Expression tool based on reconstitution of endogenous regulatory elements of yeast, its establishment method and application in recombinant expression

Abstract

The present application relates to expression tools based on yeast endogenous regulatory elements, its establishment method and application in recombinant expression. A regulatory element derived from yeast endogenous source is screened and isolated. Based on the regulatory element, a recombinant promoter can be constructed, and further matched with a synthetic transcription factor to form a new expression tool based on the whole endogenous regulatory element, which avoids the food safety approval obstacles caused by the introduction of exogenous element sequences, and realizes the recombinant expression level of brazzein and other food proteins under various carbon sources, which is significantly better than that of commercial expression system, and has industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering; more specifically, this invention relates to an expression tool based on the reconstruction of yeast endogenous regulatory elements, its establishment method, and its application in recombinant expression. Background Technology

[0002] With the continued impact of global population growth and climate change on traditional agriculture, the food protein industry, centered on alternative proteins, is experiencing explosive growth. Among these, heterogeneous production of food proteins using microorganisms as a substrate has become a research hotspot in the field due to its advantages such as low cost, high efficiency, and good safety.

[0003] Yeast recombinant expression research using Saccharomyces cerevisiae (Saccharomyces cerevisiae) S. cerevisiae ) and Pichia pastoris ( K. phaffii ,Original P. pastoris Using CRISPR / Cas9 as the core platform, Saccharomyces cerevisiae, with its clear genetic background and mature tools, was the earliest host used for recombinant insulin, hepatitis B vaccines, and other products. Pichia pastoris, relying on alcohol-induced strong promoters such as AOX1, has become a "cell factory" for industrial enzymes and therapeutic proteins (insulin, interferon, etc.) to achieve recombinant expression. Recent research has focused on CRISPR / Cas9 editing, promoter engineering, glycosylation humanization, and the construction of protease-deficient strains, and is expanding into synthetic biology scenarios such as natural product synthesis.

[0004] Pichia pastoris is recognized as a GRAS (Generally Recognized As Safe) strain by the US FDA and has been increasingly developed as a high-quality chassis host for exogenous protein expression. This strain possesses a relatively mature expression system and a good industrial foundation, making its use as a chassis for food protein production both safe and feasible. However, current methods for exogenous protein expression in Pichia pastoris still primarily rely on methanol-inducible promoters, and the expression intensity remains insufficient to meet the practical needs of large-scale industrial production of food proteins.

[0005] Traditional strategies for discovering and modifying promoter elements have proven insufficient to achieve significant breakthroughs in the expression intensity and mode of Pichia pastoris. Therefore, researchers have attempted to introduce exogenous expression regulatory elements to construct high-intensity artificial expression systems. However, in the field of food protein production, the introduction of exogenous expression regulatory elements faces stricter regulatory constraints. According to food safety regulations in various countries, production strains containing exogenous expression regulatory elements must undergo more complex food safety assessments, and the use of exogenous elements from non-edible species is even prohibited, significantly increasing product approval cycles and compliance costs.

[0006] Therefore, there is an urgent need in this field to optimize yeast recombinant expression systems or expression schemes in order to significantly improve recombinant expression capabilities while fundamentally avoiding food safety approval obstacles caused by the introduction of exogenous element sequences, thus providing an ideal technical solution for the efficient and compliant production of food proteins. Summary of the Invention

[0007] The purpose of this invention is to provide an efficient expression tool for reconstructing all endogenous regulatory elements in Pichia pastoris, its construction method, and its application in food protein expression. By screening, modifying, and reconstructing the endogenous regulatory elements of the expression host, a matching module for expression regulation is obtained. Based on this, a high-strength expression tool is further constructed, which can significantly increase the yield of food protein while avoiding safety approval issues caused by the introduction of exogenous elements.

[0008] In a first aspect of the invention, a matching module for yeast expression regulation is provided, comprising: (1) a regulatory element (t13); (2) a DNA-binding domain (or a nucleic acid encoding thereof); and (3) an activation domain of a transcription factor (or a nucleic acid encoding thereof), which interacts with the DNA-binding domain to form a synthetic transcription factor.

[0009] In one or more embodiments, (1)-(3) are present in 1, 2 or 3 expression constructs.

[0010] In one or more embodiments, (1) exists in one expression construct (expression vector); (2) and (3) exist in another expression construct (expression vector).

[0011] In one or more embodiments, in (2) and / or (3), the DNA-binding domain and / or the activation domain of the transcription factor drive the expression of its encoding gene via a yeast endogenous promoter, the yeast endogenous promoter comprising: GAP promoter, AOX1 promoter, GCW14 promoter, TEF1 Promoter; preferably, the promoter includes GAP Promoter.

[0012] In one or more embodiments, (1) further includes a promoter; the regulatory element is operatively connected to the promoter to form a recombinant promoter for driving the expression of the target gene.

[0013] In one or more embodiments, the control element is located upstream of the promoter.

[0014] In one or more embodiments, the promoter includes a core promoter.

[0015] In one or more embodiments, (1) the downstream of the recombinant promoter includes the insertion site of the target gene.

