of VHb in the three recombinant bacteria increased with culture time. Under hypoxic conditions, the expression level of the three recombinant bacteria gradually increased before 36 h. Compared with the highest expression obtained by the three bacteria under hyperxic conditions, the highest expression levels (36 h) of promoters ADH2, AM23, and AM30 under hypoxic conditions were increased by 1.4-fold, 1.5-fold, and 1.9-fold, respectively. Furthermore, under hypoxic conditions, the VHb expression levels regulated by the mutant promoters AM23 and AM30 were 1.9-fold and 3.1-fold higher than those regulated by the wild-type promoter ADH2 under the same conditions.

[0176] To gain a deeper understanding of the induction kinetics of promoter responses to oxygen environments, the transcriptional levels of the lacZ and vgb genes regulated by three different promoters were investigated. Figure 7 C and Figure 7D). Overall, under both oxygen supply conditions, the transcriptional intensity of the wild-type promoter ADH2 and the mutant promoters AM23 and AM30 also showed an intensity gradient, with the mutant promoter AM30 exhibiting the highest transcriptional intensity and the wild-type promoter ADH2 the lowest. Transcriptional levels reached their peak at 24 hours. Simultaneously, the overall transcriptional levels of all three promoters under hypoxic conditions were higher than their respective transcriptional levels under hyperxic conditions. For the lacZ gene (… Figure 7 C) Under hypoxic conditions, the highest transcriptional levels of the three promoters were increased by 1.3-fold, 1.4-fold, and 2.2-fold, respectively, compared to hyperoxia. Furthermore, under hypoxic conditions, the mutant promoters AM23 and AM30 regulated the transcriptional levels of the lacZ gene by 2.1-fold and 7.6-fold, respectively, compared to the wild-type ADH2 promoter. On the other hand, regarding the transcription of the vgb gene… Figure 7 D) Compared with hyperoxia, the transcriptional levels regulated by the wild-type ADH2 promoter and the mutant promoters AM23 and AM30 were increased by 1.5-fold, 1.7-fold, and 2.1-fold, respectively, under hypoxic conditions. Under hypoxic conditions, the transcriptional levels of the vgb gene regulated by the mutant promoters AM23 and AM30 were increased by 2.3-fold and 5.7-fold, respectively, compared with the wild-type ADH2 promoter.

[0177] Experimental results on the regulation of three reporter genes (egfp, lacZ, and vgb) by promoter mutation sequences demonstrate that, compared to hyperoxia conditions, the promoters of this invention exhibit stronger self-induction ability and greater sensitivity to hypoxia, leading to varying degrees of enhanced transcription levels and protein expression of various heterologous proteins. For the mutant promoters AM23 and AM30 under different oxygen supply conditions, compared to the wild-type ADH2 promoter, both protein expression and transcription levels were increased to varying degrees, indicating that the mutant promoters of this invention are more active in response to hypoxia induction. Furthermore, it has been demonstrated that the mutant promoters can respond to different oxygen supply conditions, spontaneously inducing expression under hypoxia without the addition of any exogenous inducers or additives, thereby inducing the expression of regulatory proteins at the transcriptional level. Moreover, the constructed promoters exhibit gradient differences in regulatory intensity under various oxygen supply conditions.

[0178] Example 4: Regulatory characteristics of the mutant promoter AM30 during shake-flask fermentation

[0179] Recombinant bacteria G / AM30g, G / AM30l, and G / AM30b containing the mutant promoter AM30 were cultured in shake flasks under conventional non-oxygen-limited conditions and fed with feed. The growth status of the recombinant bacteria and the expression and transcription levels of reporter gene proteins under the regulation of the mutant promoter were detected.