[0016] In one or more embodiments, the insertion site of the target gene is a restriction enzyme cleavage site.

[0017] In one or more embodiments, the insertion site is prepared for inserting the target gene, or the target gene has already been inserted.

[0018] In one or more embodiments, the target gene sequence is inserted downstream of the promoter sequence and is a coding gene sequence directly adjacent to the promoter sequence. Typically, the nucleic acid sequence is directly linked to the target gene sequence, or the interval is less than 1000 bp (preferably less than 500 bp; more preferably less than 200 bp; even more preferably less than 100 bp; most preferably less than 50 bp).

[0019] In one or more embodiments, the regulatory element has a nucleotide sequence shown in SEQ ID NO:1 or having more than 80% (preferably more than 85%, more than 90%, more than 95%, more than 98%, or more than 99%) sequence identity with it.

[0020] In one or more embodiments, the promoter has a nucleotide sequence shown at positions 303-404 of SEQ ID NO:3, positions 303-463 of SEQ ID NO:4, or positions 303-483 of SEQ ID NO:5, or has a sequence identity of 80% or more (preferably 85%, 90%, 95%, 98%, or 99%) with the SEQ ID NO:5.

[0021] In one or more embodiments, the recombinant promoter has a nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or has more than 80% (preferably more than 85%, more than 90%, more than 95%, more than 98%, or more than 99%) sequence identity with it.

[0022] In one or more embodiments, the DNA binding domain has the amino acid sequence shown in SEQ ID NO:2 or has 80% or more (preferably 85%, 90%, 95%, 98%, or 99%) sequence identity with the sequence shown.

[0023] In one or more embodiments, the activation domain of the transcription factor has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, or has 80% or more (preferably 85%, 90%, 95%, 98%, or 99%) sequence identity with the shown sequence.

[0024] In another aspect of the invention, a separate regulatory element (t13) is provided, having the nucleotide sequence shown in SEQ ID NO: 1 or a similar functional variant thereof.

[0025] In one or more embodiments, the regulatory element is isolated from yeast endogenous sources.

[0026] In one or more embodiments, the regulatory element serves as an upstream regulatory sequence for gene expression.

[0027] In one or more embodiments, the same functional variant includes a regulatory element having more than 80% (preferably more than 85%, more than 90%, more than 95%, more than 98%, or more than 99%) sequence identity with the illustrated sequence.

[0028] In another aspect of the invention, the use of the aforementioned regulatory element is provided for operatively linking with a promoter to form a recombinant promoter, thereby regulating the efficient expression of a target gene (improving the expression efficiency of the target gene).

[0029] In one or more embodiments, the expression is an expression in yeast.

[0030] In another aspect of the invention, a recombinant promoter is provided, including the aforementioned control element and a promoter operatively connected thereto.

[0031] In one or more embodiments, the control element is located upstream of the promoter.

[0032] In one or more embodiments, the promoter includes a core promoter.

[0033] In one or more embodiments, the regulatory element is operatively connected to the promoter to form a recombinant promoter for driving the expression of the target gene.

[0034] In one or more embodiments, the expression is an expression in yeast.

[0035] In another aspect of the invention, an activation domain of a transcription factor is provided, having an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, or having more than 80% sequence identity with the shown sequence.

[0036] In one or more embodiments, the activation domain of the transcription factor enhances the transcription initiation or transcription level of the target gene.

[0037] In another aspect of the invention, a synthetic transcription factor is provided, comprising an operatively linked DNA-binding domain and an activation domain of the transcription factor.

[0038] In one or more embodiments, the DNA binding domain has the amino acid sequence shown in SEQ ID NO:2 or has more than 80% sequence identity with the sequence shown. In one or more embodiments, the activation domain of the transcription factor has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, or has more than 80% sequence identity with the shown sequence.

[0039] In one or more embodiments, the synthetic transcription factor regulates / optimizes gene expression.

[0040] In another aspect of the present invention, a method for expressing a target gene in yeast is provided, comprising: (a) providing the matching module for yeast expression regulation, inserting the target gene downstream of the recombinant promoter, and transforming it into yeast cells; and (b) culturing yeast under a suitable carbon source to perform recombinant expression of the target gene.

[0041] In another aspect of the invention, a recombinant expression construct or a recombinant yeast engineered strain is provided, comprising: the matching module for yeast expression regulation; or the regulatory element; or the recombinant promoter.

[0042] In one or more embodiments, the target gene includes (but is not limited to): a structural gene or a functional gene; preferably, the target gene includes (but is not limited to): a reporter gene, or a food protein encoding gene; preferably, the food protein encoding gene includes a brassinosteroid encoding gene and a β-lactoglobulin encoding gene.

[0043] In one or more embodiments, the yeast includes Pichia pastoris (Pichia pastoris). Pichia (or Fafnir yeast) Komagataella )).

[0044] In one or more embodiments, the yeast expression includes expression in a culture environment using glucose, glycerol, ethanol, and / or methanol as carbon sources.

[0045] In one or more embodiments, optionally, the 5' end of the target gene is operatively linked to a signal peptide encoding a nucleic acid.

[0046] In one or more embodiments, the Pichia pastoris is Pichia pastoris ( Pichia pastoris Or Komagataella phaffii ).

[0047] In one or more embodiments, the Pichia pastoris strain includes: NRRL-Y 11430 or a derivative thereof, GS115 or a derivative thereof.

[0048] In one or more embodiments, the signal peptide includes a natural signal peptide or an exogenous signal peptide.

[0049] In another aspect of the invention, a method for efficiently expressing brassin in yeast is provided, comprising: (i) Providing yeast cells containing: expression cassette 1: comprising, from 5' to 3', the operatively linked recombinant promoter, an α-signal peptide encoding gene, and a brassinolide encoding gene; expression cassette 2: comprising, from 5' to 3', an operatively linked promoter (e.g., ... GAP The transcription factor encodes a nucleic acid via a promoter and an endogenous synthetic transcription factor, wherein the transcription factor includes a DNA-binding domain and an activation domain. (ii) Culture the yeast cells of (i) and perform recombinant expression of Brazil protein; preferably, the recombinant expression is performed using glucose, glycerol, ethanol or methanol as carbon source.

[0050] In one or more embodiments, the endogenous recombination promoter in expression cassette 1 has the nucleotide sequence shown in SEQ ID NO:3.

[0051] In one or more embodiments, the endogenous synthetic transcription factor in expression cassette 2 has the nucleotide sequence shown in SEQ ID NO: 6-8.

[0052] In one or more embodiments, the α signal peptide has the amino acid sequence shown in SEQ ID NO:14.

[0053] In one or more embodiments, the Brazilian sweet protein has the amino acid sequence shown in SEQ ID NO:15.

[0054] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0055] Figure 1 :P AOX1 The expression intensity of three endogenous recombination promoters, t13-c1, t13-c27, and t13-cA, was also measured.

[0056] Figure 2 : Expression intensity of endogenous recombination promoter t13-c1 driven by different synthetic transcriptional activators (-D for glucose as carbon source, -M for methanol as carbon source).

[0057] Figure 3 The yield of Brazilian sweet protein in expression tools sTF0016+t13-c1, sTF0222+t13-c1, and sTF0757+t13-c1 using different carbon sources; where the carbon sources are glucose (D), glycerol (G), ethanol (E), or methanol (M).

[0058] Figure 4 The expression tools sTF0016+t13-c1, sTF0222+t13-c1, and sTF0757+t13-c1 yielded β-lactoglobulin using different carbon sources; where the carbon sources were glucose (D), glycerol (G), ethanol (E), or methanol (M). Specific implementation methods

[0059] This invention focuses on the discovery and reconstruction of endogenous expression regulatory elements in yeast. Through extensive and in-depth research, a yeast-derived regulatory element was screened and isolated. Based on this regulatory element, a recombinant promoter can be constructed, which can be further matched with synthetic transcription factors to form a novel expression tool based on a fully endogenous regulatory element. This avoids food safety approval obstacles caused by the introduction of exogenous element sequences and achieves significantly better recombinant expression levels of food proteins such as Brazil nuts under various carbon source conditions than commercial expression systems, demonstrating industrial application value. This invention was completed based on these findings.

[0060] As used herein, the "matching module for expression regulation" refers to a core functional unit used to establish specific transcriptional regulatory relationships. It does not necessarily constitute a complete transcription factor or a complete promoter, but can be further extended to a complete regulatory element. It includes at least regulatory elements derived from the upstream regulatory region of the yeast endogenous promoter and transcription factors (synthetic transcription factors, including activation domains and DNA-binding domains). It is foreseeable that the regulatory element contains one or more cis-acting sites that can be specifically recognized and bound by the DNA-binding domain.

[0061] As used herein, unless otherwise stated, "regulatory element" refers to regulatory element t13, a nucleic acid sequence with transcriptional regulatory function derived from the upstream regulatory region of the yeast endogenous promoter and artificially reconstructed. This artificial reconstruction includes, but is not limited to, truncating, deleting, splicing, rearranging, combining, replacing, preserving sites, adding or removing sites, or other engineered modifications to the endogenous upstream regulatory sequence.

[0062] As used herein, a “DNA-binding domain” refers to a protein domain or functional fragment thereof derived from a transcription factor that has the ability to recognize and bind to a specific DNA sequence. In this document, the DNA-binding domain includes active variants of the DNA-binding domain, which can be naturally occurring allelic variants or non-naturally occurring variants. These variants include substitution variants, deletion variants, insertion variants, and fusion variants.

[0063] As used herein, "endogenous" refers to genetic or regulatory elements that originate naturally in yeast, rather than from exogenous species. Unless otherwise explicitly stated, the term "endogenous" in this invention emphasizes the source of the element and does not require that the element or its combination form naturally exist in natural yeast; combinations derived from natural yeast elements and artificially reconstructed still fall within the scope of "endogenous" in this invention.

[0064] As used herein, the “core promoter” refers to the minimum DNA sequence necessary to ensure the normal initiation of RNA polymerase transcription, including the transcription start site and the upstream TATA region of the transcription start site.