[0180] From the perspective of protein expression level ( Figure 8A) During fed-batch culture in shake flasks, the protein expression of the three reporter genes significantly increased within the initial 24 hours, then maintained a slow increase until the end of the culture, indicating that the promoters remained active until the end of fermentation (96 hours). Compared with the protein expression levels of their respective reporter genes at 6 hours, the yield of β-Gal increased the most during the culture, reaching a 45.8-fold increase until the end of the culture. The yields of the other two reporter genes, egfp and vgb, increased by 13.5-fold and 3.9-fold, respectively. The transcriptional levels of the three reporter genes under the regulation of the mutant promoter AM30 were then examined. Figure 8 (B) In the early stages of culture, the transcriptional levels of the three reporter genes showed a similar trend to protein expression, with a rapid increase in transcriptional levels before 24 hours. At 24 hours, the transcriptional levels of the reporter genes egfp, lacZ, and vgb reached 4.8, 7.4, and 6.2, respectively. After 24 hours, the increase in transcriptional levels slowed down and remained at a low level until the end of culture. At the end of culture, the transcriptional levels of the reporter genes egfp, lacZ, and vgb reached 6.2, 9.2, and 7.0, respectively, showing a slight increase compared to the 24-hour transcriptional levels. This result indicates that the promoter mutant sequence can consistently transcribe and express heterologous proteins under normal non-oxygen-limited conditions, and that in the later stages, as dissolved oxygen decreases, the promoter responds to the hypoxic environment by spontaneously inducing and further increasing the transcriptional and expression levels of heterologous proteins.

[0181] in conclusion

[0182] The inventors of this invention utilized sequential error-prone PCR to randomly mutate the wild-type Pichia pastoris ADH2 promoter. Using Pichia pastoris as the expression host, an effective high-throughput screening method for hypoxia-induced expression was established, resulting in a hypoxia-induced promoter library for yeast expression systems. From this library, the mutant promoters of this invention were selected. Specifically, by controlling the oxygen supply level in shake flasks, two mutant promoters, AM23 and AM30, with different activity levels, could be activated by hypoxia to induce the expression of target proteins such as egfp, lacZ, and vgb. The mutant promoters exhibit a non-strictly anaerobic oxygen response. Simulating a typical fed-batch fermentation process in shake flasks, taking AM30 as an example, as the cell mass grows, oxygen consumption increases and dissolved oxygen decreases, spontaneously activating the expression of egfp, lacZ, and vgb without additional induction conditions. This characteristic has significant application potential in large-scale industrial yeast fermentation.

[0183] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. sequence list <110> East China University of Science and Technology <120> Hypoxia-inducible promoters for yeast cells and their applications <130> 223635 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 585 <212> DNA <213> Artificial Sequence <400> 1 gagggaaaaa ccggggactc actttatcac gtaccgagaa attcttcgtt tcagcatcac 60 caccatgttg tccaattgca gcccgaagca cagtctaatg ctgaattttg atagagctca 120 tcgtgaacag ccagattcga agaaaggggg gatgagatcc gggttcatct gcaagagaca 180 cagaaaataa aaaacatacg atccgttcag ctacctggcg cttaaccagg aaaatcgctg 240 ctggagtggc cagcatgtca cgaggtggca gaatccgata atgtgtgatt gcgtgtagca 300 tcggcgcaag tcgaatctcg gtcatattcc gtgtctggat attattccac tattttttaa 360 ttttcaggt tggatgcgat tgttcccttt acgtctggac gatgcccgaa gccccaggta 420 tatataaggg gctcgaaagt cctttgacca gctggctgat ttgactttgt ttgttccttt 480 cttctttca tctactcatc actcaattgc attcgcaatt tcccattaat acacatttca 540 cttgctccac atattgcacc caattacata agtgctgcga tccat 585 <210> 2 <211> 585 <212> DNA <213> Artificial Sequence <400> 2 gagggaaaaa ccggggactc agattatcac gtaccgagaa attcttcgtt tcagcatcat 60 caccatgttg tccaattaca gcccgaagca cagtctaatg ctgaattttg atagagctca 120 tcgtgaacag ccagattcga agaaaggggg gatgagatcc gggttcatct gcaagagaca 180 cagaaaataa aaaacatacg atccgttcag ctacctggcg cttaaccagg aaaatcactg 240 ctggagtggc cagcatgtca cgaggtggca gaatccgata atgtgtgatt gcgtgtagca 300 tcggcgcaag tcgaatttcg gtcatattcc gtgtctggat attattccac tattctttaa 360 ttttcaggt tggatgcgat cgttcccttt acgtctggac gatgcctgaa gccccagtta 420 tatataaggg gctcgaaagt cctttgacca gctggttgat ttgactttgt ttgttccttt 480 ctttctttca tctactcatc actcaattgc attcgcaatt tcccattaat acatatttca 540 cttgctccac gtattacacc caaatgcata agtgctgcga tccat 585 <210> 3 <211> 47 <212> DNA <213> Artificial Sequence <400> 3 catccgcatt aggatcttcg actagtgagg gaaaaaccgg ggactca 47 <210> 4 <211> 47 <212> DNA <213> Artificial Sequence <400> 4 ttagacataa gcttgggttt gcggccgcat ggatcgcagc acttatg 47 <210> 5 <211> 37 <212> DNA <213> Artificial Sequence <400> 5 atttgcggcc gcatggtcgt tttacaacgt cgtgact 37 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <400> 6 cccaagcttt tatttttgac accagaccaa ctggtaatg 39 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 cgaagcggcc gcatggccac catgtt 26 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <400> 8 tcccaagctt gggctcgact tattcaac 28 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <400> 9 tcctccggtg gcagttctt 19 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 tccattgact cccgttttga g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <400> 11 atgaccgcca ctcaaaagac c 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <400> 12 ttagcagcac cagtggaaga tg 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 ccagttccat ggccaacctt 20 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <400> 14 acataacctt ctggcatggc ag 22 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <400> 15 atactgtcgt cgtcccctca aac 23 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 cggattctcc gtgggaacaa 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <400> 17 caaccgatga cattttggac g 21 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <400> 18 cgtacaaatc tgcttccact tg 22