[0065] As used herein, the term "transcription factor activation domain" refers to a protein domain that can recruit, bind to, or promote the function of transcription-related factors, transcription cofactors, transcription complexes, or RNA polymerase devices, thereby enhancing the transcription initiation or transcriptional level of a target gene.

[0066] As used herein, a “promoter” refers to a nucleic acid sequence, typically located upstream (5' end) of the coding sequence of a target gene, that guides the transcription of the nucleic acid sequence into mRNA. Generally, a promoter or promoter region provides recognition sites for RNA polymerase and other factors necessary for proper transcription initiation. In this document, the promoter or promoter region includes active variants of the promoter, which can be naturally occurring allelic variants or non-naturally occurring variants. These variants include substitution variants, deletion variants, insertion variants, and fusion variants.

[0067] As used herein, "operable linkage" refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region 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 region, thereby "operable linkage" of the promoter region to the nucleic acid sequence.

[0068] As used herein, an "expression cassette" refers to a gene expression system containing all the necessary elements required to express a target polypeptide, typically including the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc. These elements are operatively linked.

[0069] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0070] This invention provides a highly efficient expression tool based on the reconstruction of all endogenous regulatory elements in yeast, and applies the resulting tool to the expression of target genes. The target genes include, but are not limited to, food proteins such as brassinolide and β-lactoglobulin. More specifically, this invention focuses entirely on the discovery and modification of expression regulatory elements within yeast endogenous elements, successfully obtaining a matching module for expression regulation. Based on this, a complete recombinant promoter and synthetic transcription factor can be further constructed. The recombinant promoter can efficiently express recombinant proteins under methanol conditions, and driven by a synthetic transcription activator, a novel endogenous expression tool can be formed, further enhancing the expression levels of recombinant proteins in yeast under various carbon source conditions. The yields of food proteins such as brassinolide and β-lactoglobulin are significantly higher than those in commercially available expression systems.

[0071] In this invention, the expression regulation matching module includes (but is not limited to) regulatory elements derived from upstream regulatory sequences of yeast endogenous promoters and synthetic transcription factors. The synthetic transcription factors include a DNA-binding domain and an activation domain derived from yeast endogenous transcription factors. The regulatory elements contain one or more sites that can be specifically recognized and bound by the DNA-binding domain.

[0072] In this invention, the regulatory element is t13. t13 has the nucleotide sequence shown in SEQ ID NO:1. This invention also relates to degenerate sequences of the aforementioned polynucleotides. This invention further relates to variants of the aforementioned polynucleotides, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the DNA it encodes. This invention also relates to polynucleotides homologous to the aforementioned polynucleotides, preferably with homology of 70% or more, 80% or more, 90% or more, 93%, 95% or more, or 97% or more, whose encoded DNA also has the same function as t13.

[0073] In this invention, the DNA binding domain includes BD0836. BD0836 has the amino acid sequence shown in SEQ ID NO:2. This invention also relates to functional variants of the above-mentioned amino acid sequence, including variants formed by the substitution, deletion, or insertion of one or more amino acid residues. Those skilled in the art will understand that the above-mentioned amino acid sequence can be appropriately altered without substantially affecting its biological function. The biological function is preferably a DNA binding function, transcriptional activation function, or related regulatory function that is substantially the same as the original amino acid sequence. This invention also relates to amino acid sequences homologous to the above-mentioned amino acid sequence, preferably with a homology of 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 97% or more, and the corresponding polypeptides of these amino acid sequences also have the same function as BD0836.

[0074] In this invention, the endogenous recombination promoter includes: t13-c1, t13-c27, and t13-cA; preferably t13-c1. t13-c1 has the nucleotide sequence shown in SEQ ID NO:3. t13-c27 has the nucleotide sequence shown in SEQ ID NO:4. t13-cA has the nucleotide sequence shown in SEQ ID NO:5. This invention also relates to degenerate sequences of these polynucleotides. This invention also relates to variants of the above-mentioned polynucleotides, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the DNA it encodes. The present invention also relates to polynucleotides homologous to the above-mentioned polynucleotides, preferably with a homology of 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 97% or more, and the DNA encoded by these polynucleotides also has the same function as t13-1, t13-27 or t13-A.

[0075] In this invention, the transcription factor activation domain includes: TFAD0016, TFAD0222, TFAD0757, TFAD0503, TFAD0540, TFAD0075, TFAD0225, and TFAD0029; preferably TFAD0016 and TFAD0222. TFAD0016 has the amino acid sequence shown in SEQ ID NO:7. TFAD0222 has the amino acid sequence shown in SEQ ID NO:6. TFAD0757 has the amino acid sequence shown in SEQ ID NO:8. TFAD0503 has the amino acid sequence shown in SEQ ID NO:9. TFAD0540 has the amino acid sequence shown in SEQ ID NO:10. TFAD0075 has the amino acid sequence shown in SEQ ID NO:11. TFAD0225 has the amino acid sequence shown in SEQ ID NO:12. TFAD0029 has the amino acid sequence shown in SEQ ID NO:13. This invention also relates to functional variants of the above-mentioned amino acid sequences, including variants formed by the substitution, deletion, or insertion of one or more amino acid residues. Those skilled in the art will understand that the above-mentioned amino acid sequences can be appropriately altered without substantially affecting their biological function. The biological function is preferably a DNA-binding function, transcriptional activation function, or related regulatory function that is substantially the same as the original amino acid sequence. This invention also relates to amino acid sequences homologous to the above-mentioned amino acid sequences, preferably with a homology of 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, or 97% or more, and the polypeptides corresponding to these amino acid sequences also have the same function as the above-mentioned synthetic transcription factors.

[0076] In this invention, the expression cassette 2 includes a promoter that drives the expression of synthetic transcription factors. The promoter may be a constitutive promoter, an inducible promoter, a tissue- or organ-specific promoter, a spatiotemporally specific expression promoter, etc. Preferably, the promoter includes (but is not limited to): GAP promoter, AOX1 promoter, TEF1 promoter, GCW14 Promoter. Similarly, the expression cassette 2 also includes a suitable terminator, which is a well-known element to those skilled in the art in constructing gene expression cassettes.

[0077] In this invention, yeast cells containing the two expression cassettes can be obtained by introducing a combination of the expression cassettes of this application, or a nucleic acid construct or recombinant vector containing a combination of the expression cassettes of this application, into eukaryotic cells.

[0078] In this invention, the combination of expression cassettes can be integrated into the yeast cell genome or exist freely in the yeast cell.

[0079] 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 under conventional conditions as described in Molecular Cloning Laboratory Manual, or as recommended by the manufacturer.

[0080] Material

[0081] The plasmid construction method used was Novizan Biosciences' Seamless Cloning Kit.

[0082] All enzymes used were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Specific reaction conditions and methods were described in the product instructions.

[0083] The following commercially available plasmids and strains were used for gene cloning and protein expression: plasmid pGAP Zα, plasmid pPIC3.5K, Escherichia coli Top10, and Pichia pastoris strain GS115, all purchased from Invitrogen.

[0084] plasmid pPlacO1cAG: contains lacO -AOX1 core promoter (cA)-GFP(G) expression cassette): See Chinese patent application 201710352843.1. In this cassette, number 1 in lacO1 indicates a single copy; subsequent numbers such as 2 indicate double copies, and so on.

[0085] YP medium: 20 g / L peptone, 10 g / L yeast extract; YNB medium: 6.7 g / L YNB.

[0086] BMY medium: 20 g / L peptone, 10 g / L yeast extract, 13.4 g / L YNB, 100 mmol / L pH 6.0 phosphate buffer pair.

[0087] When preparing the above culture media, glucose should be autoclaved at 115℃ for 30 min, and other components should be autoclaved at 121℃ for 20 min. The addition amounts of glucose and glycerol should be 2%, methanol and ethanol 1%, and 2% agar powder should be added to solid culture media.

[0088] Example 1: Determination of expression intensity of endogenous recombination promoter

[0089] 1. Construction of GFP plasmid expressing endogenous recombination promoter

[0090] Using restriction endonucleases Sac Ⅰ / BamHi-I was used to linearize the plasmid pPlacO1cAG by double digestion, obtaining a vector containing the GFP sequence and plasmid backbone. Using the Pichia pastoris genome as a template, the vector was amplified using primer pair wf-AOX1-GFP-F / R (SEQ ID NO: 16-17). AOX1 The promoter fragment was used to seamlessly assemble the vector and the fragment to obtain a recombinant plasmid named pPAOX1-GFP.

[0091] Using the Pichia pastoris genome as a template, fragments were amplified using primer pairs t13-F1 / R1 (SEQ ID NO:18-19), t13-F2 / R2 (SEQ ID NO:20-21), t13-F3 / R3 (SEQ ID NO:22-23), and t13-F4 / R4 (SEQ ID NO:24-25), respectively. The regulatory element t13 was finally obtained through multiple overlap PCRs.

[0092] Using the recombinant plasmid pPAOX1-GFP as a template, a vector containing the GFP sequence and plasmid backbone was amplified using primer pair 3.5k-GFP-F / R (SEQ ID NO:26-27); using the Pichia pastoris genome as a template, the core promoter fragments c1, c27, or cA were amplified using primer pairs c1-F / R (SEQ ID NO:28-29), c27-F / R (SEQ ID NO:30-31), and cA-F / R (SEQ ID NO:32-33), respectively.

[0093] The vector, regulatory element t13, and core promoter fragment were seamlessly assembled to obtain three plasmids expressing GFP using recombinant promoters, named pPt13-c1-GFP, pPt13-c27-GFP, and pPt13-cA-GFP.

[0094] 2. Construction of strains expressing GFP using endogenous recombination promoters

[0095] The recombinant plasmids pPAOX1-GFP, pPt13-c1-GFP, pPt13-c27-GFP, and pPt13-cA-GFP were linearized and electroporated into Pichia pastoris strain GS115. The plasmids were then plated on YND solid medium and incubated at 30°C for approximately 72 hours. Single colonies were picked from the plates and transferred to YPD liquid medium, cultured at 30°C on a shaker, and the genome was extracted. PCR was used to verify the GFP expression cassette copy number, thus obtaining single-copy strains.