Claims

1. A mutant ADH2 The promoter includes a promoter selected from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; the mutant promoter is responsive to a hypoxic environment, activated by hypoxia, and drives the expression of the target gene.

2. The mutant as described in claim 1 ADH2 The promoter is characterized in that, The promoters of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 have progressively decreasing strengths in initiating the expression of the target gene.

3. The mutant as described in claim 1 ADH2 The promoter is characterized in that, The promoter of the nucleotide sequence shown in SEQ ID NO: 1 is a high-intensity enhanced promoter for hypoxia response, and the promoter of the nucleotide sequence shown in SEQ ID NO: 2 is a medium-intensity enhanced promoter for hypoxia response.

4. An expression construct containing the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as a promoter element.

5. The expression construct as described in claim 4, characterized in that, The expression construct also contains a target gene operatively linked to the promoter element.

6. The expression construct as described in claim 5, characterized in that, The target genes include: structural genes and functional genes.

7. The expression construct as described in claim 6, characterized in that, The functional genes include: reporter genes and enzymes.

8. The expression construct as described in claim 5, characterized in that, The target gene is located downstream of the promoter element and is less than 2000 bp away from the promoter.

9. A genetically engineered host cell, characterized in that, The host cell mentioned: Contains the expression construct according to any one of claims 4-8; or Its genome integrates nucleic acids with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

10. The genetically engineered host cell as described in claim 9, characterized in that, Its genome also integrates a target gene, which is operatively linked to the aforementioned promoter element.

11. The host cell as described in claim 9, characterized in that, The cells mentioned are yeast cells.

12. The mutant according to any one of claims 1-3 ADH2 The purpose of the promoter is characterized by, It is used to link with the target gene, respond to the hypoxic environment in the expression environment, and be activated by hypoxia to drive the expression of the target gene.

13. A method for driving the expression of a target gene under hypoxic conditions, characterized in that, The method includes: (a) Transforming an expression construct into a host cell, the expression construct containing the promoter of any one of claims 1-3 and a target gene operatively linked to the promoter; (b) The host cell of (a) is placed in a fermentation expression system that can create a hypoxic environment; the promoter responds to the hypoxic environment, is activated by hypoxia, and drives the expression of the target gene.

14. The method for expressing the target gene under hypoxic conditions as described in claim 13, characterized in that, The fermentation expression system is set to a low-oxygen environment in the initial expression stage, or to form a low-oxygen environment during the expression process.

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