[0096] 3. GFP fluorescence intensity measurement

[0097] Single-copy strains corresponding to each promoter were pre-cultured in YPD liquid medium. A suitable amount of bacterial culture was collected by centrifugation, washed twice with deionized water, and then transferred to YNB liquid medium containing 1% methanol for further culture. Samples were collected after 24 h and GFP fluorescence intensity was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0098] The fluorescence expression intensity results for each promoter are as follows: Figure 1 As shown. In a commercialized manner. AOX1 Using promoters as a control, it can be seen that the strengths of the three endogenous recombination promoters t13-c1, t13-c27, and t13-cA under methanol conditions can reach [values ​​missing]. AOX1 The expression intensity was significantly improved by 3.4, 3.6, and 2.6 times the promoter.

[0099] Example 2: Determination of expression intensity of novel expression tools

[0100] 1. Construction of synthetic transcription factor expression plasmids

[0101] Using plasmid pGAP Zα as a template, primer pair GAP-Zα-F / R (SEQ ID NO: 34-35) was used to amplify the plasmid containing pGAP Zα. GAP Vectors containing promoters and plasmid backbones.

[0102] Using the Pichia pastoris genome as a template, the fragment encoding the DNA-binding domain of the corresponding transcription factor was amplified using primer pair BD0836-F / R (SEQ ID NO: 36-37). Using the Pichia pastoris genome as a template, the fragment encoding the activation domain of the corresponding transcription factor was amplified using primer pair AD0016-F / R (SEQ ID NO: 38-39). The vector and fragment were seamlessly assembled to obtain the coding sequence of the synthetic transcription factor sTF0016 (including BD0836 (N-terminus) and TFAD0016 (C-terminus), linked by GGGGS), corresponding to the recombinant plasmid pGGsTF0016.

[0103] Similarly, other recombinant plasmids were constructed: pGGsTF0222 contains the coding sequence of the synthetic transcription factor sTF0222, with sTF0222 having BD0836 at the N-terminus and TFAD0222 at the C-terminus, linked by GGGGS; pGGsTF0075 contains the coding sequence of the synthetic transcription factor sTF0075, with BD0836 at the N-terminus and TFAD0075 at the C-terminus, linked by GGGGS. pGGsTF0503 contains the coding sequence of the synthetic transcription factor sTF0503, with BD0836 at the N-terminus and TFAD0503 at the C-terminus, linked by GGGGS. pGGsTF0540 contains the coding sequence of the synthetic transcription factor sTF0540, with BD0836 at the N-terminus and TFAD0540 at the C-terminus, linked by GGGGS. pGGsTF0225 contains the coding sequence of the synthetic transcription factor sTF0225, with BD0836 at the N-terminus and TFAD0225 at the C-terminus, linked by GGGGS. pGGsTF0757 contains the coding sequence of the synthetic transcription factor sTF0757, with BD0836 at the N-terminus and TFAD0757 at the C-terminus, linked by GGGGS. pGGsTF0029 contains the coding sequence of the synthetic transcription factor sTF0029, with BD0836 at the N-terminus and TFAD0029 at the C-terminus, linked by GGGGS.

[0104] 2. Construction of GFP expression strains based on novel expression systems

[0105] Recombinant plasmids corresponding to the synthetic transcription factors (pGGsTF0016, pGGsTF0222, pGGsTF0075, pGGsTF0503, pGGsTF0540, pGGsTF0225, pGGsTF0757, pGGsTF0029) were linearized and electroporated into GFP expression strains corresponding to the recombinant promoter t13-c1. The plasmids were then plated on YPD solid medium supplemented with Zeocin antibiotic and incubated at 30°C for approximately 72 hours. Single colonies were picked from the plates and transferred to YPD liquid medium, cultured at 30°C on a shaker, and the genome was extracted. PCR was used to verify the copy number of the expression cassette, and single-copy strains were obtained.

[0106] 3. GFP fluorescence intensity measurement

[0107] Based on composition GAP When driven by the promoter, the constructed expression system can be expressed at high intensity under various carbon source conditions. In the GFP assay, only the expression intensity under the conventional carbon source glucose is used as an example.

[0108] Each single-copy strain was pre-cultured in YPD liquid medium. A suitable amount of bacterial culture was centrifuged to collect the bacterial cells. After washing twice with deionized water, the cells were transferred to YPD liquid medium containing 2% glucose for further culture. Samples were taken at 24 h and 48 h and the GFP fluorescence intensity was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0109] The fluorescence expression intensity results for each synthetic transcription activator are as follows: Figure 2 As shown. In a commercialized manner. AOX1As a control, the fluorescence intensity of the promoter in YPD and YPM media can be observed. AOX1 The promoter was almost not expressed in YPD medium but was expressed in YPM medium, indicating that it is strictly induced by methanol. The expression intensity of the eight constructed expression systems was relatively high under glucose conditions. AOX1 The promoter has seen a significant improvement, and is significantly higher than... AOX1 The expression intensity of the promoter under methanol conditions can reach up to 6.7 times.

[0110] Example 3: Application of novel expression tools in the expression of carbazin

[0111] 1. Construction of Brazzein expression plasmid

[0112] Using recombinant plasmids pPAOX1-GFP and pPt13-c1-GFP as templates, vectors containing promoters and plasmid backbones were amplified using primers. The vectors, α-signal peptide fragments, and brassinolide fragments (SEQ ID NO:15) were then seamlessly assembled to obtain recombinant plasmids pPAOX1-Brazzein and pPt13-c1-Brazzein.

[0113] 2. Construction of strains expressing Brazil's sweet protein

[0114] Recombinant plasmids pPAOX1-Brazzein and pPt13-c1-Brazzein were linearized and electroporated into Pichia pastoris strain GS115, respectively. The plasmids were then plated on YND solid medium and incubated at 30°C for approximately 72 hours. Single colonies were picked from the plates and transferred to YPD liquid medium, cultured at 30°C on a shaker, and the genome was extracted. PCR was used to verify the GFP expression cassette copy number, yielding single-copy strains AOX1-Brazzein and t13-c1-Brazzein.

[0115] Recombinant plasmids pGGsTF0016, pGGsTF0222, and pGGsTF0757 were linearized and electroporated into the Brazilian sweet protein expression strains corresponding to the recombinant promoter t13-c1 (AOX1-Brazzein or t13-c1-Brazzein). The plasmids were then plated onto YPD solid medium plates supplemented with Zeocin antibiotic and incubated at 30°C for approximately 72 hours. Single colonies were picked from the plates and transferred to YPD liquid medium, cultured at 30°C on a shaker, and the genome was extracted. PCR was used to verify the copy number of the expression cassette, obtaining single-copy strains.

[0116] 3. Brazil gluten fermentation production

[0117] The above-mentioned strains were pre-cultured in YPD medium. An appropriate amount of bacterial solution was centrifuged to collect the bacterial cells. After washing twice with deionized water, strains AOX1-Brazzein and t13-c1-Brazzein were transferred to BMMY liquid medium, respectively. The strains corresponding to the three expression tools were transferred to BMY liquid medium supplemented with glucose, glycerol, ethanol or methanol, respectively. The culture time was 96 h, during which the corresponding carbon source was added to the medium every 24 h.

[0118] After fermentation, the supernatant was collected by centrifugation and the protein content was determined. The results are as follows: Figure 3 As shown, under methanol conditions, the yield of brassinoprotein corresponding to the recombinant promoter t13-c1 can reach that of commercial promoters. AOX1 The yield was 2.2 times higher than that of the commercial promoter, and the three expression tools also significantly outperformed the commercial promoter under four carbon source conditions. AOX1 The yield under methanol conditions can be up to 7.0 times higher.

[0119] This result demonstrates that the recombinant promoter and expression tool established in this invention can be successfully applied to the production of Brazilin, and its production capacity is superior to that of current commercial systems under conventional carbon sources such as glucose (D), glycerol (G), ethanol (E), and methanol (M).

[0120] Example 4: Application of the novel expression system in β-lactoglobulin expression

[0121] 1. Construction of β-lactoglobulin (BLG) expression plasmid

[0122] Using recombinant plasmids pPAOX1-GFP and pPt13-c1-GFP as templates, vectors containing promoters and plasmid backbones were amplified using primers. The vectors, α-signal peptide fragments, and β-lactoglobulin fragments (SEQ ID NO:40) were then seamlessly assembled to obtain recombinant plasmids pPAOX1-BLG and pPt13-c1-BLG.

[0123] 2. Construction of β-lactoglobulin expression strains

[0124] Recombinant plasmids pPAOX1-BLG and pPt13-c1-BLG were linearized and electroporated into Pichia pastoris strain GS115, respectively. The plasmids were then plated on YND solid medium and incubated at 30°C for approximately 72 hours. Single colonies were picked from the plates and transferred to YPD liquid medium, cultured at 30°C on a shaker, and the genome was extracted. PCR was used to verify the GFP expression cassette copy number, yielding single-copy strains AOX1-BLG and t13-c1-BLG.

[0125] Recombinant plasmids pGGsTF0016, pGGsTF0222, and pGGsTF0757 were linearized and electroporated into the β-lactoglobulin expression strains corresponding to the recombinant promoter t13-c1. The plasmids were then plated onto YPD solid medium supplemented with Zeocin antibiotic and incubated at 30°C for approximately 72 hours. Single colonies were picked from the plates and transferred to YPD liquid medium, cultured at 30°C on a shaker, and the genome was extracted. PCR was used to verify the copy number of the expression cassette, thus obtaining single-copy strains.

[0126] 3. Fermentation production of β-lactoglobulin

[0127] After fermentation, the supernatant was collected by centrifugation and the protein content was determined. The results are as follows: Figure 4 As shown in the figure, under methanol conditions, the yield of β-lactoglobulin corresponding to the synthetic promoter t13-c1 can reach that of commercial promoters. AOX1 The yield was 2.8 times higher than that of the commercial promoter, and the three expression tools also significantly outperformed the commercial promoter under four carbon source conditions in terms of β-lactoglobulin production. AOX1 The yield under methanol conditions can be up to 7.5 times higher. This result demonstrates that the recombinant promoter and expression tool established in this invention can be successfully applied to the production of β-lactoglobulin, and that the production capacity under conventional carbon sources such as glucose (D), glycerol (G), ethanol (E), or methanol (M) is superior to that of current commercial systems.

[0128] Sequence information:

Claims

1. A matching module for yeast expression regulation, comprising: (1) Control element; (2) DNA binding domain; and (3) The activation domain of a transcription factor, which forms a synthetic transcription factor with the DNA binding domain.

2. The matching module for yeast expression regulation as described in claim 1, characterized in that, (1) also includes a promoter; the regulatory element is operatively connected to the promoter to form a recombinant promoter for driving the expression of the target gene; Preferably, the control element is located upstream of the promoter; Preferably, the promoter includes a core promoter; Preferably, the downstream of the recombinant promoter includes an insertion site for the target gene; more preferably, the insertion site for the target gene is a restriction enzyme site.

3. The matching module for yeast expression regulation as described in any one of claims 1-2, characterized in that: The regulatory element has a nucleotide sequence shown in SEQ ID NO:1 or having more than 80% sequence identity with it; or The promoter has a nucleotide sequence shown in SEQ ID NO:3, positions 303-404; SEQ ID NO:4, positions 303-463; or SEQ ID NO:5, positions 303-483, or a sequence that has more than 80% sequence identity with it; or The recombinant promoter has a nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, or a sequence that has more than 80% sequence identity with it; or The DNA binding domain has the amino acid sequence shown in SEQ ID NO:2 or has more than 80% sequence identity with the sequence shown; or The activation domain of the transcription factor has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, or has more than 80% sequence identity with the shown sequence.

4. A separate regulatory element having the nucleotide sequence shown in SEQ ID NO: 1 or a functional variant thereof.

5. The use of the regulatory element according to claim 4, for operatively linking with a promoter to form a recombinant promoter, thereby regulating the efficient expression of the target gene; Preferably, the expression is an expression in yeast.

6. A recombinant promoter, comprising: The control element as described in claim 4, and the promoter operatively connected thereto; Preferably, the control element is located upstream of the promoter; Preferably, the promoter includes a core promoter; Preferably, the regulatory element is operatively linked to the promoter to form a recombinant promoter for driving the expression of the target gene; more preferably, the expression is in yeast.

7. An activation domain of a transcription factor having an amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, or having more than 80% sequence identity with the shown sequence.

8. A synthetic transcription factor comprising an operationally linked DNA-binding domain and an activation domain of the transcription factor; Preferably, the DNA binding domain has the amino acid sequence shown in SEQ ID NO:2 or has more than 80% sequence identity with the sequence shown; Preferably, the activation domain of the transcription factor has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13, or has more than 80% sequence identity with the shown sequence.

9. A method for expressing a target gene in yeast, comprising: (a) Provide a matching module for yeast expression regulation as described in any one of claims 2-3, insert a target gene downstream of the recombinant promoter, and transform it into yeast cells; (b) Culture yeast under a suitable carbon source and perform recombinant expression of the target gene; Preferably, the target gene includes: a reporter gene, or a gene encoding a food protein; More preferably, the genes encoding the food protein include the gene encoding brassinolide and the gene encoding β-lactoglobulin; Preferably, the yeast expression includes expression in a culture environment using glucose, glycerol, ethanol and / or methanol as carbon sources; Preferably, the yeast includes Pichia pastoris (Pichia pastoris). Pichia More preferably, the Pichia pastoris is Pichia pastoris (…). Pichia pastoris NRRL-Y 11430 or its derivatives, GS115 strain or its derivatives; Optionally, the 5' end of the target gene may be operatively linked to a natural or exogenous signal peptide encoding a nucleic acid.

10. A recombinant expression construct or a recombinant engineered yeast strain, comprising: The matching module for yeast expression regulation as described in any one of claims 1-3; or The control element as described in claim 4; or The recombinant promoter as described in claim 6.

11. A method for efficiently expressing brassin in yeast, comprising: (i) Providing yeast cells containing: expression cassette 1: comprising, from 5' to 3', the operatively linked recombinant promoter, an α-signal peptide encoding gene, and a brassinoprotein encoding gene; and expression cassette 2: comprising, from 5' to 3', an operatively linked promoter and an endogenous synthetic transcription factor encoding a nucleic acid, the transcription factor comprising: a DNA-binding domain and an activation domain of the transcription factor; (ii) Culture the yeast cells of (i) and perform recombinant expression of Brazil protein; preferably, glucose, glycerol, ethanol or methanol is used as the carbon source for the recombinant expression; Preferably, the endogenous recombination promoter in expression cassette 1 has the nucleotide sequence shown in SEQ ID NO:3; Preferably, the endogenous synthetic transcription factor in expression cassette 2 has the nucleotide sequence shown in SEQ ID NO: 6-8.

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